EPR17341 and A7H6R pan-TRK Immunohistochemistry Result in Highly Different Staining Patterns in a Series of Salivary Gland Tumors.

Patients with NTRK-rearranged tumors can be now treated using anti-TRK-targeted therapies making NTRK testing important for treatment choices in patients with advanced cancers. Pan-TRK immunohistochemistry (IHC) could be a valuable premolecular screening strategy in this field. The choice of 1 IHC method or another requires to investigate for intermethod comparison. A high frequency of pan-TRK positive tumors among salivary gland tumors makes these tumors particularly appropriate for such a technical study. In this work, we studied the intermethod agreement for 2 pan-TRK IHC methods (using A7H6R and EPR17341 clones) in a file of salivary gland tumors of different subtypes. Among 71 tumors, pan-TRK IHC was diagnosed as positive (ie, H score ≥5) in 23 and 18 cases using EPR17341 and A7H6R clones, respectively, with a good intermethod agreement in terms of positive/negative result (κ, 0.70) but only a moderate agreement considering the H score values themselves (intraclass correlation coefficient of 0.5399). Beyond the intensity of staining and the percentages of stained cells, major differences were also observed between the location and type of cells stained in positive cases between the 2 clones. The single NTRK-rearranged case in our series (ie, a NTRK3-rearranged salivary secretory carcinoma) was positive with the 2 pan-TRK antibodies. Future studies including molecularly proven NTRK-rearranged tumors remain required to further study and compare the performances of different pan-TRK clones in the screening of NTRK-rearranged cancers but it is now obvious that the staining patterns of A7H6R and EPR17341 clones are not strictly identical.

Testing for BRAF fusions in patients with advanced BRAF/NRAS/KIT wild-type melanomas permits to identify patients who could benefit of anti-MEK targeted therapy.

Beyond targeted therapy for patients with BRAF-mutated melanomas and immunotherapy in patients lacking BRAF mutations, anti-MEK therapy has been proposed in patients with advanced melanomas harbouring BRAF fusions. BRAF fusions diagnosis in patients with advanced melanomas is the subject of the present study. Using BRAF fluorescent in situ hybridisation (FISH), we searched for BRAF fusions in 74 samples of 66 patients with advanced BRAF/NRAS/KIT wild-type melanomas. We identified 2/66 (3%) patients with BRAF fusions in a brain metastasis of one patient and in a lymph node metastasis and in a cutaneous metastasis for the second patient with 90%-95% of tumour nuclei containing isolated 3'-BRAF FISH signals. As a result, we conclude that BRAF FISH in patients with advanced BRAF/NRAS/KIT wild-type melanomas is a valuable and easy-to-perform test to diagnose BRAF fusions and to identify patients who could benefit of anti-MEK targeted therapy.

PD-L1 Immunohistochemistry-Discrepant Results Between Synchronous Tumor Samples May Cause More Treatment Choice Dilemmas Than Molecular Heterogeneity in Patients With Advanced Non-Small Cell Lung Cancers.

Biomarker analyses have become mandatory for treatment choices in patients with advanced non-small cell lung cancers. PD-L1 expression for immunotherapy as well as oncogenic molecular alterations for targeted therapies must be analyzed in tumor samples. Intersample heterogeneity may cause dilemmas in treatment choices when faced with discrepant biomarker results. We led a retrospective study evaluating the potential impact on guideline-based therapeutic decisions of biomarker analyses performed in several synchronous tumor samples per patient. We collected retrospective data about patients with advanced non-small cell lung cancers, and synchronous tumor paired samples were analyzed for PD-L1 immunohistochemistry (IHC) and oncogene molecular statuses. Among 34 patients, none had a discrepant result between paired samples with respect to oncogene molecular statuses. At the opposite, intersample-discrepant PD-L1 IHC results may have caused treatment choice dilemmas for 6 (17.6%) patients discussing first-line therapy options (ie, immune checkpoint inhibitor therapy versus other systemic therapy with regard to the threshold of ≥50% PD-L1 IHC-positive tumor cells). In addition, for 6 (17.6%) other patients, different PD-L1 IHC results may have also influenced the choice of one therapy or another in second-line therapy (ie, with regard to the threshold of ≥1% PD-L1 IHC-positive tumor cells). In our case series, discrepant results with regard to PD-L1 IHC between paired tumor samples could generate more treatment choice dilemmas than the molecular heterogeneity of oncogenic drivers.

Avoiding non-contributive molecular results in cancer samples: proposal of a score-based approach for sample choice.

