Littérature scientifique sur le sujet « Anaplastic Large Cell Lymphoma, drug resistance, Crizotinib »

Anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL) is a subtype of non-Hodgkin lymphoma characterized by expression of the oncogenic NPM/ALK fusion protein. When resistant or relapsed to front-line chemotherapy, ALK+ ALCL prognosis is very poor. In these patients, the ALK inhibitor crizotinib achieves high response rates, however 30–40% of them develop further resistance to crizotinib monotherapy, indicating that new therapeutic approaches are needed in this population. We here investigated the efficacy of upfront rational drug combinations to prevent the rise of resistant ALCL, in vitro and in vivo. Different combinations of crizotinib with CHOP chemotherapy, decitabine and trametinib, or with second-generation ALK inhibitors, were investigated. We found that in most cases combined treatments completely suppressed the emergence of resistant cells and were more effective than single drugs in the long-term control of lymphoma cells expansion, by inducing deeper inhibition of oncogenic signaling and higher rates of apoptosis. Combinations showed strong synergism in different ALK-dependent cell lines and better tumor growth inhibition in mice. We propose that drug combinations that include an ALK inhibitor should be considered for first-line treatments in ALK+ ALCL.

Abstract Kinase inhibitors can be highly effective against cancers driven by specific oncogenic kinase proteins, but resistance usually develops after prolonged treatment. Target-dependent mechanisms, typically acquired second-site mutations that prevent drug binding, or target-independent mechanisms, such as downstream or parallel pathway activation, may mediate resistance. Prolonged exposure of kinase-dependent cell lines to inhibitors in vitro to select for resistant populations has identified clinically relevant resistance mechanisms against a number of inhibitors. ALK-positive anaplastic large-cell lymphoma (ALCL) is an uncommon form of peripheral T-cell lymphoma (PTCL) driven by the NPM-ALK fusion kinase, resulting from a t(2;5)(p23;q35) chromosomal rearrangement. ALK inhibitors developed for lung cancer, where a different ALK fusion (EML4-ALK) is found in 6% of cases, have shown promise clinically as a new treatment modality for ALK+ ALCL patients. In this study, we selected three NPM-ALK-dependent ALCL cell lines (Karpas-299, SUP-M2, and SU-DHL-1) for resistance to the ALK inhibitor crizotinib. All three lines initially were highly sensitive the drug (IC50< 100 nM) and required months of exposure at gradually increasing concentrations before acquiring resistance. Previous work has identified specific ALK kinase second-site mutations that promote crizotinib resistance, which we also observed in some resistant lines generated. A unique situation arose, however, in a sub-clone of Karpas-299, that was able to grow in crizotinib at concentrations > 1 µM (Karpas-299CR1000). This line had greatly increased viability in crizotinib at concentrations up to 1 µM, compared to untreated cells, suggesting the drug actually maintained growth. Plated in 250 nM crizotinib, Karpas-299CR1000 grows rapidly, similar to the parent line growing in drug-free media. Plated in drug-free media, however, the line’s viability drops rapidly to zero and dies, similar to the parent line plated in 250 nM crizotinib. Therefore the drug that once shut off the signaling necessary for the cells’ survival, after prolonged exposure (>9 months), became necessary to maintain it in the sub-clonal population. Melanoma cells driven by BRAF-V600E selected for vemurafenib resistance had a similar phenotype in a recent report (Das Thakur, et al., 2013). These cells amplified mutant BRAF to overcome the inhibitor but came to require the drug to counteract BRAF over-activity with amplification beyond a certain point. Preliminarily, we find a similar effect of NPM-ALK amplification in Karpas-299CR1000 cells but are actively exploring mechanism. In sum, our study shows that long-term exposure of ALK+ ALCL cells to an ALK inhibitor may induce a resistant phenotype that comes to depend on drug presence to prevent over-activity of the mutant kinase. