Heather R. Sanders

Exon scanning by reverse transcriptase–polymerase chain reaction for detection of known and novel EML4–ALK fusion variants in non–small cell lung cancer

Chromosomal inversions within chromosome 2p, resulting in fusions between the echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) genes, are a recent focus of treatment options for non-small cell lung cancer. Thirteen EML4-ALK fusion variants have been identified, affecting eight EML4 exons. We have developed an exon scanning approach using multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify known and potential variants involving the first 22 EML4 exons. A total of 55 formalin-fixed, paraffin-embedded lung cancer tumors were screened, of which 5 (9%) were positive for EML4-ALK fusions. Four positive cases harbored known fusion variants: variant 3a, 3b, or both in three cases and variant 1 in one case. The fifth positive specimen harbored two novel variants, designated 8a and 8b, involving exon 17 of EML4. Fluorescence in situ hybridization confirmed the presence of EML4-ALK fusions in three of the four RT-PCR-positive specimens with sufficient tissue for examination, and also confirmed absence of fusions in all 19 RT-PCR-negative specimens tested. Immunohistochemistry analysis confirmed ALK protein expression in the sample containing the novel 8a and 8b variants. This RT-PCR-based exon scanning approach avoids the limitations of screening only for previously identified EML4-ALK fusions and provides a simple molecular assay for fusion detection in a clinical diagnostics setting.

Somatic mutations of signaling genes in non-small-cell lung cancer

Lung cancer is the leading cause of cancer-related deaths, with non-small-cell lung cancer (NSCLC) accounting for approximately 85% of cases. A significant proportion of NSCLC cases are not diagnosed until a late stage, when aggressive treatments are required but often prolong survival only modestly. Recent advances in molecular characterization of NSCLC have enabled identification of numerous cell growth and proliferation pathways that are disrupted in these tumors. This knowledge has provided insight into the mechanisms of tumor development in various histologic subtypes of NSCLC and has pointed the way toward targeted treatment strategies. In this review, we highlight literature findings of somatic mutations in genes involved in cell growth and proliferation that are commonly found in the various subtypes of NSCLC, and we discuss how these findings may relate to treatment strategies.

Incidence and patterns of ALK FISH abnormalities seen in a large unselected series of lung carcinomas

BackgroundAnaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements have been reported in 2-13% of patients with non-small cell lung cancer (NSCLC). Patients with ALK rearrangements do not respond to EGFR-specific tyrosine kinase inhibitors (TKIs); however, they do benefit from small molecule inhibitors targeting ALK.ResultsIn this study, fluorescence in situ hybridization (FISH) using a break-apart probe for the ALK gene was performed on formalin fixed paraffin-embedded tissue to determine the incidence of ALK rearrangements and hybridization patterns in a large unselected cohort of 1387 patients with a referred diagnosis of non-small cell lung cancer (1011 of these patients had a histologic diagnosis of adenocarcinoma). The abnormal FISH signal patterns varied from a single split signal to complex patterns. Among 49 abnormal samples (49/1387, 3.5%), 32 had 1 to 3 split signals. Fifteen samples had deletions of the green 5′ end of the ALK signal, and 1 of these 15 samples showed amplification of the orange 3′ end of the ALK signal. Two patients showed a deletion of the 3′ALK signal. Thirty eight of these 49 samples (38/1011, 3.7%) were among the 1011 patients with confirmed adenocarcinoma. Five of 8 patients with ALK rearrangements detected by FISH were confirmed to have EML4-ALK fusions by multiplex RT-PCR. Among the 45 ALK-rearranged samples tested, only 1 EGFR mutation (T790M) was detected. Two KRAS mutations were detected among 24 ALK-rearranged samples tested.ConclusionsIn a large unselected series, the frequency of ALK gene rearrangement detected by FISH was approximately 3.5% of lung carcinoma, and 3.7% of patients with lung adenocarcinoma, with variant signal patterns frequently detected. Rare cases with coexisting KRAS and EGFR mutations were seen.

Circulating Ki-67 index in plasma as a biomarker and prognostic indicator in chronic lymphocytic leukemia.

Ki-67 is a nuclear antigen that is expressed in all stages of the cell cycle, except G(0), and is widely used as a marker of cellular proliferation in human tumors. We recently showed that elevated levels of Ki-67 circulating in plasma (cKi-67) are associated with shorter survival in patients with acute lymphoblastic leukemia. The current study included 194 patients with CLL and 96 healthy control subjects. cKi-67 levels in plasma were determined using an electrochemiluminescent immunoassay. We normalized the cKi-67 level to the absolute number of lymphocytes in the patients peripheral blood to establish the plasma cKi-67 index. The cKi-67 index showed significant correlation with lymph node involvement and Rai stage (P=0.05). Higher cKi-67 index values were significantly associated with shorter survival. Multivariate Cox proportional hazards regression analysis demonstrated that the association of the cKi-67 index with shorter survival was independent of IgV(H) mutation status. In a multivariate model incorporating the cKi-67 index with B2M and IgV(H), only cKi-67 index and B2M levels remained as independent predictors of survival. The results of this study suggest that the plasma cKi-67 index, along with B2M level, is a strong predictor of clinical behavior in CLL.

