Anaplastic Lymphoma Kinase Positive Non-small Cell Lung Cancer Research Articles

Recurrence of anaplastic lymphoma kinase (ALK) positive adenocarcinoma after 17 years: Case report

Abstract Introduction About four to six percent of non-small cell lung cancer (NSCLC) harbor Anaplastic lymphoma kinase rearrangement (ALK). ALK positive NSCLC has a distinct clinicopathological features. In the advanced setting ALK tyrosine kinase inhibitors are used in the first and second line of treatment. However, less is known about the outcome of stage one ALK positive NSCLC. Presentation of case Our case is a 58 year old man who presented initially with stage one ALK positive NSCLC. He relapsed 17 years later. Discussion It is very unusual for stage one NSCLC to relapse beyond 10 years. It is surprising that our patient relapsed many years after his initial diagnosis. Conclusion This may highlight a different biology and outcome. It may also mean a longer follow up is needed for this subset of patients.

Acquired Resistance to a New Alk Inhibitor, Alectinib in Lung Cancer

ABSTRACT Aim: Alectinib (CH/RO5424802) is a second generation anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor (TKI). It has been reported to overcome acquired resistance to the first generation ALK-TKI, crizotinib, in a preclinical study (Cancer Cell 19, 2011: 679-90). A phase I/II clinical trial of alectinib for ALK positive non-small cell lung cancer (NSCLC) showed an unprecedented response rate (93.5 % in phase II portion) and the drug seemed to lead to less toxicities than crizotinib (Lancet Oncol 14, 2013: 590-98). However, subsequent resistance to alectinib is expected after its approval. Thus, we need to consider the therapeutic strategy to overcome the resistance to alectinib in advance. Methods: Two alectinib-resistant H2228/CHR and ABC-11/CHR NSCLC cell lines from H2228 and ABC-11, respectively, harboring EML4-ALK fusion genes were established by continuous exposure to alectinib. We characterized the resistant cell lines using MTT assay, western blotting, immunohistochemistry, polymerase chain reaction, fluorescent in situ hybridization, phospho-receptor tyrosine kinase array, comparative genomic hybridization, RNA array, ELISA and xenograft models. Results: H2228/CHR and ABC-11/CHR cells showed 130-fold and 17-fold higher resistance to alectinib than their respective parent cells in vitro. H2228/CHR cells had lost EML4-ALK fusion gene and ALK protein expression. In addition, the resistant cells overexpressed IGF1R, and were sensitive to IGF1R-TKI (TAE684) as a result. While ABC-11/CHR cells reduced phospho-ALK in spite of preservation of total ALK protein, they activated MET with increment of its ligand (hepatocyte growth factor) as an autocrine action. Because crizotinib originally functions as MET-TKI along with ALK-TKI, ABC-11/CHR was sensitive to the drug both in vitro and in vivo. Conclusions: Loss or inactivation of ALK leads to the acquired resistance to alectinib in the two cell lines we established. In addition, signaling switch from ALK to IGF1R or MET was observed. IGF1R inhibitor or MET inhibitor was effective on such cases. Thus, ALK positive NSCLC patients refractory to alectinib might benefit from IGF1R or MET inhibitors. Disclosure: K. Hotta: Honoraria from speaker's bureau from Chugai pharmaceutical CO., LTD.; N. Takigawa: Honoraria from speaker's bureau from Pfizer Inc. Japan; K. Kiura: Honoraria from speaker's bureau from Chugai pharmaceutical CO., LTD., Pfizer Inc. Japan and Novartis Pharma K.K. All other authors have declared no conflicts of interest.

Adjuvant molecularly targeted therapy-epidermal growth factor tyrosine kinase inhibition and beyond.

In stage IV non-small cell lung cancer (NSCLC), DNA molecular testing for mutations in epidermal growth factor receptor ( EGFR ) and gene rearrangements of anaplastic lymphoma kinase ( ALK ) has become the new standard of care. This is based on the unprecedented efficacy of small molecule EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib against EGFR mutant NSCLC (1,2), and the ALK TKI crizotinib against ALK positive NSCLC (3). While these highly active drugs should conceptually be effective as a component of the curative treatment of earlier stage NSCLC, the presently available evidence is minimal as pivotal studies are either underway or still in development. This article reviews current evidence about the use of adjuvant therapy for molecular targets in NSCLC, in particular regarding the use of EGFR TKIs in the treatment of early stage NSCLC harboring EGFR mutations.