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 4862 Background: Anaplastic lymphoma kinase (ALK)-positive diffuse large B-cell lymphoma (DLBCL) displays a rare and distinct variant of DLBCL characterized by very aggressive clinical course with an estimated median survival of only 11.0 months. Crizotinib, an ALK inhibitor, has demonstrated impressive clinical efficacy in patients with alk-rearranged tumors such as NSCLC. The potential role of Crizotinib in ALK-positive DLBCL has not been established yet. Case report: A 27-year-old female patient with initial advanced-stage IV ALK-positive DLBCL was primary refractory to 6 cycles of standard chemotherapy with CHOP. She was subsequently treated with different salvage combination chemotherapy regimens including high-dose chemotherapy with autologous stem-cell transplantation. However, disease followed a very aggressive course and relapse occurred fast after each regimen. 6 weeks after transplantation patient presented with right cervical lymphadenopathy and multiple cutaneous lesions. CT scan revealed additional bilateral cervical, axillary, mediastinal and abdominal lymphadenopathy and involvement of lung and liver. LDH levels were exorbitant elevated (233 mmol/l). On an individual base, our patient received the ALK inhibitor Crizotinib 250 mg per os twice daily. No glucocorticoids or other drugs with antineoplastic activity were co-administered. Within 72 hours LDH levels were halved (89 mmol/l). On day 8 cervical palpable lymphadenopathy disappeared. Initial erythematous papular cutaneous lesion decreased from 4.0 × 2.5 cm to a residual hyperpigmented macule of 1.5 × 2.5 cm. On day 16 LDH levels were almost within normal limits (6 mmol/l). However, remission was not sustained and patient developed resistance to Crizotinib. By day 21 after beginning the treatment with Crizotinib LDH levels increased again and patient presented with a swollen left arm due to lymphatic obstruction, detected by CT scan, which showed a general progressive disease, causing death one month later. Conclusion: To our knowledge, this is the first case which reports sensitivity in ALK-positive DLBCL to ALK inhibition with Crizotinib. Despite remission duration was short our results indicate a potential role of ALK in the pathogenesis of ALK-positive DLBCL and suggest ALK inhibition as a potentially promising treatment modality in the therapy of this rare and fatal disease. Disclosures: Off Label Use: Crizotinib is an ALK inhibitor, indicated for the treatment of patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) that is anaplastic lymphoma kinase (ALK)-positive. Recently impressive clinical efficacy of Crizotinib has been reported in two patients with ALK-positive anaplastic large cell lymphoma. Based on these data we initiated the treatment with Crizotinib in our patient, who showed very aggressive clinical behavior and fulminant early recurrence of a ALK-positive diffuse large B-cell lymphoma after salvage chemotherapy regimens on an individual base.

Anaplastic large cell lymphoma (ALCL) is a type of CD30-expressing non-Hodgkin's lymphoma (NHL), which accounts for 2% to 3% of adult non-Hodgkin's lymphoma,accounting for 15% to 30% of children with large cell lymphoma. Anaplastic lymphoma kinase (ALK) positive ALCL is highly invasive, and currently it is generally based on CHOP combined with chemotherapy. The proportion of patients with complete relief of symptoms is as high as 90%, but the proportion of recurrence is also as high as 40%. Crizotinib is the first generation of ALK inhibitors that have been approved for the treatment of ALK+ ALCL. Unfortunately, most patients treated with crizotinib relapse after a significant initial response. The median progression-free survival of clinical trials was 10.5 months. Various mutations in the ALK kinase domain and amplification of the ALK gene copy number, activation of the alternative pathway, and tumor heterogeneity are major causes of crizotinib resistance. Studies have shown that IGF-1R interacts with NPM-ALK to promote ALK+ALCL transformation, proliferation and migration. GSK is a small molecule kinase inhibitor that inhibits both IGF-IR and ALK. Therefore, GSK with simultaneous inhibition of the bidirectional potential of IGF-IR and ALK has a promising prospect in the targeted therapy of NPM-ALK+ALCL. This study explored the inhibitory effects of GSK on