Break-apart Fluorescence In Situ Hybridization Research Articles

Abstract 749: Novel ALK specific mRNA in situ hybridization assay for non-small cell lung carcinoma

Abstract A recent technical advance in the mRNA in situ hybridization (mRNA-ISH) assay provides simultaneous signal amplification and background suppression with a unique probe design to achieve single molecule visualization. We assessed the utility of the mRNA-ISH assay as a diagnostic tool to detect anaplastic lymphoma receptor tyrosine kinase (ALK) mRNA in non-small cell lung carcinoma. We compared the mRNA-ISH assay with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). The study included 279 surgically resected lung adenocarcinomas and 44 transbronchial biopsied (TBB) adenocarcinomas. mRNA-ISH was conducted using the RNAscope 2.0 system which includes pre-designed detection probes for the tyrosine kinase domain of ALK mRNA. IHC was conducted on all of the 323 samples using ALK-specific antibodies. mRNA-ISH was performed on 279 surgical samples and 6 TBB samples. Break-apart FISH was used to assess cases that were mRNA-ISH or IHC positive. ALK protein expression was detected in 11/279 (3.9%) specimens. ALK mRNA was also detected by mRNA-ISH in these cases, and 9 of the 11 specimens (81%), were also positive by ALK FISH. Using the IHC results as a reference, the sensitivity and specificity of mRNA-ISH was 100%. In the TBB cohort, ALK protein expression was observed in 3/44 (6.8%) of specimens, in which ALK mRNA expression was also detected. ALK mRNA-ISH data were highly correlated with the IHC data, and ALK mRNA-ISH was able to identify every FISH-positive sample. We conclude that mRNA-ISH could play an alternative or complementary role in ALK genetic diagnosis in NSCLC. Citation Format: Noriko Hirai, Takaaki Sasaki, Yoshinobu Ohsaki. Novel ALK specific mRNA in situ hybridization assay for non-small cell lung carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 749. doi:10.1158/1538-7445.AM2017-749

The utility of ETV1, ETV4 and ETV5 RNA in-situ hybridization in the diagnosis of CIC-DUX sarcomas.

A recently characterized group of undifferentiated small round cell sarcomas harbours fusions of the genes CIC and DUX4. Studies report a distinctive gene expression profile for these sarcomas, including expression of E26 transformation-specific (ETS) family proto-oncogenic transcription factors ETV1, ETV4 and ETV5. To test the utility of an ancillary diagnostic technique for these tumours, we evaluated chromogenic RNA in-situ hybridization assays for ETV1, ETV4 and ETV5 as diagnostic adjuncts for this emerging group of highly malignant sarcomas. We tested six confirmed CIC-DUX4 sarcomas and 105 lesions in the differential, including 48 Ewing sarcomas for expression of ETV1, ETV4 and ETV5, scoring expression utilizing a previously validated scale. ETV1 and ETV4 were positive in five of six cases, while ETV5 was positive in six of six. No Ewing sarcoma or other sarcoma tested showed coexpression of these transcripts, while one ETV1/ETV4/ETV5 triple positive previously unclassified round cell sarcoma was identified as harbouring a CIC rearrangement by break-apart fluorescence in-situ hybridization (FISH). We identified overexpression of ETV1, ETV4 and ETV5 transcripts in situ in CIC-DUX4 sarcomas using a robust assay in routine archival sections. One previously unclassified round cell sarcoma showed ETV1/4/5 positivity, and was proved to harbour a CIC rearrangement by break-apart FISH. The sensitivity and specificity observed with our in-situ hybridization assay implies potential utility as an ancillary diagnostic technique, particularly when faced with limited biopsy samples.

A sensitive and high throughput TaqMan-based reverse transcription quantitative polymerase chain reaction assay efficiently discriminates ALK rearrangement from overexpression for lung cancer FFPE specimens

ObjectivesALK fusion gene is an oncogenic driver in lung cancer with low prevalence, which can be ameliorated by crizotinib. Currently, ALK fusion gene can be diagnosed by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), but inconstistnt results between the two methods are encountered regularly. To make the ALK fusion gene screening more efficient and to provide a simple solution to clarify the discrepancy between FISH and IHC results, a sensitive TaqMan-based reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay was established. Materials and methodsThe 3-plex TaqMan-based RT-qPCR assay was established and performed on 102 archived formalin-fixed, paraffin-embedded (FFPE) NSCLC samples to detect ALK rearrangement and overexpression. Break-apart FISH and automatic immunohistochemistry based ALK assays were performed side by side using tissue microarray. ResultsThe RT-qPCR was performed successfully for 80 samples and 10 of them showed positive signals. Three out of the 10 qPCR positive cases were further confirmed by FISH and IHC test. Two others were IHC positive and FISH negative, and expressed full-length ALK transcript. The rest were neither FISH nor IHC positive and their ALK expression level was significantly lower than those FISH or IHC positive cases. ConclusionOur RT-qPCR assay demonstrates that the capability and reliability of ALK detection is comparable to FISH and IHC, but it is more effective at discriminating ALK rearrangement from overexpression. The RT-qPCR assay easily clarifies the discrepancy between FISH and IHC, and can be incorporated into routine ALK screening for lung cancer.