Investigation of anaplastic lymphoma kinase (ALK) translocations in gastric and esophageal signet ring cell carcinomas.

e15106 Background: Anaplastic lymphoma kinase (ALK) fusion oncogenes are present in multiple cancer types. The inversion of echinoderm microtubule associated protein like 4 (EML4) and ALK genes on chromosome 2 is present in a subset of non-small cell lung cancer (NSCLC) patients. ALK mutated lung cancers demonstrate a significantly higher incidence of signet ring cell histology than compared to ALK-negative tumors. Based on the histological similarities of ALK positive NSCLC and signet ring cell carcinomas (SRCC) of the GI tract, we hypothesized that gastric and/or esophageal SRCC may also harbor ALK translocations. Methods: Thirty-five formalin-fixed, paraffin-embedded (FFPE) tissue specimens of SRCC originating from esophageal, GE junction or gastric locations were obtained from the Fletcher Allen Healthcare (Burlington, Vermont) tissue bank following Internal Review Board guidelines. Confirmation of SRCC or adenocarcinoma with signet ring cell features was confirmed by a board certified, gastrointestinal pathologist. SRCC specimens were analyzed by fluorescence in situ hybridization (FISH) analysis using an ALK (2p23) break-apart probe (Kreatech Diagnostics). Results: The FISH analysis revealed no evidence of ALK translocation: all thirty-five (100%) SRCC specimens showed intact (yellow) ALK FISH signals. Conclusions: These data indicate that despite histological similarities between SRCC of the GI tract and ALK positive NSCLC, ALK translocations are unlikely to be a significant contributor to gastric and esophageal SRCC molecular etiology. Further genomic investigations are on-going. This study was performed with funding received from the Lake Champlain Cancer Research Organization.

Abstract 919: AZD3463, a novel ALK/IGF1R inhibitor, overcomes multiple mechanisms of acquired resistance to crizotinib.

Abstract Genomic rearrangement of Anaplastic Lymphoma Kinase (ALK) has been observed in several tumor types including 60-80% anaplastic large cell lymphoma (ALCL) and 3-6% of non small cell lung cancer (NSCLC). Although the ALK inhibitor crizotinib has clinical efficacy in selected ALK positive NSCLC patients, the majority of patients who show initial responses eventually relapse. Various mechanisms leading to resistance have been proposed and include ALK amplification and resistance mutations, as well as alternative pathway drivers including EGFR, cKIT and, more recently, IGF1R. We have discovered a novel and potent inhibitor of ALK, AZD3463 with a Ki value of 0.75nM which also inhibits additional receptor tyrosine kinases including insulin growth factor receptor (IGF1R) with equivalent potency. AZD3463 inhibits ALK in cells as demonstrated by its ability to decrease ALK autophosphorylation in tumor cell lines containing ALK fusions including DEL (ALCL NPM-ALK), H3122 (NSCLC EML4-ALK) and H2228 (NSCLC EML4-ALK). Inhibition of ALK is associated with perturbations in downstream signaling including ERK, AKT and STAT3 pathways leading to preferential inhibition of proliferation in the ALK fusion containing cell lines in vitro. AZD3463 also demonstrates the ability to dose dependently inhibit pALK in xenograft tumors in vivo resulting in stasis (H3122) or regression (DEL, H2228). AZD3463 retains good activity against a number of clinically relevant crizotinib resistant mutations including the gatekeeper mutant L1196M where equivalent potency to wild type ALK is observed in vitro and in vivo in EML4-ALK containing BAF3 cell lines. To further assess the potential ability of AZD3463 to overcome additional resistance mechanisms, antiproliferative activity was assessed in multiple crizotinib resistant cell lines independently derived in vitro from H3122 cells as well as a patient derived crizotinib relapsed model. These resistant cell lines contain multiple resistance mechanisms including the L1196M gatekeeper and T115Ins mutations, ALK amplification and/or secondary drivers including EGFR and IGF1R. AZD3463 retains antiproliferative potency within 4 fold of parental H3122 cells for 10 out of 12 of these acquired resistance models in vitro. In summary, AZD3463 is a potent ALK inhibitor which inhibits additional kinases including IGF1R and has activity in a number of crizotinib resistant models driven by multiple resistance mechanisms. Citation Format: Lisa Drew, Jane Cheng, Jeffrey Engelman, Douglas Ferguson, Ryohei Katayama, Brenda McDermott, Jamal Saeh, Alice Shaw, Minhui Shen, Dan Widzowski, Allan Wu, Graeme Smith. AZD3463, a novel ALK/IGF1R inhibitor, overcomes multiple mechanisms of acquired resistance to crizotinib. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 919. doi:10.1158/1538-7445.AM2013-919

Abstract 4465: Concurrent roles for IGF1R and EGFR in driving acquired resistance to crizotinib and ability to overcome with a combination of the ALK/IGF1R inhibitor AZD3463 and Iressa.

