High tumour-infiltrating lymphocytes correlate with distinct gene expression profile and favourable survival in single hormone receptor-positive breast cancer.

Introduction: This study aimed to evaluate the impact of tumour-infiltrating lymphocytes (TILs) on the expression of immune-related genes and prognosis in single hormone receptor-positive breast cancer. Material and methods: Tumour-infiltrating lymphocytes were analysed according to the guidelines of the International TILs Working Group in a cohort of 206 patients with single hormone receptor-positive breast cancer. Of these, 44.7% were classified as ER+/PgR-/HER2-, 18.4% as ER+/PgR-/HER2+, 26.2% as ER-/PgR+/HER2-, and 10.7% as ER-/PgR+/HER2+. Moreover, in 52 samples the analysis of gene expression profiling was performed using nCounter technology. Results: Most cases (74.3%) showed at least 1% of stromal TILs, with a median of 4%, mean of 16.3%, and interquartile range of 0-20%. ER-/PgR+ tumours displayed significantly higher TILs density than ER+/PgR- cases (p < 0.001, Wilcoxon test), regardless of HER2 status. The abundance of TILs was positively associated with ER-/PgR+ phenotype, higher Ki-67, and higher grade, but not with age, tumour size, or regional and distant metastases at diagnosis. Additionally, in ER+/PgR- subgroup higher TILs were associated with HER2-positive status. Stromal TILs > 5% were associated with better survival in the whole group, but this effect was less prominent in ER-/PgR+ patients. We identified 50 differentially expressed genes (DEGs) between single hormone receptor-positive breast tumours with high and low TILs, including 39 up-regulated and 11 down-regulated genes in the high TILs group. Conclusions: The up-regulated expression of immune-related genes was consistent also among separately analysed single hormone receptor-positive groups (ER+/PgR- and ER-/PgR+).

Loss of Y in regulatory T lymphocytes in the tumor micro-environment of primary colorectal cancers and liver metastases.

Male sex is a risk factor for colorectal cancer (CRC) with higher illness burden and earlier onset. Thus, we hypothesized that loss of chromosome Y (LOY) in the tumor micro-environment (TME) might be involved in oncogenesis. Previous studies show that LOY in circulating leukocytes of aging men was associated with shorter survival and non-hematological cancer, as well as higher LOY in CD4 + T-lymphocytes in men with prostate cancer vs. controls. However, nothing is known about LOY in leukocytes infiltrating TME and we address this aspect here. We studied frequency and functional effects of LOY in blood, TME and non-tumorous tissue. Regulatory T-lymphocytes (Tregs) in TME had the highest frequency of LOY (22%) in comparison to CD4 + T-lymphocytes and cytotoxic CD8 + T-lymphocytes. LOY score using scRNA-seq was also linked to higher expression of PDCD1, TIGIT and IKZF2 in Tregs. PDCD1 and TIGIT encode immune checkpoint receptors involved in the regulation of Tregs function. Our study sets the direction for further functional research regarding a probable role of LOY in intensifying features related to the suppressive phenotype of Tregs in TME and consequently a possible influence on immunotherapy response in CRC patients.

Characterization of Pleural Mesothelioma by Hierarchical Clustering Analyses Using Immune Cells within Tumor Microenvironment.

