Impact of Region-of-Interest Size on Immune Profiling Using Multiplex Immunofluorescence Tyramide Signal Amplification for Paraffin-Embedded Tumor Tissues.

INTRODUCTION: Representative regions of interest (ROIs) analysis from the whole slide images (WSI) are currently being used to study immune markers by multiplex immunofluorescence (mIF) and single immunohistochemistry (IHC). However, the amount of area needed to be analyzed to be representative of the entire tumor in a WSI has not been defined. METHODS: We labeled tumor-associated immune cells by mIF and single IHC in separate cohorts of non-small cell lung cancer (NSCLC) samples and we analyzed them as whole tumor area as well as using different number of ROIs to know how much area will be need to represent the entire tumor area. RESULTS: For mIF using the InForm software and ROI of 0.33 mm2 each, we observed that the cell density data from five randomly selected ROIs is enough to achieve, in 90% of our samples, more than 0.9 of Spearman correlation coefficient and for single IHC using ScanScope tool box from Aperio and ROIs of 1 mm2 each, we found that the correlation value of more than 0.9 was achieved using 5 ROIs in a similar cohort. Additionally, we also observed that each cell phenotype in mIF influence differently the correlation between the areas analyzed by the ROIs and the WSI. Tumor tissue with high intratumor epithelial and immune cells phenotype, quality, and spatial distribution heterogeneity need more area analyzed to represent better the whole tumor area. CONCLUSION: We found that at minimum 1.65 mm2 area is enough to represent the entire tumor areas in most of our NSCLC samples using mIF.

Evaluation of PARP and PDL-1 as potential therapeutic targets for women with high-grade neuroendocrine carcinomas of the cervix.

OBJECTIVES: Women with recurrent high-grade neuroendocrine cervical cancer have few effective treatment options. The aim of this study was to identify potential therapeutic targets for women with this disease. METHODS: Specimens from patients with high-grade neuroendocrine carcinomas of the cervix were identified from pathology files at MD Anderson Cancer Center. Immunohistochemical stains for PD-L1 (DAKO, clone 22-C3), mismatch repair proteins (MLH1, MSH2, MSH6, PMS2), somatostatin, and Poly (ADP-ribose) polymerase (PARP) were performed on sections from formalin-fixed paraffin-embedded tissue blocks. Nuclear PARP-1 staining was quantified using the H-score with a score of <40 considered low, 40-100 moderate, and ≥100 high. RESULTS: Forty pathologic specimens from patients with high-grade neuroendocrine carcinomas of the cervix were examined (23 small cell, 5 large cell, 3 high-grade neuroendocrine, not otherwise specified, and 9 mixed). The mean age of the cohort was 43 years and the majority of patients (70%) were identified as white non-Hispanic. All 28 (100%) samples tested stained for mismatch repair proteins demonstrated intact expression, suggesting they were microsatellite stable tumors. Of the 31 samples tested for PD-L1 expression, only two (8%) of the 25 pure high-grade neuroendocrine carcinomas were positive whereas three (50%) of the six mixed carcinoma tumors tested positive. Of the 11 small cell specimens tested for PARP-1, 10 (91%) showed PARP expression with six (55%) demonstrating high expression and four (36%) showing moderate expression. Somatostatin staining was negative in 18 of 19 small cell cases (95%). CONCLUSIONS: Pure high-grade neuroendocrine cervical carcinomas were microsatellite stable and overwhelmingly negative for PD-L1 expression. As the majority of tumors tested expressed PARP-1, inclusion of PARP inhibitors in future clinical trials may be considered.

Pathological Response and Immune Biomarker Assessment in Non-Small-Cell Lung Carcinoma Receiving Neoadjuvant Immune Checkpoint Inhibitors.

Lung cancer is the leading cause of cancer incidence and mortality worldwide. Adjuvant and neoadjuvant chemotherapy have been used in the perioperative setting of non-small-cell carcinoma (NSCLC); however, the five-year survival rate only improves by about 5%. Neoadjuvant treatment with immune checkpoint inhibitors (ICIs) has become significant due to improved survival in advanced NSCLC patients treated with immunotherapy agents. The assessment of pathology response has been proposed as a surrogate indicator of the benefits of neaodjuvant therapy. An outline of recommendations has been published by the International Association for the Study of Lung Cancer (IASLC) for the evaluation of pathologic response (PR). However, recent studies indicate that evaluations of immune-related changes are distinct in surgical resected samples from patients treated with immunotherapy. Several clinical trials of neoadjuvant immunotherapy in resectable NSCLC have included the study of biomarkers that can predict the response of therapy and monitor the response to treatment. In this review, we provide relevant information on the current recommendations of the assessment of pathological responses in surgical resected NSCLC tumors treated with neoadjuvant immunotherapy, and we describe current and potential biomarkers to predict the benefits of neoadjuvant immunotherapy in patients with resectable NSCLC.

