Non-Small Cell Lung Cancer Testing on Reference Specimens: An Italian Multicenter Experience.

INTRODUCTION: Biomarker testing is mandatory for the clinical management of patients with advanced non-small cell lung cancer (NSCLC). Myriads of technical platforms are now available for biomarker analysis with differences in terms of multiplexing capability, analytical sensitivity, and turnaround time (TAT). We evaluated the technical performance of the diagnostic workflows of 24 representative Italian institutions performing molecular tests on a series of artificial reference specimens built to mimic routine diagnostic samples. METHODS: Sample sets of eight slides from cell blocks of artificial reference specimens harboring exon 19 EGFR (epidermal growth factor receptor) p.E746_AT50del, exon 2 KRAS (Kirsten rat sarcoma viral oncogene homologue) p.G12C, ROS1 (c-ros oncogene 1)-unknown gene fusion, and MET (MET proto-oncogene, receptor tyrosine kinase) Δ exon 14 skipping were distributed to each participating institution. Two independent cell block specimens were validated by the University of Naples Federico II before shipment. Methodological and molecular data from reference specimens were annotated. RESULTS: Overall, a median DNA concentration of 3.3 ng/µL (range 0.1-10.0 ng/µL) and 13.4 ng/µL (range 2.0-45.8 ng/µL) were obtained with automated and manual technical procedures, respectively. RNA concentrations of 5.7 ng/µL (range 0.2-11.9 ng/µL) and 9.3 ng/µL (range 0.5-18.0 ng/µL) were also detected. KRAS exon 2 p.G12C, EGFR exon 19 p.E736_A750del hotspot mutations, and ROS1 aberrant transcripts were identified in all tested cases, whereas 15 out of 16 (93.7%) centers detected MET exon 14 skipping mutation. CONCLUSIONS: Optimized technical workflows are crucial in the decision-making strategy of patients with NSCLC. Artificial reference specimens enable optimization of diagnostic workflows for predictive molecular analysis in routine clinical practice.

Real-world use of multigene signatures in early breast cancer: differences to clinical trials.

PURPOSE: In Italy, Lombardy was the first region to reimburse multigene assays (MGAs) for patients otherwise candidates for chemotherapy. This is a real-world experience of MGAs usage in six referral cancer centers in Lombardy. METHODS: Among MGAs, Oncotype DX (RS) was used in 97% of cases. Consecutive patients tested with Oncotype DX from July 2020 to July 2022 were selected. The distribution of clinicopathologic features by RS groups (low RS: 0-25, high RS: 26-100) was assessed using chi-square and compared with those of the TAILORx and RxPONDER trials. RESULTS: Out of 1,098 patients identified, 73% had low RS. Grade and Ki67 were associated with RS (p < 0.001). In patients with both G3 and Ki67 > 30%, 39% had low RS, while in patients with both G1 and Ki67 < 20%, 7% had high RS. The proportion of low RS in node-positive patients was similar to that in RxPONDER (82% vs 83%), while node-negative patients with low RS were significantly less than in TAILORx (66% vs 86%, p < 0.001). The distribution of Grade was different from registration trials, with more G3 and fewer G1 (38% and 3%) than in TAILORx (18% and 27%) and RxPONDER (10% and 24%) (p < 0.001). Patients ≤ 50 years were overrepresented in this series (41%) than in TAILORx and RxPONDER (31% and 24%, respectively) (p < 0.001) and, among them, 42% were node positive. CONCLUSIONS: In this real-world series, Oncotype DX was the test almost exclusively used. Despite reimbursement being linked to pre-test chemotherapy recommendation, almost 3/4 patients resulted in the low-RS group. The significant proportion of node-positive patients ≤ 50 years tested indicates that oncologists considered Oncotype DX informative also in this population.

Effect of Radio-Chemotherapy on PD-L1 Immunohistochemical Expression in Head and Neck Squamous Cell Carcinoma.

