Journal articles: 'Intraclonal'

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Gurrieri, Carmela, Peter McGuire, Hong Zan, Xiao-Jie Yan, Andrea Cerutti, Emilia Albesiano, Steven L. Allen, et al. "Chronic Lymphocytic Leukemia B Cells Can Undergo Somatic Hypermutation and Intraclonal Immunoglobulin VHDJH Gene Diversification." Journal of Experimental Medicine 196, no. 5 (September 2, 2002): 629–39. http://dx.doi.org/10.1084/jem.20011693.

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Chronic lymphocytic leukemia (CLL) arises from the clonal expansion of a CD5+ B lymphocyte that is thought not to undergo intraclonal diversification. Using VHDJH cDNA single strand conformation polymorphism analyses, we detected intraclonal mobility variants in 11 of 18 CLL cases. cDNA sequence analyses indicated that these variants represented unique point-mutations (1–35/patient). In nine cases, these mutations were unique to individual submembers of the CLL clone, although in two cases they occurred in a large percentage of the clonal submembers and genealogical trees could be identified. The diversification process responsible for these changes led to single nucleotide changes that favored transitions over transversions, but did not target A nucleotides and did not have the replacement/silent nucleotide change characteristics of antigen-selected B cells. Intraclonal diversification did not correlate with the original mutational load of an individual CLL case in that diversification was as frequent in CLL cells with little or no somatic mutations as in those with considerable mutations. Finally, CLL B cells that did not exhibit intraclonal diversification in vivo could be induced to mutate their VHDJH genes in vitro after stimulation. These data indicate that a somatic mutation mechanism remains functional in CLL cells and could play a role in the evolution of the clone.

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Volkheimer, Alicia D., J. Brice Weinberg, Bethany E. Beasley, John F. Whitesides, Jon P. Gockerman, Joseph O. Moore, Garnett Kelsoe, Barbara K. Goodman, and Marc C. Levesque. "Progressive immunoglobulin gene mutations in chronic lymphocytic leukemia: evidence for antigen-driven intraclonal diversification." Blood 109, no. 4 (November 2, 2006): 1559–67. http://dx.doi.org/10.1182/blood-2006-05-020644.

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Abstract Somatic mutations of immunoglobulin genes characterize mature memory B cells, and intraclonal B-cell diversification is typically associated with expansion of B-cell clones with greater affinity for antigen (antigen drive). Evidence for a role of antigen in progression of intraclonal chronic lymphocytic leukemia (CLL) cell diversification in patients with mutated immunoglobulin genes has not been previously presented. We performed a single-cell analysis of immunoglobulin heavy and light chains in 6 patients with somatically mutated CLL-cell immunoglobulin genes and identified 2 patients with multiple related (oligoclonal) subgroups of CLL cells. We constructed genealogic trees of these oligoclonal CLL-cell subgroups and assessed the effects of immunoglobulin somatic mutations on the ratios of replacement and silent amino acid changes in the framework and antigen-binding regions (CDRs) of the immunoglobulin heavy and light chains from each oligoclonal CLL-cell population. In one subject, the amino acid changes were consistent with an antigen-driven progression of clonally related CLL-cell populations. In the other subject, intraclonal diversification was associated with immunoglobulin amino acid changes that would have likely lessened antigen affinity. Taken together, these studies support the hypothesis that in some CLL cases intraclonal diversification is dependent on antigen interactions with immunoglobulin receptors.

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Rawstron, Andy C., James A. L. Fenton, Marieth Plummer, Harry O. King, Fiona L. Bennett, Andrew S. Jack, and Peter Hillmen. "Monoclonal B-Cell Lymphocytosis (MBL) and CLL Show Intraclonal Variation: Cases Classified as “Unmutated” Have the Greatest Clonal Diversity." Blood 108, no. 11 (November 16, 2006): 30. http://dx.doi.org/10.1182/blood.v108.11.30.30.

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Abstract A monoclonal B-cell lymphocytosis (MBL) is detectable in approximately 3% of the general adult population. In most cases the abnormal cells have a phenotype and genotype that is indistinguishable from indolent CLL. Individuals presenting with an MBL count over 500 cells per microlitre progress to CLL requiring treatment at a rate of approximately 1% per year. The relationship between MBL and CLL therefore appears to be similar to that of MGUS and myeloma. Intraclonal variation (ICV) in the immunoglobulin heavy chain gene (IgVH) gene occurs in approximately half of MGUS patients but is not present in myeloma. The majority MGUS patients that progress to myeloma lack intraclonal variation at the MGUS stage suggesting that clonal selection is a critical pathway for disease progression in myeloma. The aim of this study was to compare the rate of intraclonal variation in MBL and CLL and determine whether low rates of ICV are associated with progressive disease. DNA was extracted from ammonium-chloride lysed blood samples from individuals with CLL-phenotype MBL (n=20) and CLL with progressive disease (n=10). IgVH and BCL6 PCR products were directly sequenced and also cloned and at least 10 clones sequenced. Intraclonal variation was defined as the number of unique sequences as a percentage of total clones sequenced. Unmutated CLL was defined as having <2% difference in the predominant IgVH clone sequence from germline. The median IgVH mutation rate in the MBL group was 7.4% (range 1.3–13.7%) and in the progressive CLL group 0.6% (range 0–11.3%). Intraclonal variation was observed in both groups of patients: the median number of unique clones was 2/10 (range 0–7/10) in MBL patients and 3/10 (range 0–5/10) in CLL patients. Intraclonal variation was generally restricted to 1 or 2 point mutations in each sequence and for the VH gene the replacement/silent (R/S) ratio of mutations was 1.7 in the framework regions and 3.3 in the complementarity-determining regions. Independent of disease category, unmutated CLL/MBL had a higher degree of intraclonal variation than mutated CLL with a median ICV for unmutated cases of 31% and for mutated cases of 20%. The proportion of patients showing complete clonal homogeneity was lower for unmutated CLL/MBL (14%) than mutated CLL/MBL (27%). The BCL6 gene, which is also mutated during the canonical somatic hypermutation process, showed similar results with a greater degree of intraclonal variation in unmutated CLL/MBL. The results demonstrate that intraclonal variation is a frequent occurrence in both MBL and CLL particularly when non-immunoglobulin genes are also considered. Clonal heterogeneity is either independent of, or inversely related to, the immunoglobulin mutation status demonstrating that both mutated and unmutated CLL have undergone (or are continuing to undergo) somatic hypermutation. The mechanisms of disease progression in MBL/CLL are clearly biologically distinct from MGUS/Myeloma and this data provides strong evidence for an antigen-driven selection process in CLL. Disease progression in CLL may therefore be the result of continued activity of the somatic hypermutation machinery, demonstrated by AID expression in some patients. Coupled with a selective pressure to maintain the immunoglobulin gene this process may result in the selection of CLL clones with transforming mutations in non-immunoglobulin genes.

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Rajamanickam, C., and K. Rajmohan. "Intraclonal Variation in Musa (AAB) Palayankodan." International Journal of Current Microbiology and Applied Sciences 9, no. 6 (June 10, 2020): 269–75. http://dx.doi.org/10.20546/ijcmas.2020.906.034.

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Zojer, Niklas, Heinz Ludwig, Michael Fiegl, Freda K. Stevenson, and Surinder S. Sahota. "Patterns of somatic mutations in VH genes reveal pathways of clonal transformation from MGUS to multiple myeloma." Blood 101, no. 10 (May 15, 2003): 4137–39. http://dx.doi.org/10.1182/blood-2002-09-2825.

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AbstractMonoclonal gammopathy of undetermined significance (MGUS) can transform to multiple myeloma (MM). In myeloma, mutated VHgenes with sequence homogeneity reveal a postfollicular origin. Previously, some MGUS cases showed mutated VH genes with intraclonal variation, indicating an earlier stage of arrest. We investigated progression from 2 of 2 MGUS to MM, in which VH genes confirmed clonal evolution. In one MGUS case, intraclonal heterogeneity was evident, and transformation to myeloma occurred rapidly with apparent homogeneity in the emergent clone. However, residual MGUS-derived sequences were detectable at this time. Heterogeneity in MGUS does not associate with benign disease, but it indicates an origin from a tumorigenic cell, most likely surface immunoglobulin+, undergoing somatic mutation. The remaining case displayed intraclonal homogeneity at the MGUS stage, conceivably resulting from a self-cloning outgrowth from MGUS with heterogeneity. Transformation can occur at either MGUS stage, but it involves a single cell in which somatic mutation is then silent.

