Journal articles on the topic 'Two Stage Clonal Expansion Model'

Abstract Leishmania, a unicellular eukaryotic parasite, is a unique model for aneuploidy and cellular heterogeneity, along with their potential role in adaptation to environmental stresses. Somy variation within clonal populations was previously explored in a small subset of chromosomes using fluorescence hybridization methods. This phenomenon, termed mosaic aneuploidy (MA), might have important evolutionary and functional implications but remains under-explored due to technological limitations. Here, we applied and validated a high throughput single-cell genome sequencing method to study for the first time the extent and dynamics of whole karyotype heterogeneity in two clonal populations of Leishmania promastigotes representing different stages of MA evolution in vitro. We found that drastic changes in karyotypes quickly emerge in a population stemming from an almost euploid founder cell. This possibly involves polyploidization/hybridization at an early stage of population expansion, followed by assorted ploidy reduction. During further stages of expansion, MA increases by moderate and gradual karyotypic alterations, affecting a defined subset of chromosomes. Our data provide the first complete characterization of MA in Leishmania and pave the way for further functional studies.

A stochastic two-stage cancer model with clonal expansion was used to investigate the potential impact on human lung cancer incidence of some aspects of the hormesis mechanisms suggested by Feinendegen ( Health Phys. 52 663–669, 1987). The model was applied to low doses of low-LET radiation delivered at low dose rates. Non-linear responses arise in the model because radiologically induced adaptations in radical scavenging and DNA repair may reduce the biological consequences of DNA damage formed by endogenous processes and ionizing radiation. Sensitivity studies were conducted to identify critical model inputs and to help define the changes in cellular defense mechanisms necessary to produce a lifetime probability for lung cancer that deviates from a linear no-threshold (LNT) type of response. Our studies suggest that lung cancer risk predictions may be very sensitive to the induction of DNA damage by endogenous processes. For doses comparable to background radiation levels, endogenous DNA damage may account for as much as 50 to 80% of the predicted lung cancers. For an additional lifetime dose of 1 Gy from low-LET radiation, endogenous processes may still account for as much as 20% of the predicted cancers ( Fig. 2 ). When both repair and scavengers are considered as inducible, radiation must enhance DNA repair and radical scavenging in excess of 30 to 40% of the baseline values to produce lifetime probabilities for lung cancer outside the range expected for endogenous processes and background radiation.

Cancer incidence rates are significantly different all over the world. Breast cancer is affected by many factors, the most important being genetics and lifestyle. The aim of this paper is to study the mutation mechanisms of breast cancer for Japanese women by fitting the incidence data of three high-quality population areas in Japan from 1985 to 2010. To achieve this goal, we have set up multi-stage models within the mathematical model of Moolgavkar, Venzon, and Knudson. Such models take both mutation rates and clonal expansion rates as parameters in each compartment into consideration. Based on our simulation outcomes, two to twelve driver mutations are sufficient in the pathway to female breast cancer in Japan. On the other hand, a previous study demonstrated that breast cancer in American women requires two to fourteen gene mutations to get a cancerous cell. Moreover, the 3-stage mathematical model is the optimal model as it fits clinical data very nicely for all affected cases of females in Japan and the US. The genetic instability has a prominent effect on the tumorigenesis of Japanese females caused by the first four driver mutations. The calculated results for Japanese women are compared with previous works for American women.

Myelofibrosis (MF) is the deadliest subtype of myeloproliferative neoplasm (MPN) with a median survival of approximately 5 years. Ruxolitinib, a front line therapy for JAK2V617F mutant MPN, can alleviate symptoms of the disease, but does not eliminate the malignant clone and has minimal impact on BM fibrosis and overall survival. Current mouse models do not recapitulate the clinical heterogeneity, clonal genetic composition, or morphological features of MF. Most notably, these models do not generate robust reticulin fibrosis in the bone marrow, the most significant MF pathology. This lack of clinically relevant MF models presents a major barrier to deciphering the complex genetic drivers of the disease and developing effective therapies against it. We evaluated the ability of CD34+ hematopoietic stem and progenitor cells (HSPCs) from MF patients (that contain the MPN-disease initiating population) to give rise to MF in xenotransplanted NSGS mice. >5x104 FACS-sorted CD34+ HSPCs from the peripheral blood of MF patients with JAK2V617F (n=12), CALRindels (n=7) and MPLW515L (n=2) were transplanted into sublethally irradiated (200rads) NSGS mice via X-ray guided intra-tibial injection. We observed robust engraftment of patient-derived cells at 12 weeks post-transplant regardless of their genetic background or donor patient disease severity (Fig 1A). Post-transplant, BM analysis revealed robust expansion of phenotypically defined MF HSCs relative to cord blood CD34+ control recipients, suggesting a permissive niche for MF HSCs to undergo self-renewal. Remarkably, transplantation of CD34+ cells produced other hallmarks of MF in recipient animals such as splenomegaly, thrombocytosis and most importantly BM reticulin fibrosis in all recipients (Fig 1B). We assessed the clonal architecture of engrafted human cells compared to the primary disease in the donor patients through exome sequencing of CD34+ cells prior to transplantation and hCD45+ cells from MF xenografts. We found that the clonal and subclonal mutational landscape observed in CD34+ cells prior to transplantation was maintained in recipient mice (Fig 1C), suggesting that the PDX model accurately reflects the cellular composition of the primary disease. Intriguingly, in two of the xenografted patient samples, we identified additional mutations that were not detected in the primary patient samples using standard sequencing - namely TP53R248Q and EZH2Y663H respectively. Two years after we detected these mutations in PDXs, these MF patients transformed into sAML with acquisition of TP53R248Q and EZH2Y663H mutations. We performed droplet digital PCR and demonstrated that indeed rare pre-leukemic subclones containing these mutations were present at low levels (< 0.01% VAF) in chronic stage MF patients at least two years prior to sAML progression. These data also suggested that these rare subclones responsible for leukemic transformation expand significantly (>300 fold) under the selective pressure of transplantation in NSGS mice. Additional validation of these findings in a further six pre-sAML MF patient samples is currently ongoing. If successful, this model could be used to prospectively identify rising leukemic clones in chronic stage MF patients, which are below the level of detect of standard sequencing as a mechanism to stratify such patients for more aggressive treatments. While sequencing can identify ultra-rare variants, it cannot discern their functional potential for sAML transformation, which is the advantage of this approach. Finally, we harnessed this system for pre-clinical studies, initially focusing on inhibiting the JAK/STAT signaling pathway. Ruxolitinib treatment in MF PDXs produced remarkably similar phenotypes as observed in patients. We observed a small, but significant reduction in engraftment of MF cells in the BM and a sharp reduction in spleen size in Ruxolitinib-treated group compared to vehicle control. Ruxoltinib treatment however did not reduce the frequency of MF HSCs, the disease initiating population or lessen the degree of reticulin fibrosis. These data suggest that this system can be used as a reliable, clinically-relevant drug screening platform. Taken together, we offer the field a critical, previously missing biologically relevant screening system for validation of MPN drug targets identified in cell lines or genetic mouse models prior to moving forward into clinical trials. Disclosures Oh: Blueprint Medicines: Consultancy; Celgene/BMS: Consultancy; Constellation: Consultancy; CTI Biopharma: Consultancy; Disc Medicine: Consultancy; Gilead Sciences: Consultancy; Incyte Corporation: Consultancy; Kartos Therapeutics: Consultancy; Novartis: Consultancy; PharmaEssentia: Consultancy.

Abstract Background: Human epidermal growth factor receptor 2 (HER2) positivity in breast cancer portends an aggressive tumor behavior and poor prognosis and is associated with a positive response to trastuzumab treatment. Prior immunohistochemistry and RNA sequencing of breast tumor tissues suggest that trastuzumab may recruit and activate anti-tumor T cells. Tumor infiltrating lymphocytes have been associated with improved response in patients with HER2+ early breast cancer treated with neoadjuvant trastuzumab plus chemotherapy. However, these cells have not previously been characterized at the single cell level in tumor tissue or in the periphery. Assessing the T cell component in the peripheral blood via single-cell sequencing enables high sensitivity detection of rare cells, may identify T cell antigen receptors (TCR) involved in the anti-tumor response, and could lead to a non-invasive means of monitoring trastuzumab-mediated immune activity. Here we perform single cell sequencing of the blood T cell repertoire in breast cancer patients pre- and post-trastuzumab treatment. Methods: To characterize T cell response in trastuzumab plus chemotherapy treated patients, we profiled peripheral CD3+ T cells using 10x Genomics VDJ single-cell sequencing in paired samples from five patients with HER2+ breast cancer. Patients had stage IIA (n=2), stage IIIC (n=2) or stage IV (n=1) breast cancer and were treated with preoperative docetaxel, carboplatin, trastuzumab, pertuzumab (TCHP). Two patients had a pathological complete response (pCR), and three patients had partial clinical response with residual disease at surgery. Peripheral blood mononuclear cells (PMBCs) were collected at a C1D1 pre-treatment and day 1 of cycles 3, 4, or 5. Results: Eleven T cell subpopulations, including naïve and memory CD4+ and CD8+ T cells, activated CD4+ and CD8+ T effector cells, activated CD4+ T regulatory cells, were characterized in the five patients’ peripheral blood based on their transcriptional profiles. T cell subpopulation distribution and clonal expansion profiles were similar in pre- and post- treatment samples in all five donors. Large T cell clonal expansions were detected in the peripheral blood of the two patients who had a pCR, but were not detected in the three patients who had residual disease at surgery. The patients who had a pCR exhibited large expansions in activated CD8+ terminal effector memory/effector memory (TEM/EM) cells followed by expansions in activated CD4+ TEM/EM cells. A minor increasing trend in activated CD4+ Treg cells was observed across all patients upon treatment with TCHP. Conclusions: Single-cell sequencing enables high-resolution characterization of immune cell subsets and represents a promising approach to assess the immune response to trastuzumab and other cancer therapeutics. In this study, single-cell sequencing of peripheral CD3+ T cells identified clonal expansions in activated CD8+ and CD4+ T cells in HER2+ breast cancer patients who had a pCR with preoperative TCHP treatment. These data are consistent with the model that T cells play a key role in trastuzumab-mediated tumor control, and warrant further investigation in a larger sample population. Citation Format: John Vivian, Kimberly Walter, Elliott Drabek, Nicole Haaser, Maren K. Levin, Esther San Roman Rodriguez, Kendra Peck, Ngan Nguyen, Carl Millward, Jonathan Benjamin, William H. Robinson, Joyce A. O'Shaughnessy. Single-cell sequencing of the blood T cell repertoire before and after trastuzumab treatment in early stage HER2+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-11.