BACKGROUND: Programmed death-ligand 1 (PD-L1) checkpoint inhibitors represent a mainstay of therapy in head and neck squamous cell cancer (HNSCC). However, little is known about the influence of combined therapy on PD-L1 expression. The study aims to gather evidence on this topic. METHODS: A systematic search was carried out in electronic databases Pubmed-MEDLINE and Embase to retrieve studies on the comparison of PD-L1 expression before and after conventional therapy. Data were extracted and a quantitative analysis with pooled odds ratios (ORs) was performed when applicable. RESULTS: Of 5688 items, 15 were finally included. Only a minority of studies assessed PD-L1 with the recommended combined positive score (CPS). The results are highly heterogeneous, with some studies reporting an increase in PD-L1 expression and others reporting a decrease. Three studies allowed for quantitative analysis and showed a pooled OR of 0.49 (CI 0.27-0.90). CONCLUSIONS: From the present evidence, a clear conclusion towards an increase or decrease in PD-L1 expression after combined therapy cannot be drawn, but even with few studies available, a trend towards an increase in expression in tumor cells at a cutoff of 1% can be noted in patients undergoing platinum-based therapy. Future studies will provide more robust data on the effect of combined therapy on PD-L1 expression.

Pediatric BCOR-Altered Tumors From Soft Tissue/Kidney Display Specific DNA Methylation Profiles.

In the pediatric population, BCL6-correpresor gene (BCOR)-upregulated tumors include primitive myxoid mesenchymal tumors/undifferentiated sarcomas (PMMTI/UND), clear cell sarcomas of the kidney (CCSK), and high-grade neuroepithelial tumors (HG-NET). We investigated DNA methylation (DNAm) and copy number variation (CNV) profiling in these tumors (N = 34) using an Illumina EPIC BeadChip to better define the potential use of these tools to confirm diagnosis and predict outcomes. Twenty-seven tumors from 25 patients (age range, 0-10 years), showed molecular confirmation of genetic abnormalities as follows: BCOR internal tandem duplication in 14 PMMTI/UND, 8 CCSK, and 3 HG-NET and YWHAE fusions in 2 PMMTI/UND. The remaining 7 cases lacking informative molecular data were analyzed by immunophenotyping and were included in the study as a training cohort, clearly separated from the main study group. These were 4 PMMTI, 1 HG-NET, and 1 CCSK in which poor RNA preservation precluded the confirmation of BCOR rearrangements and 1 CCSK in which no rearrangements were found. DNAm data were compared with those of brain tumor and/or sarcoma classifier. Differentially methylated regions (DMRs) were analyzed in the 3 groups. Twenty-two cases of the 24 molecularly confirmed PMMTI/UND and CCSK and 3 of 6 of those with only immunophenotyping were classified within the methylation class "BCOR-altered sarcoma family" with optimal calibrated scores. PMMTI/UND and CCSK showed similar methylation profiles, whereas thousands of DMRs and significantly enriched pathways were evident between soft tissue/kidney tumors and HG-NET. The CNV analysis showed an overall flat profile in 19 of the 31 evaluable tumors (8/10 CCSK; 9/18 PMMTI/UND; 2/4 HG-NET). The most frequent CNVs were 1q gain and 9p and 10q loss. Follow-up time data were available for 20 patients: ≥2 CNV significantly correlated with a worse overall survival rate. In conclusion, soft tissue and kidney BCOR sarcomas matched with BCOR-altered sarcoma methylation class, whereas those from the brain matched with the central nervous system tumor classifier HG-NET BCOR, supporting the notion that DNAm profiling is an informative diagnostic tool. CNV alterations were associated with a more aggressive clinical behavior.

Pathology of Immunotherapy-induced Responses in Cutaneous Melanoma: Current Evidences and Future Perspectives.