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Bartholdy, Boris A., Xiahoua Wang, Xiao-Jie Yan, Marién Pascual, Manxia Fan, Jacqueline Barrientos, Steven L. Allen, et al. "CLL intraclonal fractions exhibit established and recently acquired patterns of DNA methylation." Blood Advances 4, no. 5 (March 9, 2020): 893–905. http://dx.doi.org/10.1182/bloodadvances.2019000817.

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Abstract Intraclonal subpopulations of circulating chronic lymphocytic leukemia (CLL) cells with different proliferative histories and reciprocal surface expression of CXCR4 and CD5 have been observed in the peripheral blood of CLL patients and named proliferative (PF), intermediate (IF), and resting (RF) cellular fractions. Here, we found that these intraclonal circulating fractions share persistent DNA methylation signatures largely associated with the mutation status of the immunoglobulin heavy chain locus (IGHV) and their origins from distinct stages of differentiation of antigen-experienced B cells. Increased leukemic birth rate, however, showed a very limited impact on DNA methylation of circulating CLL fractions independent of IGHV mutation status. Additionally, DNA methylation heterogeneity increased as leukemic cells advanced from PF to RF in the peripheral blood. This frequently co-occurred with heterochromatin hypomethylation and hypermethylation of Polycomb-repressed regions in the PF, suggesting accumulation of longevity-associated epigenetic features in recently born cells. On the other hand, transcriptional differences between paired intraclonal fractions confirmed their proliferative experience and further supported a linear advancement from PF to RF in the peripheral blood. Several of these differentially expressed genes showed unique associations with clinical outcome not evident in the bulk clone, supporting the pathological and therapeutic relevance of studying intraclonal CLL fractions. We conclude that independent methylation and transcriptional landscapes reflect both preexisting cell-of-origin fingerprints and more recently acquired hallmarks associated with the life cycle of circulating CLL cells.

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Hale, Anna L., J. Creighton Miller, K. Renganayaki, Alan K. Fritz, J. J. Coombs, L. M. Frank, and D. S. Douches. "Suitability of AFLP and Microsatellite Marker Analysis for Discriminating Intraclonal Variants of the Potato Cultivar Russet Norkotah." Journal of the American Society for Horticultural Science 130, no. 4 (July 2005): 624–30. http://dx.doi.org/10.21273/jashs.130.4.624.

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The objective of this study was to differentiate six intraclonal variants of the potato (Solanum tuberosum L.) cultivar Russet Norkotah. One-hundred-twelve AFLP primer combinations producing 3755 bands and 79 microsatellite primers producing over 400 bands failed to identify any reproducible polymorphisms among the intraclonal variants and `Russet Norkotah'. The inability to detect differences between clones underscores the degree of genetic similarity between them, despite differences in phenotypic expression. This inability could be due to the tetraploid nature of the clones and/or to epigenetic differences not detected by the utilized procedures.

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8

Beke, Allan, Lucie Laplane, Julie Riviere, Qin Yang, Miguel Torres-Martin, Thibault Dayris, Philippe Rameau, et al. "Multilayer intraclonal heterogeneity in chronic myelomonocytic leukemia." Haematologica 105, no. 1 (May 2, 2019): 112–23. http://dx.doi.org/10.3324/haematol.2018.208488.

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9

Aarts, Wilhelmina M., Richard J. Bende, Eric J. Steenbergen, Philip M. Kluin, Engelbert C. M. Ooms, Steven T. Pals, and Carel J. M. van Noesel. "Variable heavy chain gene analysis of follicular lymphomas: correlation between heavy chain isotype expression and somatic mutation load." Blood 95, no. 9 (May 1, 2000): 2922–29. http://dx.doi.org/10.1182/blood.v95.9.2922.009k38_2922_2929.

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The expansion of follicular lymphomas (FLs) resembles, both morphologically and functionally, normal germinal center B-cell growth. The tumor cells proliferate in networks of follicular dendritic cells and are believed to be capable of somatic hypermutation and isotype switching. To investigate the relation between somatic mutation and heavy chain isotype expression, we analyzed the variable heavy (VH) chain genes of 30 FL samples of different isotypes. The VH genes of the FLs were heavily mutated (29.3 mutations on average). In addition, isotype-switched lymphomas contained more somatic mutations than immunoglobulin M–positive lymphomas (33.8 mutations per VH gene versus 23.0, respectively). In all but one of the FLs, the ratios of replacement versus silent mutations in the framework regions were low, independent of the absolute number of somatic mutations and the level of intraclonal variation. Analysis of relapse samples of 4 FLs showed no obvious increase in somatic mutation load in most FLs and a decrease in intraclonal variation in time. In 3 of 4 cases, we obtained evidence for selection of certain subclones, rather than clonal evolution. Our findings question if intraclonal variation is always a reflection of ongoing somatic hypermutation. This may have implications for the concept of antigen-driven lymphomagenesis.

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Fagerström, T., and A. G. B. Poore. "Intraclonal Variation in Macroalgae: Causes and Evolutionary Consequences." Selection 1, no. 1-3 (January 2001): 123–34. http://dx.doi.org/10.1556/select.1.2000.1-3.12.

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11

HAGBERG, ARNE, and OLOF TEDIN. "INTER- AND INTRACLONAL CROSSES AND INBREEDING IN POTATOES." Hereditas 37, no. 1-2 (July 9, 2010): 280–87. http://dx.doi.org/10.1111/j.1601-5223.1951.tb02894.x.

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12

Jacob, Joshy, Garnett Kelsoe, Klaus Rajewsky, and Ursula Weiss. "Intraclonal generation of antibody mutants in germinal centres." Nature 354, no. 6352 (December 1991): 389–92. http://dx.doi.org/10.1038/354389a0.

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13

Pilbrough, Warren, Trent P. Munro, and Peter Gray. "Intraclonal Protein Expression Heterogeneity in Recombinant CHO Cells." PLoS ONE 4, no. 12 (December 23, 2009): e8432. http://dx.doi.org/10.1371/journal.pone.0008432.

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Benomar, Lahcen, Annie DesRochers, and Guy R. Larocque. "Changes in specific leaf area and photosynthetic nitrogen-use efficiency associated with physiological acclimation of two hybrid poplar clones to intraclonal competition." Canadian Journal of Forest Research 41, no. 7 (July 2011): 1465–76. http://dx.doi.org/10.1139/x11-073.

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Photosynthesis is the most important process driving productivity, and its acclimation to intraclonal competition is not well understood in hybrid poplars. The aim of this study was to examine the physiological response of the crown of two hybrid poplar clones, BT747215 ( Populus balsamifera L. × Populus trichocarpa Torr. & A. Gray) and MB915319 ( Populus maximowiczii A. Henry × P. balsamifera ), to intraclonal competition. We measured light-saturated photosynthesis (Amax), leaf N content on an area basis (Narea), specific leaf area (SLA), and photosynthetic N-use efficiency (PNUE) for three successive growing seasons on trees planted at three spacings (1 m × 1 m, 3 m × 3 m, and 5 m × 5 m). Crowns were sampled at 10 locations corresponding to vertical and horizontal subdivisions. Significant changes took place at the crown level in the closest spacing (1 m × 1 m) compared with the wider spacings (3 m × 3 m and 5 m × 5 m): (i) 30% decrease in Narea, (ii) 20% increase in SLA, and (iii) 40% increase in PNUE. The slope of the Amax–Narea curve was greatest in the closest spacing, indicating a greater change in Amax per unit change in Narea. The two hybrid poplar clones had a similar morphophysiological response to changes of spacing. Both clones showed physiological acclimation of their foliage in response to intraclonal competition through modulation of SLA and PNUE.

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Budeus, Bettina, Stefanie Schweigle de Reynoso, Martina Przekopowitz, Daniel Hoffmann, Marc Seifert, and Ralf Küppers. "Complexity of the human memory B-cell compartment is determined by the versatility of clonal diversification in germinal centers." Proceedings of the National Academy of Sciences 112, no. 38 (August 31, 2015): E5281—E5289. http://dx.doi.org/10.1073/pnas.1511270112.

