Anaplastic Lymphoma Kinase Translocation Research Articles

Potential Therapies for Anaplastic Lymphoma Kinase-Driven Tumors in Children: Progress to Date

Anaplastic lymphoma kinase (ALK) is an oncogenic tyrosine kinase that is deregulated due to a variety of molecular mechanisms in pediatric cancer. They include chromosomal translocations, activation mutations, and gene amplifications. Since the initial discovery of ALK as an oncogenic tyrosine kinase involved in the chromosomal translocation t(2, 5)(p23;q35) in 1994, more than 20 translocation partners of ALK have been identified in various cancers. Furthermore, deregulation of ALK tyrosine kinase activity is critical for the pathogenesis of several other pediatric tumors, including neuroblastomas and inflammatory myofibroblastic tumors. The recent discovery of ALK translocations in adult lung cancer patients (non-small cell lung cancer) has accelerated the development of inhibitors of ALK tyrosine kinase as therapeutic agents. While excellent clinical response has been observed in many patients, the acquisition of clinical resistance to ALK inhibition highlights the need for development of second-generation ALK kinase inhibitors and/or combination therapies that target downstream signaling mediators or antibody drug conjugates. This article provides an update on the spectrum of ALK-driven tumors in the pediatric population and the potential therapies which target these tumors.

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.

Multiplatform assessment of anaplastic lymphoma kinase (ALK) gene rearrangement in signet ring cell carcinoma of the gastrointestinal tract.

e14612 Background: ALK-EML4 translocation is an established driver aberration in NSCLC, with reported predilection for cases with signet ring histology. We assessed the presence of ALK gene rearrangements in signet ring cancers arising in the stomach and colon. Methods: Histologically confirmed cases of signet ring adenocarcinoma of the stomach or the colon were identified from the pathology archive of Emory University Hospital. Presence of the classic ALK and EML4 fusion gene was initially determined by fluorescence in-situ hybridization (FISH) method. Cases showing break-apart signal in more than 15% of enumerated cells were considered positive; at least 10% of enumerated cells was considered “possibly positive”. Immunohistochemistry of the fused gene product was assessed using previously validated antibodies ALK1 clone (1:100; DAKO) and 5A4 (Novocastra, Leica Biosystems) and positive controls of ALK-translocated lung cancer. Positive or possibly positive cases by FISH were further analyzed by RT-PCR using specific primers for the most common translocation pairs (EML4 Ex13/ALK Ex20 and EML4 Ex6/ALK Ex20) along with positive (H228, HEK293) and negative (H460, H1975) control cell lines. Results: We retrieved 44 cases of signet ring carcinoma diagnosed between 2001 and 2011; 26 gastric and 18 colon cancer. Median age - 63 yrs; M/F 17/27; Black - 47.5%; White - 47.5%; Others - 5%; Stage I - 21.4%; II - 31%; III - 26.2; IV - 21.4%. One of 44 cases (2.3%) was positive for ALK translocation by FISH using the standard criteria of at least 15% positive cells for the break-apart signal (50-70 cells counted per case). 7 cases (16%) were considered possibly positive [1 of 18 (6%) colon cancer and 6 of 26 (26%) gastric cancer cases]. IHC showed 0 of 44 (0%) cases positive. Molecular analysis using RT-PCR did not confirm the suspected ALK gene rearrangements in any of the 7 cases considered possibly positive by FISH. Conclusions: The overall frequency of ALK gene rearrangement detected by FISH in signet ring cancer of the GI tract was low, but appears comparable to the reported frequency in lung cancer. Signet ring histology failed to enrich for cases with ALK gene translocation in this histologic subtype.

Anaplastic lymphoma kinase (ALK): A potential oncogenic driver in triple-negative breast cancer?

1067 Background: TNBC, a molecularly heterogeneous subset of breast cancer, has an aggressive clinical course and no established targets for therapy. Wild-type ALK gene amplification by fluorescence in situ hybridization (FISH) was noted in 6/9 inflammatory TNBC specimens (Robertson et al. SABCS, P3-01-18). The status of ALK, full-length or fused, in non-inflammatory TNBC has not been described. Identification of a biomarker such as ALK in TNBC would be clinically significant, given the ongoing development of multiple ALK inhibitors. We screened for ALK expression in TNBC specimens with a novel patented qRT-PCR assay (Insight ALK Screen). Methods: We tested archived formalin-fixed, paraffin-embedded blocks of TNBC tissue. Evaluable specimens had sufficient tumor material for 10 slides with 5-micron sections. Insight ALK Screen was performed using previously described methodology (Hout, et al. AACR 2011, Abstract 2220). We subjected ALK Screen positive specimens to ALK break-apart FISH (Vysis) and direct sequencing analysis. Results: See clinical characteristics (Table). Insufficient RNA: 8/40 (20%). Insight ALK Screen-positive: 8/32 (25%). Break-apart FISH performed on these 8 specimens suggested 3/8 to be positive for ALK translocation and 2/8 to contain amplification of ALK without a translocation. The ALK-amplified specimens were subsequently confirmed with sequencing of RNA-PCR products to express full-length ALK. One additional ALK Screen-positive, FISH-negative specimen had full-length ALK expression confirmed on sequencing. Further analysis of the other 2 ALK Screen-positive cases is pending. Conclusions: ALK expression was seen in ~25% of TNBC specimens screened by Insight ALK Screen. We observed both ALK translocations and amplification/overexpression of full-length ALK. We are testing additional specimens by FISH and immunohistochemistry to evaluate the full spectrum of ALK alterations in TNBC. Updated data will be presented. [Table: see text]