NPM-ALK+ALCL and crizotinib-resistant NPM-ALK+ALCL by in vivo and in vitro experiments. In vitro experiments: The sensitivity of ALCL cell line to GSK1838705a was detected by CCK8 and flow cytometry. The expression of phosphorylation of IGF-1R and NPM-ALK signaling pathway in Karpas299 and SR786 cell lines stimulated by GSK was detected by WB method. In order to study the crizotinib resistance mutation, we established ALK+ALCL crizotinib-resistant cell lines Karpas299-R and SR786-R, and identified the resistance of Karpas299-R and SR786-R cell lines by CCK8 and flow cytometry. The drug-resistant and non-resistant strains were stimulated with gradient concentrations of crizotinib and gradient GSK, and the IC50 of the two were compared by CCK8. The WB method was used to compare the phosphorylation levels of downstream signaling pathways in drug-resistant and non-resistant strains. In vivo experiment: The ALK+ALCL and resistant-ALK+ALCL mouse model was established, and three groups of mice treated with control, GSK single drug 30 mg/kg, GSK single drug 60 mg/kg, were established. The tumor volume and body weight of the four groups were compared. Immunohistochemistry was used to compare the expression levels of key signaling molecules and apoptotic proteins in each group. SPSS statistical software draws survival curves. As the concentration of GSK gradually increases, the survival rate of ALCL cells gradually decreases. The expression of pIGF-1R, pNPM-ALK, pSTAT3, pAKT, casepase3 and other molecules decreased in the downstream signaling pathway, and the expression level of cleaved-casepase3 increased.In the crizotinib-resistant cell line, with the increase of the concentration of GSK, the apoptosis rate of the cells increased and the phosphorylation level of the downstream molecules gradually decreased. Tumor volume of three groups of mouse models: control>GSK single drug 30 mg/kg>GSK single drug 60 mg/kg. Immunohistochemistry results showed that the expression level of key signaling molecules in GSK-treated CHOP-treated mice decreased, and the expression level of apoptotic proteins increased. In this research, we explored the effects of GSK1838705A on proliferation, apoptosis, and clonogenesis of ALCL cell lines. Subsequently, we established a crizotinib-resistant cell line and noticed that GSK1838705A can effectively reduce the viability of resistant ALCL cells and significantly restrain the transmission of downstream survival signaling pathways induced by IGF1R/IR phosphorylation. Besides, we discovered that GSK1838705A inhibited the development of both crizotinib-sensitive and crizotinib-resistant ALCL tumors in the ALCL mouse model established by subcutaneous tumorigenesis. Based on the results of previous clinical trials, we put forward to use GSK1838705A as an alternative treatment strategy to overcome crizotinib-resistant ALCL. Disclosures No relevant conflicts of interest to declare.

Initially discovered in anaplastic large cell lymphoma (ALCL), the ALK anaplastic lymphoma kinase is a tyrosine kinase which is affected in lymphomas by oncogenic translocations, mainly NPM-ALK. To date, chemotherapy remains a viable option in ALCL patients with ALK translocations as it leads to remission rates of approximately 80%. However, the remaining patients do not respond to chemotherapy and some patients have drug-resistant relapses. It is therefore crucial to identify new and better treatment options. Nowadays, different classes of ALK tyrosine kinase inhibitors (TKI) are available and used exclusively for EML4-ALK (+) lung cancers. In fact, the significant toxicities of most ALK inhibitors explain the delay in their use in ALCL patients, who are predominantly children. Moreover, some ALCL patients do not respond to Crizotinib, the first generation TKI, or develop an acquired resistance months following an initial response. Combination therapy with ALK inhibitors in ALCL is the current challenge.