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Our knowledge about the clonal composition and intraclonal diversity of the human memory B-cell compartment and the relationship between memory B-cell subsets is still limited, although these are central issues for our understanding of adaptive immunity. We performed a deep sequencing analysis of rearranged immunoglobulin (Ig) heavy chain genes from biological replicates, covering more than 100,000 memory B lymphocytes from two healthy adults. We reveal a highly similar B-cell receptor repertoire among the four main human IgM+ and IgG+ memory B-cell subsets. Strikingly, in both donors, 45% of sequences could be assigned to expanded clones, demonstrating that the human memory B-cell compartment is characterized by many, often very large, B-cell clones. Twenty percent of the clones consisted of class switched and IgM+(IgD+) members, a feature that correlated significantly with clone size. Hence, we provide strong evidence that the vast majority of Ig mutated B cells—including IgM+IgD+CD27+ B cells—are post-germinal center (GC) memory B cells. Clone members showed high intraclonal sequence diversity and high intraclonal versatility in Ig class and IgG subclass composition, with particular patterns of memory B-cell clone generation in GC reactions. In conclusion, GC produce amazingly large, complex, and diverse memory B-cell clones, equipping the human immune system with a versatile and highly diverse compartment of IgM+(IgD+) and class-switched memory B cells.

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Rawstron, Andy C., Darren J. Newton, Ruth M. de Tute, Jane Shingles, Fiona L. Bennett, Paul A. S. Evans, Sheila J. M. O’Connor, Andrew S. Jack, and Peter Hillmen. "Differential Protein Expression in MBL and CLL: LAIR1 Is a Powerful Surface Marker for Identifying Cases with Adverse Cellular Features." Blood 112, no. 11 (November 16, 2008): 2076. http://dx.doi.org/10.1182/blood.v112.11.2076.2076.

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Abstract INTRODUCTION: CLL is a disorder with a wide variation in outcome. Patients with adverse cellular features are often refractory to treatment and have a short overall survival. Individuals with CLL-type MBL are unlikely to require treatment and in most cases will eventually die of an unrelated cause. Many factors that predict a poor outcome have been identified, including stage, IGHV mutation status, ZAP-70 expression, and deletions of chromosomes 17p (TP53) and/or 11q23 (ATM). Deletions and mutations in TP53 are generally not presenting features and appear to require clonal evolution. One hypothesis is that the degree of intraclonal variation in genes targeted by the somatic hypermutation machinery, e.g. IGHV and BCL6, may predict the potential for clonal evolution. We have previously tested 66 antigens for their capacity to differentiate proliferating CLL cells, resting CLL cells and normal B-cells and identified 30 potentially relevant markers, including common markers such as CD38 and less frequently used markers such as the Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1). AIM: To compare the expression of relevant cell surface markers with the degree of intraclonal variation in the IGHV and BCL6 genes and to determine if these markers can be used to differentiate CLL-type MBL and CLL with or without adverse biological features. METHODS: The cell surface phenotype was assessed by 6-colour cytometry in 133 patients: 22 CLL with deletion 17p or 11q23, 69 CLL with no adverse prognostic chromosomal abnormalities, and 42 MBL. Surface phenotype was also compared with IGHV mutation status in a cohort of 29 CLL patients (16 ≤2% IGHV mutation, 13 >2% IGHV mutation). These antigens were also assessed using 4-colour flow cytometry in 20 cases (4 MBL, 16 CLL) and compared with IGHV & BCL6 mutation status and degree of intraclonal variation (defined as the proportion of mutations that were detected in a single clone only), and with ZAP-70 (AF488-1E7.2) expression. RESULTS: CLL cases with ≤2% IGHV mutation showed increased expression of CD38 (6.8 fold, p 0.02), CD49d (4.9-fold, P = 0.04), IgD (2.0-fold, P = 0.05), ZAP-70 (1.5-fold, P=0.04) and decreased expression of LAIR-1 (6.2-fold, P = 0.003) in comparison to CLL cases with >2% IGHV mutation. CLL cases with deletions of 17p and 11q23 showed decreased expression of CCR6 (1.7-fold, P = 0.0001), IgD (1.3-fold, P = 0.03) and LAIR-1 (7.1-fold, P<0.0001) in comparison to CLL cases without deletion 17p/11q23. CLL cases showed increased expression of CD23 (1.4-fold, P = 0.04), CD25 (1.3-fold, P = 0.05) and CD62L (1.7-fold, P = 0.04) in comparison to MBL. Cases with ≤2% overall IGHV mutation showed a similar degree of intraclonal variation as cases with >2% overall IGHV mutation in both IGHV (median 0.075% vs. 0.049% unique mutations, P>0.05) and BCL6 (median 0.10% vs. 0.095% unique mutations, P>0.05). However, there was an inverse relationship between BCL6 and IGHV intraclonal variation and cases with the highest levels of BCL6 intraclonal variation showed significantly decreased expression of CD39 (1.9-fold, P = 0.04) and LAIR1 (4.7-fold, P = 0.019). CONCLUSIONS: There were no markers or marker combinations that could discriminate MBL from CLL. The key differences were decreased expression of markers that are expressed during cell cycle, i.e. CD23, and adhesion markers such as CD62L and CD49d. These markers show sequential changes with disease stage, supporting the hypothesis that cellular interactions are central to the accumulation and expansion of CLL cells. However, the marker most consistently associated with adverse biological features is LAIR1, which is weak or negative in CLL with ≤2% IGHV mutation, high levels of intraclonal variation and TP53 or ATM deletions. LAIR-1 is an inhibitory receptor involved in regulating classs-witching. LAIR1 is strongly expressed in normal circulating peripheral B-cells. As with other prognostic markers, expression is a continuous variable and therefore a suitable cutoff will need to be identified. However, fluorochrome-conjugated antibodies are readily available and expression on CLL cells is stable for several days in EDTA samples which should minimise inter-laboratory analytical variation. LAIR1 expression in CLL is more closely associated with IGHV mutation status than CD38 or ZAP-70 expression. LAIR1 is a promising prognostic marker that appears to be central to the development of aggressive CLL as there is a strong association between downregulation of LAIR1, intraclonal heterogeneity in BCL6 and development of TP53 and ATM deletions.

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Ruzickova, Sarka, Axel Pruss, Marcus Odendahl, Karsten Wolbart, Gerd-Rüdiger Burmester, Jürgen Scholze, Thomas Dörner, and Arne Hansen. "Chronic lymphocytic leukemia preceded by cold agglutinin disease: intraclonal immunoglobulin light-chain diversity inVH4-34 expressing single leukemic B cells." Blood 100, no. 9 (November 1, 2002): 3419–22. http://dx.doi.org/10.1182/blood.v100.9.3419.

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Abstract Autoimmune phenomena may precede or accompany lymphoid malignancies, especially B-chronic lymphocytic leukemia (B-CLL). We report a patient with a 7-year history of primary (idiopathic) cold agglutinin (CA) disease in whom B-CLL subsequently developed. Immunophenotyping and single-cell reverse transcription–polymerase chain reaction (RT-PCR) were applied to investigate the origin and diversification of leukemic B cells. The obtained data indicate a memory cell-type origin of the B-CLL cells. Remarkably, theIgVκ genes of the B-CLL cells showed intraclonal diversity, whereas the mutational pattern of their paired IgVH genes were invariant. Thus, the light-chain–restricted intraclonal diversity in individual leukemic B cells in this patient strongly indicates a differential regulation or selection of the ongoing mutational process. Of note, our findings suggest that this B-CLL had developed from the patient's CA-producing B-cell population.

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Sahota, Surinder S., Francesco Forconi, Christian H. Ottensmeier, Drew Provan, David G. Oscier, Terry J. Hamblin, and Freda K. Stevenson. "Typical Waldenstrom macroglobulinemia is derived from a B-cell arrested after cessation of somatic mutation but prior to isotype switch events." Blood 100, no. 4 (August 15, 2002): 1505–7. http://dx.doi.org/10.1182/blood.v100.4.1505.h81602001505_1505_1507.

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There exists a wide spectrum of IgM-secreting B-cell tumors with different clinical behavior. Knowledge of the VH gene status can reveal their origin and clonal history. For Waldenstrom macroglobulinemia (WM), a distinct subtype of lymphoplasmacytic lymphoma, early data on limited sequences showed evidence for somatic mutation. A recent report of one case demonstrated intraclonal mutational activity occurring after transformation, a characteristic of germinal center lymphomas. To extend the investigation, we have analyzed 7 cases of WM. VH genes were somatically mutated with no evidence of intraclonal variation in all cases. In contrast to IgM-secreting multiple myeloma, there was no evidence for isotype switch transcripts in any of the cases. These data support the concept that typical WM is derived from a B cell that has undergone somatic mutation prior to transformation, at a point where isotype switch events have not been initiated.