Clinical and pathological significance of ROS1 expression in intrahepatic cholangiocarcinoma.

BackgroundMore knowledge about genetic and molecular features of cholangiocarcinoma is needed to develop effective therapeutic strategies. We investigated the clinical and pathological significance of ROS1 expression in intrahepatic cholangiocarcinoma.MethodsOne hundred ninety-four patients with curatively resected intrahepatic cholangiocarcinoma were included in this study. Tumor tissue specimens were collected and analyzed for ROS1 gene rearrangement using fluorescence in situ hybridization (FISH) and ROS1 protein expression using immunohistochemistry (IHC).ResultsROS1 immunohistochemistry was positive (moderate or strong staining) in 72 tumors (37.1 %). ROS1 protein expression was significantly correlated with well differentiated tumors, papillary or mucinous histology, oncocytic/hepatoid or intestinal type tumors, and periductal infiltrating or intraductal growing tumors (vs. mass-forming cholangiocarcinoma). ROS-expressing tumors were associated with better disease-free survival (30.1 months for ROS1 expression (+) tumors vs. 9.0 months for ROS1 (−) tumors, p = 0.006). Moreover, ROS1 expression was an independent predictor of better disease-free survival in a multivariate analysis (HR 0.607, 95 % CI 0.377–0.976; p = 0.039). Although break-apart FISH was successfully performed in 102 samples, a split pattern indicative of ROS1 gene rearrangement was not found in the examined samples.ConclusionROS1 protein expression was associated with well-differentiated histology and better survival in our patients with resected intrahepatic cholangiocarcinoma. ROS1 gene rearrangement by break-apart FISH was not found in the examined samples.

Validation and utilization of a TFE3 break-apart FISH assay for Xp11.2 translocation renal cell carcinoma and alveolar soft part sarcoma.

BackgroundXp11.2 or TFE3 translocation renal cell carcinomas (RCC) and alveolar soft part sarcoma (ASPS) are characterized by chromosome translocations involving the Xp11.2 breakpoint resulting in transcription factor TFE3 gene fusions. The most common translocations documented in TFE3 RCCs are t(X;1) (p11.2;q21) and t(X;17) (p11.2;q25) which leads to fusion of TFE3 gene on Xp11.2 with PRCC or ASPL respectively. TFE3 immunohistochemistry (IHC) has been inconsistent over time due to background staining problems in part related to fixation issues. Karyotyping to detect TFE3 gene rearrangement requires typically unavailable fresh tissue. Reverse transcriptase-polymerase chain reaction (RT-PCR) is generally very challenging due to degradation of RNA in archival material. The study objective was to develop and validate a TFE3 break-apart fluorescence in situ hybridization (FISH) assay to confirm Xp11 translocation RCCs and ASPS.MethodsRepresentative sections of formalin-fixed paraffin-embedded tissue blocks were selected in 40 possible cases. Approximately 60 tumor cells were analyzed in the targeted region. The validation of TFE3 FISH was done with 11 negative and two positive cases. Cut off for a positive result was validated as >7.15 % positive nuclei with any pattern of break-apart signals. FISH evaluation was done blinded of the immunohistochemical or karyotype data.ResultsThree out of forty cases were positive for the TFE3 break-apart signals by FISH. The negative cases were reported as clear cell RCC with papillary features (10), clear cell RCC with sarcomatoid areas (2), Papillary RCC with clear cell areas (9), Chromophobe RCC (2), RCC, unclassified type (3) and renal medullary carcinoma (1). 3 of the negative cases were consultation cases for renal tumor with unknown histology. Seven negative cases were soft tissue tumor suspicious for ASPS.ConclusionOur study validates the utility of TFE3 break-apart FISH on formalin-fixed paraffin-embedded tissue sections for diagnosis and confirmation of Xp11.2 translocation RCCs and ASPS.

Abstract 4244: Comparison of different ALK tests in non-small cell lung cancer (NSCLC) patients treated with crizotinib and their clinical outcome