Abstract 4675: Detection of ALK, ROS1, and RET translocations in non-small cell lung cancer (NSCLC) patients by intragenic differential expression analysis

BACKGROUND: ALK, ROS1, and RET translocations are frequently detected in NSCLC patients. Crizotinib, a tyrosine kinase inhibitor (TKI), was approved by the FDA in 2011 to treat NSCLC in patients harboring ALK translocations as detected by an FDA-approved assay. However, the FDA-approved ALK FISH assay is technically challenging, with failures due to pre-analytic variables. Another approach, intragenic differential expression (IDE), detects translocations by comparing expression levels of the 5′ end with the 3′ end of target gene transcripts. In this study we developed and evaluated a rapid IDE assay to screen for ALK, ROS1, and RET translocations, independent of the fusion partner. METHODS: A total of 419 samples (408 randomly-selected NSCLC clinical samples, ALK positive and ROS1 positive cell lines (2 each), and 7 previously-tested RET-positive clinical samples) were used to develop and evaluate performance characteristics of the IDE assays. To determine IDE scores, levels of ALK, ROS1, and RET expression were first determined by quantitative RT-PCR measurement of the 5′- and 3′- ends of the respective transcripts. The differences in expression levels were calculated as ΔCt (Ct5′ - Ct3′). High ΔCt values indicate presumptive presence of gene translocations. 212/408 NSCLC samples were analyzed by ALK FISH and EML4-ALK RT-PCR, and 196/408 samples were analyzed by EML4-ALK RT-PCR. RESULTS: Thirty-one of the 408 (7.6%) clinical samples tested positive for ALK rearrangements by IDE. Among them, 20 were confirmed by FISH and/or EML4-ALK (true positive, 64.5%), while 11 were negative by FISH and/or EML4-ALK (false positive, 35.5%). One of 10 ALK FISH positive samples tested negative by both ALK IDE and EML4-ALK RT-PCR analysis (false negative), while one of 202 FISH-negative sample tested positive by both EML4-ALK and ALK IDE. ALK IDE exhibited 94.5% (189/200) concordance with ALK FISH and 96.0% (356/371) concordance with the EML4-ALK assay. For ROS1, both ROS1-positive cell lines and 4/408 (1.0%) NSCLC samples tested positive for ROS1 by IDE. Among the 4 IDE-positive NSCLC samples, 1 was confirmed by ROS1 FISH. For RET, all 7 known positives and 10/408 (2.5%) NSCLC samples tested positive by IDE. Three of six RET IDE positive NSCLC samples were confirmed by RET FISH. Overall, ALK, ROS1, and RET translocations were mutually exclusive in NSCLC patients. The lung IDE assay had a failure rate of 3.7%. CONCLUSION: These findings demonstrate the feasibility of using IDE to detect ALK, ROS1, and RET gene translocations. These assays may have potential as a screening tool to select patients for further confirmation by FISH for TKI-targeted therapy. The IDE concept can be applied to a wide range of somatic translocations. Citation Format: Shih-Min Cheng, Cindy Barlan, Feras Hantash, Heather R. Sanders, Patricia H. Chan, Vladimira Sulcova, Marc A. Sanidad, Kevin Qu, Joann C. Kelly, Fatih Z. Boyar, Anthony D. Sferruzza, Frederic M. Waldman. Detection of ALK, ROS1, and RET translocations in non-small cell lung cancer (NSCLC) patients by intragenic differential expression analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4675. doi:10.1158/1538-7445.AM2014-4675