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Coelho, Vânia, Sergey Krysov, Andrew Steele, Marina Sanchez Hidalgo, Peter W. Johnson, Prabhjoat S. Chana, Graham Packham, Freda K. Stevenson, and Francesco Forconi. "Identification in CLL of circulating intraclonal subgroups with varying B-cell receptor expression and function." Blood 122, no. 15 (October 10, 2013): 2664–72. http://dx.doi.org/10.1182/blood-2013-02-485425.

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Key Points The study reveals that circulating CLL cells contain intraclonal subgroups that differ in the level of signal-responsive surface IgM. Subgroups with higher surface IgM express more CXCR4, potentially predicting a dangerous ability to migrate to tissue and engage antigen.

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Dimitriadou, Evangelia, Vasiliki Karali, Maria Aggelopoulou, G. A. Pangalis, and Panayiotis Panayiotidis. "Intraclonal Immunoglobulin VDJ Diversification in Chronic Lymphocytic Leukemia Cells." Blood 104, no. 11 (November 16, 2004): 4812. http://dx.doi.org/10.1182/blood.v104.11.4812.4812.

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Abstract The monoclonal Ig expressed on CLL cells was considered to be identical in all cells of the leukemic clone in a CLL patient. However, intraclonal VDJ gene diversification was recently reported in CLL B cells using SSCP analysis and sequencing of Ig PCR products exhibiting altered mobility. We used a different approach, by cloning directly monoclonal VHDHJH PCR bands from CLL patients and sequencing a large number of clones. Peripheral blood from five patients with typical CLL was used to extract DNA.. In one patient DNA from bone marrow and a lymph node biopsy was also analysed. PCR was performed with VH family specific primers and a consensus JH primer for 30 cycles. Monoclonal bands were ligated to TOPO vector and 25 to 70 insert containing clones were sequenced from both orientations. Sequenses contigs were aligned with DNASTAR and were compared with the germ line counterpart using BLAST and IMGT data bases. Intraclonal base variation was observed in 8–33% of the clones in all five patients. One to five base point-mutations /clone were found and there were mostly transitions (in 79% of mutations). In one patient with two monoclonal Ig sequences, (one hypermutated V3-7 and one unmutated VH3-23) intraclonal variation was observed in 69% and 8% of the clones respectivelly. In two patients clones with partially shared mutations were observed indicating the further diversification of a parental subclone. In the patient that VHDHJH clones from blood/bone marrow/lymph node were obtained, a difference in the frequency of diversified clones was noted. All samples were obtained from the patient within one month to exclude the possibility of time related mutations in the monoclonal Ig of the patient. In the patient’s VH1-69 unmutated sequence, 19 of 64 (29,6%) of bone marrow derived clones had additional mutations but they were found in only 12 of 71 (16%) peripheral blood derived clones. Five of 53 (9,4%) lymph node derived clones had additional point mutations. Low R/S ratio and targeting of FR rather than CDR regions is against the hypothesis of antigen driven somatic mutations in a fraction of the CLL cells in a given patient. The difference in diversification rate between different tissues in the same patient raises the question of the role of the patient microenvironment/stroma in partcipating in these mutations observed in CLL subclones. With the analysis of large number of clones it is possible that all CLL patients may have at least some degree of intaraclonal VHDHJH gene diversification.

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Puig, Noemi, Isabel Conde, Cristina Jimenez, Maria E. Sarasquete, Ana Balanzategui, Miguel Alcoceba, Jonathan Quintero, et al. "Intraclonal Heterogeneity Associates with Clonal Stability in Multiple Myeloma." Blood 124, no. 21 (December 6, 2014): 3412. http://dx.doi.org/10.1182/blood.v124.21.3412.3412.

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Abstract Multiple myeloma (MM) pathogenesis has been explained for many years by the cancer biology dogma introduced by Peter Nowell: first, a single plasma cell would be immortalized by an error in the immunoglobulin genes rearrangement process; then, a progressive stepwise acquisition of somatic cell mutations would induce a sequential selection and domination by the fittest clone. In line with this idea of “myeloma stability”, SNP arrays studies in diagnostic-relapse paired samples have revealed the presence of common clonal characteristics. Biologically, the M-protein remains usually constant across MM evolution and further, the variable domain of the rearranged immunoglobulin heavy chain genes (or CDR3 region) has been used as a patient-specific myeloma fingerprint in minimal residual disease (MRD) studies. However, massive genome studies with Next Generation Sequencing (NGS) have challenged this concept, showing a significant intraclonal heterogeneity at diagnosis with the possible presence of several clonal progenitors or tumor-initiating cells. In this study, we have characterized and compared the CDR3 region in 52-paired samples from 26 MM patients aiming: 1) to assess mono-clonality in MM evolution through the analysis of the CDR3 sequence and, 2) to validate ASO RQ-PCR approaches for MRD in MM, based on the constancy and specificity of the CDR3 region. Samples were obtained at diagnosis and progression (19 pairs) or at 2 different timepoints of progressive disease (7 pairs). Median time between sampling was 2 years. M-protein subtype remained stable in all pairs but 1, associated with a light-chain escape phenomenon. All samples proceeded from bone marrow (BM) except for 2 pairs, composed by BM and extramedullary disease (spleen and testes). Two major cytogenetic changes were identified: increased 13q14 deletion (from 7 to 54%) in 1 pair and increased 17p (p53) deletion (from 5 to 87%) in a further one. Treatments administered between sampling included most of the current approaches used in MM (data not shown). Genomic DNA isolation, PCR amplification and sequencing were performed following conventional methods. Germline VH, DH and JH segments were identified by comparison with public databases. CDR3 region was first identified in all samples and then compared between the two samples in the 26 pairs: the sequence of nucleotides was constantly identical in each pair, including those associated with major cytogenetic changes, a light-chain escape, extramedullar vs. BM infiltration and relapsed (and therefore, treatment selected) vs. refractory disease. Therefore, we can first conclude that the main tumor clone in MM retains a specific signature across all stages of disease evolution that allows the identification of samples as evolutionary related. This major clone signature is not modified by clinical or biological changes in the disease nor under different treatment pressures and would thus identify disease relapse and progression. Our results have also a clear impact on the validity of molecular MRD techniques. The high rate of complete responses (up to 50-60%) currently achieved in MM has prompted the use of new techniques for disease assessment. Today, ASO RQ-PCR, based on the use of specific primers and probes complementary of the VDJH rearrangement, continues to be the most sensitive approach. One pitfall of this technique would be the potential instability of PCR targets over time, which would induce false negative results. In B-cell precursor ALL, this is estimated to happen in 30-40% of cases but has not been deeply evaluated in MM yet. With the present study, we can also conclude that the junction region of the VDJH rearrangement in MM constantly identifies the myeloma cells responsible for relapse and therefore can be used as a reliable target for MRD assessment by ASO RQ-PCR and more recently, by NGS methods. If the CDR3 region remains stable, the novel concept of clonal tiding in MM should not be interpreted as a poly- or oligoclonal but subclonal. In MM, tides can be subclonal, but the ocean remains monoclonal. Disclosures No relevant conflicts of interest to declare.

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22

Peacock, Lori, Vanessa Ferris, Mick Bailey, and Wendy Gibson. "Intraclonal mating occurs during tsetse transmission of Trypanosoma brucei." Parasites & Vectors 2, no. 1 (2009): 43. http://dx.doi.org/10.1186/1756-3305-2-43.

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23

ELENA, SANTIAGO F., FRANCISCO M. CODOÑER, and RAFAEL SANJUÁN. "Intraclonal variation in RNA viruses: generation, maintenance and consequences." Biological Journal of the Linnean Society 79, no. 1 (May 7, 2003): 17–26. http://dx.doi.org/10.1046/j.1095-8312.2003.00173.x.

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24

Medioli, F. S., D. B. Scott, and B. H. Abbott. "A case study of protozoan intraclonal variability; taxonomic implications." Journal of Foraminiferal Research 17, no. 1 (January 1, 1987): 28–47. http://dx.doi.org/10.2113/gsjfr.17.1.28.

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25

Kocks, C., and K. Rajewsky. "Stepwise intraclonal maturation of antibody affinity through somatic hypermutation." Proceedings of the National Academy of Sciences 85, no. 21 (November 1, 1988): 8206–10. http://dx.doi.org/10.1073/pnas.85.21.8206.