Identification of Somatic Genomic Alterations in Circulating Tumors Cells: Another Step Forward in Non–Small-Cell Lung Cancer?

The last decade has witnessed substantial progress in defining the molecular determinants of non–small-cell lung cancer (NSCLC) and demonstrating how these can be exploited in the clinic. In 2004, the identification of somatic mutations in the epidermal growth factor receptor (EGFR) gene provided the first insight into a clinically relevant NSCLC driver oncogene. EGFR mutations in NSCLC are transforming, enhance the activity of the kinase domain of EGFR, and increase the affinity for EGFR tyrosine kinase inhibitors (TKIs). In EGFR-mutated NSCLCs (ie, tumors with classic EGFR exon 19 deletions or the L858R mutation, which are found in approximately 15% of all NSCLCs), clinical and radiographic responses are achieved in most patients with the use of two commercially available reversible EGFR TKIs: gefitinib and erlotinib. These EGFR TKIs improve outcomes when compared with cytotoxic chemotherapies, and the evidence-based use of these drugs, as recommended by the American Society of Clinical Oncology and other practice and regulatory agencies, is now restricted to EGFR-mutated NSCLCs in the first-line treatment of advanced tumors. The significant palliative benefits that are offered by matching a tumor driver mutation with an appropriate inhibitor paved the way for the genomic characterization of NSCLCs and the development of novel TKIs that can target these changes. The deciphering of NSCLCs using next-generation sequencing (through whole-genome, exome, and transcriptome techniques) has identified numerous driver oncogenic events (ie, activating mutations or rearrangements) involving targetable kinases, including ROS1, ERBB2, BRAF, and RET, among others. The separation of NSCLCs into distinct, actionable subtypes is mostly clear in lung adenocarcinomas of never-smokers (Figure 1), in which almost all tumors have a mutually exclusive driver oncogene for which TKIs are either clinically available or in earlyto late-stage development. Amid the exciting series of discoveries that have occurred during the last decade, one of the most remarkable has been the story of how anaplastic lymphoma kinase (ALK) gene alterations became a biomarker and therapeutic target in NSCLC. ALK is a transmembrane tyrosine kinase that had been previously implicated in the pathogenesis of anaplastic large cell lymphoma and neuroblastoma. In 2007, a Japanese group from Jichi Medical University identified novel fusion oncogenes involving the kinase domain of ALK in patients with NSCLC. ALK rearrangements, either inversions or translocations, characterize the genomic changes observed in NSCLC. The most frequent event is an inversion in the short arm of chromosome 2 that results in the fusion of the echinoderm microtubule-associated protein-like 4 (EML4) with ALK, leading to the production of an EML4-ALK fusion tyrosine kinase. Several EML4-ALK variants have now been described, with EML4-ALK E13;A20 and E20;A20 being the most common. Few other tumor types harbor these EML4-ALK rearrangements, and it remains unclear why NSCLCs can acquire these somatic changes. Interestingly, some clinical and pathologic features are associated with an increased incidence of tumors containing ALK rearrangements. These include never or light smoking history ( 15 pack-years), young age, and adenocarcinoma histology with signet rings. The reported prevalence of ALK rearrangements in NSCLC is approximately 5%; however, in neversmokers or light smokers with lung adenocarcinoma, the prevalence may be as high as 20%. Therefore, as many as 10,000 new cases of ALK-rearranged NSCLC are expected in the United States this year. Other/unknown

Abstract 4107: Targeted re-sequencing of neuroblastoma tumors reveals chromosomal rearrangements that involve the Anaplastic Lymphoma Kinase (ALK) gene.

