The Ring Study: an international comparison of PD-L1 diagnostic assays and their interpretation in non-small cell lung cancer, head and neck squamous cell cancer and urothelial cancer.

PD-L1 immunohistochemistry has been approved as a diagnostic assay for immunotherapy. However, an international comparison across multiple cancers is lacking. This study aimed to assess the performance of PD-L1 diagnostic assays in non-small cell lung cancer (NSCLC), head and neck squamous cell cancer (HNSCC) and urothelial cancer (UC). The excisional specimens of NSCLC, HNSCC and UC were assayed by Ventana SP263 and scored at three sites in each country, including Australia, Brazil, Korea, Mexico, Russia and Taiwan. All slides were rotated to two other sites for interobserver scoring. The same cohort of NSCLC was assessed with Dako 22C3 pharmDx PD-L1 for comparison. The PD-L1 immunopositivity was scored according to the approved PD-L1 scoring algorithms which were the percentage of PD-L1-expressing tumour cell (TC) and tumour proportion score (TPS) by Ventana SP263 and Dako 22C3 staining, respectively. In NSCLC, the comparison demonstrated the comparability of the SP263 and 22C3 assays (cut-off of 1%, κ=0.71; 25%, κ=0.75; 50%, κ=0.81). The interobserver comparisons showed moderate to almost perfect agreement for SP263 in TC staining at 25% cut-off (NSCLC, κ=0.72 to 0.86; HNSCC, κ=0.60 to 0.82; UC, κ=0.68 to 0.91) and at 50% cut-off for NSCLC (κ=0.64 to 0.90). Regarding the immune cell (IC) scoring in UC, there was a lower correlation (concordance correlation coefficient=0.10 to 0.68) and poor to substantial agreements at the 1%, 5%, 10% and 25% cut-offs (κ= -0.04 to 0.76). The interchangeability of SP263 and 22C3 in NSCLC might be acceptable, especially at the 50% cut-off. In HNSCC, the performance of SP263 is comparable across five countries. In UC, there was low concordance of IC staining, which may affect treatment decisions. Overall, the study showed the reliability and reproducibility of SP263 in NSCLC, HNSCC and UC.

PCR-based analysis of PD-L1 RNA expression in lung cancer: comparison with commonly used immunohistochemical assays.

BACKGROUND: PD-L1 testing is currently performed by immunohistochemistry (IHC). We questioned whether the results of PCR-based measurement of PD-L1 RNA expression correlate with IHC scores obtained by different commercial assays. MATERIALS AND METHODS: 167 consecutive non-squamous non-small cell lung carcinomas (NSCLCs) were analyzed for PD-L1 RNA expression and 22C3, SP263, and SP142 IHC scoring using recommended cut-offs. RNA expression was divided into low, moderate, and high categories. RESULTS: RNA and protein expression demonstrated moderate correlation as continuous variables. Using prespecified RNA cut-offs, PCR testing showed a high negative predictive value towards the IHC analysis: the share of PD-L1 protein-negative tumors among cases classified as PD-L1-low by the PCR test reached 92-99% for all three antibodies. Meanwhile, about half of cases with moderate to high PD-L1 RNA expression had IHC staining in less than 1% tumor cells as determined by 22C3 or SP263 antibodies. Among the 51 discordant cases, which had <1% tumor staining by both 22C3 and SP263 clones but high RNA level, 29 (57%) showed ≥1% positive immune cells by SP263 and/or 22C3, 14 cases (27%) had detectable IHC expression in 0.1-0.9% tumor or immune cells by SP263 and/or 22C3, and 8 (16%) were entirely negative by IHC. CONCLUSION: Some NSCLCs demonstrate readily detectable PD-L1 expression on the level of RNA, but fall below commonly accepted cut-offs by IHC. It remains to be studied whether these discrepancies are attributed to technical or biological reasons. Clinical sensitivity of these tumors to immune therapy deserves additional investigations.

Agreement between PDL1 immunohistochemistry assays and polymerase chain reaction in non-small cell lung cancer: CLOVER comparison study.

The goal of the CLOVER study was to perform a pairwise comparison of four tests based on the same patient population with non-small cell lung cancer (NSCLC): three validated PDL1 immunohistochemistry (IHC) assays (Ventana SP142, Ventana SP263, Dako 22C3) and one PCR test. Four hundred seventy-three NSCLC samples were obtained from a biobank and were stained using PDL1 IHC assays. Four trained pathologists independently evaluated the percentage of tumor cells (TC) and immune cells (IC) that stained positive at any intensity. PDL1 transcripts were quantified in 437 patients by a standard Taqman RT-PCR assay using SDHA as a reference gene. A concordance analysis was performed to assess (1) the correlation of TC and IC between different assays and (2) the predictive properties of one test for another. "High" RNA expression was detected in 187 of 437 (43%) patients. The percentage of PDL1-positive cells (≥1%) was higher among the IC than the TC in all IHC three assays. The Pearson correlation coefficients (PCC) for TC were 0.71, 0.87, and 0.75 between 22C3/SP142, 22C3/SP263, and SP263/SP142, respectively. The PCC for IC were 0.45, 0.61, and 0.68 for the same pairs. A low correlation was observed between the PCR test and each of the three IHC assays; however, if a patient tested low/negative by PCR, then they were likely to test negative by any single IHC test with a high probability (92-99%). Among patients who tested positive by PCR, only 9-45% tested positive by IHC assays. There was excellent positive and negative agreement (>91%) between 22C3 and SP263 staining using the recommended individual cutoffs for first-line treatment. PCR RNA expression analysis is not equivalent to IHC. However, this method may have some potential for the identification of PDL1-negative tumors. 22C3 could be considered as a substitute for SP263 in first-line treatment.

