Advances in antitumor activity and mechanism of natural steroidal saponins: A review of advances, challenges, and future prospects.

BACKGROUND: Cancer, the second leading cause of death worldwide following cardiovascular diseases, presents a formidable challenge in clinical settings due to the extensive toxic side effects associated with primary chemotherapy drugs employed for cancer treatment. Furthermore, the emergence of drug resistance against specific chemotherapeutic agents has further complicated the situation. Consequently, there exists an urgent imperative to investigate novel anticancer drugs. Steroidal saponins, a class of natural compounds, have demonstrated notable antitumor efficacy. Nonetheless, their translation into clinical applications has remained unrealized thus far. In light of this, we conducted a comprehensive systematic review elucidating the antitumor activity, underlying mechanisms, and inherent limitations of steroidal saponins. Additionally, we propose a series of strategic approaches and recommendations to augment the antitumor potential of steroidal saponin compounds, thereby offering prospective insights for their eventual clinical implementation. PURPOSE: This review summarizes steroidal saponins' antitumor activity, mechanisms, and limitations. METHODS: The data included in this review are sourced from authoritative databases such as PubMed, Web of Science, ScienceDirect, and others. RESULTS: A comprehensive summary of over 40 steroidal saponin compounds with proven antitumor activity, including their applicable tumor types and structural characteristics, has been compiled. These steroidal saponins can be primarily classified into five categories: spirostanol, isospirostanol, furostanol, steroidal alkaloids, and cholestanol. The isospirostanol and cholestanol saponins are found to have more potent antitumor activity. The primary antitumor mechanisms of these saponins include tumor cell apoptosis, autophagy induction, inhibition of tumor migration, overcoming drug resistance, and cell cycle arrest. However, steroidal saponins have limitations, such as higher cytotoxicity and lower bioavailability. Furthermore, strategies to address these drawbacks have been proposed. CONCLUSION: In summary, isospirostanol and cholestanol steroidal saponins demonstrate notable antitumor activity and different structural categories of steroidal saponins exhibit variations in their antitumor signaling pathways. However, the clinical application of steroidal saponins in cancer treatment still faces limitations, and further research and development are necessary to advance their potential in tumor therapy.

Novel ATG7::RAF1 gene fusion in malignant glomus tumor.

Glomus tumors are classified as members of the perivascular myoid family of tumors. Nearly half of these show NOTCH-gene fusions and a smaller subset has BRAF V600E mutations. Here, we report a novel ATG7::RAF1 fusion in malignant glomus tumor occurring in a 40-year-old female which has not been reported in the malignant glomus tumor before. A 40-year-old female presented with a persistent lateral heel pain and an increase in the size of a mass along the lateral ankle for nearly 10 years. Resected specimen showed a well circumscribed lesion composed of spindled and epithelioid cells with moderate nuclear atypia and mitotic figures (7/10 high-power fields) including atypical forms without any necrosis, lymphovascular, or perineural invasion. The tumor was positive for smooth muscle actin, smooth muscle myosin heavy chain, H-caldesmon, collagen type IV, and discovered on gastronintestinal stromal tumors-1 but negative for AE1/3, desmin, S-100, CD34, and CD117. RNA sequencing showed presence of ATG7-RAF1 fusion. This fusion has not been reported in the malignant glomus tumor before. Future studies on larger cohorts are needed to ascertain the biological significance of these tumors with novel gene fusions.

New Therapies in Melanoma: Current Trends, Evolving Paradigms, and Future Perspectives.

Melanoma is an aggressive skin cancer with increasing incidence and mortality worldwide. For many years the therapeutic strategies were limited to surgery, radiotherapy, and chemotherapy. Recent advances in immunology and cancer biology have led to the discovery and development of novel therapeutics, such as immune checkpoint inhibitors (ICIs) and targeted therapies, which have revolutionized the clinical care of patients with metastatic melanoma. Despite recent successes with ICIs, many melanoma patients do not experience long-term benefits from ICI therapies, highlighting the need for alternative treatments with novel targets such as lymphocyte-activated gene 3 (LAG-3). In this review, we explore new therapeutic agents and novel combinations that are being tested in early-phase clinical trials. We discuss newer promising tools such as nanotechnology to develop nanosystems that act as drug carriers and/or light absorbents to potentially improve therapy outcomes. Finally, we also highlight challenges such as management after resistance and intervention with novel immunotherapies and the lack of predictive biomarkers to stratify patients to targeted treatments after primary treatment failure.