Abstract Neuroblastoma (NBL) is a cancer of early childhood arising from the developing sympathetic nervous system. NBL tumors display a broad clinical and biological heterogeneity, ranging from highly aggressive tumors with fatal outcome to tumors with spontaneous regression. Recurrent mutations are mainly only observed in Anaplastic Lymphoma Kinase (ALK), which is involved in the pathogenesis of both familiar and sporadic NBL. ALK encodes a tyrosine kinase receptor with importance in neuronal development and was initially characterized in anaplastic large cell lymphoma from a translocation leading to the NPM-ALK fusion protein. Subsequent studies show that additional ALK chimeras have been generated with different fusion partners in various cancers. To date no ALK-containing rearrangement has been reported in primary NBL tumors although one study has described a NBL cell line with an ALK intragenic deletion leading to constitutive ALK kinase activity. This led us to screen for and further investigate possible ALK containing translocations in our NBL material. Genomic profiling of 350 NBL samples though SNP microarrays indicated ALK-rearrangement in four primary tumors and two cell lines (IMR32 and CLB-BAR) which were further analyzed through targeted re-sequencing of the common MYCN amplicon region (Chr2;14-18Mb) and the entire ALK gene. Briefly, detection of rearrangements was performed through solution-based enrichment followed pair-end sequencing of 400-500bp libraries. Junction sites were identified by manual sequence viewing on IGV and validated through PCR followed by Sanger sequencing. With this strategy we were able to determine translocation sites for all but one sample. In one tumor ALK was found to be translocated to GAB2 (chr11q14) causing loss of the first exon of respective gene. However, ALK and GAB2 were placed head-to-head with respect to transcription direction and this novel translocation is therefore unlikely to cause a chimeric protein. Another tumor showed translocation between ALK intron 4 and chr 4 subtelomeric region without known genes. Two NBL cell lines and one tumor had both MYCN and ALK amplification and sequencing revealed high level complexity of respective amplicon with multiple rearrangements. IMR32 show involvement of at least six different 2p-regions including MYCN and ALK exon 3-4. CLB-BAR has also multiple rearrangements including an ALK intragenic translocation causing loss of exon 4-11, ultimately resulting in an ALK protein with truncated extracellular domain. Here we show that ALK translocation sites could be deduced from targeted re-sequencing without previous knowledge about fusion partner. However, no translocations resulting in chimeric protein were detected. We were also able to detect MYCN amplicon junctions, which are highly unique for each patient and could be used for detection of minimal residual disease. Citation Format: Susanne M. Fransson, Anna Djos, Niloufar Javanmardi, Rosa Noguera, Ana Berbegall, Magnus Hansson, Per Kogner, Kristina Ruuth, Ruth Palmer, Bengt Hallberg, Tommy Martinsson. Targeted re-sequencing of neuroblastoma tumors reveals chromosomal rearrangements that involve the Anaplastic Lymphoma Kinase (ALK) gene. [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 4107. doi:10.1158/1538-7445.AM2013-4107

Amplification but not translocation of anaplastic lymphoma kinase is a frequent event in oesophageal cancer

IntroductionTranslocations of anaplastic lymphoma kinase (ALK) to various fusions partners and formation of oncogenic fusions proteins have been demonstrated in a variety of human malignancies. These fusion-proteins are potential pharmaceutically targets. Aim of this study was to investigate ALK gene status in a large cohort of squamous cell carcinoma (SCC) and adenocarcinoma (AC) of the oesophagus. Materials and Methods117 SCCs and 136 ACs were included into this study. ALK and EML4 gene status were evaluated by fluorescence in situ hybridisation (FISH) using a triple colour break apart single fusion probe and a probe against 2p11. ALK and EML4 protein expression was determined by immunohistochemistry. Data on expression of ALK downstream effector tyrosine-705 phosphorylated STAT3 (pSTAT3) were available from a previous study. ResultsFISH was performed successfully in 251 cases. All cases were negative for ALK translocations, while 14/135 (12.1%) of SCCs and 14/116 (10.4%) of ACs showed ALK amplifications. Concomitant EML4 amplifications were present in 27/28 cases with ALK amplifications. Three cases (two SCC, one with additional ALK &EML4 amplification and one AC) showed EML4 translocations not involving ALK. None of the tumours with ALK amplification showed ALK protein expression, and no correlation with clinical parameters, survival or pSTAT3 expression was observed. ConclusionsWhile ALK translocations are not present in oesophageal cancer, ALK amplifications are common events with comparable rates in SCC and AC. Since ALK amplified breast cancer cells were shown to respond to ALK inhibitors, ALK amplified oesophageal cancers might be considered as possible candidates for therapies targeting ALK.