Gene rearrangements in consecutive series of pediatric inflammatory myofibroblastic tumors.

BACKGROUND: Inflammatory myofibroblastic tumors (IMTs) are exceptionally rare neoplasms, which are often driven by rearranged tyrosine kinases. METHODS: This study considered 33 consecutive patients with IMT (median age, 6.6; age range, 0.6-15.8 years). RNA and cDNA were successfully obtained in 29 cases. The molecular analysis included sequential tests for 5'/3'-end unbalanced gene expression, variant-specific PCR, and next-generation sequencing (NGS). RESULTS: 5'/3'-end unbalanced ALK expression was revealed in 15/29 (52%) IMTs. Strikingly, all these tumors demonstrated high amount of ALK protein detected by immunohistochemistry. Variant-specific PCR was capable of identifying the type of ALK rearrangement in 11/15 IMTs with 5'/3'-end unbalanced ALK expression. The remaining four tumors were analyzed by NGS; two known and two novel (CLTC-ins6del84-ALK and EEF1G-ALK) ALK rearrangements were detected. Five IMTs demonstrated 5'/3'-end unbalanced ROS1 expression, and all these tumors carried TFG-ROS1 fusion. Nine tumors, which were negative for 5'/3'-end unbalanced ALK/ROS1 expression, were subjected to further analysis. Variant-specific PCR revealed two additional tumors with gene rearrangements (TFG-ROS1 and ETV6-NTRK3). The remaining seven IMTs were tested by NGS; single instances of TFG-ROS1 and novel SRF-PDGFRb translocations were detected. CONCLUSIONS: Twenty-four of 29 IMTs (83%) were shown to have druggable rearrangements involving tyrosine kinases, 20 of these 24 gene fusions were detectable by simple and inexpensive PCR assay, which is based on the detection 5'/3'-end unbalanced gene expression.

Overall Survival of Patients With ALK-Positive Metastatic Non-Small-Cell Lung Cancer in the Russian Federation: Nationwide Cohort Study.

PURPOSE: The overall survival (OS) results in patients with ALK-positive metastatic non-small-cell lung cancer (NSCLC) have rarely been reported. The aim of this prospective-retrospective cohort study was to obtain real-world data on the use of crizotinib or chemotherapy in patients with ALK-positive metastatic NSCLC in Russia. PATIENTS AND METHODS: Patients with epidermal growth factor receptor-negative metastatic NSCLC were screened in 23 cancer centers. To be eligible, patients were required to have confirmation of ALK rearrangement. Patients were treated with crizotinib (250 mg twice daily; n = 96) or the investigator's choice of platinum-based chemotherapy (n = 53). The primary end point was OS. RESULTS: A total of 149 ALK-positive patients were included. Mean age was 53 years in both groups. Patients were predominately women (59%) and never-smokers (74%), and most patients had adenocarcinoma histology (95%). At a median follow-up time of 15 months, 79 of the 149 patients included in the analysis had died. Median OS from the start of treatment was 31 months (95% CI, 28.5 to 33.5 months) in the crizotinib group and 15.0 months (95% CI, 9.0 to 21.0 months) in the chemotherapy group (P < .001). The objective response rate was 34% in the crizotinib group. Among patients with brain metastasis, one complete response (6%) and five partial responses (31%) were achieved. Grade 3 adverse events were observed in three patients (3%) in the crizotinib group. CONCLUSION: The improved OS observed in crizotinib clinical trials in ALK-positive NSCLC was also observed in the less selective patient populations treated in daily practice in Russia. The use of standard chemotherapy in these patients remains common but seems inappropriate as a result of the effectiveness of newer treatments, such as crizotinib.

Detection of ALK rearrangements in 4002 Russian patients: The utility of different diagnostic approaches.