INTRODUCTION: Over the past decade, classifications using immune cell infiltration have been applied to many types of tumors; however, mesotheliomas have been less frequently evaluated. METHODS: In this study, 60 well-characterized pleural mesotheliomas (PMs) were evaluated immunohistochemically for the characteristics of immune cells within tumor microenvironment (TME) using 10 immunohistochemical markers: CD3, CD4, CD8, CD56, CD68, CD163, FOXP3, CD27, PD-1, and TIM-3. For further characterization of PMs, hierarchical clustering analyses using these 10 markers were performed. RESULTS: Among the immune cell markers, CD3 (p < 0.0001), CD4 (p = 0.0016), CD8 (p = 0.00094), CD163+ (p = 0.042), and FOXP3+ (p = 0.025) were significantly associated with an unfavorable clinical outcome. Immune checkpoint receptor expressions on tumor-infiltrating lymphocytes such as PD-1 (p = 0.050), CD27 (p = 0.014), and TIM-3 (p = 0.0098) were also associated with unfavorable survival. Hierarchical clustering analyses identified three groups showing specific characteristics and significant associations with patient survival (p = 0.016): the highest number of immune cells (ICHigh); the lowest number of immune cells, especially CD8+ and CD163+ cells (ICLow); and intermediate number of immune cells (ICInt). ICHigh tumors showed significantly higher expression of PD-L1 (p = 0.00038). Cox proportional hazard model identified ICHigh [hazard ratio (HR) = 2.90] and ICInt (HR = 2.97) as potential risk factors compared with ICLow. Tumor CD47 (HR = 2.36), tumor CD70 (HR = 3.04), and tumor PD-L1 (HR = 3.21) expressions were also identified as potential risk factors for PM patients. CONCLUSION: Our findings indicate immune checkpoint and/or immune cell-targeting therapies against CD70-CD27 and/or CD47-SIRPA axes may be applied for PM patients in combination with PD-L1-PD-1 targeting therapies in accordance with their tumor immune microenvironment characteristics.

Tumor Predisposing Post-Zygotic Chromosomal Alterations in Bladder Cancer-Insights from Histologically Normal Urothelium.

Bladder urothelial carcinoma (BLCA) is the 10th most common cancer with a low survival rate and strong male bias. We studied the field cancerization in BLCA using multi-sample- and multi-tissue-per-patient protocol for sensitive detection of autosomal post-zygotic chromosomal alterations and loss of chromosome Y (LOY). We analysed 277 samples of histologically normal urothelium, 145 tumors and 63 blood samples from 52 males and 15 females, using the in-house adapted Mosaic Chromosomal Alterations (MoChA) pipeline. This approach allows identification of the early aberrations in urothelium from BLCA patients. Overall, 45% of patients exhibited at least one alteration in at least one normal urothelium sample. Recurrence analysis resulted in 16 hotspots composed of either gains and copy number neutral loss of heterozygosity (CN-LOH) or deletions and CN-LOH, encompassing well-known and new BLCA cancer driver genes. Conservative assessment of LOY showed 29%, 27% and 18% of LOY-cells in tumors, blood and normal urothelium, respectively. We provide a proof of principle that our approach can characterize the earliest alterations preconditioning normal urothelium to BLCA development. Frequent LOY in blood and urothelium-derived tissues suggest its involvement in BLCA.

Primary Cutaneous CD30-Positive Lymphoproliferative Disorders-Current Therapeutic Approaches with a Focus on Brentuximab Vedotin.

One of the most common subgroups of cutaneous T-cell lymphomas is that of primary cutaneous CD30-positive lymphoproliferative disorders. The group includes lymphomatoid papulosis (LyP) and primary cutaneous anaplastic large cell lymphoma (pcALCL), as well as some borderline cases. Recently, significant progress has been made in understanding the genetics and treatment of these disorders. This review article summarises the clinical evidence supporting the current treatment options for these diseases. Recent years have seen the introduction of novel agents into clinical practice; most of these target CD30, such as anti-CD30 monoclonal antibodies and conjugated antibodies (brentuximab vedotin), bispecific antibodies and cellular therapies, particularly anti-CD30 CAR-T cells. This paper briefly reviews the biology of CD30 that makes it a good therapeutic target and describes the anti-CD30 therapies that have emerged to date.

Great mimicker - a case of occult melanoma displaying ganglioneuroblastic differentiation.