Derlin-1 Is Overexpressed in Non-Small Cell Lung Cancer and Promotes Cancer Cell Invasion via EGFR-ERK–Mediated Up-Regulation of MMP-2 and MMP-9

Previous studies indicated a role of Derlin-1 in human cancers; however, its expression pattern in non-small cell lung cancer (NSCLC) and the molecular mechanism of Derlin-1 on cancer progression have not been characterized. In the present study, Derlin-1 expression was examined in lung cancer cell lines and human tissues. Derlin-1 overexpression correlated with pTNM stage, lymph node metastasis, and poor overall survival. siRNA knockdown of Derlin-1 impaired anchorage-dependent and anchorage-independent cell growth and invasion in A549 and H1299 cell lines, and its overexpression promoted proliferation and invasion in HBE and LTE cell lines. Derlin-1 depletion decreased matrix metalloproteinase (MMP)-2/9 at both protein and mRNA levels, with decreased MAP kinase/extracellular signal-regulated kinase (ERK)/ERK phosphorylation. Derlin-1 overexpression up-regulated MMP-2/9 expression and ERK phosphorylation, which could be reversed by MAP kinase/ERK kinase inhibitor, PD98059. The effect of Derlin-1 on MMP-2/9 up-regulation was abolished in ERK1/2 siRNA-treated cells. Further analysis showed that Derlin-1 overexpression induced EGFR phosphorylation. EGFR inhibitor blocked Derlin-1-mediated up-regulation of EGFR and ERK phosphorylation. MMP-2/9 and p-ERK up-regulation by Derlin-1 was partly blocked in EGFR-depleted cells with siRNA treatment. Immunoprecipitation confirmed the association between Derlin-1 and EGFR. In summary, our results showed that Derlin-1 is overexpressed in NSCLC and promotes invasion by EGFR-ERK-mediated up-regulation of MMP-2 and MMP-9. Derlin-1 may serve as a therapeutic target for NSCLC.

Targeted therapies in development for non-small cell lung cancer.

The iterative discovery in various malignancies during the past decades that a number of aberrant tumorigenic processes and signal transduction pathways are mediated by “druggable” protein kinases has led to a revolutionary change in drug development. In non-small cell lung cancer (NSCLC), the ErbB family of receptors (e.g., EGFR [epidermal growth factor receptor], HER2 [human epidermal growth factor receptor 2]), RAS (rat sarcoma gene), BRAF (v-raf murine sarcoma viral oncogene homolog B1), MAPK (mitogen-activated protein kinase) c-MET (c-mesenchymal-epithelial transition), FGFR (fibroblast growth factor receptor), DDR2 (discoidin domain receptor 2), PIK3CA (phosphatidylinositol-4,5-bisphosphate3-kinase, catalytic subunit alpha)), PTEN (phosphatase and tensin homolog), AKT (protein kinase B), ALK (anaplastic lym phoma kinase), RET (rearranged during transfection), ROS1 (reactive oxygen species 1) and EPH (erythropoietin-producing hepatoma) are key targets of various agents currently in clinical development. These oncogenic targets exert their selective growth advantage through various intercommunicating pathways, such as through RAS/RAF/MEK, phosphoinositide 3-kinase/AKT/mammalian target of rapamycin and SRC-signal transduction and transcription signaling. The recent clinical studies, EGFR tyrosine kinase inhibitors and crizotinib were considered as strongly effective targeted therapies in metastatic NSCLC. Currently, five molecular targeted agents were approved for treatment of advanced NSCLC: Gefitinib, erlotinib and afatinib for positive EGFR mutation, crizotinib for positive echinoderm microtubule-associated protein-like 4 (EML4)-ALK translocation and bevacizumab. Moreover, oncogenic mutant proteins are subject to regulation by protein trafficking pathways, specifically through the heat shock protein 90 system. Drug combinations affecting various nodes in these signaling and intracellular processes are predicted and demonstrated to be synergistic and advantageous in overcoming treatment resistance compared with monotherapy approaches. Understanding the role of the tumor microenvironment in the development and maintenance of the malignant phenotype provided additional therapeutic approaches as well. More recently, improved knowledge on tumor immunology has set the stage for promising immunotherapies in NSCLC. This review will focus on the rationale for the development of targeted therapies in NSCLC and the various strategies employed in preventing or overcoming the inevitable occurrence of treatment resistance.

AJ-2 - Oncology Phase I Trials at Shizuoka Cancer Center

ABSTRACT To promote therapeutic development for cancer, it is mandatory to develop new effective drugs in Japan. Oncology phase I trials have been conducted to evaluate the safety, dose-limiting toxicity, and pharmacokinetics of a drug to estimate its maximum tolerated dose. Recommended doses have been determined on the basis of the concept that toxicity can be a surrogate end point for efficacy. Recently, molecular targeted drugs, which specifically inhibit a particular molecular target, have been developed actively and have contributed to improved outcomes of cancer treatment. The efficacy of several molecular targeted drugs is highly dependent on molecular changes in cancer cells, e.g. activating mutations of the EGFR (epidermal growth factor receptor) and ALK (anaplastic lymphoma kinase) translocation. Japan is now able to participate in multinational clinical studies and aims at obtaining global simultaneous approval. However, we need to keep in mind the factor of ethnic difference between Asians and Caucasians. There is a significant difference between the incidence of EGFR-mutant lung cancer in Asians and non-Asians. It is also important to clarify possible inter-ethnic differences in pharmacokinetics and pharmacodynamics. Since 2003, there have been oncology phase I trials conducted at Shizuoka Cancer Center, including the first-in-human trials. Some recent studies demand evaluation of tumor tissue samples, and we are required to find biomarkers, or surrogate markers of biological response, from early on during development. I will present several phase I trials carried out at Shizuoka Cancer Center.