Anaplastic large cell lymphoma (ALCL) expressing the anaplastic lymphoma kinase (ALK) (ALK+ ALCL) is a rare type of lymphoma which comprises 10-15% of all non-Hodgkin lymphomas in children and 2–3% in young adults. Relapsed/refractory disease occurs even more rarely (25–40% of all cases). There is as yet no standard treatment for relapsed/refractory ALK+ ALCL. Patients with ALK+ ALCL usually present at advanced stages of the disease with extranodal involvement (skin, soft tissues, bones, lungs, liver, spleen and bone marrow) and B symptoms. ALK-positive ALCL affects males more often than females. There are two morphological variants: the common type (65% of cases) and the non-common type which is associated with a poorer prognosis. ALK+ ALCLs are often associated with t(2;5) and t(1;2), resulting in the formation of the NPM-ALK and the TPM3-ALK fusion proteins, respectively. Data about the treatment of relapsed/refractory ALK+ ALCL are limited. Earlier, targeted therapies (brentuximab vedotin (BV), ALK inhibitors) and risk-adapted chemotherapy followed by hematopoietic stem cell transplantation (HSCT) for remission consolidation were shown to be highly effective. A total of 15 patients with relapsed/refractory ALK+ ALCL were treated at the R.M. Gorbacheva Research Institute starting from 2002. Fourteen (93%) patients had ALK-positive ALCL of common morphology and one (7%) patient had the non-common variant (histiocytic). The study was approved by the Independent Ethics Committee and the Scientific Council of the Pavlov University. The expression of CD3 on tumor cells was assessed (CD3 positive: n = 4 (27%), CD3 negative: n = 8 (53%), no data: n = 3 (20%). The median age at the diagnosis was 26 years (11 months– 37 years). The median follow-up from the diagnosis was 9 years (1–19 years). Nine (60%) patients were aged > 18 years and six (40%) patients were aged < 18 years. There were 10 (67%) males and 5 (33%) females. At onset, 2 (13%) patients were diagnosed with early-stage disease (stage II), while the others were diagnosed with advanced-stage disease: 2 (13%) patients had stage III disease and 11 (74%) had stage IV disease. Staging was performed according to the St. Jude staging system (in children) and the Ann Arbour staging classification (in adults). Thirteen (86%) patients had extranodal involvement. Four (27%) patients had refractory disease (progression within the first three months or the absence of complete remission after the first-line treatment) and the rest 11 (73%) patients had recurrent ALK-positive ALCL. Six patients developed early relapse (< 12 months after remission was achieved); 5 patients had late relapse (after > 12 months of remission); local (1 site) and systemic relapses were diagnosed in 7 and 4 patients, respectively. Our patients received from 2 to 7 lines of treatment (the median is 4). In the first line of therapy, the patients were treated according to NHL-BFM based regimens (n = 9; 60%), the CHOP (n = 5; 33%), and the HyperCVAD (n = 1; 7%) protocols. In the second line of therapy, 8 (53%) patients were treated according to NHL-BFM based regimens; 2 (13%) patients were treated with GDP; 1 (7%) patient received DHAP chemotherapy; 1 (7%) patient received a combination of methotrexate and vinblastine (MTX + V); 1 (7%) patient received bendamustine as a single agent. Two (13%) patients were treated with chemotherapy in combination with targeted drugs (GDP + BV, n = 1; NHL-BFM + crizotinib, n = 1). As a third or subsequent line of treatment, the patients received a variety of chemotherapy regimens (n = 5; 33%) and chemotherapy in combination with targeted drugs (n = 10; 67%). Five (33%) patients underwent ALK-inhibitor therapy (crizotinib (n = 4) and ceritinib (n = 1)). Seven (46%) patients were treated with BV (BV as a single agent (n = 4) and BV + chemotherapy (n = 3)). The median number of treatment lines before autologous HSCT (auto-HSCT) and allogeneic HSCT (allo-HSCT) was 2 (2–3) and 3 (3–4), respectively. Auto-HSCT was carried out in 11 (73%) cases. Nine (60%) patients underwent allo-HSCT (from a matched unrelated donor (n = 6), from a matched related donor (n = 2), and from a haploidentical donor (n = 1)). One patient received NK cells from a haploidentical donor as maintenance. In 5 (33%) cases, alloHSCT was carried out following auto-HSCT. The conditioning regimens (CR) used for auto-HSCT included BEAM (carmustine – 300 mg/m2, etoposide – 800 mg/m2, Cytosar – 1600 mg/m2, melphalan – 140 mg/m2) – in 5 (45%) patients; BeEAM (bendamustine – 320 mg/m2, etoposide – 800 mg/m2, Cytosar – 1600 mg/m2, melphalan – 140 mg/m2) – in 5 (45%) patients; and BuCy (Cyclophosphan – 100 mg/kg, busulfan – 14 mg/kg) – in 