Abstracts from USCAP 2021: Pulmonary, Mediastinal, Pleural, and Peritoneal Pathology (910-952)

2021 Modern Pathology  
The understanding of molecular mechanisms involved in the non-small cell lung carcinoma (NSCLC) has increased significantly in the recent years. These have helped to develop personalized management strategies by identifying specific molecular alterations such as mutations in EGFR, ROS1, BRAF, ERBB2, MET, ALK, and KRAS genes. These mutations are targetable by various therapeutic molecules, ensuring a better clinical outcome. Next generation sequencing (NGS) methodology is the recommended
more » ... e for the identification of driver mutations in the five hot-spot genes (EGFR, ALK, ROS1, MET, and BRAF) involved in the NSCLC carcinogenesis. NGS has numerous advantages (multiplexing, tissue conservation, identification of rare and novel variants, and reduced cost and combined turn-around time) over sequential single gene testing. Herein, we demonstrate the molecular mutational profiling and their clinicopathologic correlation in a large cohort of Indian NSCLC cancer patients. Design: Five fifty two Stage IV NSCLC patients (n=552; adenocarcinoma, n=490; squamous cell carcinoma, n=51; adenosquamous carcinoma, n=5; large cell carcinoma, n=2; sarcomatoid carcinoma, n=3; spindle cell carcinoma, n=1) underwent broad molecular profiling by a custom-made, targeted DNA-and RNA-based five hot-spot genes lung cancer panel (EGFR, ALK, ROS1, BRAF and MET), compatible with the NGS Ion S5 system. The mutations were correlated with the clinicopathologic characteristics. Results: Validation of the 5 gene panel yielded the following results: a) Specificity of 99.74%; b) Sensitivity of 100% for SNVs (>5% VAF), Indels (>10% VAF) and fusions; c) 100% intra-and inter-run reproducibility; d) 88% inter-laboratory agreement. Validated panel was then used to analyze clinical samples. 320 of 552 (57.97%) tumors (patients) harbored either one (302 or 54.71%) or multiple (18 or 3.26%) mutations. EGFR and BRAF V600E mutations, ALK and ROS1 rearrangements, and MET exon 14 skipping mutation were observed in in 38.41% and 2.72% patients, 12.14% and 3.62% patients, and 1.09% patients. EGFR exon 19 deletions accounted for 52.83% of all mutations, followed by L858R (35.85%), T790M (5.19%), exon 20 insertions (6.60%) and other rare mutations (G719X, L861Q, S768I) (9.91%). Concurrent EGFR with ALK, EGFR with ROS1, EGFR with MET and EGFR with BRAF were observed in 10, 4, 1, and 3 patients. Total Positive Cases 1. This is the largest cohort of NSCLC for comprehensive targeted mutational profiling. 2. The mutations are more prevalent in non-smoker females for all genes except ALK (non-smoker males). 3. MET and BRAF mutations are more common in elderly population whereas EGFR mutations, and ALK and ROS1 genes rearrangements are more prevalent in younger population. 4. The most common histopathologic subtype associated with various mutations are as follows: acinar with EGFR, solid with ALK, macronucleoli with ROS1, signet ring with MET, and micropapillary with BRAF ( Figure 1 and 2). Background: Ex-vivo lung perfusion (EVLP), is an innovative technique applied to donor lungs that could provide platform to preserve, evaluate, and repair the donor lung graft. EVLP improves organ quality making previously unsuitable lungs safe for transplant. We report our experience of histologic assessment of before and after EVLP in tissue samples from discarded human lungs. Background: Cancer immunotherapy, especially immune checkpoint inhibitors (ICIs), can have a remarkable antitumor response in non-small cell lung cancer (NSCLC). However, the majority of patients fail to respond to PD-1/PD-L1 axis inhibitors. Pembrolizumab is indicated as a first-line therapy for patients with advanced NSCLC with PD-L1 expression Tumor Proportion Score (TPS) >1%. Although expression of PD-L1 and tumor environment immune cells have been independently associated with response rates, the contribution of these factors for better prediction of the response to ICI therapy in NSCLC is still largely unclear. In a previous small pilot study, we observed that both high PD-L1 expression and the percentage and pattern of CD4/8 positive T-cells appear to be better predictive of ICI response in NSCLC than PD-L1 alone. Herein we further evaluate the predictive value of these biomarkers by using a more extensive study sample set. Design: A total of 26 NSCLC patients who received ICIs (8 responders and 18 non-responders) were identified from the departmental surgical pathology archives and response data was retrieved from the electronic medical record. Responders were defined as those achieving at least a partial response (30% decrease or more in the sum of diameters of target lesions) according to RECIST guideline v1.1. Response was assessed at 6-8 weeks after ICI therapy initiation with follow-up of at least 6 months. Immunohistochemistry (IHC) studies for PD-L1, CD4, CD8, CD21, and CD163 were performed and the results were tabulated. High PD-L1 expression was defined as TPS>50%; immune cell tumor infiltrate level was classified as high (>10%) or low (<5%). Results: In our study, non-responders mostly displayed low PD-L1 expression (mean TPS: 19.8%; range 1-70%) and low or no CD8 positive T-cell tumor infiltrate (13/18; 72.2%). In contrast, most responders displayed a high PD-L1 expression (mean TPS: 55.5%; range 1-100%) with most (7/8, 87.5%) having a high CD8 positive T-cell tumor 1090 infiltrate. The level of tumor infiltrating CD4 positive T-cells and CD163 positive macrophages was similar for both groups. No CD21 positive cells infiltrating tumor were identified in any of the specimens. Conclusions: Our study highlights the current dilemma in treating NSCLC with ICIs as over two-thirds of our patients did not respond to treatment. However, a combination of high PD-L1 expression and high CD8 expression is much more accurate in determining response to ICI therapy and should be routinely assessed to assist in predicting response. Prospective study is underway to confirm the predictive value of these combined biomarkers. Background: A minority of small cell lung carcinomas (SCLC) has minimal or absent expression of neuroendocrine (NE) markers, which can present a diagnostic challenge. Recent studies have suggested that POU2F3 -a marker of chemosensory tuft cell lineage -is expressed specifically in NE-low SCLC. Here, we aimed to examine expression of POU2F3 in SCLC with extremely low or negative NE markers and to determine its specificity relative to non-NE lung carcinomas. Design: POU2F3 expression was examined immunohistochemically in 152 SCLC and 116 non-small cell lung carcinomas (NSCLC; 53 adenocarcinomas, 63 squamous cell carcinomas). SCLC comprised 144 unselected cases and 8 additional pre-selected NE-minimal or negative SCLC. All SCLC were tested for 4 conventional NE markers (CNM; synaptophysin, chromogranin, CD56, and INSM1), and tumors with combined NE score (average H-score of 4 CNM) <50 were defined as NE-low and those with score <10 (staining isolated cells only) as NE-minimal. TTF-1 expression was also evaluated. Results: POU2F3 was expressed in 8% of unselected SCLC (11/140), but was completely negative in all 116 NSCLC. In the whole cohort, compared to POU2F3-negative cases (n=134), POU2F3-positive SCLC (n=18) had fewer positive CNM (mean 1.8 vs 3.7, p<0.0001), lower combined NE score (mean 60 vs 183, p<0.0001) and lower rate of TTF-1 expression (6% vs 81%, p<0.0001), respectively. A total of 15 SCLC were NE-low (n=10), NEminimal (n=4) or NE-entirely negative (n=1). POU2F3 was positive in 10/15 (67%) of these cases, including all 5 NE-minimal/negative SCLC. POU2F3 expression in these cases was typically strong and diffuse (mean H-score 147; range 60-235). Conclusions: POU2F3 expression is highly specific for SCLC relative to NSCLC, and is significantly enriched in NE-low SCLC, particularly in cases with minimal or negative NE marker expression. We suggest that POU2F3 represents a novel helpful diagnostic marker of SCLC. Background: Checkpoint inhibitor therapy is a known cause of lung toxicity. There have been few histologic descriptions of pathologic features of pneumonitis clinically attributed to checkpoint inhibitor therapy, and vasculitis has not yet been reported in this setting. Design: We report histologic findings of biopsies of 3 patients who developed pulmonary symptoms that were temporally associated with monoclonal antibody treatment. Clinical information and radiologic findings were obtained by chart review. Background: Distinction between mesothelial hyperplasia and mesothelioma is essential once a proliferation is proven to be mesothelial in origin. The use of small samples obtained by minimally invasive methods for first-time diagnosis is common given physical condition limitations in these patients. However, morphologic criteria for mesothelioma such as invasion of the fat are not always present in biopsy specimens. It has been suggested in animal models that disruption of the NF2 signalling is essential for the development of human mesothelioma either by NF2 gene mutation or NF2/Merlin protein inactivation by phosphorylation. We aimed to evaluate NF2 inactivation by immunohistochemistry (IHC) in cases of mesothelioma. Design: We searched our institutional archives between 2010-2020 to identify 11 mesothelioma cases. For comparison, seven cases with normal mesothelial lining or reactive mesothelial hyperplasia were included. The NF2 IHC was performed with D3S3W monoclonal antibody from Cell Signalling Technology in 1:100 dilution. In five cases MTAP (clone 2G4 from Abnova) and BAP1 (clone C-4 from Santa Cruz Biotechnology Inc.) IHC was done at an outside institution. Background: ERBB2 is an oncogene known for its sensitivity to tratuzumab and