Melanomas are known for their diverse morphological features, presenting a diagnostic challenge for pathologists. Uncommon variations of melanoma can exhibit distinct cytological and histomorphological characteristics, including ganglioneuroblastic differentiation. However, this phenomenon is extremely rare, with only a few documented cases. Here, we present a unique case of an occult metastatic melanoma with ganglioneuroblastic differentiation developing in a 76-year-old male. The diagnosis was based on histopathology, immunophenotyping, and molecular testing, which revealed SOX10 positivity and an NRAS mutation. Notably, classic melanoma markers HMB45 and melan-A were negative, highlighting the importance of considering alternative markers. This case emphasizes the significance of immunohistochemistry and molecular investigation in diagnosing melanomas with unusual features and identifying appropriate candidates for immune checkpoint therapy. Additionally, the occurrence of ganglioneuroblastic differentiation further supports a shared histogenetic origin from the neural crest. Improved understanding of such rare variants contributes to accurate diagnosis and optimal management of melanoma patients.

Prostate Cancer and Its Mimics-A Pictorial Review.

BACKGROUND: Multiparametric prostate MRI (mpMRI) is gaining wider recommendations for diagnosing and following up on prostate cancer. However, despite the high accuracy of mpMRI, false positive and false negative results are reported. Some of these may be related to normal anatomic structures, benign lesions that may mimic cancer, or poor-quality images that hamper interpretation. The aim of this review is to discuss common potential pitfalls in the interpretation of mpMRI. METHODS: mpMRI of the prostates was performed on 3T MRI scanners (Philips Achieva or Siemens Magnetom Vida) according to European Society of Urogenital Radiology (ESUR) guidelines and technical requirements. RESULTS: This pictorial review discusses normal anatomical structures such as the anterior fibromuscular stroma, periprostatic venous plexus, central zone, and benign conditions such as benign prostate hyperplasia (BPH), post-biopsy hemorrhage, prostatitis, and abscess that may imitate prostate cancer, as well as the appearance of prostate cancer occurring in these locations. Furthermore, suggestions on how to avoid these pitfalls are provided, and the impact of image quality is also discussed. CONCLUSIONS: In an era of accelerating prostate mpMRI and high demand for high-quality interpretation of the scans, radiologists should be aware of these potential pitfalls to improve their diagnostic accuracy.

Prognostic value of ALK overexpression and molecular abnormalities in high-grade serous ovarian carcinoma.

BACKGROUND: ALK receptor tyrosine kinase (ALK) aberrations have an established role in pathogenesis of many neoplasms, but their clinical significance in high grade serous ovarian carcinoma (HGSOC) is unclear. OBJECTIVE: To analyse the frequency of ALK overexpression, molecular abnormalities of ALK, and their impact on the progression-free survival (PFS) and overall survival (OS) in HGSOC. METHODS: Protein expression was examined by immunohistochemistry (IHC) using three different clones of anti-ALK antibody. The presence of translocations was analysed using fluorescent in situ hybridization. Next-generation sequencing was used for studying the copy number variation, as well as point mutation and translocations involving other commonly rearranged genes. RESULTS: ALK overexpression was demonstrated in up to 52% of tumours, whereas ALK copy gains in 8.2%, with no clear impact on survival. ALK point mutations were identified in 13 tumours (8.9%), with 3 belonging to the class IV showing significantly better OS. A trend suggesting better PFS was also noticed in these cases. Additionally, three gene fusions were found: ERBB2-GRB7, PRKCA-BRCA1 and SND1-BRAF, none of which has been previously described in HGSOC. CONCLUSIONS: HGSOC harbouring activating ALK mutations might be associated with a better survival, while ALK overexpression and ALK amplification does not impact the prognosis.

Local T cell infiltrates are predominantly associated with corneal allograft rejection.