Abstract Background: The screening algorithm for detection of ALK-rearranged NSCLC is still under investigation. The Break-Apart ALK FISH is the standard diagnostic test for the treatment with crizotinib. However, immunohistochemistry (IHC) with different antibodies shows excellent results when compared to ALK FISH. The efficacy of crizotinib is studied in relation to ALK FISH. We compared IHC outcome and the number of ALK breaks estimated with FISH with clinical outcome. Methods: Patients treated with crizotinib who had biopsies with sufficient tumor tissue were selected. Tumour response was assessed by CT using RECIST. Fluorescence in situ hybridization (FISH) was performed with Vysis LSI ALK Break Apart FISH Probe KIT (Abbott Molecular) and immunohistochemistry with Ventana ALK IHC Kit using D5F3 antibody. Results: Twenty one patients with NSCLC were selected, median (range) age 54 yrs (21-75); 19 had adenocarcinoma, 2 had large-cell carcinoma. We confirmed the presence of EML4-ALK fusions in all samples either by FISH or IHC. Median percentage of ALK breaks was 47% (6-76). Tumour response occurred in 12 pts having a median of 51 (6-76) breaks and 9 non-responders having a median of 27 (15-64) breaks. Of the 12 responders, 8 pts had positive IHC (67%), 3 were negative and 1 was not done; the FISH negative, IHC positive patient was one of the responders. Of the 9 non-responders 2 had positive IHC (22%), 3 negative (33%), and 1 was equivocal and 3 not done. Median PFS for positive IHC is 7.9 mo (95% CI., 5.5 - 10.3) and for negative IHC 1.6 mo (1.3 - 1.8), n = 17, p<0.0001. OS survival data are 6.5 mo (95% CI., 0 - 17.8) and 18.3 mo (95% CI., 11.1 - 25.6) respectively, n = 17 p = 0.052. PFS above 30% number of breaks was 7.0 mo (95% CI., 1.8 - 12.2) versus PFS below is 1.8 mo (95% CI., 0.7 - 2.9), n = 21, p = 0.05. OS survival data are 4.1 mo (95% CI., 0 - 9.3) and 16.1 mo (95% CI., 9.0 - 23.2) respectively, n = 21 p = 0.003. Therefore PFS and OS is more prolonged in those tumors who have ALK breaks above 30%. IHC difference are only significant for PFS. Conclusions: Both ALK FISH and IHC identify NSCLC patients who benefit from crizotinib treatment. Patients with ALK breaks above 30% have a better PFS and OS. IHC only differentiates for PFS. Citation Format: Anthonie J. van der Wekken, Thijo JN Hiltermann, Wim Timens, Ed Schuuring, Harry JM Groen. Comparison of different ALK tests in non-small cell lung cancer (NSCLC) patients treated with crizotinib and their clinical outcome. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4244. doi:10.1158/1538-7445.AM2015-4244

Abstract 4250: 5′ deletion on ALK break-apart FISH and risk of false positive results

Abstract Purpose: Break-apart fluorescence in situ hybridization (FISH) is the FDA-approved assay for detecting anaplastic lymphoma kinase (ALK) rearrangements in non-small cell lung cancer (NSCLC), identifying patients (pts) who may benefit from ALK tyrosine kinase inhibitors (TKIs). A case is considered positive for rearrangement either based on split 3′ and 5′ signals or loss of the 5′ signal (5′ deletion). We hypothesized that 5′ deletion on FISH may not be completely specific for presence of rearrangement, making the assay vulnerable to false positive results. Methods: An institutional IRB-approved database of NSCLC pts was queried for those with ALK-rearrangements. Clinical characteristics and response to ALK TKIs were reviewed. Immunohistochemistry (IHC) and next-generation sequencing (NGS) were obtained to further characterize ALK status. Results: Of 1614 NSCLC pts with ALK testing, 97 (6.0%) patients had abnormal ALK FISH: 30 with split signals, 24 with 5′ deletion, 3 with 3′ deletion, 1 with atypical rearrangement, and 39 with details unavailable. Pts with 5′ deletion were older (p = 0.01), had a more extensive smoking history (p = 0.03), and were more likely to harbor mutations in EGFR or KRAS (2 cases vs 0). Pts with 5′ deletion treated with ALK TKIs had a trend toward less tumor shrinkage (p = 0.07). ALK IHC and FISH were discordant in 3 of 21 pts with 5′ deletion and 0 of 26 pts with split signals (p = 0.03). NGS was concordant with ALK IHC, but not FISH, in 2 of 7 samples with 5′ deletions. All three testing modalities were concordant in 3 samples with split signals. Conclusions: NSCLC pts with 5′ deletion on ALK FISH have clinical and molecular features less typical of ALK-rearranged tumors, suggesting an unappreciated risk of false positives with this assay. While the majority of patients with 5′ deletion on ALK FISH harbor a true rearrangement and respond to ALK TKI, confirming these cases with IHC or NGS could reduce the false positive rate. Table 1.Clinicopathologic Features of NSCLC patients with positive ALK FISHSplit Signals (N = 30)5″ Deletion (N = 24)p-valueAge at Diagnosis - yearsp = 0.01MedianRange22-8228-76Age Distribution - No. (%)<40517%14%40-491033%28%50-59827%1042%60-69413%833%>70310%313%Race - No. (%)p = 0.03Caucasian2480%2188%Asian27%28%Other413%14%Smoking History - No. (%)p = 0.03Never Smoker1963%1042%Light Smoker (<10py)620%729%Heavy Smoker517%625%Presence of Co-MutationsEGFR00%14%KRAS00%14%None30100%2292%ALK Immunohistochemistryp = 0.03Positive26100%1886%Negative00%314%Next-Generation SequencingPositive3100%571%Negative00%229% Citation Format: Xin Gao, Lynette M. Sholl, Mizuki Nishino, Jennifer Heng, Pasi A. Janne, Geoffrey R. Oxnard. 5′ deletion on ALK break-apart FISH and risk of false positive results. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4250. doi:10.1158/1538-7445.AM2015-4250