1 (10%) case. Seven (78%) patients undergoing allo-HSCT received the FluBenda conditioning regimen (fludarabine – 90–150 mg/m2, bendamustine – 390 mg/m2) and post-transplant Cyclophosphan and calcineurin inhibitors for the prevention of graft-versus-host disease (GVHD) (n = 7; 78%); one (11%) patient received the FluMel regimen (fludarabine – 150 mg/m2, melphalan – 140 mg/m2) and CsA/MTX (Cyclosporin А, methotrexate) for GVHD prevention; and in 1 case (11%) data on the RC and GVHD prophylaxis were missing. Overall response to the second line of treatment was achieved in 10 (67%) patients, with complete response observed in 7 (47%) cases, and partial response –in 3 (20%) cases. Five out of the 7 patients treated with BV during different lines of therapy managed to achieve complete response. Four out of the 5 patients who had undergone treatment with ALK inhibitors, demonstrated complete response. The 10-year overall survival (OS) rate of the study patients reached 90% (95% confidence interval (CI) 47–99). The 10-year progression-free survival (PFS) rate after the second line of treatment was 39% (95% CI 13–64). The 10-year OS and PFS rates after auto-HSCT were 100% and 35% (95% CI 8–64) respectively. The 5-year OS following allo-HSCT was 85% (95% CI 33–98), while PFS was 60% (95% CI 19–85). Four out of the 11 patients who had undergone auto-HSCT relapsed, and 2 patients progressed. Median time to relapse/progression was 8 (6–27) months. Three out of the 9 patients who had been treated with allo-HSCT ended up relapsing (median time: 8 (6–17) months). Two patients achieved repeated remission (in one case, it was the result of treatment with ceritinib, while in the other case it became possible after the resection of the lesion, radiotherapy and prescription of crizotinib), and 1 study patient died as a result of disease progression 17 months after allo-HSCT. The 6 patients who had achieved complete remission before undergoing allo-HSCT, are still in CR. Five out of the 9 patients developed grade I–II acute GVHD with skin involvement but did not show any signs of chronic GVHD. The observed complications of chemotherapy and auto-HSCT were standard and manageable and were not the focus of attention in this study. Taking into account the high probability of developing resistance to ALK inhibitors and the high risk of relapse after treatment with BV, targeted therapy should be used to prepare patients for HSCT. The use of ALK inhibitors and BV in our study led to repeated remission in 80% and 71% patients respectively. It was demonstrated that in the majority of cases even heavily pretreated patients with ALK+ ALCL can be cured (which is not the case with other non-Hodgkin lymphomas), especially if allogenic HSCT (allo-HSCT) is carried out. Still, we think that auto-HSCT can also be considered for remission consolidation since OS rates following auto-HSCT and allo-HSCT are comparable (100% and 85%). Moreover, auto-HSCT can also be a valid option in the absence of a fully matched donor, i.e. when only an alternative (haploidentical or partially matched) donor is available, since the use of haploidentical HSCT in ALCL patients has not been studied well enough yet. Further research on the matter is warranted. It was demonstrated that a non-myeloablative conditioning regimen before allo-HSCT (FluBenda) could be opted for in patients with relapsed/refractory ALK+ ALCL and that it was similarly effective as myeloablative RC when compared with a historical control group. Disease status before HSCT was proven to have a statistically significant influence on PFS (the best prognosis was associated with complete remission). At the same time, other factors did not impact the prognosis. This may be explained by the relatively small number of patients included in the study. Relapsed/refractory ALK+ ALCL is a disease with a relatively good prognosis even in heavily pretreated patients. Targeted therapy is a very important and effective step in preparation for HSCT. Allo-HSCT is more effective than auto-HSCT but the latter can also be considered a valid therapeutic option.