Targeting ALK: a promising strategy for the treatment of non-small cell lung cancer, non-Hodgkin’s lymphoma, and neuroblastoma

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that affects a number of biological and biochemical functions through normal ligand-dependent signaling. It has oncogenic functions in a number of tumors including non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma, and neuroblastoma when altered by translocation or amplification or mutation. On August 2011, a small molecule inhibitor against ALK, crizotinib, was approved for therapy against NSCLC with ALK translocations. As we determine the molecular heterogeneity of tumors, the potential of ALK as a relevant therapeutic target in a number of malignancies has become apparent. This review will discuss some of the tumor types with oncogenic ALK alterations. The activity and unique toxicities of crizotinib are described, along with potential mechanisms of resistance and new therapies beyond crizotinib.

NPM-ALK: The Prototypic Member of a Family of Oncogenic Fusion Tyrosine Kinases

Anaplastic lymphoma kinase (ALK) was first identified in 1994 with the discovery that the gene encoding for this kinase was involved in the t(2;5)(p23;q35) chromosomal translocation observed in a subset of anaplastic large cell lymphoma (ALCL). The NPM-ALK fusion protein generated by this translocation is a constitutively active tyrosine kinase, and much research has focused on characterizing the signalling pathways and cellular activities this oncoprotein regulates in ALCL. We now know about the existence of nearly 20 distinct ALK translocation partners, and the fusion proteins resulting from these translocations play a critical role in the pathogenesis of a variety of cancers including subsets of large B-cell lymphomas, nonsmall cell lung carcinomas, and inflammatory myofibroblastic tumours. Moreover, the inhibition of ALK has been shown to be an effective treatment strategy in some of these malignancies. In this paper we will highlight malignancies where ALK translocations have been identified and discuss why ALK fusion proteins are constitutively active tyrosine kinases. Finally, using ALCL as an example, we will examine three key signalling pathways activated by NPM-ALK that contribute to proliferation and survival in ALCL.

Anaplastic lymphoma kinase-directed therapy in inflammatory myofibroblastic tumors

Inflammatory myofibroblastic tumors (IMTs) are indolent mesenchymal neoplasms associated with a small risk of aggressive behavior and metastasis. Surgery is the mainstay of treatment and until recently there have been limited effective treatment options for unresectable disease. This review describes the identification of anaplastic lymphoma kinase (ALK) fusion genes in approximately 50% of IMTs and the role of ALK inhibition in the treatment of this disease. A recent phase I dose-escalation trial of the selective MET/ALK inhibitor crizotinib showed a long-term partial response in a patient with IMT carrying an ALK translocation but not in a patient with ALK-negative disease. Emergence of resistance to crizotinib occurs approximately 5-8 months after initiation of therapy and has been shown to be driven by different mechanisms. Multiple second-generation ALK inhibitors are currently being investigated in the preclinical and clinical trial setting. ALK-directed therapy has emerged as a highly effective treatment option for a subset of patients with IMT and pulmonary adenocarcinoma. A number of additional malignancies, including rhabdomyosarcoma, neuroblastoma, anaplastic large cell lymphoma, renal cell carcinoma, and inflammatory breast cancer, have been shown to activate ALK expression by means of ALK fusion proteins, ALK mutations, or increased ALK copy number. Development of more selective ALK inhibitors, which can overcome emergent crizotinib resistance mutations, as well as development of combination treatments with drugs targeting compensatory pathways, will be key to achieving therapeutic success in targeting this potent and prevalent oncogenic driver.

Differential sensitivities to tyrosine kinase inhibitors in NSCLC harboring EGFR mutation and ALK translocation

Epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) translocations in non-small cell lung cancer (NSCLC) are mutually exclusive. However, several exceptional cases harboring both genetic alterations have been reported. In this study, a total of 444 patients with lung adenocarcinoma were examined for their EGFR and ALK status at Seoul National University Hospital between July 2008 and September 2011. EGFR mutations and ALK translocations were detected in 228 (51.4%) and 34 (7.7%) patients, respectively. Four patients (0.9%) had both genetic alterations and three underwent curative surgery. One patient who received both EGFR tyrosine kinase and ALK inhibitors, separately showed an objective response to the ALK inhibitor alone. Considering our and previous studies, patients harboring both EGFR mutation and ALK translocation showed differential sensitivities to both targeted therapies, suggesting a variable dependence on EGFR and ALK oncogenes.

