Patients surviving coronavirus disease of 2019 (COVID-19) often complain of skeletal muscle weakness that may be very limiting and long-lasting. There are almost no studies on the skeletal muscle of these patients, and electron microscopic data are scarce. We assessed the ultrastructural changes in the quadriceps of eight patients with COVID-19 and found a combination of features different from those reported in corticosteroid myopathy and acute relaxant-steroid myopathy. The most remarkable and constant changes involved the endothelial cells and consisted of massive amounts of pinocytotic vesicles, degenerative changes, platelet aggregates and, most characteristic of all, an increase in the external lamina thickness that seems to stem from reduplication due to successive bouts of endothelial cell damage and subsequent regeneration. Viral particles were not found in any of the cases. This distinct and quite common set of alterations defines the myopathy associated with infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This association seems to be the result of an inflammatory process that would arise in infected cells but could damage non-infected endomysial blood vessels, thus resulting in persistent changes of the microvasculature that would be related to long-standing myopathic clinical features.

Claudin 18.2 is a transmembrane protein, part of the tight-junction complex, selectively expressed in gastric epithelium. It is showing promising results as a target in advanced gastric cancer in phase 3 clinical trials using a monoclonal antibody against claudin 18.2. A systematic review on expression of claudin 18.2 in gastric cancer was performed using the PubMed database. The following search expression was used: (“Stomach Neoplasms” [Mesh]) AND ((“claudin-18[TIAB]”) OR (“CLDN18[TIAB]”)). A total of n=99 articles were retrieved. Of those, 17 preclinical studies about claudin 18.2 expression by immunohistochemistry were selected. The results of those studies showed great variability in the criteria used for defining the thresholds for positivity of the stain. The proportion of claudin 18.2 positive cases varied between 24% and 83%. In works using a positivity threshold set at >40% or >70% of cells with membranous/cytoplasmic staining at 2+/3+ intensity, the average rate of positive cases was 50% or 30%, respectively (similar with clones 43-14A and EPR19202). Positivity of claudin 18.2 was associated with advanced stage, diffuse phenotype and PD-L1 and EBV positivity in some of the studies. Variability in criteria used to define claudin 18.2 positivity, as well as methodological differences, could explain the variation in the proportion of positive cases described, as well as the inconsistency of the association with clinical, molecular, and survival variables. The upcoming anticlaudin 18.2 therapy in advanced gastric cancer should prompt pathology laboratories to adjust their staining protocols and evaluation criteria in their series of patients, to further establish the association of claudin expression with clinical and molecular variables.

Many scientific societies have issued guidelines to introduce population-based cervical cancer screening with HPV testing. The Vitro HPV Screening assay is a fully automatic multiplex real-time PCR test targeting the L1 GP5+/GP6+ region of HPV genome. The assay detects 14 high risk (HR) HPV genotypes, identifying individual HPV16 and HPV18 genotypes, and the HPV-positive samples for the other 12 HR HPV types are subsequently genotyped with the HPV Direct Flow Chip test. Following international guidelines, the aim of this study was to validate the clinical accuracy of the Vitro HPV Screening test on ThinPrep-collected samples for its use as primary cervical cancer screening, using as comparator the validated cobas® 4800 HPV test. The non-inferiority analysis showed that the clinical sensitivity and specificity of the Vitro HPV Screening assay for a diagnosis of cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) were not inferior to those of cobas® 4800 HPV (p = 0.0049 and p < 0.001 respectively). The assay has demonstrated a high intra- and inter-laboratory reproducibility, also among the individual genotypes. The Vitro HPV Screening assay is valid for cervical cancer screening and it provides genotyping information on HPV-positive samples without further sample processing in a fully automated workflow.