Abstract In hematological disorders ALK expression is present in >50% of Anaplastic Large Cell Lymphomas (ALCL) as a result of a t(2;5)(p23;q35) translocation that causes the ALK gene on chromosome 2 to fuse with the NPM gene on chromosome 5. ALK + ALCL respond to cytotoxic drugs, but relapses occur and bear a poor prognosis(Stein, Foss et al. Blood 96 3681-95 2000; Ferreri, Govi et al. Crit Rev Oncol Hematol 83 293-302 2012). ALK-positive large B-cell lymphoma (ALK+ LBCL) is a rare lymphoma with a most frequent t(2;17)(p23;q23) translocation responsible for Clathrin-ALK fusion protein(Swerdlow, Campo et al. 2 2008). Crizotinib is the first ALK inhibitor which entered clinical practice: it is an orally bioavailable small-molecule inhibitor active on the ALK and MET receptor tyrosine kinases. While the activity of crizotinib in ALK+ lung cancer is documented (Kwak, Bang et al. N Engl J Med 363 1693-703 2010)no report on long term effects of crizotinib in ALK+ lymphomas exists; impressive short-term therapeutic activity was reported in two patients (Gambacorti-Passerini, Messa et al. N Engl J Med 364 775-6 2011), but no long-term data are available. In the present study, crizotinib was administered (250 mg BID) as monotherapy to 11 ALK+ lymphoma patients, diagnosed with ALK+ Non-Hodgkin lymphoma (NHL) by immunohistochemistry and FISH. Nine patients had a ALCL histology while the remaining 2 were DLBCL Patients had a refractory or relapsed disease after at least one prior chemotherapy regimen and measurable disease. All had involvement at multiple sites (nodal and extranodal) as well as B symptoms and an ECOG performance score of 1-4. Response to therapy was assessed according to RECIST criteria (Therasse, Arbuck et al. J Natl Cancer Inst 92 205-16 2000) The Overall Response Rate (ORR) was 10/11 (91%, 95% CI: 60-99%) and included 9 CR (82%, 95% CI: 51-96%) and 1 PR. Evidence of response by PET/CAT scan was present as early as 12 days. B symptoms disappeared promptly and LDH levels normalized within 30 days after the start of crizotinib. Disease status at the latest follow-up (June 2013) is as follows: 4 patients are in CR under continuous crizotinib treatment; they also test negative by RT-PCR for NPM/ALK (Mussolin, Damm-Welk et al. Leukemia 27 416-22 2012). Three patients (2 with LBCL and 1 with ALCL) died due to disease progression; 1 patient obtained CR, relapsed after 2 months of treatment and is now in CR on continued brentuximab treatment (month 29); 1 patient obtained CR on crizotinib and after 2 months stopped treatment, received an alloBMT and is still in CR; 2 patients treated for relapses post alloBMT obtained CR and are still in CR but they stopped crizotinib after 8-10 months. The two patients with ALK+ LBCL died within 3 months; in those with ALCL the CR rate was 9/9 (100%, 95% CI, 74-100%) with a median duration of 10 months (range 2-37). The 3 years PFS and OS rates are 62% (95% CI, 35-85%) and 73% (95% CI, 40-93%) respectively, with a plateau in the curve after the initial 6 months. In two relapsed patients the kinase domain of NPM-ALK could be amplified from peripheral blood samples obtained at the time of relapse (month 5 and 2). Deep sequencing of these products revealed the presence of different mutations: Q1064R at high prevalence (95%,) in patient (pt) #2 and I1171N (33%) plus M1328I (14%) in pt #6. All these mutations were not present in samples obtained before crizotinib treatment. I1171N was already discovered in an in-vitro screening (Ceccon, Mologni et al. Mol Cancer Res 11 122-32 2012): it commands an intermediate level of resistance to crizotinib (RI: 5.8) which however is cross resistant with other anti-ALK TKI such as AP26113 and NVP-TAE684. The other two mutations were not previously described: they present a RI to crizotinib of 2.4 (M1328I) and 8.5 (Q1064R). Since these residues do not form direct contacts with crizotinib, they probably interact with different structures within the catalytic domain such as the hydrophobic R-spine (I1171N) (Ceccon, Mologni et al. Mol Cancer Res 11 122-32 2012), the activation loop (M1328I), or yet unidentified regions (Q1064R). In conclusion, these positive results extend our initial observation on two patients (Gambacorti-Passerini, Messa et al. N Engl J Med 364 775-6 2011) and provide long-term follow up data. Crizotinib exerted a potent antitumor activity in advanced ALK+ lymphoma and produced durable responses in this population of heavily pre-treated patients, with a benign safety profile. Disclosures: Gambacorti-Passerini: Pfizer: Consultancy, Research Funding; BMS: Consultancy.