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26

Markely, Lam Raga A., and Daniel I. C. Wang. "High-throughput analysis of intraclonal variability of glycoprotein sialylation." Biotechnology Progress 28, no. 2 (November 7, 2011): 591–94. http://dx.doi.org/10.1002/btpr.738.

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27

Wang, Yui-Hsi, Zhixin Zhang, Peter D. Burrows, Hiromi Kubagawa, S. Louis Bridges, Harry W. Findley, and Max D. Cooper. "V(D)J recombinatorial repertoire diversification during intraclonal pro-B to B-cell differentiation." Blood 101, no. 3 (February 1, 2003): 1030–37. http://dx.doi.org/10.1182/blood-2002-06-1828.

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Abstract The initial B-cell repertoire is generated by combinatorial immunoglobulin V(D)J gene segment rearrangements that occur in a preferential sequence. Because cellular proliferation occurs during the course of these rearrangement events, it has been proposed that intraclonal diversification occurs during this phase of B-cell development. An opportunity to examine this hypothesis directly was provided by the identification of a human acute lymphoblastic leukemic cell line that undergoes spontaneous differentiation from pro-B cell to the pre-B and B-cell stages with concomitant changes in the gene expression profile that normally occur during B-cell differentiation. After confirming the clonality of the progressively differentiating cells, an analysis of immunoglobulin genes and transcripts indicated that pro-B cell members marked by the same DJ rearrangement generated daughter B cells with multiple VH and VL gene segment rearrangements. These findings validate the principle of intraclonal V(D)J diversification during B-cell generation and define a manipulable model of human B-cell differentiation.

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28

Crouzier, R., T. Martin, and J. L. Pasquali. "Monoclonal IgM rheumatoid factor secreted by CD5-negative B cells during mixed cryoglobulinemia. Evidence for somatic mutations and intraclonal diversity of the expressed VH region gene." Journal of Immunology 154, no. 1 (January 1, 1995): 413–21. http://dx.doi.org/10.4049/jimmunol.154.1.413.

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Abstract Mixed cryoglobulinemia is usually considered to be a nonmalignant human B cell proliferation that produces a monoclonal IgM rheumatoid factor (RF). Important immunologic similarities and differences were described between the monoclonal B cells during mixed cryoglobulinemia and during malignant chronic lymphocytic leukemia (CLL):high frequency of the same VH and V kappa gene usage by both types of monoclonal B cells producing IgM with RF activity, apparent intraclonal homogeneity, but different expression of the pan T cell CD5 Ag. The description of an unusual CD5-negative B cell CLL case secreting a mutated IgM RF led the authors to suggest that the usage of non-mutated germline Ig genes is a property of cells derived from the CD5 lineage or stage of differentiation, rather than an intrinsic property of CLL or of IgM RF-producing cells in general. Because mixed cryoglobulinemia cells are usually CD5-negative, it was of interest to test for the existence of mutations in the VH and V kappa regions, as well as for the intraclonal homogeneity of the expressed Ig genes. In this study, we used the PCR technique to analyze the monoclonal rheumatoid factor (mRF) V genes from a patient with mixed cryoglobulinemia. We show that the CD5-negative monoclonal B cells express a slightly mutated V kappa 3 gene, but a more mutated VH1 gene whose genomic counterpart was shown to be the 51p1 germline gene. The sequence analysis of several independent clones shows some degree of intraclonal diversity, suggesting the existence of a clonal filiation. These results are discussed in terms of the origin of the monoclonal B cell during mixed cryoglobulinemia and CLL.

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29

Picard, F., T. Martin, F. Legras, B. Lioure, and JL Pasquali. "Molecular analysis of T-cell receptor V beta chains of human T-cell chronic lymphocytic leukemia does not show intraclonal variability: implications for immunotherapy." Blood 82, no. 7 (October 1, 1993): 2152–56. http://dx.doi.org/10.1182/blood.v82.7.2152.2152.

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Abstract Human T-cell chronic lymphocytic leukemia (T-cell CLL) is a heterogeneous disease characterized by a monoclonal malignant proliferation of T cells in which the T-cell receptors (TCRs) can be, when expressed, considered to be membrane tumor-specific antigens. Owing to the increasing number of available monoclonal antihuman TCR reagents, it could be of interest to evaluate the feasibility of anti- TCR treatment during T-cell CLL. To test the therapeutic potentiality of anti-TCR monoclonal antibodies, we first analyzed the intraclonal variability in two terminally ill patients suffering from TCR alpha beta-positive cell CLL bearing different immunophenotypes. The cDNA corresponding to the variable regions of the TCR beta chains originating from the malignant T cells were amplified, cloned into M13 phages, and sequenced. The sequence analysis of multiple independent clones showed no intraclonal variability, with no evidence for ongoing hypermutation in the V beta region genes. The relevance of these findings with regard to an anti-V beta therapy and the comparison with similar analysis during B-cell monoclonal lymphoproliferations are discussed.

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30

Picard, F., T. Martin, F. Legras, B. Lioure, and JL Pasquali. "Molecular analysis of T-cell receptor V beta chains of human T-cell chronic lymphocytic leukemia does not show intraclonal variability: implications for immunotherapy." Blood 82, no. 7 (October 1, 1993): 2152–56. http://dx.doi.org/10.1182/blood.v82.7.2152.bloodjournal8272152.

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Human T-cell chronic lymphocytic leukemia (T-cell CLL) is a heterogeneous disease characterized by a monoclonal malignant proliferation of T cells in which the T-cell receptors (TCRs) can be, when expressed, considered to be membrane tumor-specific antigens. Owing to the increasing number of available monoclonal antihuman TCR reagents, it could be of interest to evaluate the feasibility of anti- TCR treatment during T-cell CLL. To test the therapeutic potentiality of anti-TCR monoclonal antibodies, we first analyzed the intraclonal variability in two terminally ill patients suffering from TCR alpha beta-positive cell CLL bearing different immunophenotypes. The cDNA corresponding to the variable regions of the TCR beta chains originating from the malignant T cells were amplified, cloned into M13 phages, and sequenced. The sequence analysis of multiple independent clones showed no intraclonal variability, with no evidence for ongoing hypermutation in the V beta region genes. The relevance of these findings with regard to an anti-V beta therapy and the comparison with similar analysis during B-cell monoclonal lymphoproliferations are discussed.

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31

Bezuidt, Oliver KI, Jens Klockgether, Sylvie Elsen, Ina Attree, Colin F. Davenport, and Burkhard Tümmler. "Intraclonal genome diversity of Pseudomonas aeruginosa clones CHA and TB." BMC Genomics 14, no. 1 (2013): 416. http://dx.doi.org/10.1186/1471-2164-14-416.

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32

Miller, J. Creighton, George C. C. Tai, Bernard Ouellette, and Jeannie P. Miller. "Discriminating Russet Norkotah intraclonal selections using canonical and cluster analysis." American Journal of Potato Research 81, no. 3 (May 2004): 203–7. http://dx.doi.org/10.1007/bf02871750.

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33

Nassar, Atef M. K., Jihad Abdulnour, Yves Leclerc, Xiu-Qing Li, and Danielle J. Donnelly. "Intraclonal Selection for Improved Processing of NB ‘Russet Burbank’ Potato." American Journal of Potato Research 88, no. 5 (July 27, 2011): 387–97. http://dx.doi.org/10.1007/s12230-011-9204-5.

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34

Ma, Wanlong, Maya Thangavelu, Ivan De Dios, Vincent Funari, and Maher Albitar. "Interclonal and Intraclonal Heterogeneity in Patients with IDH1/2 Mutation." Blood 128, no. 22 (December 2, 2016): 1689. http://dx.doi.org/10.1182/blood.v128.22.1689.1689.