Squamous cell carcinomas with two or more coexisting clearly different histological subtypes of penile carcinomas are designated as mixed carcinomas in current classification models. They represent about 10% of all penile carcinomas. The aim of this study was to detect HPV genotypes in these unusual tumors. Tumors were selected from an international series of 1010 patients with penile carcinomas. Mixed carcinomas were grouped, according to WHO recommendations, as follows: 1. Carcinomas with warty/basaloid features mixed with HPV-independent carcinomas and 2. HPV-independent subtypes mixed with each other. HPV detection and p16INK4a immunostaining were performed. For HPV detection, whole tissue section-PCR analyses were performed by SPF10-DEIA-LiPA25 (version 1). As expected, HPV was detected more frequently in HPV-associated mixed carcinomas than in HPV-independent mixed carcinomas. Carcinomas with basaloid or warty features mixed with other SCC subtypes showed an HPV positivity rate of 46% (33 of 72 tumors) compared with 7% found in tumors with nonwarty/basaloid morphology (2 of 29 tumors). Eleven high-risk HPV genotypes were identified and the most common was HPV16 (65%) usually associated with basaloid morphology. p16INK4a immunostaining was positive in 76% of HPV-positive tumors. As in nonmixed carcinomas, although in lower proportion, a variable array of HPV genotypes was detected in mixed carcinomas. Apparently, the presence of a non-HPV component in an otherwise typical HPV-associated type tumor does adversely affect the prevalence of HPV positivity. Any amount of HPV-associated morphology superior to 20% in a mixed tumor is sufficient to classify them as HPV-associated, a WHO requirement.

Introduction/Background Endometrial cancer is the most frequent gynecological cancer in Europe. Recently, a molecular-based classification of Endometrial carcinoma (EC) has been proposed and its prognostic relevance confirmed. This classification has been incorporated into the ESGO/ESTRO/ESP guidelines for the management of patients with EC. Methodology We present a series of 131 cases of EC on which immuhistochemical stains for p53, MLH1, MSH2, MSH6 and PMS2 (clones D0–7, M1, G219–1129, 44, NB10 using Benchmark XT) and mutation analysis of POLE and TP53 (sequencing of a custom Ampliseq panel using Ion Gene Studio S5) were performed. Additional relevant pathology data was gathered. All cases were classified in prognostic risk groups according to the current ESGO/ESTRO/ESP guidelines, categorizing them both with and without the integration of molecular markers in order to study the impact of the inclusion of this information on risk stratification. Results Of the 131 cases studied, in 7 instances there was a modification in their prognostic classification due to the incorporation of molecular information. All 7 cases were Endometrioid carcinomas, 3 of them were Copy number high while the other 4 had pathogenic mutations in POLE, 2 of these were Ultramutated while the other 2 were Multiple classifiers (MCs). Of the 7 discordant cases 5 were of high histologic grade while 2 were low grade lesions. We also consider worth reporting that in the global of our series 10% of the cases were MCs. Conclusion Our series proves the feasibility of applying the molecular classification of EC in daily practice and it indicates that it is most relevant in the prognostic stratification of Endometrioid carcinomas, particularly high grade lesions, supporting the recommendation of specially studying this cases. Additionally we detected a substantially higher percentage of MCs than expected pointing to the need of a more in-depth study of the prognostic implications of this molecular group. Disclosures None.

Human papillomavirus (HPV) is detected in 30-50% of invasive penile carcinomas, and it is frequently associated with basaloid and warty morphological features. Based on this heterogeneity and different clinical behaviors, we hypothesized a variation in their HPV genotypic composition. To test this, we evaluated 177 HPV-positive cases: basaloid (114), warty-basaloid (28), and warty (condylomatous) (35) invasive carcinomas. HPV DNA detection and genotyping was performed using the SPF-10/DEIA/LiPA25 system. Nineteen HPV genotypes were detected. High-risk HPVs predominated (96%), and low-risk HPVs were rarely present. Most common genotype was HPV16 followed by HPVs 33 and 35. According to the genotypes identified, 93% of the cases would be covered with current vaccination programs. There was a significant variation in the distribution of HPV16 and non-HPV16 genotypes according to histological subtype. HPV16 was significantly frequent in basaloid (87%) and was less frequent in warty carcinomas (61%). This molecular difference, along with their distinctive macro-microscopic and prognostic features, makes basaloid and warty carcinomas unique. The gradual decreasing frequency of HPV16 demonstrated in basaloid, warty-basaloid, and warty carcinomas suggest that the basaloid cell, present in those types in decreasing proportions, may be responsible for the differences.