Abstract ALK-positive Anaplastic Large Cell Lymphoma (ALCL) is a subset of Non-Hodgkin Lymphoma that positively took advantage of crizotinib development, a selective ALK inhibitor. Crizotinib is able to block ALK kinase activity which is fundamental for cancer cell survival. Constitutively active ALK kinase triggers the activation of several pro-proliferative and pro-survival downstream pathways, such as PI3K/AKT/mTOR. Despite the positive impact of targeted therapy on ALCL treatment, resistant clones tend to be selected during therapy. Strategies to overcome resistance include the design of more potent and selective drugs and the use of combined therapy approaches that allow for the simultaneous targeting of more than one node essential for cancer cells survival. Following this strategy, we decided to investigate the effects of combined ALK/mTOR inhibition. We first observed a synergistic effect of the combination of ALK inhibitors (crizotinib, alectinib or PF-3922) with an mTOR inhibitor (temsirolimus) in proliferation assays. Interestingly, the synergistic effect was observed only in ALK+ cell lines (Karpas 299, SUDH-L1 and SUPM2), while no synergism was observed in ALK- cell line (U937) as well as in normal lymphocytes. The Combination Index (CI) ranged from 0.16 to 0.45 indicating a synergistic/strong synergistic effect, accordingly to Chou classification. (Chou, 2006) (Table 1). The positive cooperation resulted in an increased inhibition of mTOR effectors p70S6K, 4EBP1 and eIF4B compared to single agent treatment as observed in immunoblot analysis performed on Karpas 299 treated for 4 hours. At the cell cycle level, the use of the drugs in combination induced a sharp block in G0/G1 phase, as observed by propidium iodide analysis performed on Karpas 299 at 48-72 and 96 hours. Long term exposure of ALK+ cells to either alectinib or temsirolimus, led to the establishment of resistant cell lines, while the exposure to the combination prevented the selection of resistant clones. Interestingly, the cell line resistant to alectinib showed a marked increase of ALK both at mRNA and protein level. In vivo, nude mice were injected with Karpas 299 cells. As the tumor masses reached 150mm3, mice were randomized and treated for 12 days with the combination of PF-3922 (0.5 mg/kg, administered per os twice a day) and temsirolimus (i.p., 1 mg/kg every other day) or with the single agents. The combined treatment induced a faster regression of the tumor masses compared to the single agents-treated mice. Moreover, responses were sustained for a longer period of time, upon treatment stop. (Figure 1) In conclusion, our data suggest that the combination of ALK and mTOR inhibitors could be a valuable therapeutic option for patients affected by ALK+ ALCL. Table 1. combination indexes from proliferation experiments. Synergism levels calculated accordingly to Chou, Pharmacological reviews, 2006 Crizotinib - Temsirolimus Cell lines Ratio Combination Index (CI) Average CI Synergism level EC50 EC75 EC90 NPM-ALK+ Karpas 299 1:1 0.42 0.35 0.33 0.37 Synergism SUDHL1 3:1 0.49 0.45 0.42 0.45 Synergism SUPM2 1:1 0.59 0.33 0.29 0.40 Synergism NPM-ALK- U937 1:1 >10 >10 >10 >10 Antagonism HD Lymphocytes 1:1 >10 >10 >10 >10 Antagonism Alectinib - Temsirolimus Cell lines Ratio Combination Index (CI) Average CI Synergism level EC50 EC75 EC90 NPM-ALK+ Karpas 299 1:3 0.56 0.29 0.15 0.33 Synergism SUDHL1 1:3 0.53 0.45 0.38 0.45 Synergism SUPM2 1:10 0.65 0.18 0.06 0.3 Strong synergism NPM-ALK- U937 1:3 1.5 >10 >10 >10 Antagonism HD Lymphocytes 1:3 1.2 2.7 7.2 3.7 Antagonism PF06463922 - Temsirolimus Cell lines Ratio Combination Index (CI) Average CI Synergism level EC50 EC75 EC90 NPM-ALK+ Karpas 299 1:10 0.30 0.15 0.15 0.20 Strong synergism SUDHL1 1:30 0.57 0.41 0.29 0.42 Synergism SUPM2 1:3 0.30 0.12 0.06 0.16 Strong synergism NPM-ALK- U937 1:10 3.2 5.39 >10 >10 Antagonism HD Lymphocytes 1:3 1.4 8.8 >10 >10 Antagonism Figure 1. in vivo experiment for the evaluation of the combines treatment. Tumors volume measurements ±SEM are presented Figure 1. in vivo experiment for the evaluation of the combines treatment. Tumors volume measurements ±SEM are presented Disclosures No relevant conflicts of interest to declare.