BACKGROUND: Corneal transplantations (CTXs) are a vision-saving procedure. Routinely, while CTXs' survival rates remain high, the risk of graft failure increases significantly for repeated CTXs. The reason is an alloimmunization following previous CTXs and development of memory T (Tm) and B (Bm) cells. METHODS: We characterized populations of cells present in explanted human corneas from patients receiving the first CTX and marked as a primary CTX (PCTX) or the second or more CTXs and marked as a repeated CTX (RCTX). Cells extracted from resected corneas and from peripheral blood mononuclear cells (PBMCs) were analyzed by the flow cytometry method using multiple surface and intracellular markers. RESULTS: Overall, the number of cells was similar in PCTX and RCTX patients. Extracted infiltrates from PCTXs and RCTXs contained similar numbers of T cell subsets, namely CD4+, CD8+, CD4+ Tm, CD8+ Tm, CD4+Foxp3+ T regulatory (Tregs), CD8+ Treg cells, while very few B cells (all p = NS). However, when compared to peripheral blood, PCTX and RCTX corneas contained significantly higher percentages of effector memory CD4+ and CD8+ T cells (both p < 0,05). In comparison to PCTX, RCTX group had the highest levels of Foxp3 in T CD4+ Tregs (p = 0,04) but decreased percentage of Helios-positive CD4+ Tregs. CONCLUSION: PCTXs and especially RCTXs are rejected mainly by local T cells. The accumulation of effector CD4+ and CD8+ T cells, as well as CD4+ and CD8+ Tm cells is associated with the final rejection. Furthermore, local CD4+ and CD8+ Tregs expressing Foxp3 and Helios are probably insufficient to impose the acceptance of CTX.

Assessment of Cytological Changes in the Oral Mucosa in Young Hematological Patients Treated with Systemic Chemotherapy.

BACKGROUND AND OBJECTIVES: The primary objective of the undertaken study was to determine the morphological changes that occur within the oral epithelium in children undergoing chemotherapy following a diagnosis of hematological malignancies. MATERIALS AND METHODS: The study group consisted of 18 patients diagnosed with leukemia or lymphoma undergoing treatment with chemotherapy. Swabs (liquid-based cytology) were collected from the oral cavity for microscopic evaluation at baseline, during the chemotherapy cycle with oral mucositis symptoms present, and upon completion of the cycle. Both the neutrophil count and oral mucositis (OM) were registered using the WHO (World Health Organization) scale. The control group included 41 children who were generally healthy. All samples underwent microscopical analyses at the Department of Pathology, Medical University of Gdansk, Poland. RESULTS: A total of 190 cytological preparations were evaluated. The baseline preparations revealed similar cytological images, and the superficial cells of the epithelial layers were seen. A significant (p < 0.01) increase in the number of cells in the intermediate layer of the oral epithelium, as well as a decrease (p < 0.01) in the volume of cells in the superficial layers, was observed in further stages of cytostatic treatment. CONCLUSIONS: A decrease in the percentage of superficial epithelial cells with a corresponding increase in the number of intermediate epithelial cells is considered to be a result of toxic damage to the oral mucosa during chemotherapy.

Salivary Gland Secretory Carcinoma: Clinicopathologic and Genetic Characteristics of 215 Cases and Proposal for a Grading System.