RET rearrangements have emerged as a new actionable alteration in papillary thyroid cancers (PTC). These fusions may involve many partner genes, are thought to be mutually exclusive with other oncogenic drivers and their screening is recommended based on fluorescence in situ hybridization (FISH), RT-PCR or next-generation sequencing (NGS). The aim of this study is to evaluate the use of NGS as a standard molecular technique to characterize RET gene fusions. Sixty PTC cases without the BRAF p.V600E and/or TERT promoter mutations were selected retrospectively in two institutions. Molecular alterations were evaluated using the Oncomine Focus Assay, and all samples were analyzed by RT-PCR using the Idylla GeneFusion Assay. Fusion positive samples were confirmed with FISH with break-apart probes. All DNA samples were acceptable for analysis, whereas 4 RNA samples failed quality control NGS parameters. RET fusions were detected in 11 cases (18%): CCDC6::RET (C1:R12) in 8 samples, NCOA4::RET (N6:R12) in 2 samples, and 1 RET expression imbalance positive sample without gene partner characterization. All 11 cases were validated by both FISH and RT-PCR. Besides, 4 more cases presented NGS RET expression imbalance but none of them were confirmed neither by RT-PCR nor by FISH and considered false positive results. There were no false negative NGS cases identified in parallel by RT-PCR. Overall, a positive predictive value of 89% and a negative predictive value of 90% were estimated, reaching a concordance of 89% compared to NGS. Regarding DNA analysis, NRAS p.Q61R/K was the most prevalent alteration in 13 cases (22%) followed by 2 cases with KRAS mutations (p.G12S and p.Q61R). Other potentially actionable fusions found in our series were 2 ETV6::NTRK3 (E4:N14), 2 STRN::ALK (S3:A20) and 1 EML4::ALK (E6:A20). Molecular screening in non-BRAF PTC patients is useful to identify patients harboring RET fusions who may benefit from targeted therapies. As other potentially actionable gene fusions are also found in these patients, routine implementation of NGS analysis warrants a comprehensive biomarker study.

Membranous nephropathy (MN) is a common cause of nephrotic syndrome after kidney transplantation (KT); however, scarce is known regarding post-KT thrombospondin type-1 domain-containing 7A (THSD7A)-positive MN. Herein, we report on a 72-year-old woman with end-stage kidney disease due to chronic interstitial nephritis (1996). In February 2020, she received a second deceased-donor KT, achieving optimal kidney function but presenting early post-KT proteinuria, reaching up to 1800mg/24h six months after transplantation, controlled with renin–angiotensin–aldosterone system (RAAS) blockade. In July 2021, a kidney allograft biopsy revealed features consistent with MN. Immunohistochemical stains showed diffuse and granular THSD7A and C4d deposition in glomerular capillary walls and negative PLA2R and IgG4 staining. No anti-THSD7A antibodies were detected in the serum. The pre-implantation biopsy showed no MN-associated lesions and negative THSD7A staining. Secondary triggers such as malignancy were discarded. The present report illustrates a THSD7A-positive MN in a KT recipient. Despite lacking native kidney biopsy and early presentation, a recurrent MN seemed unprovable due to documented native kidney disease and a long time span between native kidney disease and MN diagnosis. We, therefore, presumed primary de novo disease. Two years after KT, kidney function remains stable, and the patient has reached complete remission of proteinuria.

Pulmonary embolism (PE) is a common cause of death in hospitalized patients. These emboli are usually related to deep venous thrombosis, but other etiologic factors may be the cause. A 60-year-old male was diagnosed with rectal adenocarcinoma and treated by surgical rectal resection. After surgery, he presented with a decreased level of consciousness and hypoxia with no signs of bleeding. PE was ruled out by pulmonary angiography. Given the hemodynamic instability, contrast-enhanced CT was performed, showing a discrete leak related to the rectal anastomosis, which required urgent surgery. The patient suffered cardiorespiratory arrest prior to surgery, and once resolved, the surgical procedure was limited to disconnecting the intestinal anastomosis, leaving the colon and rectal stumps free in the abdomen and keeping the abdominal cavity open with negative-pressure therapy. The patient suffered from another cardiorespiratory arrest after surgery, with abdominal distension and serous-hemorrhagic material discharge through the negative-pressure device. On a new surgical revision, no bleeding was identified, so the symptoms were attributed to coagulopathy. The patient died and autopsy was performed. The autopsy revealed no surgery-related complications. The lungs were increased in weight and showed a normal macroscopic appearance; in contrast, the histological study revealed multiple and bilateral thrombo-embolisms affecting small distal arteries. Those thrombi were composed of intestinal contents, including vegetal particles, mucinous and biliary material, fibrin, and bacterial structures. Fecal PE is an extremely infrequent event. Isolated cases have been described in association with communications between the digestive tract lumen and the systemic circulation, with a generally fatal prognosis.