Artificial intelligence in diagnostic pathology.

Digital pathology (DP) is being increasingly employed in cancer diagnostics, providing additional tools for faster, higher-quality, accurate diagnosis. The practice of diagnostic pathology has gone through a staggering transformation wherein new tools such as digital imaging, advanced artificial intelligence (AI) algorithms, and computer-aided diagnostic techniques are being used for assisting, augmenting and empowering the computational histopathology and AI-enabled diagnostics. This is paving the way for advancement in precision medicine in cancer. Automated whole slide imaging (WSI) scanners are now rendering diagnostic quality, high-resolution images of entire glass slides and combining these images with innovative digital pathology tools is making it possible to integrate imaging into all aspects of pathology reporting including anatomical, clinical, and molecular pathology. The recent approvals of WSI scanners for primary diagnosis by the FDA as well as the approval of prostate AI algorithm has paved the way for starting to incorporate this exciting technology for use in primary diagnosis. AI tools can provide a unique platform for innovations and advances in anatomical and clinical pathology workflows. In this review, we describe the milestones and landmark trials in the use of AI in clinical pathology with emphasis on future directions.

Comparison of four different displays for identification of select pathologic features extracted from whole slide images of surgical pathology cases.

BACKGROUND: The establishment of minimum standards for display selection for the whole slide image (WSI) interpretation has not been fully defined. Recently, pathologists have increasingly preferred using remote displays for clinical diagnostics. Our study aims to assess and compare the performance of three fixed work displays and one remote personal display in accurately identifying ten selected pathologic features integrated into WSIs. DESIGN: Hematoxylin and eosin-stained glass slides were digitized using Philips scanners. Seven practicing pathologists and three residents reviewed ninety WSIs to identify ten pathologic features using the LG, Dell, and Samsung and an optional consumer-grade display. Ten pathologic features included eosinophils, neutrophils, plasma cells, granulomas, necrosis, mucin, hemosiderin, crystals, nucleoli, and mitoses. RESULTS: The accuracy of the identification of ten features on different types of displays did not significantly differ among the three types of "fixed" workplace displays. The highest accuracy was observed for the identification of neutrophils, eosinophils, plasma cells, granuloma, and mucin. On the other hand, a lower accuracy was observed for the identification of crystals, mitoses, necrosis, hemosiderin, and nucleoli. Participant pathologists and residents preferred the use of larger displays (>30″) with a higher pixel count, resolution, and luminance. CONCLUSION: Most features can be identified using any display. However, certain features posed more challenges across the three fixed display types. Furthermore, the use of a remote personal consumer-grade display chosen according to the pathologists' preference showed similar feature identification accuracy. Several factors of display characteristics seemed to influence pathologists' display preferences such as the display size, color, contrast ratio, pixel count, and luminance calibration. This study supports the use of standard "unlocked" vendor-agnostic displays for clinical digital pathology workflow rather than purchasing "locked" and more expensive displays that are part of a digital pathology system.

Phase II Window Study of Olaparib Alone or with Cisplatin or Durvalumab in Operable Head and Neck Cancer.