Tumor suppressor DEAR1 regulates mammary epithelial cell fate and predicts early onset and metastasis in triple negative breast cancer.

Triple negative breast cancer (TNBC) is a disease of poor prognosis, with the majority classified as the basal-like subtype associated with epithelial-mesenchymal transition and metastasis. Because basal breast cancers originate from proliferative luminal progenitor-like cells upon dysregulation of proper luminal differentiation, genes regulating luminal-basal transition are critical to elucidate novel therapeutic targets to improve TNBC outcomes. Herein we demonstrate that the tumor suppressor DEAR1/TRIM62 is a critical regulator of luminal cell fate. DEAR1 loss in human mammary epithelial cells results in significantly enhanced mammosphere formation that is accelerated in the presence of TGF-ß/SMAD3 signaling. Mammospheres formed following DEAR1 loss are enriched for ALDH1A1 and CK5 expression, EpCAM-/CD49f+ and CD44high/24low basal-like epithelial cells, indicating that DEAR1 regulates stem/progenitor cell properties and luminal-basal progenitor transition. We show that DEAR1 maintains luminal differentiation as a novel ubiquitin ligase for SNAI2/SLUG, a master regulator driving stemness and generation of basal-like progenitor populations. We also identify a significant inverse correlation between DEAR1 and SNAI2 expression in a 103 TNBC case cohort and show that low DEAR1 expression significantly correlates with young age of onset and shorter time to metastasis, suggesting DEAR1 could serve as a biomarker to stratify early onset TNBCs for targeted stem cell therapies.

Expression of TRPS1 in phyllodes tumor and sarcoma of the breast.

When a sarcomatous neoplasm is identified in the breast, distinguishing metaplastic carcinoma, malignant phyllodes tumor (MPT), and primary sarcoma is a diagnostic challenge, especially on small biopsies, as all these tumors may have overlapping morphological features, thoroughly grossing with histological examination and immunohistochemical staining being the standard approach to aid in classifying these lesions. Recently, we identified a highly sensitive and specific breast carcinoma marker TRPS1 with high expression in metaplastic breast carcinoma. In the current study, we tested TRPS1 in MPTs and primary sarcoma of the breast. We found TRPS1 was highly expressed (95%) within spindle cell, chondro-osseous, and/or liposarcomatous components of MPTs, in all breast primary chondrosarcomas and extraskeletal osteosarcomas, but not in other sarcomas of the breast. In extramammary sarcomas, TRPS1 was expressed in 28% of conventional chondrosarcomas and 56% of osteosarcomas of bone, but rarely in undifferentiated pleomorphic sarcomas (UPSs), liposarcomas, and angiosarcomas. In summary, MPTs may share similar genetic background with metaplastic carcinoma exhibiting TRPS1 expression, and TRPS1 may play a role in chondro-osseous differentiation because of its expression in chondro-osseous sarcomas from both breast and extramammary sites. Our findings suggest TRPS1 may be clinically useful in distinguishing MPT and metaplastic carcinoma from primary breast sarcoma except for tumors with chondro-osseous differentiation.

Characterization of the Immune Landscape of EGFR-Mutant NSCLC Identifies CD73/Adenosine Pathway as a Potential Therapeutic Target.

INTRODUCTION: Lung adenocarcinomas harboring EGFR mutations do not respond to immune checkpoint blockade therapy and their EGFR wildtype counterpart. The mechanisms underlying this lack of clinical response have been investigated but remain incompletely understood. METHODS: We analyzed three cohorts of resected lung adenocarcinomas (Profiling of Resistance Patterns of Oncogenic Signaling Pathways in Evaluation of Cancer of Thorax, Immune Genomic Profiling of NSCLC, and The Cancer Genome Atlas) and compared tumor immune microenvironment of EGFR-mutant tumors to EGFR wildtype tumors, to identify actionable regulators to target and potentially enhance the treatment response. RESULTS: EGFR-mutant NSCLC exhibited low programmed death-ligand 1, low tumor mutational burden, decreased number of cytotoxic T cells, and low T cell receptor clonality, consistent with an immune-inert phenotype, though T cell expansion ex vivo was preserved. In an analysis of 75 immune checkpoint genes, the top up-regulated genes in the EGFR-mutant tumors (NT5E and ADORA1) belonged to the CD73/adenosine pathway. Single-cell analysis revealed that the tumor cell population expressed CD73, both in the treatment-naive and resistant tumors. Using coculture systems with EGFR-mutant NSCLC cells, T regulatory cell proportion was decreased with CD73 knockdown. In an immune-competent mouse model of EGFR-mutant lung cancer, the CD73/adenosine pathway was markedly up-regulated and CD73 blockade significantly inhibited tumor growth. CONCLUSIONS: Our work revealed that EGFR-mutant NSCLC has an immune-inert phenotype. We identified the CD73/adenosine pathway as a potential therapeutic target for EGFR-mutant NSCLC.