Droplet Digital PCR for Absolute Quantification of EML4-ALK Gene Rearrangement in Lung Adenocarcinoma

Crizotinib treatment significantly prolongs progression-free survival, increases response rates, and improves the quality of life in patients with ALK-positive non-small-cell lung cancer. Droplet Digital PCR (ddPCR), a recently developed technique with high sensitivity and specificity, was used in this study to evaluate the association between the abundance of ALK rearrangements and crizotinib effectiveness. FFPE tissues were obtained from 103 consecutive patients with lung adenocarcinoma. Fluorescent in situ hybridization (FISH) and ddPCR were performed. The results revealed that 14 (13.6%) of the 103 patients were positive by dual-color, break-apart FISH. Three variants (1, 2, and 3) of the EML4-ALK gene rearrangements were detected. Thirteen of 14 ALK-positive cases identified by FISH were confirmed by ddPCR (four with variant 1, two with variant 2, and seven with variant 3). The case missed by ddPCR was identified as KIF5B-ALK gene rearrangement by PCR-based direct sequencing. Sixteen patients were detected with low copy numbers of EML4-ALK gene rearrangement, which failed to meet the positive cutoff point of FISH. Two of them responded well to crizotinib after unsuccessful chemotherapy. Our study indicates that ddPCR can be used as a molecular analytical tool to accurately measure the EML4-ALK rearrangement copy numbers in FFPE samples of lung adenocarcinoma patients.

Newly designed break-apart and ASPL-TFE3 dual-fusion FISH assay are useful in diagnosing Xp11.2 translocation renal cell carcinoma and ASPL-TFE3 renal cell carcinoma: a STARD-compliant article.

The diagnosis of Xp11.2 translocation renal cell carcinoma (tRCC), which relies on morphology and immunohistochemistry (IHC), is often either missed in the diagnosis or misdiagnosed. To improve the accuracy of diagnosis of Xp11.2 tRCC and ASPL-TFE3 renal cell carcinoma (RCC), we investigated newly designed fluorescence in situ hybridization (FISH) probes (diagnostic accuracy study).Based on the genetic characteristics of Xp11.2 tRCC and the ASPL-TFE3 RCC, a new break-apart TFE3 FISH probe and an ASPL-TFE3 dual-fusion FISH probe were designed and applied to 65 patients with RCC who were <45 years old or showed suspicious microscopic features of Xp11.2 tRCC in our hospital. To test the accuracy of the probes, we further performed reverse transcriptase–polymerase chain reaction (PCR) on 8 cases for which frozen tissues were available.Among the 65 cases diagnosed with RCC, TFE3 IHC was positive in 24 cases. Twenty-two cases were confirmed as Xp11.2 tRCC by break-apart TFE3 FISH, and 6 of these cases were further diagnosed as ASPL-TFE3 RCC by ASPL-TFE3 dual-fusion FISH detection. Importantly, reverse transcriptase–PCR showed concordant results with the results of FISH assay in the 8 available frozen cases.The break-apart and ASPL-TFE3 dual-fusion FISH assay can accurately detect the translocation of the TFE3 gene and ASPL-TFE3 fusion gene and can thus serve as a valid complementary method for diagnosing Xp11.2 tRCC and ASPL-TFE3 RCC.

Chromosomal rearrangements and copy number abnormalities of TP63 correlate with p63 protein expression in lung adenocarcinoma

The TP63 gene encodes a member of the p53 family of transcription factors. Although TP53 is a well-known tumor suppressor gene, the role of p63 in tumorigenesis is controversial. Our group recently identified novel chromosomal rearrangements involving TP63 in approximately 6% of peripheral T-cell lymphomas, which correlated with a p63+/p40− immunohistochemical profile. As a subset of lung adenocarcinomas are p63+/p40−, we undertook the current study to examine the presence of TP63 rearrangements and correlate with p63/p40 expression. Next-generation sequencing was used to identify genomic rearrangements of TP63 in 37 adenocarcinomas. Confirmatory fluorescence in-situ hybridization (FISH) using a break-apart probe to the TP63 gene region and immunohistochemistry for p63 and p40 were performed on adenocarcinomas with TP63 rearrangements identified by mate-pair sequencing. Immunohistochemistry for p63 and p40 was performed on 45 additional adenocarcinomas, and FISH was performed on all adenocarcinomas with p63 positivity. TP63 rearrangement was identified in two adenocarcinoma specimens from a single patient. The rearrangement resulted in a complex rearrangement of 3q that fused B3GALNT1 at the 3′ intron to TP63. FISH confirmed the rearrangement in both tumors. Immunohistochemistry staining for p63 was diffuse (>80% cells+) and p40 was negative. Of the 44 additional adenocarcinomas, 13 (30%) showed p63 expression; p40 was negative in all cases. No case showed rearrangement of TP63 by a break-apart FISH. However, extra copies of the intact TP63 locus were seen in the p63-positive areas of all 12 cases, with copy numbers ranging from three to seven. We have identified a novel chromosomal rearrangement involving TP63 in a p63+/p40− lung adenocarcinoma. Break-apart FISH testing can be used to diagnose this finding. Immunohistochemistry for p63 was not specific for this rearrangement, as nearly 33% of adenocarcinomas expressed p63. Additional copies of the intact TP63 locus were also a common finding and correlated with immunohistochemistry positivity for p63.