Purpose: We conducted a phase II randomized noncomparative window of opportunity (WOO) trial to evaluate the inhibition of cellular proliferation and the modulation of immune microenvironment after treatment with olaparib alone or in combination with cisplatin or durvalumab in patients with operable head and neck squamous cell carcinoma (HNSCC). Experimental Design: Forty-one patients with HNSCC were randomized to cisplatin plus olaparib (arm A), olaparib alone (arm B), no treatment (arm C) or durvalumab plus olaparib (arm D). The primary endpoint was to evaluate the percentage of patients in each arm that achieved a reduction of at least 25% in Ki67. Secondary endpoints included objective response rate (ORR), safety, and pathologic complete response (pCR) rate. Paired baseline and resection tumor biopsies and blood samples were evaluated for prespecified biomarkers. Results: A decrease in Ki67 of at least 25% was observed in 44.8% of treated patients, as measured by quantitative immunofluorescence. The ORR among treated patients was 12.1%. pCR was observed in 2 patients. Two serious adverse events occurred in 2 patients.Programmed death ligand 1 (PD-L1) levels [combined positive score (CPS)] were significantly higher after treatment in arms A and D. Expression of CD163 and colony-stimulating factor 1 receptor (CSF1R) genes, markers of M2 macrophages, increased significantly posttreatment whereas the expression of CD80, a marker of M1 macrophages, decreased. Conclusion: Preoperative olaparib with cisplatin or alone or with durvalumab was safe in the preoperative setting and led to decrease in Ki67 of at least 25% in 44.8% of treated patients. Olaparib-based treatment modulates the tumor microenvironment leading to upregulation of PD-L1 and induction of protumor features of macrophages. Significance: HNSCC is characterized by defective DNA repair pathways and immunosuppressive tumor microenvironment. PARP inhibitors, which promote DNA damage and "reset" the inflammatory tumor microenvironment, can establish an effective antitumor response. This phase II WOO trial in HNSCC demonstrated the immunomodulatory effects of PARP inhibitor-induced DNA damage. In this chemo-naïve population, PARP inhibitor-based treatment, reduced tumor cell proliferation and modulated tumor microenvironment. After olaparib upregulation of PD-L1 and macrophages, suggests that combinatorial treatment might be beneficial. Synopsis: Our WOO study demonstrates that preoperative olaparib results in a reduction in Ki67, upregulation of PD-L1 CPS, and induction of protumor features of macrophages in HNSCC.

Whole Slide Imaging Technology and Its Applications: Current and Emerging Perspectives.

Background. Whole slide imaging (WSI) represents a paradigm shift in pathology, serving as a necessary first step for a wide array of digital tools to enter the field. It utilizes virtual microscopy wherein glass slides are converted into digital slides and are viewed by pathologists by automated image analysis. Its impact on pathology workflow, reproducibility, dissemination of educational material, expansion of service to underprivileged areas, and institutional collaboration exemplifies a significant innovative movement. The recent US Food and Drug Administration approval to WSI for its use in primary surgical pathology diagnosis has opened opportunities for wider application of this technology in routine practice. Main Text. The ongoing technological advances in digital scanners, image visualization methods, and the integration of artificial intelligence-derived algorithms with these systems provide avenues to exploit its applications. Its benefits are innumerable such as ease of access through the internet, avoidance of physical storage space, and no risk of deterioration of staining quality or breakage of slides to name a few. Although the benefits of WSI to pathology practices are many, the complexities of implementation remain an obstacle to widespread adoption. Some barriers including the high cost, technical glitches, and most importantly professional hesitation to adopt a new technology have hindered its use in routine pathology. Conclusions. In this review, we summarize the technical aspects of WSI, its applications in diagnostic pathology, training, and research along with future perspectives. It also highlights improved understanding of the current challenges to implementation, as well as the benefits and successes of the technology. WSI provides a golden opportunity for pathologists to guide its evolution, standardization, and implementation to better acquaint them with the key aspects of this technology and its judicial use. Also, implementation of routine digital pathology is an extra step requiring resources which (currently) does not usually result increased efficiency or payment.

Pleomorphic Dermal Sarcoma With Metastasis to the Lung: A Case Report.