Abstract 3728: Two novel EML4-ALK fusion variants involving EML4 exon 17 identified in lung cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: Recently, anaplastic lymphoma kinase (ALK) inhibitors have been used successfully in patients harboring gene fusions between echinoderm microtubule-associated protein-like 4 (EML4) and ALK. These fusions result from a paracentric inversion on chromosome 2 inversion [inv(2)(p21;p23)] and have been identified in 3-7% of all non-small cell lung cancer (NSCLC) cases. To date, 11 variants have been published involving 7 different EML4 exons and invariably involving exon 20 of ALK. To screen for EML4-ALK abnormalities, we developed a multiplex RT-PCR exon screening approach that can detect variants initiating at any of the first 22 EML4 exons. Using this approach we detected both known and novel EML4-ALK fusion variants. We identified 2 novel EML4-ALK fusion variants involving exon 17 of EML4 from a single patient. Methods: Sixty-one samples were analyzed with the multiplex assay: 56 formalin-fixed paraffin-embedded (FFPE) NSCLC samples, 4 cell line samples (3 NSCLCs; 1 Prostate carcinoma), and 1 control RNA (Human Total RNA, Applied Biosystems). After RNA extraction, RT-PCR was performed using the RNA UltraSense™ One-Step qRT-PCR System (Invitrogen). Twenty-three primers (22 unlabeled EML4 forward; 1 FAM- labeled ALK reverse) were included in 4 master mixes to amplify EML4-ALK fusions initiating within the first 22 EML4 exons to ALK exon 20; 1 endogenous control (beta-2-microglobulin) primer set was included in a separate reaction. After RT-PCR, the PCR products were separated by capillary electrophoresis on a genetic analyzer (ABI 3730, Applied Biosystems) and the fusions identified based on size (bp). Samples with positive results were further analyzed by singleplex RT-PCR to confirm exon involvement and novel fusions were confirmed by sequencing. Results: One NSCLC cell line was positive for EML4-ALK variant 3a and 3b and 2 NSCLC cell lines were negative, consistent with literature findings. The prostate cancer and control RNA were negative. Two unexpected peaks resulted from multiplex and singleplex RT-PCR reactions containing EML4 exon 17 forward and ALK exon 20 reverse primers. Sequencing revealed 2 previously undescribed variants. One variant (8a) consisted of a complete EML4 exon 17 fused to a partial intron 19-20 and complete exon 20 of ALK. The second variant (8b) consisted of a complete exon 17 with partial intron 17-18 of EML4 fused to the same ALK region as in variant 8a. Overall, 9% (5/56) of lung cancer tumor tissue (FFPE) were positive for EML4-ALK fusions (variants 3a and 3b; 2/56); variant 3a only (1/56), variant 1 (1/56), and novel variants 8a and 8b (1/56)). Conclusions: This study demonstrates that proper testing for EML4-ALK fusion in lung cancer should encompass at least most of the first 22 exons of the EML4 gene. Encompassing multiplex RT-PCR assays may increase the detection prevalence of EML4-ALK fusion with the detection of new fusion variants as demonstrated in our small series of 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 3728.

Abstract 3745: Detection of various ALK translocations using intragenic differential expression (IDE) in patients with non-small cell lung cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: Efforts to target EML4-ALK fusions with ALK inhibitors in non-small cell lung cancer (NSCLC) have shown promising results in patients harboring a paracentric inversion on chromosome 2, inv(2)(p21p23). In effect, the clinical utility of these drugs is dependent on the presence of ALK gene activation. Reliable testing for ALK activation by translocation is important for selecting patients for this therapy. Although 11 variants for translocation are known, new variants have recently been reported. The aim of this study was to develop a reliable molecular assay to detect translocation of the ALK gene irrespective of the breakpoint on the EML4 gene or the partner gene. To achieve this, we used intragenic differential expression (IDE) of ALK gene comparing expression levels of the 5′ end with the 3′ end. Methods: ALK IDE was determined by measuring the levels of both 5′ and 3′ transcript regions by quantitative RT-PCR. IDE scores were obtained by calculating the endogenous control (ABL1) normalized differences in 5′ and 3′ ALK levels (IDE = 5′ALK/ABL1 - 3′ALK/ABL1). High IDE score, relative to normal, indicates the presence of ALK rearrangement. Relative expression of ALK (independent of rearrangement) was also established. A subset of samples were also analyzed by fluorescence in situ hybridization (FISH). All study samples were analyzed for direct detection of EML4-ALK by RT-PCR performed in a parallel study. Results: The ALK IDE value of an EML4-ALK fusion-positive cell line (NCI-H2228) was 0.7 whereas it was 0.0 for 2 EML4-ALK-negative NSCLC cell lines (NCI H838 and NCI H1299). The positive control value (0.7) was set as the cutoff to distinguish positive vs. negative ALK rearrangement. Eleven percent (6/56) of the lung cancer tissue samples were IDE positive. Eighty-three percent (5/6) of these positives were confirmed by direct detection of EML4-ALK fusion transcript by RT-PCR, including one specimen harboring the previously undescribed variants 8a and 8b. Five IDE-positive samples and 5 with slightly-to-moderately elevated levels of ALK transcript (3′ ALK > 0.1) were further analyzed by FISH. Of these 10 samples, 80% (8/10) showed ALK rearrangement and/or gene amplification. All samples interpreted as having ALK rearrangements by FISH were also positive by IDE (3/3). Two IDE positive (1 confirmed, 1 unconfirmed by RT-PCR) were interpreted as rearrangement negative by FISH. Conclusions: ALK IDE accurately categorized all FISH-confirmed rearrangements as positive and detected rearrangements in at least 1 other confirmed case not identified by FISH. This method is useful for detection of EML4-ALK rearrangements and may function as a universal molecular assay for determining ALK rearrangements in multiple tumor types. 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 3745.