Over the last years, immune checkpoint inhibitors (ICIs) have demonstrated remarkable anti-tumor activity and beneficial effects in patients with early and advanced melanoma. However, ICIs provide clinical benefit only in a minority of patients due to primary and/or acquired resistance mechanisms. Immunotherapy resistance is a complex phenomenon relying on genetic and epigenetic factors, which ultimately influence the interplay between cancer cells and the tumor microenvironment. Information is accumulating on the cellular and molecular mechanisms underlying the production of resistance and the resulting diminished therapeutic efficacy. In addition, current knowledge on predictors of response and toxicity to immunotherapy and on biomarkers that reliably identify resistant patients is in progress. In this review, we will focus on the tumor microenvironment changes induced by ICIs in melanoma, summarizing the available evidence of clinical trials in the neoadjuvant and metastatic setting. We will also overview the role of potential biomarkers in predicting disease response to ICIs, providing insight into current and future research in this field.

High inter-follicular spatial co-localization of CD8+FOXP3+ with CD4+CD8+ cells predicts favorable outcome in follicular lymphoma.

The spatial architecture of the lymphoid tissue in follicular lymphoma (FL) presents unique challenges to studying its immune microenvironment. We investigated the spatial interplay of T cells, macrophages, myeloid cells and natural killer T cells using multispectral immunofluorescence images of diagnostic biopsies of 32 patients. A deep learning-based image analysis pipeline was tailored to the needs of follicular lymphoma spatial histology research, enabling the identification of different immune cells within and outside neoplastic follicles. We analyzed the density and spatial co-localization of immune cells in the inter-follicular and intra-follicular regions of follicular lymphoma. Low inter-follicular density of CD8+FOXP3+ cells and co-localization of CD8+FOXP3+ with CD4+CD8+ cells were significantly associated with relapse (p = 0.0057 and p = 0.0019, respectively) and shorter time to progression after first-line treatment (Logrank p = 0.0097 and log-rank p = 0.0093, respectively). A low inter-follicular density of CD8+FOXP3+ cells is associated with increased risk of relapse independent of follicular lymphoma international prognostic index (FLIPI) (p = 0.038, Hazard ratio (HR) = 0.42 [0.19, 0.95], but not independent of co-localization of CD8+FOXP3+ with CD4+CD8+ cells (p = 0.43). Co-localization of CD8+FOXP3+ with CD4+CD8+ cells is predictors of time to relapse independent of the FLIPI score and density of CD8+FOXP3+ cells (p = 0.027, HR = 0.0019 [7.19 × 10-6 , 0.49], This suggests a potential role of inter-follicular CD8+FOXP3+ and CD4+CD8+ cells in the disease progression of FL, warranting further validation on larger patient cohorts.

The Accuracy of Transurethral Bladder Resection in Detecting Bladder Cancer Histological Variants and Their Prognostic Value at Radical Cystectomy.

OBJECTIVES: to investigate the accuracy of transurethral resection of bladder tumours (TURBT) in detecting histological variants (BHV) at radical cystectomy (RC) and to evaluate the impact of TURBT before cystectomy on oncological outcomes. METHODS: Data of 410 consecutive RCs were assessed. Positive and negative predictive values were used to assess the accuracy of TURBT in detecting BHV. Cohen's Kappa coefficient was used to calculate the agreement grade. Logistic regression analysis predicted features based on the presence of BHV at TURBT. Multivariable backward conditional Cox regression analysis was used to estimate oncological outcomes. RESULTS: A total of 73 patients (17.8%) showed BHV at TURBT as compared to 108 (26.3%) at RC. A moderate agreement in histological diagnosis was found between TURBT and RC (0.58). However, sensitivity and specificity in detecting BHV were 56% and 96%, respectively. Furthermore, positive predictive value (PPV) was 84.7% and negative predictive value (NPV) was 84.6%. Presence of BHV at TURBT was an independent predictor for pathologic upstage, albeit not a predictor for positive nodes or positive surgical margins. However, at multivariable analysis adjusted for all confounders, presence of BHV at TURBT was an independent predictor for recurrence after RC, but not for survival. Conversely, the presence of BHV at RC was an independent predictor for both recurrence and survival. CONCLUSION: There was a moderate agreement between TURBT and RC histopathological findings. TURBT, alone, could not provide an accurate and definitive histological diagnosis. Detection of BHV in TURBT specimens is not an independent predictor of oncological outcomes; indeed, only pathological features at RC are associated with worse survival. However, BHV presence in cystectomy specimens resulted as an independent predictor of both cancer-specific and overall mortality.