ABSTRACT: Atypical fibroxanthoma and pleomorphic dermal sarcoma (PDS) are dermal malignant mesenchymal tumors that lie at the ends of the same disease spectrum. Clinically indistinguishable from atypical fibroxanthoma, PDS has a more aggressive course with significantly higher rate of local recurrence and metastases. Histological findings that favor a PDS include subcutaneous invasion, tumor necrosis, lymphovascular invasion, and/or perineural infiltration. Herein, we report a case of PDS with metastasis to the lung. Our report highlights the risk of local recurrence and metastatic spread in this cutaneous tumor and the importance of distinguishing this entity from its less aggressive counterpart.

Comprehensive molecular profile of primary cutaneous epithelioid rhabdomyosarcoma: A tumor genomically and molecularly related to malignant melanoma.

The histogenesis of the rare primary cutaneous epithelioid rhabdomyosarcoma (PCERMS) remains unclear, with the morphological and immunophenotypic appearance of a rhabdomyosarcoma but a genomic profile consistent with sarcomatoid undifferentiated malignant melanoma (SUMM). Here, we provide comprehensive clinical, histopathological, and genomic analysis of a putative PCERMS presenting in an elderly patient. Histopathologic examination revealed an ulcerative tumefactive lesion with diffuse replacement of the dermis by sheets of malignant epithelioid cells with a rhabdoid appearance. By immunohistochemistry, the tumor cells were strongly and diffusely positive for desmin and myogenin. Comprehensive genomic analysis with a 542 gene DNA-based sequencing panel revealed likely biallelic NF1 inactivation (mutation and deletion), TERT promoter mutation, and a high tumor mutation burden (>100 mutations/mB) with features of a UV-mutational signature, which are all genomic features that can be seen in undifferentiated malignant melanoma. This case provides evidence of a close relationship at a molecular level between PCERMS and SUMM. Molecular genomic characterization of a larger cohort of PCERMS is warranted for further elucidation.

Image analysis-based tumor infiltrating lymphocytes measurement predicts breast cancer pathologic complete response in SWOG S0800 neoadjuvant chemotherapy trial.

We assessed the predictive value of an image analysis-based tumor-infiltrating lymphocytes (TILs) score for pathologic complete response (pCR) and event-free survival in breast cancer (BC). About 113 pretreatment samples were analyzed from patients with stage IIB-IIIC HER-2-negative BC randomized to neoadjuvant chemotherapy ± bevacizumab. TILs quantification was performed on full sections using QuPath open-source software with a convolutional neural network cell classifier (CNN11). We used easTILs% as a digital metric of TILs score defined as [sum of lymphocytes area (mm2)/stromal area(mm2)] × 100. Pathologist-read stromal TILs score (sTILs%) was determined following published guidelines. Mean pretreatment easTILs% was significantly higher in cases with pCR compared to residual disease (median 36.1 vs.14.8%, p < 0.001). We observed a strong positive correlation (r = 0.606, p < 0.0001) between easTILs% and sTILs%. The area under the prediction curve (AUC) was higher for easTILs% than sTILs%, 0.709 and 0.627, respectively. Image analysis-based TILs quantification is predictive of pCR in BC and had better response discrimination than pathologist-read sTILs%.

Digital Spatial Profiling Links Beta-2-microglobulin Expression with Immune Checkpoint Blockade Outcomes in Head and Neck Squamous Cell Carcinoma.

Programmed cell death protein-1 (PD-1)-targeted immunotherapy is approved for recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC) treatment. Although its efficacy correlates with PD-L1 expression, response is limited even among positive cases. We employed digital spatial profiling (DSP) to discover potential biomarkers of immunotherapy outcomes in HNSCC. Fifty prospectively collected, pretreatment biopsy samples from patients with anti-PD-1-treated R/M HNSCC, were assessed using DSP, for 71 proteins in four molecularly defined compartments (tumor, leukocyte, macrophage, and stroma). Markers were evaluated for associations with progression-free (PFS) and overall survival (OS). High beta-2 microglobulin (B2M), LAG-3, CD25, and 4-1BB in tumor; high B2M, CD45, CD4 in stroma, and low fibronectin in the macrophage compartment, correlated with prolonged PFS. Improved PFS and OS were observed for cases with high B2M by quantitative and mRNA. Findings were validated in an independent cohort for PFS (HR, 0.41; 95% confidence interval, 0.19-0.93; P = 0.034). B2M-high tumors showed enrichment with immune cell and immune checkpoint markers. Our study illustrates B2M expression is associated with improved survival for immune checkpoint inhibitor (ICI)-treated HNSCC. Significance: In the current study, DSP revealed the positive association of B2M expression in the tumor compartment with immunotherapy outcomes in R/M HNSCC.