Abstract Genomic rearrangement of Anaplastic Lymphoma Kinase (ALK) has been observed in several tumor types including 3-6% of non small cell lung cancer (NSCLC). Although the ALK inhibitor crizotinib has clinical efficacy in selected ALK positive NSCLC patients, the majority of patients who show initial responses eventually relapse. Various mechanisms leading to resistance have been proposed and include ALK amplification and resistance mutations, as well as alternative pathway drivers including EGFR and cKIT. To further evaluate potential mechanisms of acquired resistance to crizotinib and potential therapeutic strategies to overcome them, we have developed multiple independently generated cell line derivatives. H3122 cells, a NSCLC line with an EML4-ALK fusion, was selected in increasing concentrations of crizotinib up to 1uM over a period of 4 to 6 months to generate H3122-CR lines. The cell lines generated were confirmed to be resistance to crizotinib as well as the selective ALK inhibitor, CH5424802, in cell proliferation experiments. Evaluation of the levels of potential secondary receptor tyrosine kinase (RTK) drivers by phosphoRTK arrays and western blotting revealed increased levels of total and phosphorylated ALK (pALK), along with increased phosphorylated insulin-like growth factor 1 receptor (pIGF1R). ALK levels were also elevated at the mRNA level. To evaluate potential strategies to most effectively overcome resistance in these cell lines, selective inhibitors of ALK CH5424802, IGF1R OSI-906 and epidermal growth factor receptor (EGFR) Iressa were used alone and in combination to evaluate ability to inhibit growth of two of the H3122-CR lines. In both CR lines, OSI-906 and Iressa had no effect alone on cell growth, however a combination of CH5424802 and OSI-906 or CH5424802 and Iressa increased sensitivity to CH5424802. Most interestingly however, a combination of CH5424802, OSI-906 and Iressa had maximal sensitization effect with complete reversal of resistance observed. A similar finding was obtained with a combination of the ALK/IGF1R inhibitor AZD3463 with Iressa. Further, we were able to demonstrate pathway dependency shifts in the CR lines with OSI-906 inhibiting AKT and Iressa inhibiting ERK and a combination of CH5424802, OSI-906 and Iressa required to effectively repress these main pathways to the level observed with CH5424802 alone in the H3122 parental line. Together, these data support a potential role for both EGFR and IGF1R as secondary RTK drivers occurring concurrently to drive resistance to crizotinib. A combination of approaches inhibiting ALK, IGF1R and EGFR together may therefore be required to most effectively treat crizotinib relapsed patients and delay onset of resistance. This could be achieved by combining multiple selective agents or agents with polypharmacology such as AZD3463 along with Iressa. Citation Format: Lisa Drew, Jeffrey Engelman, Ryohei Katayama, Brenda McDermott, Jamal Saeh, Meghan Scarpitti, Alice Shaw, Minhui Shen, Suping Wang, Graeme Smith. Concurrent roles for IGF1R and EGFR in driving acquired resistance to crizotinib and ability to overcome with a combination of the ALK/IGF1R inhibitor AZD3463 and Iressa. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4465. doi:10.1158/1538-7445.AM2013-4465

Frequency of anaplastic lymphoma kinase (ALK) positive tumors among African American non-small cell lung cancer (NSCLC) patients.