Salivary gland secretory carcinoma (SC), previously mammary analog SC, is a low-grade malignancy characterized by well-defined morphology and an immunohistochemical and genetic profile identical to SC of the breast. Translocation t(12;15)(p13;q25) resulting in the ETV6 :: NTRK3 gene fusion is a characteristic feature of SC along with S100 protein and mammaglobin immunopositivity. The spectrum of genetic alterations for SC continues to evolve. The aim of this retrospective study was to collect data of salivary gland SCs and to correlate their histologic, immunohistochemical, and molecular genetic data with clinical behavior and long-term follow-up. In this large retrospective study, we aimed to establish a histologic grading scheme and scoring system. A total of 215 cases of salivary gland SCs diagnosed between 1994 and 2021 were obtained from the tumor registries of the authors. Eighty cases were originally diagnosed as something other than SC, most frequently acinic cell carcinoma. Lymph node metastases were identified in 17.1% (20/117 cases with available data), with distant metastasis in 5.1% (6/117). Disease recurrence was seen in 15% (n=17/113 cases with available data). The molecular genetic profile showed ETV6 :: NTRK3 gene fusion in 95.4%, including 1 case with a dual fusion of ETV6 :: NTRK3 and MYB :: SMR3B . Less frequent fusion transcripts included ETV6 :: RET (n=12) and VIM :: RET (n=1). A 3-tiered grading scheme using 6 pathologic parameters (prevailing architecture, pleomorphism, tumor necrosis, perineural invasion (PNI), lymphovascular invasion (LVI), and mitotic count and/or Ki-67 labeling index) was applied. Grade 1 histology was observed in 44.7% (n=96), grade 2 in 41.9% (n=90), and grade 3 in 13.5% (n=29) of cases. Compared with low-grade and intermediate-grade SC, high-grade tumors were associated with a solid architecture, more prominent hyalinization, infiltrative tumor borders, nuclear pleomorphism, presence of PNI and/or LVI, and Ki-67 proliferative index >30%. High-grade transformation, a subset of grade 2 or 3 tumors, seen in 8.8% (n=19), was defined as an abrupt transformation of conventional SC into high-grade morphology, sheet-like growth, and a tumor lacking distinctive features of SC. Both overall survival and disease-free survival (5 and 10 y) were negatively affected by tumor grade, stage, and TNM status (each P <0.0001). SC is a low-grade malignancy with predominantly solid-microcystic growth patterns, driven by a gene fusion, most commonly ETV6 :: NTRK3 . There is a low risk for local recurrence and a good overall long-term survival, with a low risk for distant metastasis but a higher risk for locoregional lymph node metastasis. The presence of tumor necrosis, hyalinization, PNI and/or LVI, and positive resection margins correlate with higher tumor grade, less favorable prognosis, and increased mortality. The statistical results allowed us to design a 3-tiered grading system for salivary SC.

Diagnostic test accuracy meta-analysis of PRAME in distinguishing primary cutaneous melanomas from benign melanocytic lesions.

PRAME is a novel immunohistochemical marker that aids the diagnosis of melanocytic lesions. Diffuse PRAME positivity suggests melanoma, whereas benign naevi are negative or only weakly positive. However, the factual diagnostic accuracy of PRAME is not well established. Moreover, some studies have suggested that the threshold of 3+/50% positive cells may be more useful in practice than the most widely used cut-off (4+/75% of positive cells). Hence, we performed a systematic review and diagnostic accuracy meta-analysis to evaluate sensitivity, specificity, likelihood ratios and optimal threshold for PRAME in distinguishing benign melanocytic proliferations from melanomas. Twenty-six studies were enrolled into the meta-analysis. A total of 2915 melanocytic lesions were analysed. The optimal threshold for PRAME positivity was estimated at 3.11, which translates into 3+ in practice. Sensitivity and specificity calculated from SROC at the 3+ threshold were 0.735 (0.631-0.818) and 0.915 (0.834-0.958), respectively, compared to 0.679 (0.559-0.957) and 0.957 (0.908-0.981) at the 4+ cut-off. In subgroup analysis, the spitzoid subgroup was characterised by the lowest sensitivity and diagnostic odds ratio of PRAME. Our findings indicate that PRAME immunohistochemistry may serve as an ancillary marker to support the diagnosis of melanoma. Nevertheless, the accuracy of PRAME may be lower in spitzoid neoplasms. Our meta-analysis suggests that the 3+/50% threshold might be more useful in practice than the 4+/75% cut-off, as it shows higher sensitivity with retained satisfactory specificity.

Comparison of Three Transcytotic Pathways for Distribution to Brain Metastases of Breast Cancer.