Supplementary Figure 4 shows that NRG1 enhances pro-migratory capacity of breast cancer cells as well as the induction of early markers from the EMT process. Moreover, it also shows that trastuzumab plus pertuzumab, but not trastuzumab alone, are able to prevent NRG1-derived effects.

Supplementary Figure 3 shows how trastuzumab plus pertuzumab, but not trastuzumab alone, overcome NRG1 enhanced pro-survival signaling in HER2-positive breast cancer cells and in ex vivo tumors.

Stromal tumour infiltrating lymphocytes (sTIL) in haematoxylin and eosin (H & E) stained sections has been linked to better outcomes and better responses to neoadjuvant therapy in triple-negative and HER2-positive breast cancer (TNBC and HER2 +). However, the infiltrate includes different cell populations that have specific roles in the tumour immune microenvironment. Various studies have found high concordance between sTIL visual quantification and computational assessment, but specific data on the individual prognostic impact of plasma cells or lymphocytes within sTIL on patient prognosis is still unknown. In this study, we validated a deep-learning breast cancer sTIL scoring model (smsTIL) based on the segmentation of tumour cells, benign ductal cells, lymphocytes, plasma cells, necrosis, and 'other' cells in whole slide images (WSI). Focusing on HER2 + and TNBC patient samples, we assessed the concordance between sTIL visual scoring and the smsTIL in 130 WSI. Furthermore, we analysed 175 WSI to correlate smsTIL with clinical data and patient outcomes. We found a high correlation between sTIL values scored visually and semi-automatically (R = 0.76; P = 2.2e-16). Patients with higher smsTIL had better overall survival (OS) in TNBC (P = 0.0021). In the TNBC cohort, smsTIL was as an independent prognostic factor for OS. As part of this work, we introduce a new segmentation dataset of H & E-stained WSI.

Stromal tumor-infiltrating lymphocytes (sTILs) are a robust prognostic and predictive biomarker in triple-negative breast carcinoma. However, the sTIL compartment comprises different cell populations. The aim of the study is to characterize the distribution of T cells (CD3+ and CD8+), B cells, and plasma cells and explore their association with outcome in the surgical specimen of 62 patients. Furthermore, programmed death ligand 1 expression and the presence of tertiary lymphoid structures (TLSs) are explored. Patients with higher sTILs achieve better progression-free survival (PFS) (P = .0013), and tumors have more plasma cells in the infiltrate. Specifically, higher counts of T cells (both CD3+ and CD8+) have better PFS (P = .002 and P = .0086, respectively) as it is observed in tumors with higher infiltration of CD8+ T cells in the tumor core (P = .035). Higher infiltration by B cells and plasma cells shows a positive tendency toward increased PFS (P = .06 and P = .058). Programmed death ligand 1 (SP142) is positive in 56% of tumors. Tumors with at least 1 TLS (42%) show higher CD8+ T cell infiltration in the tumor core and the sTIL value doubles compared to tumors devoid of TLSs [sTIL mean: 36 ± 11% and 18 ± 5% (CI [Confidence Interval]: 95%), respectively]. Our study demonstrates that the characterization of the immune cell infiltration is as relevant as its distribution. Moreover, the importance of considering different immune cell types for classification is emphasized. Therefore, a new classification of triple-negative breast carcinoma immune infiltration with CD8+ T cell and plasma cell densities in the tumor core and infiltrative margin is proposed.