Somatic mutation spectrum of non-small cell lung cancers (NSCLCs) from African Americans (AAs).

6038 Background: In the AA population, previous studies have presented conflicting data on the frequency of EGFR mutations (Reinersman JTO 2011;Leidner JCO 2009), while frequencies of other gene mutations and translocations, including anaplastic lymphoma kinase (ALK), have not been described. Methods: 161 archival FFPE tumor specimens from self reported AA patients with any stage NSCLC from 1997-2010 were collected from 3 sites in Tennessee (132 samples) and one site in Michigan (29 samples). Samples were evaluated for known recurrent driver mutations in EGFR, KRAS, BRAF, NRAS, AKT1, PI3KCA, PTEN, HER-2, MEK1 by standard SNaPshot/sizing assays, and translocations in ALK by FISH. Clinical data was collected on 119 patients. Chi-square was used to compare the frequency of mutations in subgroups and Kaplan-Meier and log rank were used to calculate and compare PFS between groups. Results: 5.0% of tumors had EGFR mutations, 14.9% had KRAS mutations, 0.6% had a BRAF, AKT1, PI3KCA, or HER2 mutation, and 0% had NRAS, PTEN, or MEK1 mutations. Of 35 ‘pan-negative’ non-squamous specimens, 0 had ALK translocations. PFS was the same in those with and without KRAS mutation (p=0.74) and showed a trend towards improvement in those with EGFR mutation (p=0.08). The frequency of EGFR mutations was higher in samples from Detroit versus those from Tennessee (17% vs 2.3%, p<0.01), as was the frequency of adenocarcinoma (62% vs 44%, p<0.05). The frequency of EGFR mutations in never smokers was higher in the samples from Detroit versus Tennessee (83% vs 7.1%, p<0.01). Conclusions: In the largest tumor mutational profiling study of NSCLC from AAs to date, EGFR mutations occurred less frequently than would be expected from a North American population. We noted a regional difference, with fewer EGFR mutations in Tennessee than in Michigan, a finding that may have been the result of more adenocarcinoma samples from Michigan. The rates of other mutations and translocations including ALK were low. While lung cancer tumors should continue to undergo routine molecular testing to prioritize therapy, future comprehensive genotyping efforts should focus on identifying novel driver mutations in this population. Funding: 5RC1CA162260 R01CA060691 R01CA87895.

Targeted therapies for the treatment of non-small cell lung cancer: Bull’s eye or missed target?

3074 Background: Targeted drugs approved for advanced non-small cell lung cancer (NSCLC) include bevacizumab [(monoclonal antibody directed against vascular endothelial growth factor-A (VEGF-A)], erlotinib [(an epidermal growth factor receptor kinase (EGFR) inhibitor)] and crizotinib [(an anaplastic lymphoma kinase (ALK) inhibitor)]. The development programs for erlotinib and bevacizumab did not prospectively employ in vitro diagnostic tests to select patients (pts). Post-hoc exploratory analyses for bevacizumab based on VEGF-A levels have not identified subgroups with differential treatment effects: however for erlotinib, pts with activating EGFR mutations had greater improvement in overall response rates (ORR) and progression-free survival. Crizotinib is the first targeted therapy for NSCLC in which pts were selected using an analytically validated investigational test for ALK translocations. Crizotinib received accelerated approval on August 26, 2011 for the treatment of pts with locally advanced or metastatic ALK-positive NSCLC as detected by an FDA-approved test (Vysis ALK Break Apart FISH Probe Kit) that was approved by FDA on the same day. Methods: NDA file for crizotinib was examined. Results: Two single arm trials assessing efficacy of crizotinib monotherapy in ALK positive pts showed ORR of 61% and 50%. At FDA’s request, a cohort of 23 pts that tested negative for the ALK translocation evaluated showed ORR of 26%. Two additional pts had a single assessment and, if confirmed, the response rate would be 37% in this “marker negative” subgroup. Further exploration of this subgroup is a postmarketing commitment. Conclusions: Evaluation of target status (positive or negative) in all pts provides optimal information to guide clinical practice. As observed with crizotinib, responses may be observed in the “marker negative” subgroup. This unexpected finding may reflect the drug’s activity via alternate pathways, selection of a suboptimal biomarker for assessing ALK activation, or selection of a suboptimal cut-point for positive/negative. Accurate diagnostic tests will facilitate drug development with possible greater observed treatment effect and potentially smaller clinical trials.

Efficacy of crizotinib in children with relapsed/refractory ALK-driven tumors including anaplastic large cell lymphoma and neuroblastoma: A Children's Oncology Group phase I consortium study.