Prevalence of chromosomal rearrangements involving non-ETS genes in prostate cancer.

Prostate cancer is characterized by structural rearrangements, most frequently including translocations between androgen-dependent genes and members of the ETS family of transcription factor like TMPRSS2:ERG. In a recent whole genome sequencing study we identified 140 gene fusions that were unrelated to ETS genes in 11 prostate cancers. The aim of the present study was to estimate the prevalence of non-ETS gene fusions. We randomly selected 27 of these rearrangements and analyzed them by fluorescence in situ hybridization (FISH) in a tissue microarray format containing 500 prostate cancers. Using break-apart FISH probes for one fusion partner each, we found rearrangements of 13 (48%) of the 27 analyzed genes in 300-400 analyzable cancers per gene. Recurrent breakage, often accompanied by partial deletion of the genes, was found for NCKAP5, SH3BGR and TTC3 in 3 (0.8%) tumors each, as well as for ARNTL2 and ENOX1 in 2 (0.5%) cancers each. One rearranged tumor sample was observed for each of VCL, ZNF578, IMMP2L, SLC16A12, PANK1, GPHN, LRP1 and ZHX2. Balanced rearrangements, indicating possible gene fusion, were found for ZNF578, SH3BGR, LPR12 and ZHX2 in individual cancers only. The results of the present study confirm that rearrangements involving non-ETS genes occur in prostate cancer, but demonstrate that they are highly individual and typically non-recurrent.

Automation of ALK gene rearrangement testing with fluorescence in situ hybridization (FISH): A feasibility study

In the past several years we have observed a significant increase in our understanding of molecular mechanisms that drive lung cancer. Specifically in the non-small cell lung cancer sub-types, ALK gene rearrangements represent a sub-group of tumors that are targetable by the tyrosine kinase inhibitor Crizotinib, resulting in significant reductions in tumor burden. Phase II and III clinical trials were performed using an ALK break-apart FISH probe kit, making FISH the gold standard for identifying ALK rearrangements in patients. FISH is often considered a labor and cost intensive molecular technique, and in this study we aimed to demonstrate feasibility for automation of ALK FISH testing, to improve laboratory workflow and ease of testing. This involved automation of the pre-treatment steps of the ALK assay using various protocols on the VP 2000 instrument, and facilitating automated scanning of the fluorescent FISH specimens for simplified enumeration on various backend scanning and analysis systems. The results indicated that ALK FISH can be automated. Significantly, both the Ikoniscope and BioView system of automated FISH scanning and analysis systems provided a robust analysis algorithm to define ALK rearrangements. In addition, the BioView system facilitated consultation of difficult cases via the internet.

Intact or broken-apart RNA: an alternative concept for ALK fusion screening in non-small cell lung cancer (NSCLC).

Anaplastic lymphoma kinase (ALK) break-apart fluorescent in situ hybridization (FISH) is currently used in diagnostics for the selection of non-small cell lung cancer (NSCLC) patients to receive crizotinib. We evaluated ALK status in NSCLC with a novel ALK mRNA test based on the break-apart FISH concept, which we called break-apart transcript (BAT) test. ALK5' and ALK3' transcript patterns were established with qPCR for ALK-expressing controls including fusion-negative neuroblastomas, as well as fusion-positive anaplastic large cell lymphomas and NSCLC. The BAT test was evaluated on 271 RNA samples from routinely processed paraffin NSCLC tissues. Test results were compared with ALK FISH (n=121), immunohistochemical (IHC) analysis (n=86), and automated quantitative analysis (AQUA, n=83). On the basis of the nonoverlapping ALK BAT patterns in ALK-expressing controls (P<0.0001), 8/174 adenocarcinomas (4.6%) among 259 informative NSCLC were predicted as fusion positive. Overall concordance for paired method results was high (94.1% to 98.8%) but mainly concerned negative prediction because of the limited availability of positive-matched cases. Tumors with 100% cytoplasmic IHC staining of any intensity (n=3) were positive for AQUA, FISH, and BAT test; tumors with lower IHC positivity and different staining patterns were AQUA-negative. Upon multiple reevaluations, ALK gene status was considered as originally misinterpreted by FISH in 3/121 cases (2.5%). Tumors with >4 ALK gene copies were associated with longer overall survival upon first-line chemotherapy. In conclusion, application of the ALK BAT test on routinely processed NSCLC tissues yields the same fusion partner independent information as ALK break-apart FISH but is more robust and cost-effective. The BAT concept may be considered for the development of further drug-predictive translocation tests.