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Abstract Background: DNA methylation in AML/MDS plays a major role in the pathogenesis of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). The major genes involved in DNA methylation in AML/MDS are IDH1 and 2, TET2 and DNMT3A. Mutations in IDH1/2 result in the production of an aberrant metabolite, 2-hydroxyglutarate, which acts as a competitive inhibitor of a-ketoglutarate and inhibits TET2 oxidation of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Mutations in TET2 or IDH1/2 are associated with reduced levels of 5hmC and genomic hypermethylation. TET2 mutations and IDH1/IDH2 mutations are believed to be mutually exclusive. In addition, DNMT3A as a DNA methyltransferase enzyme is commonly mutated in AML/MDS and its mutation is believed to lead to hypomethylation. Understanding the interaction between these genes may influence therapy with IDH1/2 inhibitors. Toward better understanding of interaction between these genes, we analyzed the mutation profile of these genes in patients with AML/MDS. Methods: A total of 1182 bone marrow (BM) aspirate samples were tested by the commercially available TruSight Myeloid Next Generation Sequencing Panel (Illumina, San Diego, CA). We extracted DNA from bone marrow aspirate using the QIAamp DNA Mini Kit. This NGS panel covers hot spot mutations in 54 genes. The average depth of sequencing was 10,000X. Results: IDH1/2 mutations were detected in 201 of the 1182 (17%). IDH1 was detected in 87 (7.4%) and IDH2 was detected in 120 (10.1%). This included 6 patients who had mutations in both IDH1 and IDH2. Variant (mutant) allele frequency (VAF) was significantly higher (P=0.01) in IDH2 as compared to IDH1 (median of 43.35% vs 35.0%, respectively). Thirteen patients (6.5%) had mutant VAF >50% suggesting homozygosity, 11 of which had IDH2 mutation. Two of the 6 patients with both IDH1 and IDH2 mutations had VAF <20%, raising the possibility of two independent clones. TET2 mutations were detected in 15 (7.5%) of the patients with IDH1/2 mutations. There was significant difference (P=0.03) in VAF between IDH1/2 and TET2. Nine of these patients showed comparable VAF while the other 6 patients showed completely different VAF, suggesting subclonal heterogeneity. In addition, 58 (29%) patients showed mutations in IDH1/2 and DNMT3A. While there was no significant difference in VAF between IDH1/2 and DNMT3A, VAF in IDH1/2 was >50% in 6 of these patient and in DNMT3A in 3 patients. Twenty four patients had TP53 mutation, of which 16 had IDH1 mutation and 8 had IDH2 mutation, which is disproportional with the prevalence of IDH1 mutation. There was no statistically significant difference in VAF between TP53 and IDH1/2, but 4 of these patients had both DNMT3 and IDH2 mutations and one had both IDH1 and IDH2 mutations. None of the patients with TP53 mutation had TET2 mutation. Conclusions: IDH2 mutations may coexist with IDH1 and TET2 mutations. This co-mutation appears to be in the same clone in some patients and in a separate clone in others. The presence of VAF>50% in 6.5% of patients, which suggests homozygosity, along with co-presence of IDH1 and IDH2 and TET2 mutations suggests possible dosage effects in the biology of MDS/AML. The high rate (29%) of co-presence of DNMT3A with IDH/1/2 mutations also suggests cooperation between the two mechanisms in influencing DNA methylation and leukemogenesis. The relatively high incidence of TP53 mutation in IDH1 patients suggests that IDH1 mutation might be associated with more aggressive disease than IDH2. This data suggests that there is interaction and significant interclonal and intraclonal heterogeneity in DNA methylation genes in AML/MDS. Complete profiling of these genes is necessary for better understanding and proper prediction of clinical behavior particularly when patients treated with DNA methylation inhibitors. Figure Figure. Disclosures Ma: Neogenomics Laboratories: Employment. Thangavelu:Neogenomics Laboratories: Employment. De Dios:Neogenomics Laboratories: Employment. Funari:Neogenomics Laboratories: Employment. Albitar:Neogenomics Laboratories: Employment, Equity Ownership.

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35

Meneses, I., B. Santelices, and P. Sánchez. "Growth-related intraclonal genetic changes in Gracilaria chilensis (Gracilariales: Rhodophyta)." Marine Biology 135, no. 3 (December 4, 1999): 391–97. http://dx.doi.org/10.1007/s002270050639.

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36

Fischer, Sebastian, Jens Klockgether, Patricia Morán Losada, Philippe Chouvarine, Nina Cramer, Colin F. Davenport, Sarah Dethlefsen, et al. "Intraclonal genome diversity of the majorPseudomonas aeruginosaclones C and PA14." Environmental Microbiology Reports 8, no. 2 (January 28, 2016): 227–34. http://dx.doi.org/10.1111/1758-2229.12372.

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37

Gibson, W., K. Winters, G. Mizen, J. Kearns, and M. Bailey. "Intraclonal mating in Trypanosoma brucei is associated with out-crossing." Microbiology 143, no. 3 (March 1, 1997): 909–20. http://dx.doi.org/10.1099/00221287-143-3-909.

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38

Comenzo, RL, SC Jhanwar, SD Nimer, P. Zhou, A. Boruchov, P. Lu, E. Bonvini, and H. Hassoun. "B500 CD32B Expression Reflects Intraclonal Functional Heterogeity in Multiple Myeloma." Clinical Lymphoma and Myeloma 9 (February 2009): S141. http://dx.doi.org/10.1016/s1557-9190(11)70751-x.

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39

Dimitriadou, Evangelia M., Panayiotis Panayiotidis, Vasiliki Karali, Konstantinos Lilakos, Maria K. Angelopoulou, Marina P. Siakantaris, and Gerassimos A. Pangalis. "Complex Paterns of Monoclonal Ig Diversification in B-CLL Cells from Different Tissue Compartments." Blood 106, no. 11 (November 16, 2005): 5021. http://dx.doi.org/10.1182/blood.v106.11.5021.5021.

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Abstract It has recently been demonstrated that a considerable subset of CLL patients (>50%) displays intraclonal diversification in their monoclonal Ig sequence, suggesting that the mechanism of ongoing somatic mutations is retained in a significant proportion of B CLL cells. The complexity of studying this phenomenon relies on the enclaved risk of misinterpreting superimposed mutations originating from experimental artifacts. We are still lacking a more detailed picture of inraclonal diversification regarding its frequency and developmental pattern in disease compartments other than blood. In order to clarify some of these questions, we analyzed a total of 675 Ig clones from 11 CLL patients. DNA was extracted from blood in 6 patients (1st group: pt.1 to 6) or from blood/marrow (BM)/lymph nodes (LN) obtained at the same in 5 patients (2nd group: pt.7-11). PCR was performed for 40 cycles with Taq Platinum polymerase and leader or FR1 VH1-6 primers and a consensus JH primer. Monoclonal PCR products were ligated to TA vector and 20 to 70 clones were sequenced. from both orientations. Polymerase mediated base error was 4.4x10−4 after 40 PCR cycles. Base differences observed in two or more clones out of 20, were verified twice and assigned as true intraclonal diversification. This was observed in 6 (pt 2, 6, 7, 9, 10, 11) out of 11 pts (54.5%). Pts 2, 6, 10, 11 were mutated and pt. 7, 9 unmutated in their Ig sequence. Intraclonal diversification was observed in pt. 2 and 6, whereas pt.7, 9, 10, 11 in the 2nd group, displayed intraclonal diversification in some or in all compartments. Base point mutations that were unique in one clone only were observed at the polymerase mediated error rate and were not verified by a second idependent experiment. In the 2nd group pt. 8 displayed lack of diversification both in blood and marrow. Pt.7 displayed diversification only in BM clones (2/64, 3.1%) but not in 71 blood derived or in 53 LN derived clones. Pt. 9 had diversified clones in blood only (2/26, 7.6%) and no diversified clones in 25 BM clones. Pt.10 had diversified clones both in blood and marrow with a distinct pattern: in blood (62 clones) 4 different patterns of diversification were observed in 54 clones (84%); 17/54 were evolving clones. In BM two of the 4 patterns of diversification present in blood were observed in 28/57 clones (51.7%); ten were evolving clones. In pt.11, no diversified clones were present in blood (50 clones) whereas marrow had 4/60 diversified clones (6,66%, two patterns of diversification) and LN had 20/40 diversified clones (50%, two patterns of diversification); 8/20 diversified were evolving clones. The patterns of diversification were different bettween LN and BM in this patient. Base changes in 110 diversified clones were in FRs rather than CDRs (16:1) and resulted in S(silent) rather than R(replacements), S/R 2:1. In conclusion, intraclonal diversification must be considered true if identical base substitutions are observed in minimum two clones. Pattern and frequency are not identical in B CLL cells from different sites (blood vs BM vs LN). Preferance of FRs v CDRs and SvR substitutions implies that diversification procces is not antigen driven. The presence of a functional mechanism of ongoing somatic mutations in leukemic clones and the ability of diversification to target with altered frequency/patterns various disease compartments implies a role of tissue millieu in CLL clone evolution.

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40

Tarín, Fabián, Francisco López-Castaño, Carmen García-Hernández, Paola Beneit, Héctor Sarmiento, Pablo Manresa, Olga Alda, et al. "Multiparameter Flow Cytometry Identification of Neoplastic Subclones: A New Biomarker in Monoclonal Gammopathy of Undetermined Significance and Multiple Myeloma." Acta Haematologica 141, no. 1 (November 14, 2018): 1–6. http://dx.doi.org/10.1159/000493568.