Evidence of vertical transmission of SARS-CoV-2 and interstitial pneumonia in second-trimester twin stillbirth in asymptomatic woman. Case report and review of the literature.

Data on the vertical transmission rate of COVID-19 in pregnancy are limited, although data reporting mother-fetal transmission in the second trimester of pregnancy are controversial. We described a case of second-trimester twin stillbirth in a woman with SARS-CoV-2 infection in which placental and fetal markers of infection were detected, despite the absence of respiratory syndrome. The patient developed clinical chorioamnionitis and spontaneously delivered 2 stillborn infants. Placental histology and immunohistochemistry demonstrated SARS-CoV-2 infection mostly within the syncytiotrophoblast, and fetal autopsy showed the development of interstitial pneumonia. Our findings demonstrated that in utero vertical transmission is possible in asymptomatic pregnant women with SARS-CoV-2 infection and that infection can lead to severe morbidity in the second trimester of pregnancy.

RNAscope in situ hybridization and RT-PCR for detection of SARS-CoV-2 in chilblain-like lesions: A clinical, laboratory and histopathological study.

BACKGROUND: Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, an increasing number of chilblain-like lesions (ChLL) have been increasingly reported worldwide. To date, the causal link between ChLL and SARS-CoV-2 infection has not been unequivocally established. METHODS: In this case series, we present demographic, clinical, laboratory, and histopathological information regarding 27 young patients with a clinical diagnosis of ChLL who referred to the Dermatology Unit of Papa Giovanni XXIII Hospital, Bergamo, Italy, from 1 April 2020 to 1 June 2020. RESULTS: The mean age was 14.2 years, and 21 patients (78%) experienced mild systemic symptoms a median of 28 days before the onset of cutaneous lesions. ChLL mostly involved the feet (20 patients - 74%). Among acral lesions, we identified three different clinical patterns: (i) chilblains in 20 patients (74%); (ii) fixed erythematous macules in 4 children (15%); (iii) erythrocyanosis in 3 female patients (11%). Blood examinations and viral serologies, including parvovirus B19, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and coxsackievirus were normal in all. Three patients (11%) underwent nasopharyngeal swab for RT-PCR for SARS-CoV-2 showing only 1 positive. Histopathological examinations of 7 skin biopsies confirmed the clinical diagnosis of chilblains; vessel thrombi were observed only in 1 case. Our findings failed to demonstrate the direct presence of SARS-CoV-2 RNA in skin biopsies, both with real-time polymerase chain reaction (RT-PCR) and RNAscope in situ hybridization (ISH). LIMITATIONS: Limited number of cases, unavailability of laboratory confirmation of COVID-19 in all patients, potential methodological weakness, and latency of skin biopsies in comparison to cutaneous lesions onset. CONCLUSIONS: These observations may support the hypothesis of an inflammatory pathogenesis rather than the presence of peripheral viral particles. Although, we could not exclude an early phase of viral endothelial damage followed by an IFN-I or complement-mediated inflammatory phase. Further observations on a large number of patients are needed to confirm this hypothesis.

Oncological outcomes of concomitant carcinoma in situ at radical cystectomy in pure urothelial bladder cancer and in histological variants.