7593 Background: ALK gene rearrangement is a recently identified molecular alteration in the tumors of a small proportion of NSCLC patients. Previous reports have shown that the rate of EGFR gene mutations may vary in NSCLC patients of different ethnic background. It is unclear if similar differences exist with regards to ALK positivity. We analyzed ALK expression in tumors of AA NSCLC patients and also assessed the epidemiologic features and outcomes of these patients, as well as the occurrence of known oncogene mutations. Methods: We identified 260 tumor tissues of AA NSCLC patients, enrolled on several epidemiologic studies conducted at our institution. Immunohistochemistry, using the anti-Alk D5F3 antibody (Cell Signaling Technology) was used to visualize ALK expression. Fifty-three of these tumors also underwent mutation analysis using the OncoCarta Panel V1 (Sequenom) to evaluate 238 mutations in 19 oncogenes. Results: Of the 260 tumors analyzed, 6 (2.3%, 95% CI- 0.5-4.1%) were ALK positive. All ALK positive patients had adenocarcinoma histology (6/157) while no patients with other NSCLC histologies were ALK positive, p=0.04. There was no difference in the rate of positivity based on sex, 1% (1/99) in males versus 3.1% (5/161) in females, p=0.27. Consistent with prior studies, patients with ALK positive tumors were slightly younger (median age 56 years vs 61 years, p = 0.27) and the positive rate was higher in never smokers 11% (2/18) than in ever smokers 1.6%, p=0.06. There was no correlation of ALK positivity with the stage of the disease at diagnosis. The survival of ALK positive and negative patients, adjusted for age, sex and stage, was similar (HR=0.77; 95% CI 0.19-3.13). Fifty-three tumors have been assessed for oncogene mutations in addition to ALK expression. Of these, 2 (3.8%) were ALK positive and neither of these tumors carried other tested mutations such as EGFR, Kras, PIK3 or AKT. Conclusions: The rate of ALK positive tumors among African-American NSCLC patients is similar to the general population and the demographic features of ALK positive AA patients is also similar to the general ALK positive NSCLC patient population.

Abstract LB-309: EML4-ALK signaling is required for the maintenance of neoplastic phenotype of non-small cell lung cancer cells: Novel strategy for lung cancer-tailored therapies

Abstract Lung cancer is the most common cancer in the world, and is lethal in 90% of the cases. In non-small cell lung cancer (NSCLC), deregulated receptor tyrosine kinases (RTKs) stand out among the causal dominant oncogenes. Consequently, the genomic and/or pharmacological ablation of RTK signaling has emerged as a novel tailored therapeutic strategy. Nevertheless, this approach has serious constrains, probably due to the frequent co-activation of multiple RTKs in a considerable subsets of solid tumors. It has become also evident that during TK inhibitor-based regimens, RTK switching and/or the selection of resistant clones can frequently occur representing a serious and problematic drawback. In a subset of NSCLC, the Anaplastic Lymphoma Kinase (ALK) gene has been described to be translocated and fused to EML4. This determines the ALK constitutive dimerization and autophosphorylation leading to cellular transformation. Here, we investigated the ALK oncogenic addiction of human NSCLC and studied the putative co-operative role of other kinases. We first demonstrated that the ectopic expression of EML4-ALK in ALK positive NSCLC cell lines (H2228 and H3122) resulted in the activation of multiple signaling pathways, in manner similar to that described for other known ALK fusions. Although EML4-ALK can induce transformation in lung in vitro and in vivo, ALK inhibition via shRNA or small molecule inhibitors (CEPs, Cephalon) induced only the apoptosis of H3122 cells, whereas in H2228 it caused cell growth arrest. Moreover, the treatment with ALK inhibitors led to tumor regression, but not tumor eradication, in vivo. Based on Phosphoproteomic analyses we demonstrated that the phosphorylation status of several tyrosine kinases (such as, EGFR, Met, FGFR, Jak1 or IGFR) was affected by the ALK inhibition only in H2228 cell line, but not in H3122. Notably, the combined treatment with anti-ALK (CEP14083, CEP2550, CEP28122) and -EGFR inhibitors resulted in an increased cell death of H2228 cells to values similar to those observed for ALK treated H3122 cells. Finally, gene expression analyses showed that known EGFR substrates were specifically down regulated upon the combined treatment in ALK positive H2228 cells. Our findings suggest that ALK signaling is required for the maintenance of the neoplastic phenotype of some ALK positive NSCLC cells and that its abrogation could represent a novel strategy for the treatment of a well-defined subset of human lung cancer (complete ALK addiction). More importantly, we showed that the tumor survival and maintenance of ALK positive neoplastic cells might relay on the concomitant activation of multiple RTKs. These findings further support the notion that molecular and functional signatures are required for designing molecular-based “patient specific” therapeutic protocols in lung cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-309.