Mutational analyses have become crucial for therapeutic choices in patients with advanced lung cancer, colorectal cancer and melanoma. Short turnaround times for molecular analyses are necessary to match the patient's therapeutic management. Non-contributive molecular analyses may increase the delay in reaching a relevant mutational status. We attempted to identify criteria in samples associated with non-contributive molecular results to better anticipate them and select samples with contributive analyses. We compared several criteria such as cancer type, sample type, organ of origin and percentage of tumour cells between samples with non-contributive or contributive EGFR, KRAS, NRAS and BRAF mutation analyses. Among two sets of 3367 and 554 tumour samples analysed in 2015-2017 and 2018, respectively, 11.7% and 15.7% of sample analyses were non-contributive for at least one oncogene. Lung cancer and melanoma cancer subtypes [odds ratio (OR)=7.2], cytological (OR=1.8) or bone samples (OR=8.5) and a percentage of tumour cells ≤20% (OR=41.4) were significantly associated with non-contributive results. By combining these parameters in a scoring system, we were able to predict the contributive or non-contributive result of a molecular analysis with sensitivity and specificity higher than 80% in a validation set of samples. Predicting the contributive or non-contributive result of a molecular analysis is feasible in samples on the basis of simple features. A combination of these features could be used to better choose samples to analyse in order to reduce the rate of non-contributive molecular results and related treatment delays and costs in patients with advanced cancers.

Decalcification can cause the failure of BRAF molecular analyses and anti-BRAFV600E VE1 immunohistochemistry.

BRAF mutation detection is worthwhile for the management of patients with some advanced cancers. The tumor samples are sometimes difficult to analyze using DNA-based molecular methods because of poor tumor DNA quality or quantity. Anti-BRAFV600E VE1 immunohistochemistry (IHC) has been proposed as a valuable ancillary tool to analyze some "molecularly challenging" tumor samples. In this technical study, we focused on its application in the field of decalcified tumor samples. We selected four patients with known BRAFV600E-mutated cancer (3 metastatic melanomas and 1 hairy cell leukemia) and paired non-decalcified/decalcified tumor samples. Molecular analyses failed in the four decalcified samples (3 bone metastases and 1 osteo-medullar biopsy) with non-contributive mutation status. Whereas non-decalcified tumor samples were all positive using anti-BRAFV600E VE1 IHC, the four decalcified samples were concluded negative. Because decalcified tumor samples are difficult to analyze from a molecular point of view, it is tempting to use IHC instead of DNA-based methods searching for BRAFV600E mutations in these samples. Nevertheless, the decalcification process may also cause false-negative results using VE1 IHC. Decalcified samples require specific and optimized IHC and molecular protocols and quality controls.

Combining ALK Fluorescent In Situ Hybridization and Immunohistochemistry to Analyze Multiple Non-Small Cell Lung Carcinoma Samples per Patient Reveals Intermethod and Intersample-discrepant Results.

ALK inhibitors have improved the therapeutic management of patients with ALK-rearranged advanced non-small cell lung cancers (NSCLC). Several diagnostic methods, mainly fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC), can be used as single or combined tests to detect the so-called "ALK-positive" NSCLC. Intersample and intermethod discrepancies could cause issues with therapeutic consequences in ALK testing. In this article, we report a case series of our real-life experience and issues in combining FISH and IHC in multiple tumor samples per patient to diagnose and treat a subset of "ALK-positive" patients with NSCLC. Among analyses conducted in 40 samples of 18 patients with advanced lung adenocarcinomas, we retrospectively encountered 10 patients with intersample-concordant ALK FISH and IHC results. Discrepant results about FISH and/or IHC were noted between different samples in 8 patients. Therapeutic responses were observed in 5 of 10 crizotinib-treated patients including 1 patient with ALK FISH+ IHC- status and 1 patient with ALK FISH- IHC+ status. Our data highlight the difficulty to predict the response/nonresponse to crizotinib therapy, in patients with advanced NSCLC, not only on the basis of single and multiple tumor samples, but also on the basis of single and combined diagnostic methods.

Evaluation of a Dual ALK/ROS1 Fluorescent In Situ Hybridization Test in Non-Small-cell Lung Cancer.

BACKGROUND: Several therapeutics targets have emerged to treat patients with non-small-cell lung carcinoma (NSCLC), with numerous biomarkers available to test for treatment choices. Minimum tumor wastage is necessary to permit the analysis of every potentially relevant target. Searching for targetable ALK and ROS1 rearrangements is now mandatory in NSCLC. In the present study, we evaluated the performance of a dual ALK/ROS1 fluorescent in situ hybridization (FISH) probe that concurrently analyzed the 2 oncogenes on a same FISH slide. MATERIALS AND METHODS: We used the FlexISH ALK/ROS1 DistinguISH Probe (Zytovision, Bremerhaven, Germany) to analyze a set of 28 formalin-fixed paraffin-embedded NSCLC tumor samples enriched in tumors with ALK- and ROS1-rearranged status. RESULTS: The dual ALK/ROS1 FISH probe test results were fully concordant with the results of previous single ALK and ROS1 FISH tests (15 ALK and 3 ROS1 rearrangements) without any false-positive results. Dual- and single-probe FISH test results were also concordant regarding the unusual ALK FISH patterns. These included 1 sample that had negative FISH results with diffuse single 5'-ALK signals and positive ALK immunohistochemistry findings in a patient with a response to crizotinib, 2 paired samples with high percentages of ALK FISH-rearranged nuclei despite negative ALK immunohistochemistry findings, and ALK FISH-positive samples from 2 patients lacking a response to crizotinib therapy despite concordant ALK FISH and immunohistochemistry-positive results. CONCLUSION: The dual ALK/ROS1 FISH probe test is a valuable tool to search concurrently for both ALK and ROS1 rearrangements on a same FISH slide and could help to spare tumor tissue for other biomarkers tests.