Detection of NFKB2 3’end Loos By Quantitative PCR (QPCR) or Detection of NFKB2 Rearrangements Correlate with Bortezomib Response in Multiple Myeloma

Background: Multiple myeloma is a malignant plasma cell cancer that is increasing in frequency in today’s aging population. While proteasome inhibitors such as bortezomib have improved the outcome of treatment, many patients fail to achieve a complete remission and ultimately the disease progresses. With these new treatments come higher associated toxicities and significant healthcare costs for the drugs. Several new cancer therapies with companion diagnostics have been approved in parallel with the drug to guide physicians in selecting those patients who would specifically benefit from the drug. No such test exists for the use of bortezomib in multiple myeloma. We have previously demonstrated that the 3’ end of the nuclear factor kappa-B 2 (NFKB2/p100) gene, a member of the NFKB/Rel gene family, functions as a mediator of bortezomib apoptotic effect through the induction of caspase 8 activation.Methodology: We have investigated the loss of NF-kB2 3’end loss by quantitative PCR (QPCR) in 56 MM patients and by break-apart Fluorescent In-Situ Hybridization in the whole bone marrow of 31 patients as well as in sorted cells of 24 MM patients treated with bortezomib containing regimens. Quality of response was assessed after 4 cycles of treatment initiation. QPCR was measured in peripheral blood lymphocyte and CD138 positive cells from the bone marrow to generate a QPCR ratio.Results: 37 of the 56 newly diagnosed patient with MM achieved >partial response (PR), while 16 remained stable or progress to treatment. The mean QPCR ratio in responders was 1.69 ± 0.13, while in non-responder was 0.43 ± 0.1 (p<0.001). Based on this finding, we assessed the influence of the loss of NF-kB 3’end on the clinical response. Univariable analysis demonstrated that low CD38(+) NF-kB2 3’end mRNA levels (odds ratio [OR] of 10.8; 95% CI 1.99 to 58.16, p=0.0057) and history of MGUS (OR: 7.6; 95% CI of 1.2 to 47.6, p<0.05) were significantly associated with poor outcome. However, multivariate analysis indicated that low CD38(+) NF-kB2 3’end levels was an independent risk factor for poor response to bortezomib and dexamethasone (VD, HR:21.47, 95% CI: 2.99 to 154.399, p <0.01, supplemental Table 2). To further evaluate for the presence of a structural rearrangement, we designed a NF-kB2 break-apart FISH assay. We first performed FISH on whole bone marrow for 31 patients treated with VD. As expected the percentage of broken signals in unsorted samples was low, but when we considered the proportion of plasma cells in each sample, the total percentage of broken signals was statistically significantly higher in the nonresponder (nonresonder: 9.12± 4.2 vs Responder:2.37 ± 0.7, p<0.035). Using a cut off detected by AUC curve, we identified NF-kB2 structural rearrangement in 9 patients (30%) patients of which 6/10 were present in VD-non responders and 3/21 of VD-responders. To validate these findings we performed FISH on CD38+ sorted cell from 24 new patients treated with VD or carfilzomib, an irreversible inhibitor of the proteasome, and found consistency with our previous results, in which 7 patients demonstrated a NFKB2 positive break signal. Six out of 7 samples of non-responders patients demonstrated a positive break signal while 1/16 were positive in the responders, indicating a chromosome translocation or inversion had disrupted the NFKB2 locus and that this disruption predispose tumors to respond poorly to proteasome inhibitors. We tested multiple cell lines carrying NF-kB2 rearrangement and identified by target sequencing that HUT88 presents a balance translocation between chromosomes 10 and 3. Taking advantage of this finding we compared the sensitivity of detection between 3’end NFKB2 QPCR and the break-apart FISH. To this end, we mixed cell line with normal NF-kB2 as it is in MM1S with titrating doses of HUT88. Quantitative PCR was able to detect a minimum of 3% HUT88 cells in the mix, while NFKB2 break-apart FISH was able to detect when the percent of HUT88 reach 10%. The correlation between both test was r2:0.98, P<0.02.Conclusion: loss of NF-kB2 3’ end is a potential companion diagnostic test for bortezomib. QPCR is more sensitive to detect NFKB2 3’end abnormalities. DisclosuresKelkar:Empire Genomics: Employment. Kaplan:Empire Genomics: Employment. Lonial:Millennium: The Takeda Oncology Company: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding.