Quantitative, Spatially Defined Expression of Leukocyte-associated Immunoglobulin-like Receptor in Non-small Cell Lung Cancer.

Targeting the interaction of leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) and its ligands has been shown to reinstate antitumor immunity. In addition, the introduction of the LAIR-1 decoy protein, LAIR-2, sensitizes previously resistant lung tumors to programmed death-1 (PD-1) blockade, indicating the potential of LAIR-1 as an alternative marker for anti-PD-1 resistance in lung cancer. Here, we assessed LAIR-1 as compared with programmed death-ligand 1 (PD-L1) expression in various tumors, with a focus on non-small cell lung cancer (NSCLC) and its histologic subtypes using multiplexed quantitative immunofluorescence (mQIF) in 287 (discovery cohort) and 144 (validation cohort) patients with NSCLC. In addition, using multispectral imaging technology on mQIF images, we evaluated the localization of LAIR-1 on various cell types. We observed that CD14+, CD68+, and CD163+ monocytes and CK+ tumor cells predominantly expressed LAIR-1 more than other cell types. Furthermore, LAIR-1 expression in the tumor compartment was significantly higher in patients with lung adenocarcinoma (LUAD) than those with lung squamous cell carcinoma subtype (**, P = 0.003). Our results indicated that high tumor LAIR-1 expression in patients with LUAD is negatively associated with OS (overall survival, HR = 2.4; *, P = 0.02) highlighting its prognostic value in LUAD but not in other subtypes. The Pearson correlation between LAIR-1 and PD-L1 is 0.31; however, mutual exclusive staining pattern (i.e., several cases were positive for LAIR-1 and negative for PD-L1) was observed. Altogether, our data suggest that the combination therapy of anti-PD-1/PD-L1 with anti-LAIR-1 or the anti-LAIR-1 monotherapy alone may be promising cancer immunotherapeutic strategies. Significance: The spatial, quantitative assessment of LAIR-1 in NSCLC shows positive association of OS with high LAIR-1+/CD68+ cell densities and negative association of OS with high LAIR-1 expression in LUAD tumor subtype.

HER2 Gene Protein Assay: A Robust Tool for Evaluating HER2 Status and Intratumoral Heterogeneity in Endometrial Cancers.

OBJECTIVES: Human epidermal growth factor receptor 2 (HER2) status in endometrial cancer is usually determined by immunohistochemistry and/or in situ hybridization. We employed a novel HER2 gene protein assay (GPA) to simultaneously assesses HER2 gene amplification and protein expression in high-grade endometrial cancers. METHODS: We performed GPA in 180 endometrial cancers, including 106 serous carcinomas, 34 carcinosarcomas, and 40 mixed epithelial carcinomas. HER2 status was determined using the 2018 HER2 guidelines for breast carcinoma, and HER2 intratumoral heterogeneity (ITH) was examined. Clinicopathologic characteristics were collected and correlated with HER2 status. RESULTS: HER2 positivity was noted in 32% of serous carcinomas, significantly higher than in carcinosarcomas (5.9%) and mixed carcinomas (12.5%). HER2 ITH was detected in 32% of serous carcinomas, significantly greater than in carcinosarcomas (8.8%) and mixed carcinomas (10%). Patients with carcinosarcoma had a significantly lower overall survival than patients with serous or mixed epithelial carcinoma, but HER2 status caused no difference in survival in patients with serous carcinoma. CONCLUSIONS: HER2 GPA can be used to accurately determine HER2 status in endometrial cancers and is a highly valuable tool for identifying HER2 heterogeneity.