INTRODUCTION: The presence of carcinoma in situ at transurethral resection is known to increase the risk of recurrence and progression to invasive disease. However, the evidence regarding the prognostic role of concomitant carcinoma in situ after radical cystectomy due to bladder cancer is controversial. Moreover, concomitant carcinoma in situ was found to be significantly associated with bladder histological variants. The aim of our study is to evaluate whether the presence of concomitant carcinoma in situ at radical cystectomy, impacts on recurrence and survival outcomes in pure urothelial bladder cancer, compared to histological variants. METHODS: We evaluated 410 consecutive patients diagnosed with non-metastatic bladder cancer and treated with radical cystectomy at a single tertiary referral centre between January 2009 and May 2019. Patients were stratified according to the presence of carcinoma in situ. The Kaplan-Meier method was used to compare recurrence free, cancer specific and overall survival in pure urothelial and histological variants. Cox proportional hazards regression analyses model was used to predict recurrence, cancer specific and overall mortality in pure urothelial and histological variants bladder cancer, according to pathological stage. RESULTS: Median age was 71 years. 340 patients (82%) were male. At a median follow-up of 32 months, disease recurrence, cancer specific mortality and overall mortality were, 37% (155 patients), 32.9% (135 patients) and 46.6% (191 patients), respectively. Concomitant and pure carcinoma in situ were found in 39% and 19% of radical cystectomy specimens, respectively. Concomitant carcinoma in situ was more frequent in patients with histological variants (50.9%) compared to pure urothelial bladder cancer (35.4%) (P-value <.001) and was associated with worst pathological features (lymphovascular invasion, lymph node involvement and non-organ confined disease). Recurrence free survival at Kaplan-Meyer analyses was significantly higher in patients with pure carcinoma in situ compared to those with concomitant or no carcinoma in situ (all P <.001), similarly for patients without carcinoma in situ compared with those with concomitant Cis (P =.02) at radical cystectomy. Cancer specific and overall survival were significantly higher in patients with pure carcinoma in situ compared to those with concomitant or no carcinoma in situ (all P <.001). Conversely no significant difference was found between patients without carcinoma in situ and with concomitant carcinoma in situ (P>0.1) at radical cystectomy Moreover, concomitant carcinoma in situ at radical cystectomy in histological variants is associated with higher free recurrence rate compared to the other groups. At multivariate Cox proportional hazards regression analyses the presence of carcinoma in situ at radical cystectomy was not associated with any survival effect or recurrence (all P > .05) in the overall population and when patients are stratified according to histology. However, concomitant carcinoma in situ represents an independent predictor of recurrence in the subgroup of patients with organ confined disease in case of urothelial bladder cancer and histological variants. CONCLUSION: Concomitant carcinoma in situ should be considered a proxy of aggressiveness in bladder cancer after radical cystectomy. Based on its prognostic implications, concomitant carcinoma in situ should be considered for strict follow-up in patients with organ confined disease which may deserve adjuvant treatment both in pure urothelial bladder cancer and histological variants.

Differential expression and regulation of MS4A family members in myeloid cells in physiological and pathological conditions.

The MS4A gene family encodes 18 tetraspanin-like proteins, most of which with unknown function. MS4A1 (CD20), MS4A2 (FcεRIß), MS4A3 (HTm4), and MS4A4A play important roles in immunity, whereas expression and function of other members of the family are unknown. The present investigation was designed to obtain an expression fingerprint of MS4A family members, using bioinformatics analysis of public databases, RT-PCR, and protein analysis when possible. MS4A3, MS4A4A, MS4A4E, MS4A6A, MS4A7, and MS4A14 were expressed by myeloid cells. MS4A6A and MS4A14 were expressed in circulating monocytes and decreased during monocyte-to-Mϕ differentiation in parallel with an increase in MS4A4A expression. Analysis of gene expression regulation revealed a strong induction of MS4A4A, MS4A6A, MS4A7, and MS4A4E by glucocorticoid hormones. Consistently with in vitro findings, MS4A4A and MS4A7 were expressed in tissue Mϕs from COVID-19 and rheumatoid arthritis patients. Interestingly, MS4A3, selectively expressed in myeloid precursors, was found to be a marker of immature circulating neutrophils, a cellular population associated to COVID-19 severe disease. The results reported here show that members of the MS4A family are differentially expressed and regulated during myelomonocytic differentiation, and call for assessment of their functional role and value as therapeutic targets.

Correlation of BRAF mutational status with clinical characteristics and survival outcomes of patients with ameloblastoma: the experience of 11 Italian centres.