Abstract Abstract 309 Background: The granulomonocytic (GM) hyperplasia of CMML has been attributed to GM-CSF hypersensitivity triggered by mutations in the CBL/RAS pathway according to the prevailing model in juvenile myelomonocytic leukemias (Kotecha Cancer Cell 2008). Recurrent mutations affecting epigenetic (eg TET2 and ASXL1) and splicing (eg SRSF2) machineries, or cytokine signaling (N/KRAS, CBL, JAK2) are present in most CMML cases, but none is specific of CMML. In 224 CMML patients (pts), we found TET2 (58%), SRSF2 (47%) and ASXL1 (38%) to be the most frequently mutated genes; only 66 (35%) cases had mutations in cytokine signaling genes (CBL, N/KRAS, JAK2, FLT3, KIT) (abstract submitted). We analyzed the differentiation of CD34+populations from genetically annotated CMML pts to address the mechanisms of GM hyperplasia in CMML. Methods: CD34+ populations (hematopoietic stem cells [HSC]; multipotent [MPP]; common myeloid [CMP] and granulomonocytic progenitors [GMP] defined by the CD34/CD38/CD90/CD123/CD45RA panel; Majeti Cell Stem Cell 2007) from 28 genetically annotated CMML and TET2 mutated MPN (n=8) or MDS (n=5) cases were cloned and genotyped for each mutation identified in mature CD14+ cells, and differentiated in vitro. Results: Early clonal dominance, with at least one mutation in > 75% of HSC/MPP clones, was found in all cases. In 18/19 pts with ≥2 mutations, a linear succession of mutations was found, with signaling mutations often following TET2 or ASXL1 mutations. Contrasting with the dominance of first events in HSC/MPP, second events reached clonal dominance in GMP, suggesting that they provide a selective advantage during the early steps of myeloid differentiation. We next analyzed the clonogenicity of peripheral blood (PB) CD34+ cells in the presence of GM-CSF (10 ng/mL) in 20 CMML cases and 4 controls. GM-CSF hypersensitivity (clonogenicity > mean+2SD of controls) was found in 7 (35%) cases. A mutation in a signaling gene was found in 6/7 pts (1 homozygous JAK2, 1 homozygous CBL, 4 heterozygous N/KRAS mutations), compared to 3/13 in pts without GM-CSF hypersensitivity (2 JAK2, 1 CBL, all heterozygous; P=.02) Median WBC was 29.2 and 11.4 G/L in pts with and without GM-CSF hypersensitivity, respectively (P=.08). The proportion of GMP in bone marrow (BM) CD34+cells was not significantly different in 33 CMML pts compared to 15 age-matched controls. Clonogenicity of GMP was similar in CMML and controls, except for a trend toward increased clonogenicity in pts with mutations in signaling genes. In contrast, the proportion of MPP and CMP was higher in CMML than in controls (P<.01 and P <.05, resp.). In erythromyeloid conditions (SCF, IL-3, G-CSF & EPO), both CMP and to a lesser extent MPP had an increased ability to form GM colonies at the expense of erythroid colonies (P <.001 and P<.01, resp.). Compared to healthy CMP, CMML CMP had and increased ability to mature into GMP in short-term culture, and increased PU.1 mRNA expression (P<.05), without significant changes in the levels of GATA1, CEBPA and CEBPB. Finally, in 16 pts, the proportion of GM colonies differentiating from CMP at the expense of erythroid colonies was inversely correlated to patient hemoglobin level (P=.002). Thus, premature GM differentiation of CMP, and to a lesser extent MPP, appears as the dominant mechanism of GM hyperplasia in CMML, whereas GM-CSF hypersensitivity and GMP expansion contribute only in the minority of patients with mutations in signaling genes. We next explored a possible link between early clonal dominance of TET2 mutations and premature GM differentiation. In TET2 mutated MPN (n=8) or MDS (n=5), the PB monocyte count was significantly correlated to the size of the TET2-mutated clone in the CD34+/CD38− (P=.006) rather than in the CD34+/CD38+ population (P=.08). Finally, functional invalidation by shRNA of TET2 in CD34+/CD38− followed by culture in the presence of SCF, IL-3, G-CSF & EPO caused a GM expansion that was not observed in CD34+/CD38+ cells. Similar analyses are underway for ASXL1. Conclusion: Our results suggest that early clonal dominance of mutations affecting the epigenetic machinery leading to premature GM differentiation of multipotent progenitors, rather than GM-CSF hypersensitivity, is the main mechanism of GM hyperplasia in CMML. This suggests a model whereby a single mutation can lead to different phenotypes, depending on the stage of differentiation at which the mutation has gained clonal dominance. Disclosures: No relevant conflicts of interest to declare.

Abstract INTRODUCTION: Breast cancer are the main cause of related deaths cancer among women, corresponding to 25% of new cases each year. When diagnosed at early stages, it has an overall five-year survival rate of up to 90%. However, in more advanced stages, survival is reduced to about 24%, with 90% of women in stage IV dying as a result of complications related to metastases. Considering that brain metastasis is an unfavorable prognostic site, and the identification of genetic-molecular profiles in primary tumors and in metastatic sites are a subject poorly described in the literature, we understand that the identification of mutational profiles may contribute to elucidate the genetic-molecular mechanisms associated with tumor progression. AIM: The aim of this study was to identify clonal and subclonal driver mutations that lead to evolution of metastatic clones from a breast cancer progression model. MATERIAL AND METHODS: For tumor progression model, automated extraction of DNA from buffy coat and paraffin samples of breast tumors and paired brain metastases (n=9) was performed. In the present work, we used a subclonal reconstruction model based on the combination of machine learning and population genetics concepts. This proposal is based on the frequency spectrum of each somatic mutation (SNVs or indels), considering VAF (Variant Allele Frequency - ratio of mapped reads of the mutant allele) in relation to the coverage of variant locus, as known as CCF (Cancer Cell Fraction). CCF is defined as the proportion of neoplastic cells that have a certain set of mutations and then is normalized considering the sample purity and the segments with changes in number of DNA copies (Copy Number Alterations). Then, a statistical model based on finite Dirichlet mixtures with mixed distributions is applied. In this model, Beta components capture clonal expansions and population genetics concepts were applied to mutant alleles in each population considering principles of cancer evolution. Finally, confidence was computed using both parametric and non-parametric bootstraps. The functions for building the model are implemented at https://caravagnalab.github.io/mobster/, and for visualization and construction of graphs, packages in R statistical-mathematical environment were used, such as ggplot2, sads, cli, clisymbols, cowplot, crayon, ctree, dndscv, dplyr, magrittr, reshape2, and tidyr. RESULTS: With this model was possible to identify the distribution of clones according to somatic alterations in patients with breast cancer and brain metastasis. It was possible to observe common patterns, such as alterations of the SF3B1 gene as a common ancestral clone in both conditions and the frequency of the AKT1 gene in a subclonal condition. Other genes relevant to breast cancer carcinogenesis, such as PIK3CA and TP53, are found in a different clonal hierarchical pattern between the two conditions. CONCLUSION: This data suggests a model of clonal evolution capable to identify which drivers clones and subclones are involved in the metastatic process. Citation Format: Muriele B. Varuzza, Adriane F. Evangelista, Cristiano P. Souza, Márcia C. Marques. TUMOR PROGRESSION MODEL IN BREAST CANCER WITH BRAIN METASTASIS [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-13-01.

Photodynamic therapy (PDT) is a two-stage treatment relying on cytotoxicity induced by photoexcitation of a nontoxic dye, called photosensitizer (PS). Using 5-aminolevulinic acid (5-ALA), the pro-drug of PS protoporphyrin IX, we investigated the impact of PDT on hepatocellular carcinoma (HCC). Optimal 5-ALA PDT dose was determined on three HCC cell lines by analyzing cell death after treatment with varying doses. HCC-patient-derived tumor hepatocytes and healthy donor liver myofibroblasts were treated with optimal 5-ALA PDT doses. The proliferation of cancer cells and healthy donor immune cells cultured with 5-ALA-PDT-treated conditioned media was analyzed. Finally, therapy efficacy on humanized SCID mice model of HCC was investigated. 5-ALA PDT induced a dose-dependent decrease in viability, with an up-to-four-fold reduction in viability of patient tumor hepatocytes. The 5-ALA PDT treated conditioned media induced immune cell clonal expansion. 5-ALA PDT has no impact on myofibroblasts in terms of viability, while their activation decreased cancer cell proliferation and reduced the tumor growth rate of the in vivo model. For the first time, 5-ALA PDT has been validated on primary patient tumor hepatocytes and donor healthy liver myofibroblasts. 5-ALA PDT may be an effective anti-HCC therapy, which might induce an anti-tumor immune response.

Colorectal adenoma are precursor lesions on the pathway to cancer. Their removal in screening colonoscopies has markedly reduced rates of cancer incidence and death. Generic models of adenoma growth and transition to cancer can guide the implementation of screening strategies. But adenoma shape has rarely featured as a relevant risk factor. Against this backdrop we aim to demonstrate that shape influences growth dynamics and cancer risk. Stochastic cell-based models are applied to a data set of 197,347 Bavarian outpatients who had colonoscopies from 2006-2009, 50,649 patients were reported with adenoma and 296 patients had cancer. For multi-stage clonal expansion (MSCE) models with up to three initiating stages parameters were estimated by fits to data sets of all shapes combined, and of sessile (70% of all adenoma), peduncular (17%) and flat (13%) adenoma separately for both sexes. Pertinent features of adenoma growth present themselves in contrast to previous assumptions. Stem cells with initial molecular changes residing in early adenoma predominantly multiply within two-dimensional structures such as crypts. For these cells mutation and division rates decrease with age. The absolute number of initiated cells in an adenoma of size 1 cm is small around 103, related to all bulk cells they constitute a share of about 10−5. The notion of very few proliferating stem cells with age-decreasing division rates is supported by cell marker experiments. The probability for adenoma transiting to cancer increases with squared linear size and shows a shape dependence. Compared to peduncular and flat adenoma, it is twice as high for sessile adenoma of the same size. We present a simple mathematical expression for the hazard ratio of interval cancers which provides a mechanistic understanding of this important quality indicator. We conclude that adenoma shape deserves closer consideration in screening strategies and as risk factor for transition to cancer.

JAK2-V617F is the most frequently recurring somatic mutation in patients with myeloproliferative neoplasm (MPN), but it can also be found in healthy individuals with clonal hematopoiesis of indeterminate potential (CHIP) with a frequency much higher than the incidence of MPN. This suggests that the acquisition of the JAK2-V617F is not the rate-limiting step and other factors might be required for the expansion of the JAK2 mutated clone and initiation of MPN disease. Chronic inflammation is a hallmark of advanced MPN and is associated with progression to myelofibrosis and AML. Interleukin-1β (IL-1β) is one of the master regulators of the inflammatory state and its aberrant activity has been implicated in various pathological diseases including MPN. Here we focused on the early stages of MPN disease initiation and examined the role of IL-1β in this context. We hypothesized that IL-1β mediated inflammation may promote early expansion of the JAK2 mutant clone to reach a critical clone size capable of initiating MPN. We used a genetic approach and crossed IL-1β knockout (IL1β-/-) mice with our tamoxifen inducible SclCreER;JAK2-V617F (VF) mice, generating a triple mutant SclCreER;JAK2-V617F;IL-1β-/-(VF;IL1β-/-) line. We then transplanted two million bone marrow (BM) cells from VF and VF;IL1β-/- mice into lethally irradiated wildtype (WT) orIL1β-/- recipients. Complete blood counts monitored every 4 weeks for up to 32 weeks post transplantation showed reduced platelet, neutrophil, leukocyte and monocyte counts in mice transplanted with VF;IL1β-/-as compared to VF. Furthermore, terminal analysis at week 16 and 32 revealed reduced splenomegaly and bone marrow fibrosis in the mice receiving VF cells lacking IL1β. This experiment shows that IL1β plays an important role in MPN pathogenesis in this mouse model. To test the hypothesis that IL1β favors clonal expansion during MPN disease initiation, we performed competitive dilution assays by mixing BM cells from VF or VF;IL1β-/-mice that also co-express the GFP protein as a reporter (VF;GFP or VF;IL1β-/-;GFP) with BM cells from IL1β-/- mice in 1:100 ratio and transplanted into lethally irradiated WT recipients (Figure 1A). Successful engraftment was defined by presence of &gt;1% GFP+ cells within Gr-1+ granulocytes in peripheral blood (PB) at week 18 after transplantation. In mice transplanted with VF;GFP, we found engraftment in 25 of 29 (86%) recipients whereas in mice transplanted with VF;IL1β-/-;GFP, only 18 of 29 (62%) recipients showed engraftment. Moreover, 10 of 25 (40%) mice engrafted with VF;GFP developed MPN at 24 weeks after transplantation as compared to only 2 of 18 (11%) mice engrafted with VF;IL1β-/-;GFP cells. GFP chimerism measured every 6 weeks in peripheral blood (PB) from erythroid (Ter119+), megakaryocytic (CD61+) and granulocytic lineages (Gr-1+) was significantly reduced in mice transplanted with VF;IL1β-/-;GFP compared to mice transplanted with VF;GFP cells (Figure 1A), suggesting the capacity to produce IL-1β protein by the VF cells was promoting the expansion of the clone and MPN manifestation.To define the relative contributions of hematopoietic and non-hematopoietic cell derived IL-1β in promoting MPN initiation, we performed competitive dilution assays in IL1β-/-recipients (Figure 1B). We detected engraftment in 27 of 30 (90%) IL1β-/-recipients transplanted with VF;GFP and 27 of 33 (82%) mice transplanted with VF;IL1β-/-;GFP. Furthermore, 9 of 27 (33%) mice engrafted with either VF;GFP or VF;IL1β-/-;GFP developed MPN at 24 weeks after transplantation. However GFP chimerism in Ter119, Gr-1 and CD61 was lower in mice transplanted with VF;IL1β-/-;GFP compared to mice transplanted with VF;GFP (Figure 1B). We further looked at plasma IL-1β protein levels by ELISA (Figure 1C). Interestingly, we found that IL-1β protein levels were also reduced in WT mice transplanted with VF;IL1β-/-;GFP donor cells, indicating that the non-hematopoietic WT cell cannot compensate for the deficiency of IL-1β in the VF clone. Overall, our results demonstrate that IL-1β favors early clonal expansion and show that IL-1β produced by the JAK2 mutant cells is required for optimal MPN disease initiation. Disclosures No relevant conflicts of interest to declare.