lapatinib in breast cancer. Nonsmall cell lung carcinomas (NSCLCs) uncommonly harbor ERBB2 mutations, with trials underway to assess for targeted response. We sought to characterize three well known HER2 immunohistochemical (IHC) clones in the detection of ERBB2 protein expression in NSCLC with ERBB2 alterations, detected by next-generation sequencing (NGS). Expression was graded using the breast and gastric HER2 scoring systems to determine correlation with mutations. We assessed for cross-reactivity among additional molecular mutations, including BRAF, KRAS, MET, NRAS, and EGFR, along with reactivity to specimens without reportable mutations by NGS ("pan-negative"). Design: NSCLC specimens were retrieved with the following criteria: 1) Eight cases had a mutation/amplification of the ERBB2 gene detected by NGS and 2) Twenty-two pan-negative control specimens were stained, along with twenty-four specimens with a mutation in BRAF, KRAS, MET, NRAS, or EGFR allowing for determination of specificity and sensitivity. Three HER2 IHC clones were analyzed: Dako-AutoStainer Link 48: HercepTest™, Roche-Ventana: clone4B5, and Leica-Bond: cloneCB11. Each clone was graded by both the breast and gastric scoring systems as described by the College of American Pathologist (CAP). Correlations were assessed using Barnard's test, and confidence intervals (95%) were obtained using Wilson's method. Background: Small cell lung carcinoma (SCLC) is characterized by unique genetic alterations, which often involve the mTOR pathway. Amplification of RICTOR, an essential component of mTORC2, has recently been identified as the most common targetable molecular alteration in SCLC. Selection of SCLC patients who may benefit from mTOR inhibitor therapy is currently problematic. The goal of our study was to address this issue by comparing various methods available to assess RICTOR amplification and mTORC2 activity. Design: Droplet Digital PCR and fluorescence in situ hybridization (FISH) were used to detect RICTOR gene amplification, and immunohistochemistry (IHC) and Western blot analysis were used to assess mTOR pathway protein expression in SCLC cell lines (n=9). FISH for RICTOR amplification and IHC for Rictor and p-Akt expression were also performed on SCLC samples (n=100). Results : RICTOR amplification was detected in 2 SCLC cell lines (22%) and 15 SCLC cases (15%). Expression of mTORC2 pathway proteins correlated with RICTOR amplification. As compared to RICTOR FISH, the sensitivity 1116 and specificity of Rictor IHC for the detection of RICTOR amplification was 93% and 73%, respectively. A high negative predictive value (98%) was also observed. Conclusions: Our data suggest that RICTOR amplification results in Rictor overexpression and mTORC2 hyperactivation in SCLC. Rictor IHC can be used as a cost-effective screening method to select patients for molecular testing and mTOR inhibitor therapy. Conclusions: In our cohort, METex14 skipping alterations were predominantly observed in adenocarcinomas in older age group. We also identified a novel 3 base pair deletion in intron 13 at -16 leading to METex 14 skipping. Due to diverse genomic mutations leading to METex14 skipping, some of the alterations may not be identified using DNA based sequencing alone due to incomplete coverage of the intronic regions, leading to false negative results. We recommend the use of RNA sequencing panels in NSCLC for METex14 detection. Background: Major histocompatibility complex (MHC) class I functions in presentation of peptide antigens. In the tumor immune environment, MHC class I enables activation of cytotoxic T-cells, and loss of expression is a proposed mechanism of failure of immune checkpoint inhibitor (ICI) therapy. We hereby examine the expression of PD-L1 and MHC class I in patients with non-small cell lung cancer (NSCLC) who received anti-PD-1/PD-L1 ICI therapy. Design: The institutional lung cancer registry was queried to identify patients with stage IV or recurrent NSCLC diagnosed between 2008 and 2018. Clinical and radiologic data were abstracted from the medical record. Patients who received at least 2 consecutive doses of an anti-PD-1/PD-L1 ICI (pembrolizumab, atezolizumab, nivolumab) were selected. Immunohistochemistry (IHC) for PD-L1 (SP142) and MHC class I (Abcam, EMR8-5) was performed on diagnostic FFPE tissue for each case. Tumor cell PD-L1 staining was scored as negative (<1%), low-positive (1-50%), or high-positive (>50%). MHC class I staining was classified as intact, partial loss, or complete loss. Results: 60 cases were identified: 34 adenocarcinomas, 20 squamous cell carcinomas, 1 adenosquamous carcinoma, and 5 NSCLC not otherwise specified. 44 patients received an ICI in isolation; 16 received an ICI in combination with chemotherapy. 42 cases (70%) demonstrated intact MHC class I expression; 4 (7%)
doi:10.1038/s41379-021-00772-3 pmid:33707674 fatcat:nje74myalzhxhkjc3ms22cqys4