AIMS: Ameloblastoma is a rare odontogenic tumour with an aggressive local behaviour. Mutations in the mitogen-activated protein kinase pathway, namely BRAF V600E mutations, are a common finding. To date, there is no clear correlation between BRAF V600 mutation and clinical outcome. METHODS: We retrospectively reviewed the medical records of patients who underwent surgery for ameloblastoma between May 1998 and June 2018, at 11 participating Italian centres. BRAF mutational status was evaluated by quantitative PCR/pyrosequencing. The primary end points were to determine BRAF mutational status in primitive and recurrent ameloblastoma, and to assess the relapse-free interval (RFI); the secondary end point was to investigate the correlation of BRAF mutational status with the clinical features of the tumour and survival outcomes. RESULTS: Overall, 74 patients were included: 33 (44.5%) were BRAF wild type and 41 (55.4%) BRAF V600 mutated. BRAF V600 mutated ameloblastomas occurred more frequently in younger patients (p=0.0031), were located at the mandible (p=0.0009) and presented with unicystic variant. After a median follow-up of 60 months, 21 (28.3%) patients relapsed (30.3% and 26.8% in the BRAF wild type and BRAF mutated group, respectively). At univariable Cox models, none of the investigated variables, including microscopic margin involvement, was associated with RFI. CONCLUSIONS: Local recurrence occurs in 30% of patients with ameloblastoma. BRAFV600 mutation is associated with younger age, mandibular localisation and with unicystic ameloblastoma. Neither BRAF mutation nor microscopically positive surgical margins were associated with RFI. Further studies are needed to elucidate outcomes of this rare disease according to clinical, histopathological and comprehensive molecular features.

COVID-19 and myocarditis: a systematic review and overview of current challenges.

Myocardial inflammation in COVID-19 has been documented. Its pathogenesis is not fully elucidated, but the two main theories foresee a direct role of ACE2 receptor and a hyperimmune response, which may also lead to isolated presentation of COVID-19-mediated myocarditis. The frequency and prognostic impact of COVID-19-mediated myocarditis is unknown. This review aims to summarise current evidence on this topic. We performed a systematic review of MEDLINE and Cochrane Library (1/12/19-30/09/20). We also searched clinicaltrials.gov for unpublished studies testing therapies with potential implication for COVID-19-mediated cardiovascular complication. Eligible studies had laboratory confirmed COVID-19 and a clinical and/or histological diagnosis of myocarditis by ESC or WHO/ISFC criteria. Reports of 38 cases were included (26 male patients, 24 aged < 50 years). The first histologically proven case was a virus-negative lymphocytic myocarditis; however, biopsy evidence of myocarditis secondary to SARS-CoV-2 cardiotropism has been recently demonstrated. Histological data was found in 12 cases (8 EMB and 4 autopsies) and CMR was the main imaging modality to confirm a diagnosis of myocarditis (25 patients). There was a substantial variability in biventricular systolic function during the acute episode and in therapeutic regimen used. Five patients died in hospital. Cause-effect relationship between SARS-CoV-2 infection and myocarditis is difficult to demonstrate. However, current evidence demonstrates myocardial inflammation with or without direct cardiomyocyte damage, suggesting different pathophysiology mechanisms responsible of COVID-mediated myocarditis. Established clinical approaches should be pursued until future evidence support different actions. Large multicentre registries are advisable to elucidate further.

Pre-Transplant Alpha-Fetoprotein > 25.5 and Its Dynamic on Waitlist Are Predictors of HCC Recurrence after Liver Transplantation for Patients Meeting Milan Criteria.