Introduction Large-scale sequencing studies have unraveled the mutational landscape of myelofibrosis (MF), demonstrating clonal heterogeneity and importance of genetically defined subgroups in disease prognosis and progression. In order to elucidate the genetics of MF progression and its molecular drivers during JAK inhibition therapy, we performed in-depth genetic studies on longitudinal blood samples from 15 MF patients covering a disease span of 3 to 5 years after initiation of ruxolitinib. Methods Sequential samples from 15 MF patients (PMF n=8; post-ET/PV-MF n=7) accounting for a total of 42 time points representing 58.5 years of ruxolitinib treatment were investigated by whole-exome sequencing (WES). Additionally, we performed targeted deep sequencing of patient-specific mutations in flow-sorted cell fractions to study clonal repartition within the hematopoietic differentiation tree. Finally, we genotyped more than 5000 Lin-CD34+ progenitor cells using a single-cell multiplexed qPCR approach on a micro-fluidic platform (Fluidigm) to infer MF phylogeny. Results WES identified a median of 14 non-silent somatic mutations per patient at initiation of ruxolitinib treatment (=baseline WES; Figure 1A). When comparing mutations between first and last investigated time points, the majority of baseline mutations (162/201=81%) could be detected also at a later disease stage. A total of 39 mutations were lost and 80 new mutations were detected at the last time point. All patients showed at least one gained/ lost mutation in sequential samples. We noted frequent acquisition of mutations in genes of the RAS/RTK pathways in one third of patients. Two patients with a JAK2 V617F mutation achieved a molecular remission at a level of persisting residual disease of 1x10-3 with ruxolitinib therapy. In one of them, a total of 13 mutations were detected at baseline. In the second sample, taken three years later, a completely different set of mutations was identified and at the last time point, four years after initiation of therapy, none of the mutations were detected. This likely represents genetic drift during neutral evolution as a consequence of a rapid expansion after JAK inhibition. All other 13 patients showed only a modest - if any - decrease of 10-20% JAK2/CALR allele burden which was often accompanied with the expansion of JAK2/CALR-wildtype clones due to positive selection and/or freed clonal space under treatment. However, in some patients with durable response to ruxolitinib, we noted opposing dynamics of clones questioning a common origin. The three patients who progressed to leukemia showed a higher number of mutations at baseline and all of them acquired mutations in KRAS or NRAS over time. As one example, MPN18 harbored mutations in ASXL1, ETV6, and SRSF2 at baseline. Thereafter, and in addition to other driver genes (IDH2,KRAS) a second JAK2 Mutation at codon R867 was acquired, which has been reported to confer treatment resistance to JAK Inhibitors (Marty, Blood 2014). Mutation analysis in flow-sorted cell fractions showed a higher allelic mutation load in the myeloid compared to the lymphoid compartment with only few mutations being detected at low allele frequency in lymphocytes. Interestingly, some patients showed evidence of differential expansion among different myeloid cell lineages (Figure 1B). Next, we sorted 480 CD34+ single-cells per sample from 12 time points from 8 patients which allowed identification of subclones at ≥2% frequency based on priori power calculations. Sorting errors (e.g.cell doublets, empty wells)determined the mean cell sorting failure rate to be 12.5%. We employed a heuristic search algorithm to select a phylogenetic tree with Maximum Likelihood under a finite site model of evolution. Loss of heterozygosity (LOH) events were found in 7/8 patients and were not restricted to the JAK2 locus. In some patients, LOH of JAK2 occurred independently in two subclones, a phenomenon of convergent evolution (Figure 1C). We also noted cases with multiple 9pUPDs, of which one got selected during therapy. LOH events gave rise to both, a mutant homozygous but also reversion to a wildtype genotype. Conclusions Comprehensive serial genotyping of MF patients treated with ruxolitinib revealed heterogeneous patterns of clonal composition and evolution. Our data support LOH as a major determination factor for clonal diversification in MF. EM, KY, and MF contributed equally Figure 1 Disclosures Zenz: Abbvie: Consultancy, Honoraria, Other: Travel support; Roche: Consultancy, Other: Travel support; Janssen: Consultancy; Takeda: Consultancy; Gilead: Honoraria. Bullinger:Pfizer: Honoraria; Astellas: Honoraria; Amgen: Honoraria; Abbvie: Honoraria; Bayer: Other: Financing of scientific research; Seattle Genetics: Honoraria; Sanofi: Honoraria; Novartis: Honoraria; Menarini: Honoraria; Jazz Pharmaceuticals: Honoraria; Janssen: Honoraria; Hexal: Honoraria; Gilead: Honoraria; Daiichi Sankyo: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria. Le Coutre:Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau. Ogawa:Kan Research Laboratory, Inc.: Consultancy; Asahi Genomics: Equity Ownership; Qiagen Corporation: Patents & Royalties; RegCell Corporation: Equity Ownership; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding. Damm:Novartis: Research Funding; AbbVie: Other: Travel support.