Canadian Anaplastic Lymphoma Kinase Study: A Model for Multicenter Standardization and Optimization of ALK Testing in Lung Cancer

Fluorescence in situ hybridization (FISH) is currently the standard for diagnosing anaplastic lymphoma kinase (ALK)-rearranged (ALK+) lung cancers for ALK inhibitor therapies. ALK immunohistochemistry (IHC) may serve as a screening and alternative diagnostic method. The Canadian ALK (CALK) study was initiated to implement a multicenter optimization and standardization of laboratory developed ALK IHC and FISH tests across 14 hospitals. Twenty-eight lung adenocarcinomas with known ALK status were used as blinded study samples. Thirteen laboratories performed IHC using locally developed staining protocols for 5A4, ALK1, or D5F3 antibodies; results were assessed by H-score. Twelve centers conducted FISH using protocols based on Vysis' ALK break-apart FISH kit. Initial IHC results were used to optimize local IHC protocols, followed by a repeat IHC study to assess the results of standardization. Three laboratories conducted a prospective parallel IHC and FISH analysis on 411 consecutive clinical samples using post-validation optimized assays. Among study samples, FISH demonstrated 22 consensus ALK+ and six ALK wild type tumors. Preoptimization IHC scores from 12 centers with 5A4 and the percent abnormal cells by FISH from 12 centers showed intraclass correlation coefficients of 0.83 and 0.68, respectively. IHC optimization improved the intraclass correlation coefficients to 0.94. Factors affecting FISH scoring and outliers were identified. Post-optimization concurrent IHC/FISH testing in 373 informative cases revealed 100% sensitivity and specificity for IHC versus FISH. Multicenter standardization study may accelerate the implementation of ALK testing protocols across a country/region. Our data support the use of an appropriately validated IHC assay to screen for ALK+ lung cancers.

Ros1 Rearrangements and Copy Number Alterations in Nsclc Patients: High Frequency of Ros1 Deletions

ABSTRACT Aim: Recently, c-ros oncogene 1, receptor tyrosine kinase (ROS1, 6q22) has been identified as a driver oncogene in non-small-cell lung carcinomas (NSCLC). Patients carrying ROS1 translocations are candidates to targeted therapy with crizotinib®. We aimed to determine the prevalence, patterns and fusion partners in ROS1-rearranged NSCLC samples and to characterize ROS1 copy number alterations (CNAs) by fluorescence in situ hybridization (FISH). Methods: A total of 216 NSCLC patients (median age 64 years, 66% males, 51% smokers, 73% adenocarcinomas (ADC), 35% stage 4), were screened for ROS1 abnormalities using a break-apart FISH probe (Abbott Molecular). To characterize ROS1 partners, clones from Human 32K BAC Re-Array Library were used to develop CD74, SLC34A2 and EZR probes. Immunohistochemistry (IHC) with ROS1 D4D6 antibody (Cell Signalling Technology) was also performed in ROS1-translocated cases. Correlation with clinico-pathologic information was investigated. Results: Four samples were ROS1 translocated (1.9%): three showed deletion of the non-translocated allele and one had a 5'ROS1 deletion. One harbored a SLC34A2-ROS1 fusion and another a CD74-ROS1. No fusion partner was identified in the other two samples. All cases were positive for ROS1 IHC staining, three presenting a cytoplasmic pattern and one with membranous pattern. ROS1-translocated tumors were ADC, all EGFR, KRAS, and ALK wt. We observed an association with non-smoking habit (P=0.036). Regarding CNAs in ROS1 non-rearranged cases, 43.5% had gains (3-6 copies) and 26.4% had deletions. Conclusions: We confirm the low prevalence of ROS1-translocated lung ADC in a Spanish population. Moreover, our study reveals a significant frequency of ROS1 deletion as a potentially relevant aberration, also in ROS1-translocated patients. Disclosure: All authors have declared no conflicts of interest.

Extraskeletal Myxoid Chondrosarcoma of the Vulva With PLAG1 Gene Activation

Extraskeletal myxoid chondrosarcoma (EMC) is a rare mesenchymal neoplasm, rarely reported in the genitourinary tract with only 5 cases reported in the vulva. We investigated 2 cases of vulvar sarcomas whose morphologic appearance and immunohistochemical profiles were consistent with EMC using fluorescence in situ hybridization (FISH), reverse-transcription polymerase chain reaction, and a whole genome expression array. FISH and reverse-transcription polymerase chain reaction assays showed no EWSR1 and NR4A3 loci rearrangements. Microarray-based analysis also revealed no changes in NR4A3 and EWSR1 gene transcription levels. Microarray data showed a significant downregulation of the muscle-related genes (eg, myosin heavy chain family, actins, myoglobin, desmin, creatine kinase, troponins) and cytokeratins (KRT6A, 6B, 13, 14, and 78), upregulation of several neuron-specific genes [neural cell adhesion molecule 1 (NCAM-1/CD56), neurofilament (NEFH)], along with some well-characterized tumor biomarkers [carbonic anhydrase IX (CA-9), topoisomerase IIα (TOP2A), matrix metalloproteinases (MMP-7, MMP-9), CDKN2 gene (p16-INK4a), checkpoint homolog 2 (CHEK2)]. Notably, both tumors showed upregulation of the pleomorphic adenoma gene 1 (PLAG1), and in 1 case PLAG1 gene rearrangement was detected by break-apart FISH. Some vulvar tumors with morphologic and immunohistochemical characteristics of EMC may represent a molecular genetic entity separate from EMCs arising in other locations. PLAG1 gene activation appears to be involved in the development of these neoplasms.