BACKGROUND: Clinical guidelines highly recommended the detection of potentially targetable genetic aberrations such as anaplastic lymphoma kinase (ALK) rearrangements in patients with non-small cell lung cancer (NSCLC). Few methods, such as the ALK break apart FISH assay and IHC for ALK protein, are approved for routine diagnostics. However, some challenges exist in selecting the most reliable, robust and cost-effective algorithm, especially for large-scale screening of NSCLC patients. MATERIALS AND METHODS: A total of 4002 FFPE samples from Russian patients with NSCLC were tested for ALK rearrangement using two algorithms: FISH testing only (2334 samples) and IHC screening supported by FISH in positive or equivocal cases (1546 samples). Cross validation of the methods was performed blindly in 122 specimens. All discrepant IHC/FISH cases were examined for unbalanced 5'/3'-end ALK expression and variant-specific RT PCR. RESULTS: The sensitivity and specificity of IHC compared to FISH was 100% and 99%, respectively, therefore initial IHC screening was strongly supported. The prevalence of ALK rearrangements was estimated to be 7.8% and 6.6% for the FISH and IHC/FISH groups, respectively, with significant correlations for female gender, non-smoking status and age below 60. The use of FISH after IHC screening revealed 10 additional positive cases among equivocal samples (13.4%). Seven IHC/FISH cases (0.5% of the total group) characterized as discordant were reevaluated, and four were reclassified as truly discrepant. The PCR-based investigation revealed chimeric transcripts in IHC-/FISH+ and IHC+/FISH borderline samples, while no transcript was found in two IHC+/FISH- cases. CONCLUSION: These results reveal the utility of the two-step testing algorithm for the evaluation of potentially complicated cases with preanalytic defects, providing additional information for IHC equivocal cases without a significant increase in cost. The evaluation of discrepant cases appears to be necessary to better understand ALK biology and should be included in the routine testing algorithm.

Nuclear Protein of the Testis Midline Carcinoma Masquerading as a Primary Mediastinal Seminoma.

Nuclear protein of the testis (NUT) midline carcinomas are rare aggressive carcinomas characterized by chromosomal rearrangements that involve the gene encoding the NUT. This article reviews the clinicopathologic features and the differential diagnosis of these malignancies.

Immunohistochemistry, fluorescence in situ hybridization, and reverse transcription-polymerase chain reaction for the detection of anaplastic lymphoma kinase gene rearrangements in patients with non-small cell lung cancer: potential advantages and methodologic pitfalls.

CONTEXT: Echinoderm microtubule-associated protein-like 4 gene (EML4) and anaplastic lymphoma kinase gene (ALK) fusion was shown to be the driver of tumorigenesis in approximately 3% to 5% of patients with non-small cell lung cancer (NSCLC) and is associated with response to inhibition with crizotinib. However, no complete agreement regarding the best diagnostic test for identification of ALK rearrangements has been achieved yet. OBJECTIVE: To investigate the concordance, sensitivity, and specificity of immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and reverse transcription-polymerase chain reaction (RT-PCR) for detection of ALK rearrangements. DESIGN: Thirty-six prospectively tested patients with NSCLC who had adenocarcinoma and 10 ALK-positive samples were included in the study. All samples were tested by IHC (ALK1 clone, 5A4 clone, D5F3 clone), FISH (LSI ALK Break Apart and ALK FISH Probe), and multiplexed RT-PCR. RESULTS: Immunohistochemistry staining was successful in all samples.. Clone D5F3 showed the best sensitivity and specificity of 100%; clones ALK1 and 5A4 showed sensitivities of 91% with specificity of 100%. Both FISH probes showed concordance with sensitivity and specificity of 100%. Hybridization and RT-PCR were successful in 98% and 93.4% of samples, respectively, with sensitivity of 88% and specificity of 100%. Frequent artifacts leading to misinterpretation were observed with all 3 methodologies. CONCLUSIONS: All 3 methodologies showed good sensitivity, specificity, and concordance, when artifacts were characterized and excluded. However, all ambiguous cases have to be confirmed as ALK rearranged by at least 2 of the 3 methods.

Histological heterogeneity of Ewing's sarcoma/PNET: an immunohistochemical analysis of 415 genetically confirmed cases with clinical support.

Ewing's sarcoma (ES)/peripheral neuroectodermal tumor (PNET) are malignant neoplasms affecting children and young adults. We performed a study to typify the histological diversity and evaluate antibodies that may offer diagnostic/prognostic support. In total, 415 cases of genetically confirmed paraffin-embedded ES/PNET were analyzed on whole sections and in tissue microarrays. This study confirms the structural heterogeneity of ES/PNET, distinguishing three major subtypes: conventional ES (280 cases); PNET (53 cases); and atypical ES/PNET (80), including large cells, vascular-like patterns, spindle pattern, and adamantinoma-like configuration. All cases presented positivity for at least three of the four tested antibodies (CD99, FLI1, HNK1, and CAV1). CAV1 appeared as a diagnostic immunomarker of ES/PNET being positive in CD99-negative cases. Hence, the immunohistochemical analysis confirmed the diagnostic value of all four antibodies, which together cover more than 99% of the tumors, independently of the histological variety. The univariate analysis for survival revealed atypical ES as the only histological parameter apparently associated with less favorable clinical outcome, particularly in the subgroup of patients treated with surgery. In conclusion, the diagnosis of atypical ES is a challenge for the pathologist and needs support from molecular techniques to perform an optimal differential diagnosis with other small round cell tumors.