Evaluation of a Rapid, Fully Automated Platform for Detection of BRAF and NRAS Mutations in Melanoma.

BRAF and NRAS genetic analyses are time-consuming and can delay treatment choices in patients with metastatic melanomas presenting with acute deterioration. We compared the rapid, real-time, fully automated molecular diagnosis platform Idylla™ with next-generation sequencing (NGS) and immunohistochemistry for detection of BRAF and NRAS mutations in 36 patients with metastatic melanomas. The Idylla™ NRAS-BRAF-EGFRS492R mutation assay (110 min per sample) detected BRAF and NRAS mutations in 15 and 17 samples, respectively. One NRAS mutation was different between NGS and Idylla™ (NRASG13C vs. NRASG12A/D). Four samples were BRAF and NRAS wild-type. The global concordance between NGS and Idylla™ assays was 97.2% (35/36 cases). Immunohistochemistry was positive only in 9/9 BRAFV600E- and 6/6 NRASQ61R-mutated samples with VE1 and SP174 antibodies, respectively. The Idylla™ platform is a valuable rapid molecular diagnosis tool to reduce the delay in BRAF and NRAS analyses-related treatment choices for patients with metastatic melanoma presenting with acute deterioration.

Mismatch repair-deficient colorectal cancer: a model of immunogenic and immune cell-rich tumor despite nonsignificant programmed cell death ligand-1 expression in tumor cells.

Mismatch repair-deficient (dMMR) colorectal cancers (CRCs) are good responders to anti-programmed cell death ligand-1 (PD-L1) immunotherapy, but the value of PD-L1 testing remains unclear. We studied PD-L1 expression and the tumor immune microenvironment in dMMR CRC as a model of good responders to immunotherapy. We examined 35 dMMR and 34 mismatch repair-proficient (pMMR) CRCs using immune cell markers (CD3, CD4, CD8, CD20, CD68, and FOXP3) as well as programmed cell death receptor-1 (PD-1) and PD-L1 immunohistochemistry staining in whole tumor specimens and tissue microarray slides to compare 4 PD-L1 immunohistochemistry clones (SP142, E1L3N, 22C3, and 28.8). We observed no significant difference in PD-L1 expression between dMMR and pMMR CRCs. Only 2 dMMR tumors had membranous PD-L1 staining. Expression of PD-L1 was greater in stromal immune cells of dMMR CRC, which also contained more numerous intraepithelial (CD3+, CD8+, FOXP3+, and PD-1+) and stromal (CD8+, PD-1+) lymphocytes than did pMMR tumors. Immune cell quantification discriminated better between dMMR and pMMR tumors than did PD-L1 expression. Tumor heterogeneity and variations in PD-L1 expression were noted with different antibodies, especially for PD-L1+ immune cells, which were more numerous at the invasion margin. Given the poor correlation with mismatch repair status and technical limitations, the value of PD-L1 testing to accompany the development of anti-PD-1/PD-L1 immunotherapy remains unclear. Further clinical trials are required to determine which parameters are valuable predictive biomarkers of the response to immunotherapy among mismatch repair status, PD-L1 expression, and immune cell quantification in CRC.

Translocation t(2;7)(p11;q21) associated with the CDK6/IGK rearrangement is a rare but recurrent abnormality in B-cell lymphoproliferative malignancies.

Structural abnormalities of chromosome 7q have been regularly reported in chronic B-cell lymphoproliferative disorders. They include chromosomal translocations involving 7q21, leading to overexpression of the CDK6 gene. Three different translocations, t(7;14)(q21;q32), t(7;22)(q21;q11), and t(2;7)(p11;q21), leading to the juxtaposition of the CDK6 gene with a immunoglobulin gene enhancer during B-cell differentiation, have been described. In the past 2 years, we identified three patients with lymphoproliferative malignancy associated with a t(2;7)(p11;q21). Fluorescent in situ hybridization using an IGK probe and a library of bacterial artificial chromosome (BAC) clones located in bands 7q21.2 and 7q21.3, containing CDK6, revealed that the telomeric part of the IGK probe was translocated on the der(7) within a 51-kb region upstream of the transcriptional start site of CDK6. A total of 23 patients with indolent B-cell lymphoproliferative disorders and juxtaposition of the IG and CDK6 genes, including 20 with IGK and CDK6 juxtaposition, have been reported thus far. This rearrangement leads to the overexpression of CDK6, which encodes a cyclin-dependent protein kinase involved in cell cycle G1 phase progression and G1/S transition.