Advances in drug treatments for brain metastases of breast cancer have improved progression-free survival but new, more efficacious strategies are needed. Most chemotherapeutic drugs infiltrate brain metastases by moving between brain capillary endothelial cells, paracellular distribution, resulting in heterogeneous distribution, lower than that of systemic metastases. Herein, we tested three well-known transcytotic pathways through brain capillary endothelial cells as potential avenues for drug access: transferrin receptor (TfR) peptide, low-density lipoprotein receptor 1 (LRP1) peptide, albumin. Each was far-red labeled, injected into two hematogenous models of brain metastases, circulated for two different times, and their uptake quantified in metastases and uninvolved (nonmetastatic) brain. Surprisingly, all three pathways demonstrated distinct distribution patterns in vivo. Two were suboptimal: TfR distributed to uninvolved brain but poorly in metastases, while LRP1 was poorly distributed. Albumin distributed to virtually all metastases in both model systems, significantly greater than in uninvolved brain (P < 0.0001). Further experiments revealed that albumin entered both macrometastases and micrometastases, the targets of treatment and prevention translational strategies. Albumin uptake into brain metastases was not correlated with the uptake of a paracellular probe (biocytin). We identified a novel mechanism of albumin endocytosis through the endothelia of brain metastases consistent with clathrin-independent endocytosis (CIE), involving the neonatal Fc receptor, galectin-3, and glycosphingolipids. Components of the CIE process were found on metastatic endothelial cells in human craniotomies. The data suggest a reconsideration of albumin as a translational mechanism for improved drug delivery to brain metastases and possibly other central nervous system (CNS) cancers.In conclusion, drug therapy for brain metastasis needs improvement. We surveyed three transcytotic pathways as potential delivery systems in brain-tropic models and found that albumin has optimal properties. Albumin used a novel endocytic mechanism.

CD70 and PD-L1 (CD274) co-expression predicts poor clinical outcomes in patients with pleural mesothelioma.

Diffuse pleural mesothelioma (PM) is a highly aggressive tumour typically associated with short survival. Recently, the effectiveness of first-line immune checkpoint inhibitors in patients with unresectable PM was reported. CD70-CD27 signalling plays a co-stimulatory role in promoting T cell expansion and differentiation through the nuclear factor κB (NF-κB) pathway. Conversely, the PD-L1 (CD274)-PD-1 (PDCD1) pathway is crucial for the modulation of immune responses in normal conditions. Nevertheless, pathological activation of both the CD70-CD27 and PD-L1-PD-1 pathways by aberrantly expressed CD70 and PD-L1 participates in the immune evasion of tumour cells. In this study, 171 well-characterised PMs including epithelioid (n = 144), biphasic (n = 15), and sarcomatoid (n = 12) histotypes were evaluated immunohistochemically for CD70, PD-L1, and immune cell markers such as CD3, CD4, CD8, CD56, PD-1, FOXP3, CD68, and CD163. Eight percent (14/171) of mesotheliomas simultaneously expressed CD70 and PD-L1 on the tumour cell membrane. PMs co-expressing CD70 and PD-L1 contained significantly higher numbers of CD8+ (p = 0.0016), FOXP3+ (p = 0.00075), and CD163+ (p = 0.0011) immune cells within their microenvironments. Overall survival was significantly decreased in the cohort of patients with PM co-expressing CD70 and PD-L1 (p < 0.0001). In vitro experiments revealed that PD-L1 and CD70 additively enhanced the motility and invasiveness of PM cells. In contrast, PM cell proliferation was suppressed by PD-L1. PD-L1 enhanced mesenchymal phenotypes such as N-cadherin up-regulation. Collectively, these findings suggest that CD70 and PD-L1 both enhance the malignant phenotypes of PM and diminish anti-tumour immune responses. Based on our observations, combination therapy targeting these signalling pathways might be useful in patients with PM.

Analysis of BRCA1 and BRCA2 alternative splicing in predisposition to ovarian cancer.

BACKGROUND: The mRNA splicing is regulated on multiple levels, resulting in the proper distribution of genes' transcripts in each cell and maintaining cell homeostasis. At the same time, the expression of alternative transcripts can change in response to underlying genetic variants, often missed during routine diagnostics. AIM: The main aim of this study was to define the frequency of aberrant splicing in BRCA1 and BRCA2 genes in blood RNA extracted from ovarian cancer patients who were previously found negative for the presence of pathogenic alterations in the 25 most commonly analysed ovarian cancer genes, including BRCA1 and BRCA2. MATERIAL AND METHODS: Frequency and spectrum of splicing alterations in BRCA1 and BRCA2 genes were analysed in blood RNA from 101 ovarian cancer patients and healthy controls (80 healthy women) using PCR followed by gel electrophoresis and Sanger sequencing. The expression of splicing events was examined using RT-qPCR. RESULTS: We did not identify any novel, potentially pathogenic splicing alterations. Nevertheless, we detected six naturally occurring transcripts, named BRCA1ΔE9-10, BRCA1ΔE11, BRCA1ΔE11q, and BRCA2ΔE3, BRCA2ΔE12 and BRCA2ΔE17-18 of which three (BRCA1ΔE11q, BRCA1ΔE11 and BRCA2ΔE3) were significantly higher expressed in the ovarian cancer cohort than in healthy controls (p ≤ 0.0001). CONCLUSIONS: This observation indicates that the upregulation of selected naturally occurring transcripts can be stimulated by non-genetic mechanisms and be a potential systemic response to disease progression and/or treatment. However, this hypothesis requires further examination.