Wnt Family Member 9b (Wnt9b) Is a Sensitive and Specific Marker for Triple-negative Breast Carcinoma Including Metaplastic Carcinoma.

Wnt9b was recently identified as a highly sensitive and specific marker for breast carcinomas. Due to the limited number of triple-negative breast carcinomas (TNBCs) in previous study, we further explored Wnt9b's utility in breast carcinoma, especially in TNBCs including metaplastic carcinomas. We systematically evaluated Wnt9b expression on tissue microarrays (TMAs) from 413 breast carcinomas, 208 urothelial carcinomas, 102 endometrial carcinomas, 109 cholangiocarcinomas, 192 ovarian carcinomas, 48 lung adenocarcinomas, 69 colorectal adenocarcinomas, and 78 melanomas, and whole tissue section (WTS) from 20 human epidermal growth factor receptor 2-positive, 34 nonmetaplastic TNBCs, and 67 invasive metaplastic carcinomas. The results showed Wnt9b was highly expressed in breast carcinomas (91% on TMA and 98% on WTS) and in nonmetaplastic TNBCs (91% on TMA and 97% on WTS), but almost completely negative in other tested tumor types. Wnt9b was also highly expressed in metaplastic carcinomas (80%), significantly higher than GATA3 (56%) and SOX10 (48%), but slightly lower than TRPS1 (90%). In summary, our results demonstrate that Wnt9b is a highly sensitive marker for breast carcinomas, including TNBCs and metaplastic carcinomas. Further, we compared its utility with other breast markers including TRPS1, GATA3, and SOX10 in metaplastic carcinomas.

Baseline gene expression profiling determines long-term benefit to programmed cell death protein 1 axis blockade.

Treatment with immune checkpoint inhibitors has altered the course of malignant melanoma, with approximately half of the patients with advanced disease surviving for more than 5 years after diagnosis. Currently, there are no biomarker methods for predicting outcome from immunotherapy. Here, we obtained transcriptomic information from a total of 105 baseline tumor samples comprising two cohorts of patients with advanced melanoma treated with programmed cell death protein 1 (PD-1)-based immunotherapies. Gene expression profiles were correlated with progression-free survival (PFS) within consecutive clinical benefit intervals (i.e., 6, 12, 18, and 24 months). Elastic net binomial regression models with cross validation were utilized to compare the predictive value of distinct genes across time. Lasso regression was used to generate a signature predicting long-term benefit (LTB), defined as patients who remain alive and free of disease progression at 24 months post treatment initiation. We show that baseline gene expression profiles were consistently able to predict long-term immunotherapy outcomes with high accuracy. The predictive value of different genes fluctuated across consecutive clinical benefit intervals, with a distinct set of genes defining benefit at 24 months compared to earlier outcomes. A 12-gene signature was able to predict LTB following anti-PD-1 therapy with an area under the curve (AUC) equal to 0.92 and 0.74 in the training and validation set, respectively. Evaluation of LTB, via a unique signature may complement objective response classification and characterize the logistics of sustained antitumor immune responses.

Integrating and validating automated digital imaging analysis of estrogen receptor immunohistochemistry in a fully digital workflow for clinical use.

Background: The Visiopharm automated estrogen receptor (ER) digital imaging analysis (DIA) algorithm assesses digitized ER immunohistochemistry (IHC) by segmenting tumor nuclei and detecting stained nuclei automatically. We aimed to integrate and validate this algorithm in a digital pathology workflow for clinical use. Design: The study cohort consisted of a serial collection of 97 invasive breast carcinoma specimens including 73 biopsies and 24 resections. ER IHC slides were scanned into Philips Image Management System (IMS) during our routine digital workflow and digital images were directly streamed into Visiopharm platform and analyzed using automated ER algorithm to obtain the positively stained tumor nuclei and staining intensity. ER DIA scores were compared with pathologists' manual scores. Results: The overall concordance between pathologists' reads and DIA reads was excellent (91/97, 93.8%). Pearson Correlation Coefficient of the percentage of ER positive nuclei between the original reads and VIS reads was 0.72. Six cases (3 ER-negative and 3 ER-positive) had discordant results. All 3 false negative cases had very weak ER staining and no more than 10% positivity. The causes for false positive DIA were mainly pre-analytic/pre-imaging and included intermixed benign glands in tumor area, ductal carcinoma in-situ (DCIS) components, and tissue folding. Conclusions: Automated ER DIA demonstrates excellent concordance with pathologists' scores and accurately discriminates ER positive from negative cases. Furthermore, integrating automated biomarker DIA into a busy clinical digital workflow is feasible and may save time and labor for pathologists.