Abstract Clonal evolution of DCIS to invasion Ductal carcinoma in situ (DCIS) is the most common form of preinvasive breast cancer and, despite treatment, a small fraction (5-10%) of DCIS patients develop subsequent invasive breast cancer (IBC). If not treated, at least 3 out of 4 women with DCIS will not develop IBC1-3. This implies many women with non-progressive, low-risk DCIS are likely to carry the burden of overtreatment. To solve this DCIS dilemma, two fundamental questions need to be answered. The first question is, how the subsequent IBC is related to the initial DCIS lesion. The second question is how to distinguish high- from low-risk DCIS at the time of diagnosis. This is essential to take well-informed DCIS management decisions, i.e., surgery, followed by radiotherapy in case of breast conserving treatment with or without subsequent endocrine treatment, or test whether active surveillance for low-risk DCIS is safe. How is the subsequent IBC related to the initial DCIS? The high genomic concordance in DNA aberrations between DCIS and IBC suggest that most driver mutations and CNA events are acquired at the earliest stages of DCIS initiation. It has therefore been assumed that most solid tumours arise from a single cell and that the probability of two independent tumours arising from the same tissue is low4-6. However, lineage tracing and genomic studies strongly suggest both direct and independent clonal lineages during the initiation of DCIS and evolution to IBC. In these processes, mammary stem cells have been implicated in DCIS initiation. Role of mammary stem cells in DCIS initiation Lineage tracing mouse model experiments have shown the fate of individual cells and lineages that acquire mutations before a tumour is established7-9. This is also relevant for DCIS initiation10,11, as different pools of MaSCs drive the growth and development of the ductal network and are considered the cell of origin for breast cancers9,10. The ductal trees remain quiescent until puberty, during which extension, branching and termination of terminal end buds (TEBs) leads to its expansion throughout the fat pad7,12,13. Any oncogenic mutation that occurs in a fetal MaSC will spread throughout the ductal network to a large part of the ductal tree, leading to sick lobes9. By contrast, oncogenic mutations acquired by a single MaSC during puberty spread to a smaller number of offspring located in small clusters in a part of the ductal network8,14. Direct lineage models for DCIS progression Direct lineage models postulate that DCIS has a single cell of origin that acquires mutations and progresses to IBC15-18. This is also supported by the high genomic concordance of CNAs and mutations in synchronous DCIS–IBC regions6,15,17,19-21 and the results of a recent large longitudinal study that profiled pure DCIS and recurrent IBC using multiple sequencing techniques, which estimated direct clonal lineages in approximately ~80% of patients18. Two distinct direct lineage models have been proposed: the evolutionary bottleneck model and the multiclonal invasion model. In the evolutionary bottleneckmodel, a single clone (or a limited number of clones) with an invasive genotype is selected and breaks through the basement membrane to migrate into surrounding tissues15,16,22, while other clones are unable to escape the ducts21-28. The multiclonal invasion model posits that most or all subclones can escape the basement membrane, establishing invasive disease6,16,17,20. The multiclonal model has not been studied widely in pure DCIS and recurrent IBC samples. Independent lineage model for DCIS progression DCIS lesions and IBCs can arise from different initiating cells in the same breast independently5,20,29-32. An analysis of sequential DCIS–IBC pairs in a unique, large-scale, in-depth study of 95 matched pure DCIS and recurrent IBC showed that ~20% of the IBC recurrences were indeed clonally unrelated to the primary DCIS18, as is also supported by some mathematical model studies33. The potential role of a field effect IBC can develop in the same breast as an initial DCIS even after treatment, which could be explained by the presence of a field effect34-37. Alternatively, the sick lobe hypothesis proposes that a single lobe harbours first-hit mutations, acquired in utero or during early mammary development37-42. This could also explain the restriction of IBC to the ipsilateral side of the breast39,43,44. Germline mutations may also explain the emergence of independent lineages in DCIS and IBC patients, lowering the threshold for cancer development32,43-46. Convergent evolution model of DCIS progression A third model for the emergence of IBC from DCIS is convergent evolution, in which the same mutations and CNA are selected and expanded during tumour growth such that environmental factors fuel competition between distinct clones and push them towards a similar genotype. Ultimately, two independent clonal lineages from different ancestral cells then happen to share multiple genomic aberrations or driver mutations across regions47-49. Although independent lineages are considered uncommon (~20%) in ipsilateral recurrences, they occur at much higher frequencies in contralateral recurrences (&gt;80%), in which single-nucleotide polymorphism and comparative genomic hybridization microarrays show few (or no) genomic alterations shared in tumours from the contralateral breast cancer18,50,51. How to distinguish high- from low-risk DCIS at the time of diagnosis? The genomic and transcriptomic profile present at the time of DCIS diagnosis may contain crucial information on the risk of progression of DCIS to IBC. Thus far, it has been unclear whether prognostic gene expression markers can be used to separate indolent DCIS from potentially progressive DCIS. To this end, microarrays and RNA-seq have been applied for the comparison of bulk RNA from microdissected DCIS and IBC tissue. In synchronous DCIS–IBC, a limited number of transcriptional differences have been found and the few events discovered often varied extensively across different tumours52-56. Although these differences were strong, the added value of these studies is uncertain as they are often confounded by small sample size, lack of matched receptor status data, and low sample purity. Despite these limitations, these studies have implicated the epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) remodelling pathways as potentially relevant for the progression of DCIS to IBC55-62. We studied two large DCIS cohorts: the Sloane cohort, a prospective breast screening cohort from the UK (median follow-up of 12.5 years), and a Dutch population-based cohort (NKI, median follow-up of 13 years). FFPE tissue specimens from patients with pure primary DCIS after breast-conserving surgery (BCS) +/- RT that did develop a subsequent ipsilateral event (DCIS or invasive) were considered as cases, whereas patients that did not develop any form of recurrence up to the last follow-up or death were considered as controls. We performed copy number analysis (CNA) and RNAseq analysis on 229 cases (149 IBC recurrences and 80 DCIS recurrences) and 344 controls. We classified DCIS into the PAM50 subtypes using RNAseq data which revealed an enrichment of luminal A phenotype in DCIS that did not recur (P = 0.01, Fisher Exact test). No single copy number aberration was more common in cases compared to controls. RNAseq data did not reveal any genes significantly over/under expressed in cases versus controls after false discovery rate (FDR) correction. However, by limiting the analysis to samples that had not had RT and excluding pure DCIS recurrences we developed a penalized Cox model from RNAseq data. The model was trained on weighted samples (to correct for the biased sampling of the case control dataset) from the NKI series with double loop cross validation. Using this predicted hazard ratio, the samples were split into high, medium and low risk quantiles, with a recurrence risk of 20%, 9% and 2.5%, respectively at 5 years (p&lt;0.001, Wald test). The NKI-trained predictor was independently validated in the Sloane No RT cohort (p = 0.02, Wald test). GSEA analysis revealed proliferation hallmarks enriched in the recurrence predictor (FDR = 0.058). The NKI-RNAseq predictor was more predictive of invasive recurrence than PAM50, clinical features (Grade, Her2 and ER) and the 12-gene Oncotype DCIS score (p &lt; 0.001, permutation test using the Wald statistic) in both the NKI and Sloane series. In the methylation analysis, 50 controls were compared with 35 cases. We could identify Variably Methylation Regions (VMRs) and Differentially Methylated Regions (DMRs) between cases and controls. Interestingly, VMRs were enriched in cell adhesion pathways Conclusion The recently acquired knowledge described above on how often the subsequent IBC is directly related to the initial DCIS and on molecular markers predicting the risk of DCIS progression is essential for accurate DCIS risk assessment. This is essential to aid accurate clinical decision making to personalize DCIS management in the near future. References 1. Falk, R. S., Hofvind, S., Skaane, P. & Haldorsen, T. Second events following ductal carcinoma in situ of the breast: a register-based cohort study. Breast Cancer Res Treat 129, 929-938, doi:10.1007/s10549-011-1531-1 (2011). 2. Ryser, M. D. et al. Cancer Outcomes in DCIS Patients Without Locoregional Treatment. Jnci J National Cancer Inst 111, 952-960, doi:10.1093/jnci/djy220 (2019). 3. Maxwell, A. J. et al. Unresected screen detected Ductal Carcinoma in Situ: outcomes of 311 women in the Forget-me–not 2 study. Breast 61, 145-155, doi:10.1016/j.breast.2022.01.001 (2022). 4. Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 144, 646-674, doi:10.1016/j.cell.2011.02.013 (2011). 5. Kim, H., Kim, C. Y., Park, K. H. & Kim, A. Clonality analysis of multifocal ipsilateral breast carcinomas using X-chromosome inactivation patterns. Hum Pathol 78, 106-114, doi:10.1016/j.humpath.2018.04.016 (2018). 6. Bergholtz, H. et al. Comparable cancer-relevant mutation profiles in synchronous ductal carcinoma in situ and invasive breast cancer. Cancer Rep (Hoboken) 3, e1248, doi:10.1002/cnr2.1248 (2020). 7. Giraddi, R. R. et al. Stem and progenitor cell division kinetics during postnatal mouse mammary gland development. Nat Commun 6, 8487, doi:10.1038/ncomms9487 (2015). 8. Scheele, C. L. et al. Identity and dynamics of mammary stem cells during branching morphogenesis. Nature 542, 313-317, doi:10.1038/nature21046 (2017). 9. Ying, Z. & Beronja, S. Embryonic Barcoding of Equipotent Mammary Progenitors Functionally Identifies Breast Cancer Drivers. Cell Stem Cell 26, 403-419.e404, doi:10.1016/j.stem.2020.01.009 (2020). 10. Zhou, J. et al. Stem Cells and Cellular Origins of Breast Cancer: Updates in the Rationale, Controversies, and Therapeutic Implications. Front Oncol 9, 820, doi:10.3389/fonc.2019.00820 (2019). 11. Watson, C. J. & Khaled, W. T. Mammary development in the embryo and adult: new insights into the journey of morphogenesis and commitment. Development 147, doi:10.1242/dev.169862 (2020). 12. Williams, J. M. & Daniel, C. W. Mammary ductal elongation: differentiation of myoepithelium and basal lamina during branching morphogenesis. Dev Biol 97, 274-290, doi:10.1016/0012-1606(83)90086-6 (1983). 13. Silberstein, G. B. & Daniel, C. W. Glycosaminoglycans in the basal lamina and extracellular matrix of serially aged mouse mammary ducts. Mech Ageing Dev 24, 151-162, doi:10.1016/0047-6374(84)90067-8 (1984). 14. Davis, F. M. et al. Single-cell lineage tracing in the mammary gland reveals stochastic clonal dispersion of stem/progenitor cell progeny. Nat Commun 7, 13053, doi:10.1038/ncomms13053 (2016). 15. Hernandez, L. et al. Genomic and mutational profiling of ductal carcinomas in situ and matched adjacent invasive breast cancers reveals intra-tumour genetic heterogeneity and clonal selection. J Pathol 227, 42-52, doi:10.1002/path.3990 (2012). 16. Casasent, A. K., Edgerton, M. & Navin, N. E. Genome evolution in ductal carcinoma in situ: invasion of the clones. J Pathol 241, 208-218, doi:10.1002/path.4840 (2017). 17. Casasent, A. K. et al. Multiclonal Invasion in Breast Tumors Identified by Topographic Single Cell Sequencing. Cell 172, 205-217 e212, doi:10.1016/j.cell.2017.12.007 (2018). 18. Lips, E. H. et al. Genomic analysis defines clonal relationships of ductal carcinoma in situ and recurrent invasive breast cancer. Nat Genet 54, 850–860, doi:10.1038/s41588-022-01082-3 (2022). 19. Miron, A. et al. PIK3CA mutations in in situ and invasive breast carcinomas. Cancer Res 70, 5674-5678, doi:10.1158/0008-5472.CAN-08-2660 (2010). 20. Yates, L. R. et al. Subclonal diversification of primary breast cancer revealed by multiregion sequencing. Nat Med 21, 751-759, doi:10.1038/nm.3886 (2015). 21. Pareja, F. et al. Whole-Exome Sequencing Analysis of the Progression from Non-Low-Grade Ductal Carcinoma In Situ to Invasive Ductal Carcinoma. Clin Cancer Res 26, 3682-3693, doi:10.1158/1078-0432.CCR-19-2563 (2020). 22. Trinh, A. et al. Genomic Alterations during the In Situ to Invasive Ductal Breast Carcinoma Transition Shaped by the Immune System. Mol Cancer Res 19, 623-635, doi:10.1158/1541-7786.MCR-20-0949 (2021). 23. Poste, G. & Fidler, I. J. The pathogenesis of cancer metastasis. Nature 283, 139-146, doi:10.1038/283139a0 (1980). 24. Greaves, M. & Maley, C. C. Clonal evolution in cancer. Nature 481, 306-313, doi:10.1038/nature10762 (2012). 25. Kroigard, A. B. et al. Clonal expansion and linear genome evolution through breast cancer progression from pre-invasive stages to asynchronous metastasis. Oncotarget 6, 5634-5649, doi:10.18632/oncotarget.3111 (2015). 26. Martelotto, L. G. et al. Whole-genome single-cell copy number profiling from formalin-fixed paraffin-embedded samples. Nat Med 23, 376-385, doi:10.1038/nm.4279 (2017). 27. Walens, A. et al. Adaptation and selection shape clonal evolution of tumors during residual disease and recurrence. Nat Commun 11, 5017, doi:10.1038/s41467-020-18730-z (2020). 28. Welter, L. et al. Treatment response and tumor evolution: lessons from an extended series of multianalyte liquid biopsies in a metastatic breast cancer patient. Cold Spring Harb Mol Case Stud 6, doi:10.1101/mcs.a005819 (2020). 29. Maggrah, A. et al. Paired ductal carcinoma in situ and invasive breast cancer lesions in the D-loop of the mitochondrial genome indicate a cancerization field effect. Biomed Res Int 2013, 379438, doi:10.1155/2013/379438 (2013). 30. Desmedt, C. et al. Uncovering the genomic heterogeneity of multifocal breast cancer. J Pathol 236, 457-466, doi:10.1002/path.4540 (2015). 31. Visser, L. L. et al. Discordant Marker Expression Between Invasive Breast Carcinoma and Corresponding Synchronous and Preceding DCIS. Am J Surg Pathol 43, 1574-1582, doi:10.1097/PAS.0000000000001306 (2019). 32. McCrorie, A. D. et al. Multifocal breast cancers are more prevalent in BRCA2 versus BRCA1 mutation carriers. J Pathol Clin Res 6, 146-153, doi:10.1002/cjp2.155 (2020). 33. Sontag, L. & Axelrod, D. E. Evaluation of pathways for progression of heterogeneous breast tumors. J Theor Biol 232, 179-189, doi:10.1016/j.jtbi.2004.08.002 (2005). 34. Mai, K. T. Morphological evidence for field effect as a mechanism for tumour spread in mammary Paget’s disease. Histopathology 35, 567-576, doi:10.1046/j.1365-2559.1999.00788.x (1999). 35. Foschini, M. P. et al. Genetic clonal mapping of in situ and invasive ductal carcinoma indicates the field cancerization phenomenon in the breast. Hum Pathol 44, 1310-1319, doi:10.1016/j.humpath.2012.09.022 (2013). 36. Asioli, S., Morandi, L., Cavatorta, C., Cucchi, M. C. & Foschini, M. P. The impact of field cancerization on the extent of duct carcinoma in situ (DCIS) in breast tissue after conservative excision. Eur J Surg Oncol 42, 1806-1813, doi:10.1016/j.ejso.2016.07.005 (2016). 37. Tan, M. P. Integration of ’sick lobe hypothesis’ with concept of field cancerisation for a personalised surgical margin for breast conserving surgery. J Surg Oncol 116, 954-955, doi:10.1002/jso.24728 (2017). 38. Going, J. J. & Mohun, T. J. Human breast duct anatomy, the ’sick lobe’ hypothesis and intraductal approaches to breast cancer. Breast Cancer Res Treat 97, 285-291, doi:10.1007/s10549-005-9122-7 (2006). 39. Tot, T. The theory of the sick breast lobe and the possible consequences. Int J Surg Pathol 15, 369-375, doi:10.1177/1066896907302225 (2007). 40. Dooley, W., Bong, J. & Parker, J. Redefining lumpectomy using a modification of the "sick lobe" hypothesis and ductal anatomy. Int J Breast Cancer 2011, 726384, doi:10.4061/2011/726384 (2011). 41. Tan, M. P. & Tot, T. The sick lobe hypothesis, field cancerisation and the new era of precision breast surgery. Gland Surg 7, 611-618, doi:10.21037/gs.2018.09.08 (2018). 42. Petrova, S. C. et al. Regulation of breast cancer oncogenesis by the cell of origin’s differentiation state. Oncotarget 11, 3832-3848, doi:10.18632/oncotarget.27783 (2020). 43. Knudson, A. G., Jr. Heredity and human cancer. Am J Pathol 77, 77-84 (1974). 44. Park, S., Supek, F. & Lehner, B. Systematic discovery of germline cancer predisposition genes through the identification of somatic second hits. Nat Commun 9, 2601, doi:10.1038/s41467-018-04900-7 (2018). 45. Konishi, H. et al. Mutation of a single allele of the cancer susceptibility gene BRCA1 leads to genomic instability in human breast epithelial cells. Proc Natl Acad Sci U S A 108, 17773-17778, doi:10.1073/pnas.1110969108 (2011). 46. Mazzola, E., Cheng, S. C. & Parmigiani, G. The penetrance of ductal carcinoma in situ among BRCA1 and BRCA2 mutation carriers. Breast Cancer Res Treat 137, 315-318, doi:10.1007/s10549-012-2345-5 (2013). 47. Tegze, B. et al. Parallel evolution under chemotherapy pressure in 29 breast cancer cell lines results in dissimilar mechanisms of resistance. PLoS One 7, e30804, doi:10.1371/journal.pone.0030804 (2012). 48. Gao, Y. et al. Single-cell sequencing deciphers a convergent evolution of copy number alterations from primary to circulating tumor cells. Genome Res 27, 1312-1322, doi:10.1101/gr.216788.116 (2017). 49. Wang, F. et al. MEDALT: single-cell copy number lineage tracing enabling gene discovery. Genome Biol 22, 70, doi:10.1186/s13059-021-02291-5 (2021). 50. Brommesson, S. et al. Tiling array-CGH for the assessment of genomic similarities among synchronous unilateral and bilateral invasive breast cancer tumor pairs. BMC Clin Pathol 8, 6, doi:10.1186/1472-6890-8-6 (2008). 51. Regitnig, P., Ploner, F., Maderbacher, M. & Lax, S. F. Bilateral carcinomas of the breast with local recurrence: analysis of genetic relationship of the tumors. Mod Pathol 17, 597-602, doi:10.1038/modpathol.3800089 (2004). 52. Ma, X. J. et al. Gene expression profiles of human breast cancer progression. Proc Natl Acad Sci U S A 100, 5974-5979, doi:10.1073/pnas.0931261100 (2003). 53. Porter, D. et al. Molecular markers in ductal carcinoma in situ of the breast. Mol Cancer Res 1, 362-375 (2003). 54. Castro, N. P. et al. Evidence that molecular changes in cells occur before morphological alterations during the progression of breast ductal carcinoma. Breast Cancer Research 10, doi:ARTN R87 10.1186/bcr2157 (2008). 55. Dettogni, R. S. et al. Potential biomarkers of ductal carcinoma in situ progression. BMC Cancer 20, 119, doi:10.1186/s12885-020-6608-y (2020). 56. Song, G. et al. Identification of aberrant gene expression during breast ductal carcinoma in situ progression to invasive ductal carcinoma. J Int Med Res 48, 300060518815364, doi:10.1177/0300060518815364 (2020). 57. Abba, M. C. et al. Transcriptomic changes in human breast cancer progression as determined by serial analysis of gene expression. Breast Cancer Res 6, R499-513, doi:10.1186/bcr899 (2004). 58. Schuetz, C. S. et al. Progression-specific genes identified by expression profiling of matched ductal carcinomas in situ and invasive breast tumors, combining laser capture microdissection and oligonucleotide microarray analysis. Cancer Res 66, 5278-5286, doi:10.1158/0008-5472.CAN-05-4610 (2006). 59. Lee, S. et al. Differentially expressed genes regulating the progression of ductal carcinoma in situ to invasive breast cancer. Cancer Res 72, 4574-4586, doi:10.1158/0008-5472.CAN-12-0636 (2012). 60. Coradini, D., Boracchi, P., Ambrogi, F., Biganzoli, E. & Oriana, S. Cell polarity, epithelial-mesenchymal transition, and cell-fate decision gene expression in ductal carcinoma in situ. Int J Surg Oncol 2012, 984346, doi:10.1155/2012/984346 (2012). 61. Knudsen, E. S. et al. Progression of ductal carcinoma in situ to invasive breast cancer is associated with gene expression programs of EMT and myoepithelia. Breast Cancer Res Treat 133, 1009-1024, doi:10.1007/s10549-011-1894-3 (2012). 62. Krstic, M. et al. TBX3 promotes progression of pre-invasive breast cancer cells by inducing EMT and directly up-regulating SLUG. Journal of Pathology 248, 191-203, doi:10.1002/path.5245 (2019). Citation Format: Jelle Wesseling. Clonal evolution of DCIS to invasion [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr F1-2.