Assessment of RANK/RANK-L prevalence and clinical significance in NSCLC European Thoracic Oncology Platform Lungscape cohort and SPLENDOUR randomized clinical trial.

BACKGROUND: The primary objective of this study is to evaluate the clinical significance of RANK/L expression, in both a retrospective cohort of surgically resected stage I-III NSCLC (Lungscape) and a randomized clinical trial-cohort (SPLENDOUR) of advanced NSCLC treated with chemotherapy alone or in combination with denosumab. METHODS: RANK-L expression was assessed on tissue microarrays (TMAs) in Lungscape and whole sections in SPLENDOUR, using immunohistochemistry, with H-scores values > 0 indicating positivity. Prevalence of RANK positivity and its association with clinicopathological characteristics, and patient outcome was explored in a subset of the ETOP Lungscape cohort and in SPLENDOUR. Also investigated were the prevalence of RANK overexpression (proportion of positive cancer cells ≥ 50%) in the Lungscape cohort, and RANK-L in the SPLENDOUR trial. RESULTS: In the Lungscape cohort, RANK expression was assessed at a median follow-up of 46 months (N = 488 patients; 4 centers); 35% were female, 44/49/6% adenocarcinomas (AC)/squamous cell carcinomas (SCC)/other, 48/27/25% with stage I/II/III. Median RFS/TTR/OS were 58/Not reached/74 months. Prevalence of RANK expression was 31% (95%CI:27%-35%); significantly higher in AC: 50% (95%CI:43%-57%) vs SCC: 12% (95%CI:8%-16%) (p < 0.001); more frequent in females (42% vs 25%, p < 0.001) and tumors ≤ 4 cm (35.3% vs 23.3%, p = 0.0065). No association with outcome was found. In the SPLENDOUR trial (463 patients), the prevalence of membranous and cytoplasmic RANK positivity was 34% (95%CI:30%-38%) and 9% (95%CI:7%-12%), respectively, while prevalence for RANK-L was 5% (95%CI:3%-7%) and 36% (95%CI:31%-40%), respectively. Cytoplasmic RANK-L positivity was more common among females (47% vs 31%, p = 0.001) and in non-SCC histology (45% vs 10%, p < 0.0001). At the pre-specified 1% significance level, no prognostic or predictive effect was found. CONCLUSIONS: Both cohorts indicate that RANK expression is more common in adenocarcinoma/non-squamous NSCLC and in female patients. No prognostic effect is found, and in the clinical trial involving addition of denosumab to chemotherapy no predictive effect is detected.

Immune checkpoint receptor VISTA on immune cells is associated with expression of T-cell exhaustion marker TOX and worse prognosis in renal cell carcinoma with venous tumor thrombus.