9500 Background: Genetic aberrations in the anaplastic lymphoma kinase (ALK) gene are found in anaplastic large cell lymphoma (ALCL), neuroblastoma (NB) and other tumors.Crizotinib,a small molecule inhibitor of ALK and c-Met, is active in non-small cell lung cancers (NSCLC) harboring an ALK translocation. We performed a phase 1 dose-escalation and pharmacokinetic (PK) trial of crizotinib in patients (pts) with refractory solid tumors and ALCL. Methods: Crizotinib was administered bid without interruption in 28 day cycles using the rolling-six design. Six dose levels (100, 130, 165, 215, 280, 365 mg/m2/dose) have been evaluated (A1). Pts with confirmed ALK fusion proteins, mutations or amplification (A2) could enroll at one dose level lower than part A1 and those with NB could enroll on a separate stratum (A3). PK studies were performed on day 1 and at steady state (SS). ALK genomic status in tumor tissue was evaluated and qPCR was used to measure NPM-ALK fusion transcript in bone marrow and blood samples of ALCL pts. Results: 70 pts were enrolled, 57 fully evaluable for toxicity, [median (range) age 9.9 yrs (1.1–21.3)]: 29 on A1, 18 on A2, and 10 on A3. In A1, 2/7 pts developed DLT (grade 3 dizziness, grade 5 intra-tumoral hemorrhage) at 215 mg/m2 and 1/6 pts developed DLT (grade 4 liver enzyme elevation) at 365 mg/m2. In A2, 1 grade 4 DLT (neutropenia) occurred at 165 mg/m2; in A3, no DLTs occurred. Mean (±SD) Cave (=AUC0-12h/12h) of crizotinib at SS was 466±114 ng/mL at 215 mg/m2/dose (n=5), 443±121 ng/mL at 280 mg/m2/dose (n=8), and 720±230 ng/mL at 365mg/m2/dose (n=4). Response data for pts with ALCL (six at 165 mg/m2, two at 280 mg/m2) approved for release by the Data Safety Monitoring Committee demonstrates 7/8 (88%) complete response (CR) rate to date. RT-PCR data for 6 of these pts at 57 time points was obtained and will be described. In addition, 2 pts with NB have had CRs, one with a documented ALK mutation. One patient with an inflammatory myofibroblastic tumor and one with NSCLC had PRs. Conclusions: Inhibition of ALK in pediatric pts with ALK-driven tumors occurs with minimal toxicity and is associated with objective anti-tumor activity.

Abstract 1108: Subtype specific expression of immnune-modulating miR-155 and miR-146a in anaplastic large cell lymphoma

Abstract Anaplastic large-cell lymphoma (ALCL) is a mature T-cell malignancy that is subdivided further into two distinct disease entities based on the presence or absence of the anaplastic lymphoma kinase (ALK) fusion protein. ALK+ ALCL bears the t(2;5) (p23;q35) translocation in greater than 80% of cases, which results in the expression of the chimeric nucleophosmin (NPM)-ALK. This leads to the activation of many different growth-promoting and anti-apoptotic pathways, including PI3K/Akt1/mTOR, Jak/Stat and AP-1. Recently, micro-RNAs (miRNAs) have emerged as tiny but potent molecules to regulate cell differentiation and proliferation. In a recent publication we have reported that miR-155 is up-regulated in ALK+ and down-regulated in ALK- ALCL. We have now identified an additional miRNA, miR-146a, that is significantly higher expressed in ALK- ALCL. Both miRNAs are strongly induced by T-cell activation. We report here that in ALCL cell lines that highly express miR-155 (like ALK- Mac1 and Mac2a) its target protein CCAAT-enhancer-binding protein ≤ (C/EBPβ) is suppressed and moreover reintroduction of miR-155 in cell lines that have low miR-155 levels (Karpas-299, SR786, SU-DHL-1, SUP-M2) can actively down-regulate C/EBPβ. Conversely, high expression of miR-146a in primary ALK- ALCL FFPE specimens corresponds to low expression of its target protein IL1 receptor-associated kinase 1 in ALK - cell lines. Due to the known roles of miR-155 and miR-146a we assessed cytokine release in a set of ALCL cell lines with and without the ALK translocation. Interestingly, we found expression of cytokines including IL-6, IL-10, IL-21 and IL-22 in ALCL cell lines. Inflammatory cytokines IL-6, IL-21 and IL-22 were 4 to 10-fold higher in ALK- cell lines, whereas anti-inflammatory IL-10 levels were not related to ALK status. Reintroduction of miR-155 into the ALK+ cell lines Karpas-299 and SR786 was able to enhance expression of IL-22. To test the influence of miR-155 on ALK- ALCL tumor growth in vivo we injected 5x10^6 Mac1 cells with low (transfected with anti-miR) or high miR-155 (transfected with pre-miR) into the flanks of BALB/c mice. High miR-155 led to a significantly enhanced tumor growth and increased IL-22 in the serum, suggesting miR-155 as tumor driver and potential therapeutic target in ALK- ALCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1108. doi:1538-7445.AM2012-1108