Extraskeletal myxoid chondrosarcoma with a t(9;16)(q22;p11.2) resulting in a NR4A3-FUS fusion

Extraskeletal myxoid chondrosarcoma (EMC) is a rare neoplasm characterized by rearrangement of NR4A3. A t(9;22)(q22;q12), creating a fusion protein of EWSR1 and NR4A3, has been reported as a unique, recurring translocation in most cases. Reported variant translocations have resulted in fusion of NR4A3 with three other genes: TAF15, TCF12, and TFG. We report a case of EMC in a 59-year-old man who presented with a 6-month history of an enlarging mass in the proximal right thigh. The karyotype of fresh tissue from tumor taken at incisional biopsy revealed a t(9;16)(q22;p11.2). There was no evidence of an EWSR1 rearrangement by dual-color break-apart fluorescence in situ hybridization (FISH). Dual-color FISH probes revealed fusion of NR4A3 and FUS, a member of the TET family of genes, which includes EWSR1 and TAF15. Break-apart FISH probe results confirmed rearrangement of FUS. These findings show that a fusion product of FUS and NR4A3 may be an additional pathway to development of EMC.

ALK Status Testing in Non–Small-Cell Lung Carcinoma by FISH on ThinPrep Slides with Cytology Material

Oncogenic anaplastic lymphoma kinase (ALK) gene rearrangements in non-small-cell lung carcinomas (NSCLC) provide the basis for targeted therapy with crizotinib and other specific ALK inhibitors. Treatment eligibility is conventionally determined by the Food and Drug Administration-approved companion diagnostic fluorescence in situ hybridization (FISH) assay on paraffin-embedded tissue (PET). On limited samples such as fine needle aspiration-derived cytoblocks, FISH for ALK is often uninformative. FISH performed on liquid-based ThinPrep slides (ThinPrep-FISH) may represent a robust alternative. Two hundred thirty cytology samples from 217 patients with advanced NSCLC, including a consecutive series of 179 specimens, were used to generate matched ThinPrep slides and paraffin cytoblocks. The same ThinPrep slides used for cytologic diagnosis were assessed by standard ALK break-apart two-color probe FISH, after etching of tumor areas. Ultrasensitive ALK immunohistochemistry (IHC) on corresponding cytoblocks [D5F3 antibody, OptiView signal amplification] served as the reference data set. ThinPrep-FISH ALK signals were robust in 228 of 230 cases and not compromised by nuclear truncation inherent in paraffin-embedded tissue-FISH; only two samples displayed no signals. Nine of 178 informative cases (5%) in the consecutive series and 18 of 228 informative cases (7.8%) overall were ALK rearranged by ThinPrep-FISH. In 154 informative matched ThinPrep-FISH and cytoblock-IHC samples, 152 were concordant (10, 6.5% ALK status positive; 142, 92.2% ALK status negative), and two (1.3%) were ThinPrep-FISH positive but IHC negative (sensitivity 100%, specificity 98.6%, overall agreement 98.7%). Detection of ALK gene rearrangements in liquid cytology ThinPrep slides derived from patients with NSCLC can be confidently used for clinical ALK molecular testing.

Micro-cost Analysis of ALK Rearrangement Testing by FISH to Determine Eligibility for Crizotinib Therapy in NSCLC: Implications for Cost Effectiveness of Testing and Treatment.

Break-apart fluorescence in situ hybridization (FISH) is the gold standard test for anaplastic lymphoma kinase (ALK) gene rearrangement. However, this methodology often is assumed to be expensive and potentially cost-prohibitive given the low prevalence of ALK-positive non-small cell lung cancer (NSCLC) cases. To more accurately estimate the cost of ALK testing by FISH, we developed a micro-cost model that accounts for all cost elements of the assay, including laboratory reagents, supplies, capital equipment, technical and pathologist labor, and the acquisition cost of the commercial test and associated reagent kits and controls. By applying a set of real-world base-case parameter values, we determined that the cost of a single ALK break-apart FISH test result is $278.01. Sensitivity analysis on the parameters of batch size, testing efficiency, and the cost of the commercial diagnostic testing products revealed that the cost per result is highly sensitive to batch size, but much less so to efficiency or product cost. This implies that ALK testing by FISH will be most cost effective when performed in high-volume centers. Our results indicate that testing cost may not be the primary determinant of crizotinib (Xalkori®) treatment cost effectiveness, and suggest that testing cost is an insufficient reason to limit the use of FISH testing for ALK rearrangement.