PURPOSE: The study aimed to determine the expression of VISTA and TOX within venous tumor thrombus and primary clear cell renal cell carcinoma (ccRCC) and to assess their prognostic value. METHODS: The study enrolled 82 patients with ccRCC and coexisting venous tumor thrombus treated radically from 2012 to 2019 in two tertiary centers. Tissue microarrays were prepared and stained with respective antibodies. The expression of markers was assessed separately on tumor cells (TCs) and/or tumor-associated immune cells (TAICs). RESULTS: TOX expression was positively correlated with the percentage of VISTA-positive TAICs in venous thrombus (p = 0.011), but not in the primary tumor (p = 0.674). High TOX expression was associated with a higher percentage of PD-L1-positive TAICs in both compartments (p = 0.001, p = 0.011, respectively). Positive expression of VISTA on TAICs was associated with PD-L1 expression on TCs (p = 0.005) and TAICs (p = 0.004) in the primary tumor, and only with PD-L1 on TAICs in thrombus (p = 0.006). The presence of VISTA-positive TAICs in venous thrombus was significantly more common in females (p = 0.034), and positively correlated with metastases (p = 0.028), and tumor necrosis (p = 0.013). The cases with VISTA-positive TAICs in venous tumor thrombi had significantly shorter OS than VISTA-negative cases (p = 0.041). CONCLUSION: For the first time, we demonstrated the expression of VISTA- and TOX-positive TAICs in the venous tumor thrombus. We found the association between immune checkpoint receptors and T cell exhaustion markers in both tumor mass and venous thrombus. Finally, we demonstrated that abundance of VISTA-positive TAICs in venous tumor thrombus correlates with worse outcomes in ccRCC.

Diagnostically misleading aberrant terminal deoxynucleotidyl transferase expression in germ cell tumors.

Terminal deoxynucleotidyl transferase (TdT) is a unique type of DNA polymerase predominantly expressed in precursor lymphoid cells and acute lymphoblastic leukemia. It participates in the junctional diversity of T-cell receptors and immunoglobulins. Recently, aberrant TdT expression was found in seminomas. Here, we evaluated the expression of TdT in our cohort of germ cell tumors (GCTs) with two anti-TdT antibody clones. We included 173 cases of testicular GCTs, 5 ovarian dysgerminomas, and one gonadoblastoma in the study. Tissue microarrays containing representative tumor samples were constructed and subsequently stained with anti-TdT monoclonal rabbit antibody EP266 (Dako) and TdT rabbit polyclonal antibody (Cell Marque). Expression was assessed with the H-score. No specific nuclear reaction was observed for the polyclonal anti-TdT antibody. The H-score values varied between the histological subtypes for the EP266 antibody. Positive nuclear staining was consistently seen in germ cell neoplasia in situ , seminoma, dysgerminoma, and embryonal carcinoma. Pure tumors had higher TdT H-scores than the mixed ones. Teratomas, yolk sac tumors, and choriocarcinomas were almost uniformly negative. Our study confirms that aberrant expression of TdT by testicular and ovarian GCTs exemplifies a potential diagnostic pitfall in histopathological diagnostics.

Pathway-level mutation analysis in primary high-grade serous ovarian cancer and matched brain metastases.

Brain metastases (BMs) in ovarian cancer (OC) are a rare event. BMs occur most frequently in high-grade serous (HGS) OC. The molecular features of BMs in HGSOC are poorly understood. We performed a whole-exome sequencing analysis of ten matched pairs of formalin-fixed paraffin-embedded samples from primary HGSOC and corresponding BMs. Enrichment significance (p value; false discovery rate) was computed using the Reactome, the Kyoto Encyclopedia of Genes and Genomes pathway collections, and the Gene Ontology Biological Processes. Germline DNA damage repair variants were found in seven cases (70%) and involved the BRCA1, BRCA2, ATM, RAD50, ERCC4, RPA1, MLHI, and ATR genes. Somatic mutations of TP53 were found in nine cases (90%) and were the only stable mutations between the primary tumor and BMs. Disturbed pathways in BMs versus primary HGSOC constituted a complex network and included the cell cycle, the degradation of the extracellular matrix, cell junction organization, nucleotide metabolism, lipid metabolism, the immune system, G-protein-coupled receptors, intracellular vesicular transport, and reaction to chemical stimuli (Golgi vesicle transport and olfactory signaling). Pathway analysis approaches allow for a more intuitive interpretation of the data as compared to considering single-gene aberrations and provide an opportunity to identify clinically informative alterations in HGSOC BM.