Abstract Why do we get cancer? Why is cancer highly associated with old age, and why are insults like smoking, obesity, and sunlight associated with increased risk of cancers? Of course, these contexts all cause mutations, and some of these mutations can contribute to malignant phenotypes. But we now understand that carcinogenesis is much more complex than originally appreciated. In particular, there are microenvironmental forces that both impede and promote cancer evolution. Just as organismal evolution is known to be driven by environmental changes, somatic evolution in our bodies is similarly driven by changes in tissue environments, whether caused by the normal process of aging, by lifestyle choices or by extrinsic exposures. Environmental change promotes selection for new phenotypes that are adaptive to the new context. In our tissues, aging or insult-driven alterations in tissues drives selection for adaptive mutations, and some of these mutations can confer malignant phenotypes. We will discuss the evidence from many labs for microenvironmental change driven selection in the evolution of cancer, additional support from epidemiological and clinical studies, as well as the limitations, inconsistencies and unknowns that require the attention of cancer researchers. My own lab has been using mouse models of cancer initiation, mathematical models of clonal evolution, and analyses of human tissue samples to better understand the evolutionary forces that control somatic cell evolution and thus cancer risk. Using Monte Carlo modeling, we have explored the impact of key somatic evolutionary parameters on multi-stage carcinogenesis, revealing that two additional major mechanisms, aging-dependent somatic selection and life history-dependent evolution of species-specific tumor suppressor mechanisms, need to be incorporated into the multi-stage model of carcinogenesis to make it capable of generalizing cancer incidence across tissues and species. Using mouse models, we have shown that aging and inflammation dependent changes in tissue microenvironments dramatically impact the fitness impact of oncogenic mutations, leading to aging and inflammation driven clonal expansions in the hematopoietic compartment. These studies have also uncovered molecular mechanisms underlying oncogene-driven adaptation to aged and inflammatory tissue environments. In all, these studies indicate that strategies to prevent or treat cancers will need to incorporate interventions that alter tissue microenvironments. While we largely cannot prevent mutation accumulation through our lives, we do have the ability to manipulate tissue microenvironments so as to change the evolutionary trajectories of oncogenically-mutated cells. Citation Format: James V. DeGregori. Changes in adaptive landscapes and somatic evolution as we age [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY24-01.

Abstract Gene expression analyses established at least two molecular subtypes of pancreatic adenocarcinoma (PDAC), the classical (or glandular), and the basal (or mesenchymal), each of which is associated with distinct prognoses and sensitivity to chemotherapy. It remains unclear, however, whether the basal carcinoma cells arise from a separate cell-of-origin, or are emerging from the pre-existing well-differentiated “classical” PDAC cells. To distinguish these alternatives, we conducted single-cell transcriptome analyses and virtual lineage tracing comparing cellular populations at pre-malignant stages in basal versus classical PDAC mouse models. We previously reported that chemical or genetic inhibition of the cholesterol biosynthetic pathway in KrasG12D; Trp53 (KPPC) mice predisposes to basal rather than glandular PDAC development because of the pancreas-specific increased sterol response element-binding protein 1 (SREBP1) activity and TGFβ signaling that induces cancer cell stemness and the EMT (PMID: 32976774). Pancreas-selective knockout of a conditional allele of cholesterol pathway gene, Nsdhl (NAD(P)-dependent steroid dehydrogenase-like), renders pancreatic epithelial cells cholesterol auxotrophs and drives basal PDAC in the majority of animals (KPPCN mice). At 5-6 weeks of age, grossly and microscopically tumor-free pancreatic tissues were selected for single-cell isolation and single-cell RNA sequencing (scRNA seq) using the 10X platform. After standard filtering and sample normalization procedures, downstream analyses included identification of relevant cell clusters using Seurat, lineage tracing algorithms, and in silico modeling of autocrine and paracrine signaling interactions between subsets of PDAC and non-malignant cells. Our key findings are as follows: 1) premalignant KPPCN pancreata exhibit a massive expansion of cancer-associated fibroblasts (CAFs) of predominantly inflammatory differentiation (iCAFs); 2) despite relatively fewer ADM and PanIN pre-malignant lesions in KPPCN compared to KPPC, scRNA seq identifies the significant expansion of epithelial cells with features of centroacinar and stem-like cells (increased expression of Aldh1a2, Nes, Sox9, Ly6a, Cxcl12, and Met); these centroacinar-like cells, while retaining epithelial identity (Epcam, Cdh1), also exhibit features of pluripotency by co-expression of Ins2 and other stem cell markers; 3) alignment with basal PDAC (KPPCN) and classical (KPPC) carcinoma cell populations strongly suggests the continuity of clonal evolution of the centroacinar-like cells towards the basal PDAC. While our genetic model does not recapitulate the multiple alternative pathways leading to basal PDAC development, cholesterol auxotrophy via SREBP1 may be a factor governing the expansion of undifferentiated precursors, which via interactions with cancer-promoting iCAFs, drive basal PDAC development. Citation Format: Michael Kotliar, Aizhan Surumbayeva, Linara Gabitova, Suraj Peri, Diana Restifo, Kathy Q. Cai, Artem Barski, Igor Astsaturov. Cholesterol auxotrophy promotes the expansion of centroacinar cells giving rise to the basal subtype of pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-068.

Abstract Abstract 669 Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world but its pathogenesis remains largely unknown. More recently, a widely prevalent premalignant condition termed monoclonal B cell lymphocytosis (MBL) has been defined and similarly involves expansion of a CD19+CD5+ population of B cells. MBL has been the focus of a wide number of recent studies in hopes it can provide insight into the early pathological events that lead to clonal expansion of a pre-leukemic CLL-like clone. Previously, we and others identified the transcription factor lymphocyte enhancer-binding factor-1 (LEF-1) as one of several genes significantly over expressed in CLL B cells relative to blood CD19+ B cells from healthy adults. LEF-1 is crucial for the proliferation and survival of pro-B cells during B cell development and deregulated LEF-1 activation has been directly linked with leukemogenesis in a transgenic murine model. In the present study, we addressed three critical questions concerning LEF-1 and CLL: 1) do CD5+ normal B cells express LEF-1; 2) at what stage of transformation does LEF-1 expression first appear; and 3) what mechanism(s) underlies LEF-1 expression in leukemic B cells. Methodology: The goals of our study were to: 1) determine the LEF-1 expression status of normal CD 19+/CD5+ human B cells; 2) determine the expression status of LEF-1 in clonal B cells present in patients with MBL; and 3) identify regulatory mechanisms that control aberrant expression of LEF-1 in CLL B cells. In order to achieve the first two goals, a 3-color flow cytometry assay for CD19, CD5, and intracellular LEF-1 was developed. To achieve the third goal, we performed in silico analysis of the LEF-1 locus and correlated this with publicly available genome wide methylated CpG island recovery assay (MIRA) data describing the methylome of normal human B cells (Rauch, T. A. Proc Natl Acad Sci U S A. 2009; 106(3): 671-8). Results: Analysis of human umbilical cord blood B cells, a rich source of the CD19+/CD5+ B cell subset, revealed that all normal CD 19+/CD5+ human B cells are negative for LEF-1 protein expression. These data demonstrate LEF-1 expression by CLL B cells is truly aberrant and does not simply reflect the phenotype of CD5+ lineage B cells. We next tested if the CD19+/CD5+ cells obtained from patients with MBL express LEF-1 protein. In these analyses, we restricted our study to MBL patients with absolute B cell counts of less than 2.5 ×10 9 cells/(L) (range: 0.756 - 2.44 × 10 9 cells/(L)), which is significantly below the upper limit that defines the MBL to CLL transition. Of interest, each MBL sample analyzed (n=6) revealed the presence of two populations of cells: 1) CD19+/CD5+ B cells expressing LEF-1; and 2) CD19+/CD5- B cells lacking expression of LEF-1. To confirm the clonal nature of the CD19+/CD5+ cells from MBL patients we demonstrated that the cells were light chain restricted. These data clearly indicated that LEF-1 expression becomes deregulated in the premalignant state of MBL and may therefore represent an early event in CLL leukemogenesis. In order to identify differential pathways of LEF-1 regulation that may be altered in CLL and MBL B cells, in silico promoter analysis of LEF-1 was performed. We identified a number of potential transcription factor binding sites as well as a putative CpG island in the 5' promoter region of LEF-1. Using data from a human B cell genome wide methylation array, we were able to determine that this same putative LEF-1 promoter CpG island was highly methylated in normal human B cells. Methylation of promoter region CpG islands is known to play an important role in developmental regulation of gene expression and may be the operative mechanism underlying silencing of LEF-1 expression in normal B cells. Conclusions: LEF-1 expression is deregulated in MBL and appears to be an early and possibly key event in the transition of normal B cells into a premalignant/malignant state. Our data suggest that loss of epigenetic regulation of this developmentally important locus may play a role in aberrant LEF-1 expression in MBL and CLL B cells. Ongoing studies are aimed at determining the methylation status of the LEF-1 promoter in CLL B cells and the functional role of this protein in transcriptional regulation and survival of CLL and MBL cells. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2433 Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of B lymphocytes which typically express CD19 and CD5. The disease remains incurable and recurrence often occurs after current standard therapies due to residual disease or probably due to the presence of therapy-resistant CLL precursors. Based on the growing evidence for the existence of leukemia stem cells, this study was designed to search for putative CLL precursors/stem cells based on the co-expression of CLL cell markers (CD19/CD5) with the hematopoietic stem cell marker (CD34). Forty seven CLL patients and 17 healthy persons were enrolled in the study. Twenty four patients had no previous treatment and 23 had pre-therapy. Twenty two patients were in Binet stage C and 25 patients in B. Twenty two patients had unmutated and 18 mutated IgVH gene (7: ND). Cytogenetic analysis by FISH showed that 14 patients had del 13q, 8 had del 11q, 4 had del 17p and 9 had trisomy 12. Peripheral blood and bone marrow mononuclear cells were subjected to multi-colour FACS analysis using anti-human antibodies against CD34, CD19 and CD5 surface antigens. The results revealed the presence of triple positive CD34+/CD19+/CD5+ cells in CLL samples (mean 0.13%; range 0.01–0.41) and in healthy donors (0.31%; range 0.02–0.6) within the CD19+ B cells. However, due to the high leukocyte count in CLL patients, the absolute number of these cells was significantly higher in CLL samples (mean: 78.7; range 2.5–295 cells /μL blood) compared to healthy persons (mean: 0.45: range 0.04–2.5 cells/μl)(p<0,001). These triple positive “putative CLL stem cells” (PCLLSC) co-express CD133 (67%), CD38 (87%), CD127 (52%), CD10 (49%), CD20 (61%), CD23 (96%), CD44 (98%) and CD49d (74%). FISH analysis on 4 patients with documented chromosomal abnormalities detected the corresponding chromosomal aberrations of the mature clone in the sorted CD34+/CD5+/CD19+ and/or CD34+/CD19-/CD5- cells but not in the CD3+ T cells. Multiplex RT-PCR analysis using IgVH family specific primer sets confirmed the clonality of these cells. Morphologically, PCLLSC appeared larger than lymphocytes with narrow cytoplasm and showed polarity and motility in co-culture with human bone marrow stromal cells. Using our co-culture microenvironment model (Shehata et al, Blood 2010), sorted cell fractions (A: CD34+/19+/5+, B: CD34+/19-/5- or C: CD34-/CD19+/5+) from 4 patients were co-cultured with primary autologous human stromal cells. PCLLSC could be expanded in the co-culture to more than 90% purity from fraction A and B but not from fraction C. These cells remained in close contact or migrated through the stromal cells. PCLLSC required the contact with stromal cells for survival and died within 1–3 days in suspension culture suggesting their dependence on bone marrow microenvironment or stem cell niches. RT-PCR demonstrated that these cells belong to the established CLL clone. They also eexpress Pax5, IL-7R, Notch1, Notch2 and PTEN mRNA which are known to play a key role in the early stages of B cells development and might be relevant to the early development of the malignant clone in CLL. Using NOD/SCID/IL2R-gamma-null (NOG) xenogeneic mouse system we co-transplanted CLL cells from 3 patients (5 million PBMC/mouse) together with autologous bone marrow stromal cells (Ratio: 10:1). The percentage of PCLLSC in the transplanted PBMC was 0.18% (range 0.06–0.34%). Using human-specific antibodies, human CD45+ cells were detected in peripharal blood of the mice (mean 0.9 % range 0.47–1.63%) after 2 months of transplantation. More than 90% of the human cells were positive for CD45 and CD5. Among this population, 26% (range 15–35%) of the cells co-expressed CD45, CD19, CD5 and CD34 and thus correspond to the PCLLSC. In conclusion, our data suggest the existence of putative CLL precursors/stem cells which reside within the CD34+ hematopoietic stem cell compartment and carry the chromosomal aberrations of the established CLL clone. These cells could be expanded in vitro in a bone marrow stroma-dependent manner and could be engrafted and significantly enriched in vivo in NOG xenotransplant system. Further characterization and selective targeting and eradication of these cells may pave the way for designing curative therapeutic strategies for CLL. Disclosures: No relevant conflicts of interest to declare.