BACKGROUND AND AIM: Hepatocellular carcinoma (HCC) recurrence rates after liver transplantation (LT) range between 8 and 20%. Alpha-fetoprotein (AFP) levels at transplant can predict HCC recurrence, however a defined cut-off value is needed to better stratify patients. The aim of this study was to evaluate the rate of HCC recurrence at our centre and to identify predictors, focusing on AFP. METHODS: We retrospectively analysed 236 consecutive patients that were waitlisted for HCC who all met the Milan criteria from January 2001 to December 2017 at our liver transplant centre. A total of twenty-nine patients dropped out while they were waitlisted, and 207 patients were included in the final analysis. All survival analyses included the competing-risk model. RESULTS: The mean age was 56.8 ± 6.8 years. A total of 14% were female (n = 29/207). The median MELD (model for end-stage liver disease) at LT was 12 (9-16). The median time on the waitlist was 92 (41-170) days. The HCC recurrence rate was 16.4% (n = 34/208). The mean time to recurrence was 3.3 ± 2.8 years. The median AFP levels at transplant were higher in patients with HCC recurrence (p < 0.001). At multivariate analysis, the AFP value at transplant that was greater than 25.5 ng/mL (AUC 0.69) was a strong predictor of HCC recurrence after LT [sHR 3.3 (1.6-6.81); p = 0.001]. The HCC cumulative incidence function (CIF) of recurrence at 10 years from LT was significantly higher in patients with AFP > 25.5 ng/mL [34.3% vs. 11.5% (p = 0.001)]. Moreover, an increase in AFP > 20.8%, was significantly associated with HCC recurrence (p = 0.034). CONCLUSIONS: In conclusion, in our retrospective study, the AFP level at transplant > 25.5 ng/mL and its increase greater than 20.8% on the waitlist were strong predictors of HCC recurrence after LT in a cohort of patients that were waitlisted within the Milan criteria. However further studies are needed to validate these data.

NTRK Gene Fusion Detection in Atypical Spitz Tumors.

Atypical Spitz tumors (AST) deviate from stereotypical Spitz nevi for one or more atypical features and are now regarded as an intermediate category of melanocytic tumors with uncertain malignant potential. Activating NTRK1/NTRK3 fusions elicit oncogenic events in Spitz lesions and are targetable with kinase inhibitors. However, their prevalence among ASTs and the optimal approach for their detection is yet to be determined. A series of 180 ASTs were screened with pan-TRK immunohistochemistry and the presence of NTRK fusions was confirmed using FISH, two different RNA-based NGS panels for solid tumors, and a specific real time RT-PCR panel. Overall, 26 ASTs showed pan-TRK immunostaining. NTRK1 fusions were detected in 15 of these cases showing cytoplasmic immunoreaction, whereas NTRK3 was detected in one case showing nuclear immunoreaction. Molecular tests resulted all positive in only two ASTs (included the NTRK3 translocated), RNA-based NGS and real time RT-PCR were both positive in three cases, and FISH and real time RT-PCR in another two cases. In seven ASTs NTRK1 fusions were detected only by FISH and in two cases only by real time RT-PCR. The frequency of NTRK fusions in ASTs is 9%, with a clear prevalence of NTRK1 compared to NTRK3 alterations. Pan-TRK immunohistochemistry is an excellent screening test. Confirmation of NTRK fusions may require the use of different molecular techniques.

A 57-year-old man with rapidly progressive pulmonary hypertension.

Pulmonary Tumor Thrombotic Microangiopathy (PTTM) is a rare condition associated with neoplastic disorders, predominantly gastric cancer, leading to pre-capillary Pulmonary Hypertension (PH). The pathologic mechanism involved is a fibrocellular intimal proliferation of small pulmonary vessels sustained by nests of carcinomatous cells lodged in pulmonary vasculature. Clinical presentation is nonspecific, including progressive dyspnea and dry cough. Diagnosis of PTTM is extremely challenging ante-mortem and prognosis is poor. Here we describe the case of a middle-aged man, without known previous cancer history. The clinical course was rapidly unfavorable, with progressive dyspnea and PH associated with hemodynamic instability, eventually culminating in patient's death. PTTM diagnosis was made post-mortem. PTTM should be considered in any patient presenting with unexplained PH, especially if it is rapidly progressive, poorly responsive to standard approaches or there is suspected history of malignancy. A prompt diagnosis of PTTM could help in bringing light into this still under-recognized condition.