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Multiparameter flow cytometry (MFC)-based clonality assessment is a powerful method of diagnosis and follow-up in monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). However, the relevance of intraclonal heterogeneity in immunophenotypic studies remains poorly understood. The main objective of this work was to characterize the different immunophenotypic subclones in MGUS and MM patients and to investigate their correlation with disease stages. An 8-color MFC protocol with 17 markers was used to identify the subclones within the neoplastic compartment of 56 MGUS subjects, 151 newly diagnosed MM patients, 30 MM subjects in complete remission with detectable minimal residual disease, and 36 relapsed/refractory MM patients. Two or more clusters were observed in > 85% of MGUS subjects, 75% of stage I MM patients, and < 15% in stage III. Likewise, a significant correlation between the dominant subclone size, secondary cytogenetic features, and changes in the expression of CD27, CD44, and CD81 was detected. The loss of intraclonal equilibrium may be an important factor related with kinetics and risk of progression not well considered to date in MFC studies. The MFC strategy used in this work can provide useful biomarkers in MGUS and MM.

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41

Kriangkum, Jitra, Brian J. Taylor, Erin Strachan, Michael J. Mant, Tony Reiman, Andrew R. Belch, and Linda M. Pilarski. "Impaired class switch recombination (CSR) in Waldenström macroglobulinemia (WM) despite apparently normal CSR machinery." Blood 107, no. 7 (April 1, 2006): 2920–27. http://dx.doi.org/10.1182/blood-2005-09-3613.

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AbstractAnalysis of clonotypic isotype class switching (CSR) in Waldenström macroglobulinemia (WM) and IgM monoclonal gammopathy of undetermined significance (MGUS) reveals a normal initial phase of B-cell activation as determined by constitutive and inducible expression of activation-induced cytidine deaminase (AID). Switch μ (Sμ) analysis shows that large deletions are not common in WM or IgM MGUS. In CD40L/IL-4-stimulated WM cultures from 2 patients, we observed clonotypic IgG exhibiting intraclonal homogeneity associated with multiple hybrid Sμ/Sγ junctions. This suggests CSR had occurred within WM cells. Nevertheless, the estimated IgG/IgM-cell frequency was relatively low (1/1600 cells). Thus, for the majority of WM B cells, CSR does not occur even when stimulated in vitro, suggesting that the WM cell is constitutively unable to or being prevented from carrying out CSR. In contrast to WM, the majority of IgM MGUS clones exhibit intraclonal heterogeneity of IgH VDJ. Furthermore, most IgM MGUS accumulate more mutations in the upstream Sμ region than do WM, making them unlikely WM progenitors. These observations suggest that switch sequence analysis may identify the subset of patients with IgM MGUS who are at risk of progression to WM.

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42

Go, Jai Hyang, Dae Shick Kim, Tae Jin Kim, Young H. Ko, Hyun Ki Ra, Jong Chul Rhee, Seon Woo Kim, and Howe J. Ree. "Comparative Studies of Somatic and Ongoing Mutations in Immunoglobulin Heavy-Chain Variable Region Genes in Diffuse Large B-Cell Lymphomas of the Stomach and the Small Intestine." Archives of Pathology & Laboratory Medicine 127, no. 11 (November 1, 2003): 1443–50. http://dx.doi.org/10.5858/2003-127-1443-csosao.

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Abstract Context.—Many diffuse large B-cell lymphomas (DLBCLs) of the stomach are believed to represent high-grade transformation of low-grade marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue type, which is of memory B-cell origin, displaying evidence for positive antigen selection and a low level of ongoing somatic mutation of the rearranged immunoglobulin heavy-chain variable region (VH) genes. The pattern of somatic mutation has been studied little in intestinal DLBCLs. Objective.—To assess evidence for antigen selection and the levels of ongoing mutation, we analyzed the ratio of replacement to silent mutations, as well as the frequency of intraclonal sequence variation in gastric and small intestinal DLBCLs that showed no concomitant low-grade component. Design.—Genomic DNA was extracted from formalin-fixed paraffin blocks of gastric (n = 6) and small intestinal (n = 6) DLBCLs. The complementarity-determining region 2 and framework region 3 sequences (<200 base pairs) of the rearranged immunoglobulin VH gene were obtained from polymerase chain reaction–amplified product, and the ratio of replacement-to-silent mutations and the frequency of intraclonal sequence variation were determined. Results.—Clustering of replacement mutations in complementarity-determining region 2 with a high (>2.9) ratio of replacement-to-silent mutations was observed in 5 gastric DLBCLs, whereas it was recognized in only 1 intestinal DLBCL. Intraclonal sequence variation was observed in 6 intestinal and 5 gastric DLBCLs. The frequency of ongoing mutation was much higher in the intestinal (median, 0.33%) than in the gastric DLBCLs (median, 0.13%), but the difference was not statistically significant (P = .09). Conclusions.—The mutation pattern was consistent with positive antigen selection in gastric DLBCLs, but not in the intestinal tumors. Ongoing mutation was much more frequent in the intestinal than in the gastric DLBCLs. These findings suggest that positive antigen selection plays a major role in a significant proportion of gastric tumors, whereas germinal center reaction with aberrant mutation is important in small intestinal DLBCLs.

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Le, Thuc-vy L., Tea Hyun Kim, and David D. Chaplin. "Intraclonal Competition Inhibits the Formation of High-Affinity Antibody-Secreting Cells." Journal of Immunology 181, no. 9 (October 20, 2008): 6027–37. http://dx.doi.org/10.4049/jimmunol.181.9.6027.

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44

Palucka, Karolina A., Eva Knaust, Dawei Xu, Barbara Macnamara, Anna Porwit-MacDonald, Astrid Gruber, Curt Peterson, Magnus Björkholm, and Pavel Pisa. "Intraclonal Heterogeneity in theIn VitroDaunorubicin-Induced Apoptosis in Acute Myeloid Leukemia." Leukemia & Lymphoma 32, no. 3-4 (January 1999): 309–16. http://dx.doi.org/10.3109/10428199909167391.

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45

Clarke, S. H., K. Huppi, D. Ruezinsky, L. Staudt, W. Gerhard, and M. Weigert. "Inter- and intraclonal diversity in the antibody response to influenza hemagglutinin." Journal of Experimental Medicine 161, no. 4 (April 1, 1985): 687–704. http://dx.doi.org/10.1084/jem.161.4.687.

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This study focuses on 10 BALB/c anti-influenza virus (A/PR/8/34) hemagglutinin antibodies that have light chains encoded by the same variable region kappa chain (V kappa) gene, V kappa 21C. A comparison of antibodies from lymphocytes of independent origin reveals the contribution of germline diversity (combinatorial joining and association) to this response. Although combinatorial joining and association contribute to sequence diversity, they appear to have little effect on the fine specificity of these antibodies. Somatic mutation, in addition to contributing to the sequence diversity of these antibodies, creates differences in their fine specificity. The extent of mutation and its effect on fine specificity can be seen by comparing antibodies of lymphocytes from the same clone. These intraclonal comparisons also indicate that somatic mutation is an ongoing process occurring at a high rate (estimated to be at least 10(-3) mutations per base pair per division) in the expressed V region heavy chain (VH) and V kappa genes. Furthermore, both the nature and distribution of these mutations suggest that amino acid replacement mutations in the light but not the heavy chain are selected for by antigen.

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Tschumper, Renee C., Yan W. Asmann, Asif Hossain, Paul M. Huddleston, Xiaosheng Wu, Angela Dispenzieri, Bruce W. Eckloff, and Diane F. Jelinek. "Assessment of Multiple Myeloma IGHV Intraclonal Variation by Massively Parallel Pyrosequencing." Blood 118, no. 21 (November 18, 2011): 1814. http://dx.doi.org/10.1182/blood.v118.21.1814.1814.