Abstract A somatic gain-of-function mutation of the Janus kinase 2 (JAK2) gene is found in most patients with polycythemia vera (PV) and in about half of those with essential thrombocythemia (ET) or chronic idiopathic myelofibrosis (CIMF). In this study, we investigated the time course of the proportion of mutant alleles during the clinical course of the disease, and its relationship with clinical phenotype and treatment. Using a quantitative real-time polymerase chain reaction (qRT-PCR)-based allelic discrimination assay for the detection of the V617F mutation, we studied 522 patients diagnosed according to WHO criteria; 289 subjects were at diagnosis and 233 were at follow-up. Sequential studies were performed in 113 patients. Of these, 42 individuals were JAK2 (V617F)-negative at the first evaluation (6 with PV, 20 with ET and 16 with CIMF), and all of them remained negative after a median interval of 20 months (range 6–43 months). The remaining 71 patients carried JAK2 (V617F): 31 had PV, 13 had ET, 20 had CIMF and 7 had post-PV myelofibrosis. A statistically significant increase in the proportion of mutant alleles between the two time points was found only in PV (P=0.02). Taking into account the effect of time on the proportion of JAK2 (V617F) mutant alleles, we studied the slope between sequential assessments. The slope calculation was based on the difference between sequential assessments and the time elapsed. Values obtained in patients who received phlebotomy or anti-platelet therapy exclusively were considered as associated with the natural history of the disease. Median values for slopes were positive in all conditions: the highest increase was found in the PV subgroup (0.33% increase in the proportion of mutant alleles per month) while the lowest increment was observed in ET (0.08% increase in the proportion of mutant alleles per month). This observation suggests that the rate of clonal expansion of JAK2 (V617F)-positive cells is much slower in ET than in PV. This conclusion is also supported by findings in patients belonging to the follow-up subgroup: in 42 of 43 ET patients the proportion of mutant alleles did not exceed 50% after a median disease duration of 5.8 years. In order to investigate the impact of cytoreductive therapy on the time course of the proportion of JAK2 (V617F) mutant alleles, factorial ANOVA was performed using cytoreductive therapy and diagnosis as independent factors, and linear slope of the proportion of JAK2 (V617F) mutant alleles as a dependent variable. The interaction between diagnosis and treatment was also included in the model. Being on cytoreductive treatment was associated with a lower increase in the proportion of JAK2 mutant alleles (p=0.03). We also studied five JAK2 (V617F)-positive patients who needed sequential diagnostic reevaluation for clinical reasons. Three patients went through different stages of myelofibrosis and two patients with PV developed myelofibrosis. All these patients showed significant increases in the proportion of JAK2 (V617F) mutant alleles. In conclusion, JAK2 (V617F)-positive and JAK2 (V617F)-negative myeloproliferative disorders are likely distinct nosological entities. Within JAK2 (V617F)-positive conditions, the proportion of mutant alleles is relatively stable in ET while it tends to increase in PV. Cytoreductive agents may reduce the rate of clonal expansion of JAK2 (V617F)-positive cells.

Abstract The entire blood system is supported throughout life by a small number of hematopoietic stem and progenitor cells (HSPCs) that are produced exclusively during embryonic development. These stem cells are generated from the hemogenic endothelium of the dorsal aorta, then migrate to the fetal liver where they expand, before making a final migration to the fetal bone marrow. After seeding the bone marrow, the stem cell pool stabilizes and the total number of HSPCs remains relatively constant. Very little is known about this early stage of bone marrow niche colonization. A better understanding of native stem cell pool establishment will likely lead to improved clinical HSPC transplantation that depends on repopulation of the bone marrow niche. Currently, imaging technology does not allow direct visualization of the bone marrow niche during colonization because it occurs in utero in the long bones of the fetus. The zebrafish is an advantageous model because the embryos develop externally and are transparent. To quantify the early stem cell pool, we employed long term fate mapping with clonal analysis using the multicolor Zebrabow system, which imparts a unique fluorescent hue to stem cells and their progeny. Our findings reveal that 21 HSPC clones exist prior to HSPC emergence (24 hours post fertilization) and 30 clones are present during peak production from the aorta (48 hours post fertilization). Seeding of the presumptive adult marrow niche in zebrafish begins 4-5 days post fertilization, versus 16.5 days in the mouse. We previously described a transgenic zebrafish line (Runx:mCherry) that marks long term repopulating HSPCs throughout development and into adulthood. HSPC-specific expression is driven by the well-characterized Runx1 +23 kb mouse enhancer element. We used this line to directly observe the earliest immigration events of HSPCs as they arrive in the marrow. To achieve this, we immobilized the zebrafish by injection of the snake venom protein alpha-bungarotoxin directly into circulation. This allowed long term live imaging of the niche (~16 hours) so we could quantify the dynamics of HSPC colonization and expansion. To rapidly acquire high resolution imaging data for this deep tissue we applied lightsheet microscopy. By simultaneously illuminating the sample in the X plane, while taking images in the Z plane, hundreds of optical sections can be captured in seconds. The high pixel and temporal resolution of lightsheet microscopy in a large volume of tissue provides a highly dynamic view of the entire marrow niche. We could assess the localization of HSPCs in relation to other cell types within the niche. For example, HSPCs were closely associated with endothelial cells in a perivascular niche, similar to what has been described in mammalian bone marrow. Furthermore, we could quantify single Runx+ nuclei over time on one side of the bilateral kidney marrow. During this early stage of niche colonization, we found the number of HSPCs per side was ~50 (so ~100 total) and that remained relatively constant. This was in fact a dynamic equilibrium achieved by ingress and egress of cells, as well as occasional cell divisions. This cell number was independently validated using another transgenic zebrafish line, cd41:GFP, that also marks HSPCs. This quantification, combined with our data from earlier development, suggests that HSPCs undergo around two population doublings between emergence from the aorta and engraftment in the marrow. This unique platform for the quantification of a total stem cell pool will allow further functional and mechanistic studies using both genetics and chemical biology. Our goal is to gain insights into the establishment of the stem cell pool within the niche microenvironment and how this could improve clinical transplantation outcomes. Disclosures Zon: Marauder Therapeutics: Equity Ownership, Other: Founder; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Abstract T cell acute lymphoblastic leukaemias (T-ALL) are highly aggressive malignancies representing 10–15% of paediatric and 25% adult ALLs. T-ALL was considered to arise as a consequence of clonal expansion of early thymocytes. However, progress towards increasing our understanding of the biology of this disease has been hampered by lack of appropriate culture systems to study primary cells and use of murine model systems that often do not accurately reflect human disease. Traditional xenograft models of leukaemia have involved implantation of malignant cells or immortalised leukaemic cell lines with either intraperitoneal or subcutaneous localisation of leukaemia. These models do not mimic the normal pathophysiology of the disease and may therefore provide misleading data. Since evaluation of new agents in paediatric malignancies is limited by the small number of children eligible for clinical trails, there is a need for a predictive preclinical model of paediatric ALL. We have previously used a long-term suspension culture system to evaluate proliferation of T-ALL cells in vitro and demonstrated these cells had a CD34+/CD4−, CD7− phenotype. T-ALL cells with this phenotype were also capable of engrafting NOD/SCID mice, suggesting the disease may arise in a more primitive cell. In this study we have attempted to further characterise T-ALL cells with long-term proliferative ability in vivo and to investigate the kinetics of engraftment. Unsorted cells and cells sorted for the expression of CD133 and CD7 from 5 T-ALL patients were inoculated into sublethally irradiated NOD/SCID mice. Peripheral blood samples were taken at weekly intervals from the lateral tail vein from two weeks post inoculation onwards. BM samples were analysed from 4 weeks post inoculation and all animals were sacrificed no later than 10 weeks post inoculation. Human CD45+ cells were first detected at day 17-post inoculation (1.54–3.8% CD45+). By week 4, this had increased to 4.4–21% CD45+ in PB samples, while levels in BM aspirates were significantly higher at this stage (24–47% CD45+). This pattern of tissue dissemination closely mimics that observed in the patients. The levels of CD45+ cells continued to rise with time and had exceeded 85% in the BM of animals injected with cells from 3 patients by week 7-post inoculation. FISH and flow cytometric analyses showed the engrafted cells had a similar karyotype and phenotype to the patient at diagnosis and there was no evidence of myeloid cell engraftment. Cells harvested from these animals have been used in secondary, tertiary and quaternary transplants with no loss of NOD/SCID repopulating potential and similar engraftment kinetics. Quinary transplants are currently underway. In the sorted cell populations, only the CD133+/CD7− subfraction was capable of engrafting, 0.5–54% CD45+, with as few as 1.4x103–5x103 cells. There was no engraftment with the other subfractions despite injecting 10 to 1000-fold more cells. These engrafted cells expressed high levels of CD34, CD2, CD4 and CD7 and very low levels of CD133. This phenotype was similar to that of the patients at diagnosis, implying they had differentiated in vivo. These data add to the evidence that T-ALL may arise in a cell with a more primitive phenotype, rather than committed thymocytes. These cells may be the most relevant targets for emerging therapeutic strategies and we describe a robust, reproducible in vivo leukaemia model which could be used to investigate the efficacy of novel agents for the treatment of paediatric T-ALL.