Abstract 1715: High ALK gene copy number as a predictor of response to crizotinib in non-small cell lung cancer cell lines

Abstract Introduction: The anaplastic lymphoma kinase (ALK) gene translocation is a rare event, described in approximately 4-6% of lung adenocarcinoma. ALK translocation is a robust predictive factor for ALK inhibitors sensitivity. However, increased ALK gene copy number seems to be a frequent event, described in 13-17% of Non-small cell lung cancer (NSCLC). The goal of this study was to explore the predictive value of ALK High copy number for 2 ALK inhibitors, crizotinib and TAE684 in NSCLC cell lines. Methods: 27 NSCLC cell lines were screened for ALK copy number by FISH after cytospin. ALK high copy number (HCN) was defined as the presence of ≤ 5 copies of ALK. FISH with CEP2 was performed to determine the ploïdy status in cell lines with HCN. As crizotinib is a dual c-MET and ALK inhibitor, FISH c-MET was performed in selected cell lines for in vitro studies. C-MET scoring was based on Cappuzzo criteria. In vitro sensitivity was evaluated through WST1 assays and clonogenic tests. Kelly, an ALK mutated neuroblastic cell line, was added as a positive control. Cells were seeded in triplicates in 96-well plates 18h before the treatment with TAE 684 or crizotinib with concentrations varying from 50 to 4000nM and 100 to 10000nM respectively. The half maximal inhibitory concentration of the two drugs (IC50) was estimated after 72h of treatment with 4 and 5 parameters logistic regression models. In clonogenic tests, crizotinib was tested with three schedules of concentrations: low dose (50nM), intermediate (200nM) and high (1000nM). Results: Among NSCLC cell lines, 6 (22%) displayed more than 5 copies of ALK, 19 (70%) presented a gain of 3 or 4 ALK copy number, only one cell line exhibited normal ALK copies and one harbored EML4-ALK translocation. FISH with CEP2 revealed a polysomy of chromosome 2 in cases with ALK HCN. C-MET amplification was observed in only three cell lines. Based on ALK FISH status, 9 NSCLC cell lines were selected to undergo in vitro assays, 6 exhibiting ALK HCN (H2030, H661, A427, H3255, BEN, H1299) and 3 exhibiting low ALK copy number (LCN) (H1975, H1651, H1650). Kelly and A549 cell lines were added as sensitive and resistant controls respectively. In ALK HCN cell lines, two (H661 and A427) were as sensitive as Kelly for crizotinib through clonogenic and WST1 assays. In addition, ALK HCN cell lines exhibited lower IC50 for anti-ALK drugs than cell lines with ALK LCN (median IC50 with crizotinib values: 1750nM [300-2800nM] in ALK HCN cell lines versus 4500nM [800-8000nM] in ALK LCN cell lines, p=0.35). The trend of crizotinib activity was not due to c-MET inhibition. Conclusion: The in vitro assays suggest that ALK HCN may be a predictive marker for sensitivity to crizotinib. The predictive value of ALK HCN would be investigated in in vivo models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1715. doi:1538-7445.AM2012-1715

Immunohistochemical screening for anaplastic lymphoma kinase (ALK) rearrangement in advanced non-small cell lung cancer patients

Fluorescence in situ hybridization (FISH) is currently used to detect non-small cell lung cancer (NSCLC) patients with anaplastic lymphoma kinase (ALK) gene rearrangement, who are candidates for ALK inhibitor therapy. However, FISH may not be a practical method for screening for ALK-positive patients in a large population due to its cost and difficulty in interpretation. We investigated the role of immunohistochemistry (IHC) to screen for ALK rearrangement in advanced NSCLC. We identified 1,166 stage IIIB or IV NSCLC patients with non-squamous histology from the Seoul National University Hospital NSCLC database. To enrich ALK-positive cases, we selected 262 patients who were either EGFR wild-type or non-responders to previous EGFR tyrosine kinase inhibitors (TKI). ALK IHC and ALK FISH were performed on formalin-fixed, paraffin-embedded tissue. ALK protein was expressed in 28 (10.7%) tumors in 262 patients. ALK FISH was positive in 25 (9.5%) cases. All patients with IHC score of 3 (n=9) were FISH-positive and all patients with score of 0 (n=234) were FISH-negative. Among patients with IHC scores of 1 and 2, five (83.3%, 5/6) and eleven (84.6%, 11/13) were FISH-positive, respectively. The sensitivity and specificity of ALK IHC with intensity score of 1 or more were 100% and 98.7%, respectively. IHC can be a useful test for screening ALK FISH-positive cases in advanced NSCLC. FISH testing should be considered for advanced NSCLC patients with tumors showing mild to moderate staining for ALK by IHC to confirm ALK translocation.