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Abstract Abstract 1814 Multiple myeloma (MM) is an incurable hematologic disorder characterized by the accumulation of plasma cells (PCs) in the bone marrow (BM). Like normal B cells and PCs, MM cells express a distinctive immunoglobulin (Ig) molecular fingerprint resulting from heavy and light chain gene rearrangements and unique mutations introduced by the somatic hypermutation (SHM) process. Detailed examination of the Ig molecule may offer insight into the etiology of B cell malignancies as well as the ontogeny of malignant transformation and ongoing genetic evolution. Because prior studies of the Ig fingerprint of MM cells have been limited in size and scope, our current understanding of MM Ig repertoire is minimal. We used massively parallel sequencing of normal and malignant PCs to interrogate: 1) DNA versus RNA (cDNA) as starting material for studies of Ig repertoire; 2) the Ig repertoire of normal and malignant PCs; and 3) intraclonal heterogeneity in MM. Bone marrow PCs (BMPCs) from an untreated MM patient and from a normal control subject were isolated by magnetic bead separation. DNA and RNA were isolated from purified cells and amplified in a multiplex PCR reaction with primers designed to capture the entire IGHV region including the heavy chain complementarity determining region 3 (HCDR3) necessary for defining clonal cells. Using Roche 454 GS-FLX Titanium chemistry and a GS FLX Titanium Genome Sequencer, over 30,000 sequence reads were obtained from each sample. Following a detailed algorithm to minimize Taq and sequencing related errors and removal of non-Ig or non-productive Ig sequences, the resulting sequences were grouped based on HCDR3 amino acid identity. Each unique HCDR3 represented one member of the repertoire and all the sequences with the same HCDR3 defined the clone size. This measurement showed that there was nearly a five-fold higher level of unique HCDR3s in normal BMPCs when the starting PCR template was DNA vs. cDNA. Thus, DNA is clearly a better starting template for repertoire analysis using deep sequencing methods. Thorough analysis of the MM DNA sample identified PCs expressing 37 different IGHV genes, and predictably, the vast majority of sequence reads (83%) had exact nucleotide identity in the IGHV and HCDR3 region (IGHV3-74 with 18 mutations in the IGHV region) thereby defining the dominant MM clone. There were also ∼5000 additional sequences that either shared an identical HCDR3 region but varied within the IGHV3-74 gene sequence (n=3975) or were identical within the IGHV3-74 sequence yet varied within the HCDR3 (n=862). These sequences were further analyzed, with sequences not present 10 or more times or not found in both the 5' and 3' directions eliminated from further scrutiny. This process revealed a total of 64 putative subclones with single point mutations in the IGHV or HCDR3 region (10–56 copies of each unique sequence representing a total of 1583 clonally related sequences). When we used a more stringent subclone frequency of ≥0.1%, 22 subclones remained. Of note, conventional Ig repertoire analysis would require sequencing ≥1000 colonies to detect MM subclones present at this frequency. Finally, 11 subclones displayed a significant number of IGHV gene nucleotide differences from the dominant MM clone despite exhibiting an identical HCDR3 region. Notably, a number of these distinguishing IGHV gene mutations are silent and thus may preserve the antigenic specificity of these clonally related cells. These subclones failed our frequency criteria but the type and quality of deviations from the clonal sequence suggest that they are not artifact and warrant further investigation. Notably, all of the putative subclones were also found in the MM cDNA cohort providing further evidence as genuine subclones. The very large number of identical MM clonal sequences is consistent with the idea that MM is a post germinal center malignancy that can no longer undergo SHM. However, we present clear evidence of MM-related subclones albeit relatively small in number. The subclone sequence deviations discovered may reflect on-going genetic evolution independent of the formal SHM mechanism. Alternatively, these subclones may indeed reflect “sister” cells generated during the original germinal center reaction that resulted in the malignant clone. In summary, our studies demonstrate the extraordinary potential of this methodology to track clonal evolution in MM and its precursor conditions over time. Disclosures: No relevant conflicts of interest to declare.

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Monro, Keyne, and Alistair G. B. Poore. "Selection in Modular Organisms: Is Intraclonal Variation in Macroalgae Evolutionarily Important?" American Naturalist 163, no. 4 (April 2004): 564–78. http://dx.doi.org/10.1086/382551.

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Thangavelu, Maya, Wanlong Ma, Steven Brodie, Christopher Mixon, Wayne Chen, Sally Agersborg, and Maher Albitar. "Interclonal and Intraclonal Heterogeneity in Patients with Early Myelodysplastic Syndrome (MDS)." Blood 126, no. 23 (December 3, 2015): 1670. http://dx.doi.org/10.1182/blood.v126.23.1670.1670.

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Abstract Introduction: Recent data suggest that MDS evolves by accumulating mutations. Early mutations may involve genes that require additional mutations prior to clinical manifestation as MDS. We explored if mutant allele burden and the relative mutation of one gene to another gene could provide information on the interclonal and intraclonal progression of MDS using next generation sequencing (NGS) in patients with early MDS. Methods: NGS data was generated from 96 patients diagnosed with MDS with marrow blast count <5% using a targeted sequencing covering mutations in the following genes: TET2, SF3B1, ASXL1, DNMT3A, SRSF2, RUNX1, NRAS, ZRSR2, EZH2, ETV6, TP53, CBL, NPM1, JAK2, U2AF1, IDH1, KRAS, IDH2, FLT3, PTPN11, SETBP1, and BCOR. The average depth of sequencing was 10,000X. Differences in mutant allele frequency between two genes in the same sample were considered significant if they were >10%. A difference of 10% to 20% was considered mild, 20%-30% moderate, and >30% severe. A heat map reflecting these differences in mutant allele frequency was generated. Results: In this group of early MDS patients, 63 patients (66%) had more than one gene mutated and 38 (40%) had a significant (>10%) difference in allele frequency. The median number of genes mutated was 2 (range 1 to 5). Difference in mutant allele frequency was severe in 15 patients (16%), intermediate in 15 patients (16%), and mild in 13 patients (14%). TET2 was the most commonly mutated gene (43 patients, 45%) and was rarely the sole mutation with most cases exhibiting a mutation in a second gene (39 patients, 91%). The mutant allele burden was highest in TET2 in 26 of these 39 patients (67%), reflecting early event in the tumorigenic process. Of the 13 cases with TET2 mutation and allele burden less than the companion gene, 6 had a mutation in SF3B1, 3 had significant cytogenetic abnormalities (monosomy 5, del(7q), and trisomy 8), 2 had a mutation in SRSF2, 1 had a mutation in ZRSR2 and 1 had a mutation in ASXL1, which suggests that these abnormalities might be the initiating event. A second TET mutation (biallelic mutation) was detected in 16 of the 39 patients. SF3B1 was the most common gene having a solitary mutation (10% of all patients), although mutation in SF3B1 was detected in 27 patients (26% of all patients). All solitary SF3B1 mutations were associated with normal karyotypes, except for one patient with del(11q). JAK2 was mutated with SF3B1 in two cases diagnosed as RARS-T (refractory anemia with ring sideroblasts and thrombocytosis). In one case, the JAK2 and SF3B1 mutation allele frequencies were similar, but in the other, the JAK2 mutant allele frequency was 23% higher, suggesting that a myeloproliferative neoplasm was the initiating process. ASXL1 was mutated in 14 cases, 13 of which had additional mutations. DNMT3A gene was mutated in 18 cases, 5 of which were solitary; two of these five showed cytogenetic abnormalities. TP53 was mutated in 13 cases, but except for one case, all had either mutation in another gene or a cytogenetic abnormality. Conclusion: These data suggest that in patients with clinically confirmed early MDS, TET2 mutations are most likely the initiating oncogenic event, but mutations in other genes or cytogenetic abnormalities most likely lead to clinically confirmed MDS. In contrast, patients with SF3B1 mutation can have clinical disease without additional mutations. Our data suggest that SRSF2, ZRSR2, and ASXL1 may initiate mutagenesis in patients with MDS. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Santelices, Bernabe, Juan A. Correa, Isabel Meneses, Diego Aedo, and Daniel Varela. "SPORELING COALESCENCE AND INTRACLONAL VARIATION IN GRACILARIA CHILENSIS (GRACILARIALES, RHODOPHYTA)1." Journal of Phycology 32, no. 2 (April 1996): 313–22. http://dx.doi.org/10.1111/j.0022-3646.1996.00313.x.

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Vasconcelos, Zilton, Sabina Müller, Delphine Guipouy, Wong Yu, Claire Christophe, Sébastien Gadat, Salvatore Valitutti, and Loïc Dupré. "Individual Human Cytotoxic T Lymphocytes Exhibit Intraclonal Heterogeneity during Sustained Killing." Cell Reports 11, no. 9 (June 2015): 1474–85. http://dx.doi.org/10.1016/j.celrep.2015.05.002.

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Journal articles on the topic 'Intraclonal'

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