Abstract Known genetic loci influencing blood cell production account for <10% of platelet and red blood cell variability, and thrombopoietin (Tpo)/c-MPL liganding is dispensable for definitive thrombopoiesis establishing that fundamentally important modifier loci remain unelucidated. We completed RNASeq of highly-purified platelets from 7 essential thrombocythemia (ET) subjects [4 harbored the JAK2V617F mutation and 3 were genotypically normal] and 5 healthy controls, and developed an iterative algorithm to identify 33 non-synonymous SNVs that are putatively linked to the ET phenotype. Transcripts possessing these candidate SNVs were enriched on average in early-stage megakaryocyte/erythroid progenitors (MEPs), and became more restricted in terminally-differentiating megakaryocytes and erythroblasts. Genotypic studies using an expanded ET cohort (N = 36), followed by statistical association analyses using controls from the 1000 Human Genomes Project and an independently-genotyped cohort of healthy controls (N = 208), established that 5 SNVs (excluding JAK2V617F) were associated with ET. A single mutation (BLVRBS111L; p = 0.0006) retained its significance as a thrombocytosis risk allele using genotypic data from a secondary cohort with reactive (non-clonal) thrombocytosis (RT, N = 53), suggesting a function as an independent driver mutation of enhanced thrombopoiesis. TheBLVRB (biliverdin IXβ reductase) gene functions downstream of heme oxygenase(s)-1 (inducible HMOX1) and -2 (constitutive HMOX2) within the heme degradation pathway to catalyze reduction of biliverdin (BV) tetrapyrrole(s) as an intermediary redox substrate in Bilirubin (BR) generation. Bacterially-expressed and purified recombinant BLVRBS111L showed defective enzymatic activity [compared to BLVRBWT (wild-type)] using flavin mononucleotide [flavin reductase (FR) activity; p <0.0001] and BV IXβ dimethyl esters (biliverdin reductase (BVR) activity; p <0.0001), the latter specifically generated by coupled heme oxidation as verdin-restricted BLVRB activity probes. The loss-of-mutation BLVRBS111L NAD(P)H-dependent redox coupling caused higher baseline ROS (reactive oxygen species) accumulation in lentivirus-transduced CD34+-derived induced pluripotent stem cells (iPSC/BLVRBS111L), results sharply contrasting with ROS neutralization in iPSC/BLVRBWT exposed to tert-butyl hydroperoxide (TBHP) as the oxidant stress source (p < 0.00001). Disparate redox coupling/ROS handling in genetically-modified primary CD34+ multipotential progenitor cultures established disproportionate expansion of primitive CFU-GEMMs in CD34+/BLVRBS111L (p = 0.001), and an absolute increase of BFU-E in CD34+/BLVRBWT (p = 0.001). Collagen-based progenitor cultures demonstrated a statistically-significant increase of CD41+ CFU-MKs in CD34+/BLVRBS111L cells (p <0.01) with no increased CFU-MKs in CD34+/BLVRBWT cells. Cumulative distribution plots of parallel Tpo-suspension cultures confirmed divergent ROS accumulation between MK/BLVRBWT and MK/BLVRBS111L, differences that were identifiable pre-terminal differentiation (Day 0, p = 0.0007), most pronounced at Day 5 corresponding to peak MK BLVRB expression across genotypes (p = 8 x 10-7), and persistent at terminal differentiation (Day 10; p = 0.05). Maximally disparate ROS accumulation (Day 5) corresponded to greatest size disparity and temporally-earlier (and sustained at Day 10) CD41 expression in MK/BLVRBS111L (p = 0.03). A bilineage (Tpo/Epo) differentiation model, designed to characterize erythroid/megakaryocyte (E/Meg) progenitor balance arising from common MEPs demonstrated no evidence for differential Mk (CD41+/Glycophorin A-) lineage balance, suggesting that BLVRBS111L ROS-promoting effects accelerate post-commitment expansion downstream of MEP lineage fate decisions. These data provide the first evidence linking redox coupling to MK lineage fate and expansion in humans, either via activity as a flavin reductase, in partnered electron exchange with an unidentified protein, or as a verdin-regulated redox coupler regulated by heme degradation and isomer-restricted BV IXβ (or IXδ, IXγ) generation; in principle, development of BLVRB-specific redox inhibitors represent innovative approaches to selectively alter a regulatory pathway controlling MK lineage expansion and human platelet counts. Disclosures No relevant conflicts of interest to declare.

AbstractGenetic intra-tumour heterogeneity fuels clonal evolution, but our understanding of clinically relevant clonal dynamics remain limited. We investigated spatial and temporal features of clonal diversification in clear cell renal cell carcinoma through a combination of modelling and real tumour analysis. We observe that the mode of tumour growth, surface or volume, impacts the extent of subclonal diversification, enabling interpretation of clonal diversity in patient tumours. Specific patterns of proliferation and necrosis explain clonal expansion and emergence of parallel evolution and microdiversity in tumours. In silico time-course studies reveal the appearance of budding structures before detectable subclonal diversification. Intriguingly, we observe radiological evidence of budding structures in early-stage clear cell renal cell carcinoma, indicating that future clonal evolution may be predictable from imaging. Our findings offer a window into the temporal and spatial features of clinically relevant clonal evolution.

e20582 Background: Chemotherapy ± immunotherapy has demonstrated meaningful clinical benefit to patients (pts) with extensive-stage small cell lung cancer (ES-SCLC); however, chemotherapy-induced damage to the immune system can potentially diminish treatment efficacy. Trilaciclib (T) is an intravenous cyclin-dependent kinase 4/6 inhibitor that protects hematopoietic stem and progenitor cells from chemotherapy-induced damage (myeloprotection) and may directly enhance antitumor immunity. Here, we evaluated the immune effects of T in pts with ES-SCLC receiving T or placebo (P) prior to first-line etoposide plus carboplatin (E/C) or E/C plus atezolizumab (E/C/A) in two phase 2 clinical trials. Methods: Genomic DNA, extracted from peripheral blood mononuclear cells (baseline and on treatment) and archival tumor tissue (baseline), was analyzed using the immunoSEQ® Assay (Adaptive Biotechnologies). T-cell receptor (TCR) β CDR3 regions were amplified and sequenced to identify and quantitate the abundance of each unique TCRβ CDR3. Clonal frequencies were compared at baseline and on treatment, and statistical differences between T and P were determined by Wilcoxon rank sum test. Antitumor response was defined as complete/partial response. Results: In both studies, peripheral T-cell clonal expansion was greater among pts receiving T versus P. Among pts receiving E/C, those in the T/E/C group with an antitumor response had significantly more peripheral clonal expansion than P responders (median 23 vs 12 clones; P= 0.04) and a greater number of tumor-associated expanded clones ( P= 0.03). T responders had more newly detected expanded peripheral clones compared with P responders (6 vs 1.5 clones; P= 0.06) and T nonresponders ( P= 0.02). Increased clonal expansion in T responders was more evident after two cycles of E/C versus four, suggesting that T results in a rapid T-cell response. Similarly, among pts receiving E/C/A, those in the T/E/C/A group with an antitumor response had significantly more peripheral clonal expansion than P responders (median 90 vs 43 clones; P= 0.002) and T nonresponders ( P= 0.016). T responders also had more newly expanded peripheral clones compared with P responders (68 vs 11 clones; P= 0.003) and T nonresponders ( P= 0.02). There was no increase in tumor-associated expanded clones among T responders compared to P responders, possibly due to the time point at which clonal expansion was assessed (after four cycles) or the addition of atezolizumab. Associations between peripheral and tumor-associated clonal expansion and survival will be presented. Conclusions: The data suggest that, among pts treated with T/E/C or T/E/C/A, increased clonal expansion is associated with clinical response, indicating that T may enhance antitumor immunity in pts with ES-SCLC treated with chemotherapy.

Abstract Background and hypothesis: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy, comprising of B lineage ALL and T-cell lineage ALL (about 85% and 15%, respectively). In most cases, treatment regimens for ALL patients have proved efficient and prognosis is usually encouraging. However, a recently described subgroup of T-cell ALL patients have been found to have a poor prognosis when treated with conventional therapy. These higher risk T-cell leukemias have a cell of origin thought to resemble an early T-cell precursor (ETP), which retains the capability to differentiate into both myeloid or T-lineage cells, and are therefore termed ETP ALL. ETP ALL is characterized by lack of or low expression of typical T-cell surface markers, aberrant expression of myeloid and hematopoietic stem cell (HSC) surface markers and a gene expression signature resembling that of murine ETP cells. Recently, ETP ALL patients were found to have a unique mutational landscape that includes acquired mutations in genes typically associated with myeloid differentiation. The ambiguous lineage attributes of ETP ALL may explain the ineffectiveness of current therapies, stressing the need for establishing pre-clinical animal models. Here we show that co-expression of Nup98-Hoxd13 (NHD13) fusion gene and a mutant IDH2 gene (IDH2 R140Q) triggers an ETP-like leukemia in mice. Expression of NHD13 has been reported in MDS and AML patients, and NHD13 mice have been previously shown to develop MDS, AML, and T-cell ALL. Mutations in IDH1/2 genes occur in several cancers, including AML; the most common IDH mutation in AML patients is the IDH2 R140Q. However, expression of mutant IDH1/2 in mice has been reported to induce an expansion of the HSC compartment but was not sufficient for establishment of leukemia. Targeted sequencing of NHD13 mice has identified recurrent mutations in IDH1, suggesting that these two aberrations may collaborate to cause leukemia. Study Design and Methods: IDH2 R140Q mice were crossed with NHD13 mice and the progeny were monitored for survival. Classification of leukemic samples was done using flow cytometry and PCR analysis of TCRβ gene rearrangements. Global expression and mutational patterns of IDH2/NHD13 leukemias were analyzed using gene chip arrays and whole exome sequencing (WES). Results and conclusions: Transgenic IDH2/NHD13 mice showed decreased survival compared to all control groups. Detailed analysis of surface marker expression revealed that most double Tg cases (10/11 tested) developed leukemias resembling ETP (cKit+CD44+CD25-) or double negative (DN) 2 cells (cKit+CD44+CD25+). PCR analysis of the TCRβ locus revealed that unlike NHD13 T-cell leukemias, IDH2/NHD13 leukemias do not have clonal VDJ rearrangements. However, most of IDH2/NHD13 leukemias have clonal or oligoclonal DJ rearrangements of the TCRβ locus, suggesting that these leukemias emerged during an early stage in T cell development. Expression analysis revealed that the IDH2/NHD13 leukemia expression signature is enriched in genes that are upregulated in ETP or DN2 cells. In addition, we found that the IDH2/NHD13 leukemia expression signature is also enriched in genes uniquely expressed in ETP ALL patients. Finally, WES showed correspondence between genes mutated in the mouse ETP-like leukemia and genes recurrently mutated in ETP ALL patients. These results indicate that IDH2/NHD13 leukemias resemble ETP ALL in terms of gene expression, immunophenotype, and cooperative mutations. Relevance and importance: The poor prognosis of ETP ALL may be related to incomplete effectiveness of treatment strategies, emphasizing the need for novel therapies. IDH2/NHD13 mice can serve as an excellent pre-clinical model to study ETP ALL and to test potential treatments. Disclosures Aplan: NIH Office of Technology Transfer: Patents & Royalties.