INTRODUCTION: Anaplastic large cell lymphomas (ALCL) frequently carry oncogenic fusion proteins as a consequence of chromosomal translocations of the anaplastic lymphoma kinase (ALK) gene. The fusion protein resulting from the translocation between dimerization domain of nucleophosmin (NPM) and intracellular tyrosine kinase domain of ALK activates several signaling pathways, promoting cell growth, transformation, migration, and survival of the cells. Chemotherapy has been used as a standard treatment approach for ALCL patients, yet about 30% of patients relapse. A more specific treatment method is based on targeting ALK tyrosine kinase using tyrosine kinase inhibitors (TKIs). Crizotinib is an ALK TKI that is approved for the treatment of ALK-rearranged lung cancer and has received Breakthrough Therapy designation for lymphoma because of its high activity in chemo refractory ALCL. However, as for lung cancer, also ALCL patient develop crizotinib resistance due to ALK mutations or unknown mechanisms. In this study, we aimed at elucidating unknown by-pass mechanisms of crizotinib resistance in ALCL. METHODS: We used Genome-wide CRISPR-Cas9 Knockout Screening (GeCKO) to identify candidate genes that contribute to resistance to crizotinib. Four different ALCL cell lines were infected with Lenti-GeCKO libraries. After treatment with crizotinib for 14 days, DNA isolation and next generation sequencing was performed on crizotinib resistant cells to identify candidate genes depleted by the GeCKO screening. Top candidates were selected for validation assays and further analyses. RESULTS: We identified two phosphatases, PTPN1 and PTPN2, in different ALCL cell lines as consistent top hits. Functional validation of these candidate genes showed that single loss of either PTPN1 or PTPN2 generate immediate resistance to crizotinib in ALCL cell lines. Analysis of downstream pathways showed that while loss of PTPN1 activates primarily the MAPK pathway, loss of PTPN2 promotes persistent STAT3 and MAPK activation in ALK inhibited cells. Remarkably, in PTPN1 knockout cells we observed hyperactivation of SHP2, an oncogenic phosphatase that positively regulates the RAS-MAPK pathway. On the other hand, over-expression of PTPN1 and PTPN2 partially inhibited SHP2 phosphorylation. A treatment that combined crizotinib and the recently developed SHP2 inhibitor completely blocked the sustained ERK phosphorylation and reverted the crizotinib resistance observed in PTPN1 and PTPN2 deficient lymphoma cells. CONCLUSIONS: GeCKO library screen identified PTPN1 and PTPN2 as specific genes that mediate crizotinib resistance in ALCL cell lines. Loss of PTPN1 and PTPN2 drives resistance by activating MAPK and/or JAK-STAT pathway. Combined inhibition of SHP2 is a potent therapeutic approach to overcome resistance to crizotinib in ALCL cells. Disclosures Gambacorti-Passerini: Bristol-Meyers Squibb: Consultancy; Pfizer: Honoraria, Research Funding.

Clinical pharmacists are important contributors to the care of patients with cancer; it is therefore critical for oncology clinical pharmacists to stay current with new anticancer therapies. This review summarizes the epidemiology and pathogenesis of non-small cell lung cancer, including the most common genetic alterations, as well as the mechanism of action, clinical development, pharmacodynamics and pharmacokinetics of the anaplastic lymphoma kinase inhibitor ceritinib for the treatment of patients with anaplastic lymphoma kinase-positive non-small cell lung cancer. Targeted therapies based on the presence of specific mutations are an important development in the treatment of non-small cell lung cancer. However, acquired resistance to the first anaplastic lymphoma kinase-inhibitor approved by the U.S. Food and Drug Administration, crizotinib, is observed in almost half of patients treated with it. Ceritinib is an oral anaplastic lymphoma kinase-inhibitor that has demonstrated more potent antitumor activity than crizotinib in preclinical models. It was granted accelerated approval in 2014 to treat anaplastic lymphoma kinase-positive metastatic non-small cell lung cancer patients who have progressed on or are intolerant to crizotinib. Ceritinib represents an important alternative second-line therapy for patients with metastatic non-small cell lung cancer who have traditionally limited treatment options.

The dual ALK/MET inhibitor Crizotinib was recently approved for the treatment of metastatic and late stage ALK+ NSCLC, and is currently in clinical trial for other ALK‐related diseases. As predicted after other TKIs clinical experience, the first mutations that confer resistance to Crizotinib have been described in Non Small Cell Lung Cancer (NSCLC) and in one Inflammatory Myofibroblastic Tumor (IMT) patients. Here we focused our attention on Anaplastic Large Cell Lymphoma (ALCL), where the oncogenic fusion protein NPM-ALK, responsible for 70-80% of cases, represents an ideal Crizotinib target. We selected and characterized two human NPM-ALK+ ALCL cell lines, KARPAS299 and SUP-M2, able to survive and proliferate at different Crizotinib concentrations. Sequencing of ALK kinase domain revealed that a single mutation became predominant at high Crizotinib doses in each cell line, namely L1196Q and I1171N in Karpas299 and SUP-M2 cells, respectively. These mutations also conferred resistance to Crizotinib in Ba/F3 cells expressing human NPM‐ALK. The resistant cell populations, as well as mutated Ba/F3 cells, were characterized for sensitivity to two additional ALK inhibitors: the dual ALK/EGFR inhibitor AP26113 and NVPTAE684. While L1196Q-positive cell lines were sensitive to both inhibitors, cells carrying I1171N substitution showed cross-resistance to all ALK inhibitors tested. This study provides potential relevant information for the management of ALCL patients that may relapse after Crizotinib treatment.