Comprehensive analysis of clinicopathologic features and p53 mutation in neuroendocrine neoplasms of the breast: experience from a large academic center.

PURPOSE: The recent WHO classification of breast cancer (2019) categorizes breast carcinoma with neuroendocrine (NE) differentiation into three morphologically distinct subtypes: well-differentiated neuroendocrine tumor (NET), poorly differentiated neuroendocrine carcinoma (NEC), and invasive breast carcinoma, no special type with neuroendocrine differentiation (IBC-NST-NE). Data regarding the prognostic significance of neuroendocrine differentiation are conflicting and an association, if any, between p53 mutation and neuroendocrine differentiation is largely unknown. METHODS: We examined p53 expression and other clinicopathologic characteristics in three types of invasive breast carcinoma with NE differentiation in a cohort of sixty-three patients, including 45 IBC-NST with NE differentiation, 10 NETs, and 8 NECs. RESULTS: No significant difference of clinicopathologic feature was observed between IBC-NST with NE differentiation and NET, but NECs showed significantly lower expressions of hormone receptors, more mutated p53, and higher frequency of distant metastases than IBC-NST with NE differentiation and NETs. CONCLUSION: NECs of the breast are genetically and clinically different from IBC-NST-NEs and NETs of the breast.

Objective assessment of tumor infiltrating lymphocytes as a prognostic marker in melanoma using machine learning algorithms.

BACKGROUND: The prognostic value of tumor-infiltrating lymphocytes (TILs) assessed by machine learning algorithms in melanoma patients has been previously demonstrated but has not been widely adopted in the clinic. We evaluated the prognostic value of objective automated electronic TILs (eTILs) quantification to define a subset of melanoma patients with a low risk of relapse after surgical treatment. METHODS: We analyzed data for 785 patients from 5 independent cohorts from multiple institutions to validate our previous finding that automated TIL score is prognostic in clinically-localized primary melanoma patients. Using serial tissue sections of the Yale TMA-76 melanoma cohort, both immunofluorescence and Hematoxylin-and-Eosin (H&E) staining were performed to understand the molecular characteristics of each TIL phenotype and their associations with survival outcomes. FINDINGS: Five previously-described TIL variables were each significantly associated with overall survival (p<0.0001). Assessing the receiver operating characteristic (ROC) curves by comparing the clinical impact of two models suggests that etTILs (electronic total TILs) (AUC: 0.793, specificity: 0.627, sensitivity: 0.938) outperformed eTILs (AUC: 0.77, specificity: 0.51, sensitivity: 0.938). We also found that the specific molecular subtype of cells representing TILs includes predominantly cells that are CD3+ and CD8+ or CD4+ T cells. INTERPRETATION: eTIL% and etTILs scores are robust prognostic markers in patients with primary melanoma and may identify a subgroup of stage II patients at high risk of recurrence who may benefit from adjuvant therapy. We also show the molecular correlates behind these scores. Our data support the need for prospective testing of this algorithm in a clinical trial. FUNDING: This work was also supported by a sponsored research agreements from Navigate Biopharma and NextCure and by grants from the NIH including the Yale SPORE in in Skin Cancer, P50 CA121974, the Yale SPORE in Lung Cancer, P50 CA196530, NYU SPORE in Skin Cancer P50CA225450 and the Yale Cancer Center Support Grant, P30CA016359.