Abstract Follicular lymphoma (FL) is one of the most common B-cell lymphoma, and remains virtually incurable despite its relatively indolent nature. T(14;18)(q32;q21), the genetic hallmark and early initiating event of FL pathogenesis, is also present at low frequency (10−5–10−7) in blood from healthy individuals (HI), indicating that t(14;18) and the ensuing BCL2 overexpression is necessary but not sufficient for malignant transformation. It has long been assumed that in HI, t(14;18) is carried by circulating quiescent naïve B-cells, where its oncogenic potential would be restrained. Yet, several reports, including long-term persistence and immunomodulation of t(14;18)+ cells in lymphoma-free individuals, led us to question this model and investigate the status of circulating t(14;18)+ cells in HI. We first determined if t(14;18)+ cells are naïve B-cells by assessing class-switch recombination (CSR) on the translocated allele. Using 2 long-range PCR assays designed to amplify unswitched BCL2/Sμ and switched BCL2/Sg regions, DNA samples from 6 HI with t(14;18) were tested. Contrary to previous assumptions, our data clearly show that most peripheral t(14;18)+ cells already underwent CSR (n=5/6) and therefore that most t(14;18)+ cells are not naïve B-cells. Are they then memory B cells? Naïve and memory B cell subsets from 9 HI were isolated by cell sorting according to IgD and CD27 markers, and the rate of t(14;18) analyzed in each subset relatively to that of the total B cells. Strikingly, while the level of naïve t(14;18)+ cells remained at baseline for all individuals, memory B-cells tightly accounted for the wide modulation of t(14;18) frequencies observed between individuals. In addition, sequence analysis of t(14;18) clones revealed that this wide modulation was not due to the accumulation of clonally unrelated t(14;18) naïve B-cells, but rather to the clonal expansion of t(14;18)-bearing memory B-cells. To further define the t(14;18)+ cells, we next examined the repartition of the translocation in the IgD−/CD27+ and IgD+/CD27+ memory B-cell subsets. Unexpectedly, we found that the IgD+/CD27+ subset contained significantly higher rates of translocation than the IgD−/CD27+, both in terms of prevalence and frequency. Thus, while CSR is found in the majority of translocated alleles (~75%), most t(14;18)+ memory B cells have not switched their productive allele (~70%) and express an IgM/D. Most importantly, although atypical among physiological peripheral B-cells, this “allelic paradox” is a specific hallmark of FL, and suggests the presence of the same selective pressure in favor of sIgM expression on a B-cell population that is at the same time permanently driven to switch. In line with B-cell hyperplasia in BCL2 transgenic mice slowly progressing to low grade lymphoma, it is likely that “FL-like” cells in HI are rescued by BCL2 from apoptosis, and “frozen” at a differentiation stage in which constitutive AID expression drives continuous somatic hypermutation and CSR activity, two mechanisms conferring a high propensity for genomic instability. Altogether, our findings identify a novel intermediate step in early lymphomagenesis, and strongly impact both on the current understanding of disease progression from potent pre-malignant niches, and on the proper handling of t(14;18) frequency in blood as a potential early biomarker for lymphoma.

Abstract Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells (PCs) and aberrant production of monoclonal immunoglobulin detected as an M spike using serum protein electrophoresis. In the United States, MM represents ~15% of hematologic malignancies and is one of the few cancers increasing in incidence (e.g., 14,400 in 1996 to 30,770 in 2018, from SEER). Previously, a Vk*MYC mouse model was described, in which AID-dependent activation of MYC transgene in germinal center (GC) B cells catalyzes a highly penetrant, indolent MM after a prolonged latency, suggesting that additional genetic mutations are required for the malignant MM progression. Recent sequencing of paired tumor/normal samples from advanced or refractory MM patients identified that constitutive activation of Ras signaling pathway (KRAS: 23%; NRAS:20%; BRAF: 8%) associates with MM progression and therapy resistance. To determine whether oncogenic Nras promotes the progression of Myc-induced indolent MM to a malignant stage, we generated NrasLSL Q61R/+; Vk*MYC; IgG1-Cre (VQ) mice along with single mutant mice Vk*MYC; IgG1-Cre or NrasLSL Q61R/+; IgG1-Cre. To boost NrasQ61R expression in GC B cells, 6-7 weeks old mice were immunized with NP-CGG. A significant fraction of VQ mice developed M-spike after immunization and subsequently died of a highly aggressive MM, which was characterized by high proliferative index, hyperactivation of AKT and ERK pathways, and disease hallmarks (e.g. osteolytic lesions, anemia, and kidney injury). VQ myeloma was readily transplantable into serial syngeneic recipients. In our preliminary study, combined Bortezomib and AZD6244 treatment attenuated MM phenotypes and prolonged the survival of VQ recipients. To facilitate easy molecular or genetic manipulations of VQ MM cells, we established two cell lines from primary cells. These cells could be easily infected (see another abstract from our co-author, Dr. Asimakopoulos's group). The cultured MM cells also express high levels of CD155, a ligand of immune checkpoint TIGIT, which has been recently reported to mediate a significant immune checkpoint blockade in MM patients. We further investigated the transcriptional signature of VQ MM cells (from bone marrow [BM] and from lymph nodes [LN]) vs wild-type (WT) plasma cells using the Illumina Bio-Rad's Single-Cell Sequencing platform. Illumina's Sure Cell app was used to de-multiplex samples, process barcodes and perform single-cell 3' gene counting. The processed data were then analyzed using Seurat R package to yield cluster information and their associated enriched genes. Our results revealed distinct clusters of CD138+ MM and WT cells, and also showed high similarity & overlap between BM & LN MM samples. Consistent with the genetic changes introduced to VQ MM cells, both Myc and Nras expression levels were significantly elevated in MM clusters compared to WT clusters. We also found that transcriptional levels of several potential therapeutic targets were significantly elevated in MM clusters, including Integrin alpha 4 and metabolic enzymes. Currently we are validating the scRNA-Seq results. Taken together, we generated a novel mouse model in which activation of Vk*MYC and oncogenic Nras in GC B cells results in a highly malignant, transplantable MM. The VQ mouse model represents an important innovation that will serve as a platform to investigate pathogenesis of resistant and refractory multiple myeloma and allow for testing the efficacy of novel therapeutic agents. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 2397 B cells can undergo at least two differentiation pathways, dependent of T cells or not, starting from follicular or marginal zone B cells respectively. The T-independent response, less understood than the germinal center reaction, is triggered by specific antigens and arises from marginal zone B cells. During this development, some B cells undergo somatic hypermutation (SHM) and class switch recombination (CSR), triggered by the same DNA editing enzyme called Activation Induced Cytidine Deaminase (AID). The splenic marginal zone lymphoma (SMZL) is a rare lymphoproliferative disorder characterized by a clonal expansion of B cells in the marginal zone of the spleen. These B-cells underwent SHM in roughly 60% of the cases but nearly none underwent CSR. These observations suggest that tumor clones originate from a particular activated B cell subset not transiting through the germinal center. In order to confirm this hypothesis, we focused our work on the status and impact of AID in this disease and worked on purified B cells extracted from spleen of well-characterized SMZL cases. We determined AID status by quantitative RT-PCR analysis on 27 SMZL samples and compared it with 5 controls. In the SMZL group the relative level of expression of AID is heterogeneous but two subgroups could be distinguished: one considered as expressing AID (14 cases out of the 27 analyzed), the remaining considered as not expressing AID. When we compared AID expression rate with occurrence of SHM and CSR, no clear correlation between AID expression and presence of SHM or CSR could be observed suggesting that AID, when expressed, is dysfunctional. To address this hypothesis, we first analyzed AID protein by immunohistochemistry and a good correlation between IHC signal and AID mRNA expression level has been observed. As AID gene was not mutated, we next focused our work on AID mRNA splicing variants as these variants exhibit different functions according to the domain of the protein they contain in a murine model. We found that SMZL B cells express various splicing variants of AID mRNA, some of those variants corresponding to the full length isoform (n = 6/17), and other variants corresponding to AID-ΔE4a (n = 2/17) or AID-ΔE4 (n = 7/17) isoforms known to be expressed in normal germinal center B cells as well as in Chronic Lymphocytic and Acute Lymphoblastic Leukemia. These findings indicate that although expressed at the mRNA and protein levels, AID may not be fully functional in SMZL cases. Finally we addressed the potential clinical significance of AID expression. We identified for that purpose a group of “progressive SMZL” patients that had received immuno-chemotherapy after splenectomy because of a significant risk of progression or transformation into aggressive large B cell lymphoma (n = 8/27) pre-empting outcome differences. We found a higher proportion of AID expressing patients in the defined “progressive SMZL” group (n = 7/8) as compared to the proportion found in the “indolent SMZL” group (n = 5/14, p = 0,03). Altogether, this data suggest that the B cell clone leading to SMZL originate from the marginal zone and support the hypothesis of a lymphoproliferative disorder affecting the T-independent response. AID expression in SMZL may reflect an advanced stage of the disease and could be correlated with the evolution of the lymphoma into a more clinically or pathologically aggressive form. Disclosures: No relevant conflicts of interest to declare.

The volumetric changes and variable porosity due to the concentration expansion of the solid phase in the synthesis of zirconium nitride (ZrN) are studied. The model of two-stage reactor based on spark plasma sintering (SPS) is proposed. At the first stage the synthesis for the given kinetics is simulated. At the second stage the densification of ZrN using the Olevsky’s sintering model [1-5] is applied. The synthesis and densification processes using the prescribed heat sources, at the given positions inside the reactor is simulated. The generalization of the two-temperature model [6] and the formula of the porosity in the densification using calculation of the solid concentration expansion and thermal dispersion is proposed. The concentration expansion coefficients in the process of zirconium nitrogenating at a given initial density values and coefficients of expansion of reagents .is studied The temperature at the stage of ZrN synthesis and porosity variation at the stage of densification are in satisfactory agreement with experimental results [2,7,8]

Abstract CTL are well studied in mammals, however less is known about the dynamics of memory CTL expansion and exhaustion in teleost fish. Here we provide insights into teleost CTL dynamics using our model alloantigen-specific catfish clonal CTL cell line called TS32.15. Briefly, TS32.15 was cloned from peripheral blood leukocytes isolated from fish immunized with allogenic 3B11 B cells. This clonal line is not synchronized and consists of small resting T cells and larger granular effectors. TS32.15 cells exhibit strict target specificity for 3B11s and they require stimulation with irradiated 3B11 B cells every 7–10 days for their continuous growth. When exposed to irradiated targets, the small T cells initially do not lyse targets, but replicate and differentiate to morphologically distinct cytotoxic effectors. These effectors do not replicate, but efficiently lyse 51Cr-labeled 3B11s. After lysing targets, or with a prolonged absence of targets, effector cells transition to a non-cytolytic exhausted stage and become apoptotic. Late stage apoptotic CTL bind IgM on their surface and are preferentially cleared by myelocyte-enriched head kidney leukocytes. These results indicate teleost CTL likely undergo clonal expansion and differentiation in response to antigen similar to their mammalian counterparts, and establish binding of exogenous IgM is a marker for monitoring exhausted CTL in catfish. Additionally, our studies show that repeated stimulation of a TS32.15 culture mimics a chronic infection and results in CTL exhaustion.

Changes in the structure and function of antibodies occur during the course of an immune response due to variable (V) region gene somatic mutation and isotype switch recombination. While the end products of both these processes are now well documented, their mechanisms, timing, and regulation during clonal expansion remain unclear. Here I describe the characterization of antibodies expressed by a large number of hybridomas derived from single B cell clones at an intermediate stage of an immune response. These data provide new insights into the mechanism, relative timing, and potential of V gene mutation and isotype switching. The data suggest that somatic mutation and isotype switching are completely independent processes that may, but need not, occur simultaneously during clonal expansion. In addition, the results of this analysis demonstrate that individual B cell clones are far more efficient than previously imagined at generating and fixing particular V region somatic mutations that result in increased affinity for the eliciting epitope. Models to account for this high efficiency are discussed. Taken together with previous data, the results of this analysis also suggest that the "somatic evolution" of V region structure to a single epitope takes place in two stages; the first in which particular mutations are sustained and fixed by antigen selection in the CDR regions of the V region genes expressed in a clone over a short period of clonal expansion, and the second in which these selected CDR mutations are maintained in the growing clone, deleterious mutations are lost, and selectively neutral mutations accumulate throughout the length of V genes over long periods of clonal expansion.

AbstractUnderstanding cancer evolution provides a clue to tackle therapeutic difficulties in colorectal cancer. In this review, together with related works, we will introduce a series of our studies, in which we constructed an evolutionary model of colorectal cancer by combining genomic analysis and mathematical modeling. In our model, multiple subclones were generated by driver mutation acquisition and subsequent clonal expansion in early-stage tumors. Among the subclones, the one obtaining driver copy number alterations is endowed with malignant potentials to constitute a late-stage tumor in which extensive intratumor heterogeneity is generated by the accumulation of neutral mutations. We will also discuss how to translate our understanding of cancer evolution to a solution to the problem related to therapeutic resistance: mathematical modeling suggests that relapse caused by acquired resistance could be suppressed by utilizing clonal competition between sensitive and resistant clones. Considering the current rate of technological development, modeling cancer evolution by combining genomic analysis and mathematical modeling will be an increasingly important approach for understanding and overcoming cancer.

An anisotropic cosmological model with expansion and rotation and the Bianchi type IX metric has been constructed within the framework of general relativity theory. The first inflation stage of the Universe filled with a scalar field and an anisotropic fluid is considered. The model describes the Friedman stage of cosmological evolution with subsequent transition to accelerated exponential expansion observed in the present epoch. The model has two rotating fluids: the anisotropic fluid and dust-like fluid. In the approach realized in the model, the anisotropic fluid describes the rotating dark energy.