Molecular Pathology Analysis of SARS-CoV-2 in Syncytiotrophoblast and Hofbauer Cells in Placenta from a Pregnant Woman and Fetus with COVID-19.

A small number of neonates delivered to women with SARS-CoV-2 infection have been found to become infected through intrauterine transplacental transmission. These cases are associated with a group of unusual placental pathology abnormalities that include chronic histiocytic intervillositis, syncytiotrophoblast necrosis, and positivity of the syncytiotrophoblast for SARS-CoV-2 antigen or RNA. Hofbauer cells constitute a heterogeneous group of immunologically active macrophages that have been involved in transplacental infections that include such viral agents as Zika virus and human immunodeficiency virus. The role of Hofbauer cells in placental infection with SARS-CoV-2 and maternal-fetal transmission is unknown. This study uses molecular pathology techniques to evaluate the placenta from a neonate infected with SARS-CoV-2 via the transplacental route to determine whether Hofbauer cells have evidence of infection. We found that the placenta had chronic histiocytic intervillositis and syncytiotrophoblast necrosis, with the syncytiotrophoblast demonstrating intense positive staining for SARS-CoV-2. Immunohistochemistry using the macrophage marker CD163, SARS-CoV-2 nucleocapsid protein, and double staining for SARS-CoV-2 with RNAscope and anti-CD163 antibody, revealed that no demonstrable virus could be identified within Hofbauer cells, despite these cells closely approaching the basement membrane zone of the infected trophoblast. Unlike some other viruses, there was no evidence from this transmitting placenta for infection of Hofbauer cells with SARS-CoV-2.

Digital Immunophenotyping Predicts Disease Free and Overall Survival in Early Stage Melanoma Patients.

BACKGROUND: the prognostic significance of tumor infiltrating lymphocytes (TILs) in intermediate/thick primary cutaneous melanoma (PCM) remains controversial, partially because conventional evaluation is not reliable, due to inter-observer variability and diverse scoring methods. We aimed to assess the prognostic impact of the density and spatial distribution of immune cells in early stage intermediate/thick PCM. MATERIALS AND METHODS: digital image acquisition and quantitative analysis of tissue immune biomarkers (CD3, CD4, CD8, CD68, PD-L1, CD163, FOX-P3, and PD-1) was carried out in a training cohort, which included patients with primary PCM ≥ 2 mm diagnosed, treated, and followed-up prospectively in three Italian centers. Results were validated in an independent Italian cohort. RESULTS: in the training cohort, 100 Stage II-III melanoma patients were valuable. At multivariable analysis, a longer disease free survival (DFS) was statistically associated with higher levels of CD4+ intratumoral T-cells (aHR [100 cell/mm2 increase] 0.98, 95%CI 0.95-1.00, p = 0.041) and CD163+ inner peritumoral (aHR [high vs. low] 0.56, 95%CI 0.32-0.99, p = 0.047). A statistically significant longer DFS (aHR [high-high vs. low-low] 0.52, 95%CI 0.28-0.99, p = 0.047) and overall survival (OS) (aHR [high-high vs. low-low] 0.39, 95%CI 0.18-0.85, p = 0.018) was found in patients with a high density of both intratumoral CD8+ T-cells and CD68+ macrophages as compared to those with low density of both intratumoral CD8+ T-cells and CD68+ macrophages. Consistently, in the validation cohort, patients with high density of both intratumoral CD8+ and CD3+ T-cells were associated to a statistically better DFS (aHR[high-high vs. low-low] 0.24, 95%CI 0.10-0.56, p < 0.001) and those with high density of both intratumoral CD8+ and CD68+ were associated to a statistically longer OS (aHR[high-high vs. low-low] 0.28, 95%CI 0.09-0.86, p = 0.025). CONCLUSION: our findings suggest that a specific preexisting profile of T cells and macrophages distribution in melanomas may predict the risk of recurrence and death with potential implications for the stratification of stage II-III melanoma patients.