Genomic analysis of spermatocytic tumors demonstrates recurrent molecular alterations in cases with malignant clinical behavior.

Spermatocytic tumor (ST) is a rare type of germ cell tumor that occurs exclusively in the postpubertal testis and typically affects elderly men. Most STs are benign, but rare cases exhibit aggressive clinical behavior, often in association with transition to sarcomatoid histology. Limited molecular analyses have been performed on STs; therefore, their genomic and epigenomic features remain incompletely described. Twenty-seven samples from 25 individual patients were analyzed with a combination of DNA sequencing panels, genomic methylation profiling, SNP array, isochromosome (12p) [i(12p)] FISH, and immunohistochemistry. The series included five metastasizing tumors (three with sarcomatoid transformation, one anaplastic, and one conventional) and 20 non-metastasizing tumors (14 anaplastic and six conventional). Anaplastic tumors comprised a monomorphic population of intermediate-sized neoplastic cells, as previously described. Multiomic analyses demonstrated that there were two genomic subgroups of STs: one with diploid genomes and hotspot RAS/RAF variants and the other with global ploidy shift and absence of recurrent mutations. Relative gain of chromosome 9 was a consistent finding in both subgroups. A comparison of metastasizing and non-metastasizing cases demonstrated that aggressive behavior was associated with the acquisition of pathogenic TP53 mutations and/or relative gains of 12p/i(12p). In cases with sarcomatoid transformation, TP53 mutations seem to underlie the transition to sarcomatoid histology. Genomic methylation analysis demonstrated that aggressive cases with gains of 12p cluster closer to pure seminomas than to STs without gains of 12p. In conclusion, STs include two genomic subgroups, characterized by global ploidy shifts without recurrent mutations and diploid genomes with RAS/RAF hotspot mutations, respectively. Biologic progression was associated with relative gains of 12p and TP53 mutations. The findings in STs with relative gains of 12p suggest that they may exhibit biologic characteristics akin to those seen in germ cell neoplasia in situ-related germ cell tumors rather than non-germ cell neoplasia in situ-derived STs. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

ß-Catenin alterations in testicular Leydig cell tumour: a immunohistochemical and molecular analysis.

BACKGROUND: Testicular Leydig cell tumours (LCTs) are the most common type of sex cord-stromal tumour in men, representing 1%-3% of all testicular neoplasms. Among testicular sex cord-stromal tumours, CTNNB1 mutations and nuclear expression of ß-catenin have been typically associated with Sertoli cell tumour. Recent genomic analyses have shown that CTNNB1 variants are also identified in a subset of LCTs; however, the frequency and clinicopathologic associations of ß-catenin alterations remain incompletely understood in this tumour type. METHODS: In this study, we evaluated 32 LCTs (five malignant/metastasizing, 27 nonmetastasizing) using ß-catenin immunohistochemistry and DNA sequencing. RESULTS: Immunohistochemistry revealed focal or multifocal nuclear ß-catenin expression in 47% of the tumours. Diffuse nuclear ß-catenin expression (in >50% of the tumour cells) was not detected in any of the cases analysed herein. Comparison of ß-catenin-positive and ß-catenin-negative cases did not show significant differences in the frequency of adverse histopathologic findings or malignant clinical behaviour. DNA sequencing performed de novo on a subset of seven cases revealed the presence of exon 3 CTNNB1 variants in four of them (4/7, 57%), with variant allele frequencies (VAF) ranging from 7 to 33%. Two additional ß-catenin-positive cases that had been sequenced as part of a previous study harboured exon 3 CTNNB1 variants at VAF of 28% and 7%, respectively. CONCLUSION: These results demonstrate that ß-catenin alterations are relatively common in LCT, most likely occurring as subclonal events that are not enriched in cases with aggressive features. Further studies are needed to clarify the oncogenic role of ß-catenin in this tumour type.

FoxA2 is a reliable marker for the diagnosis of yolk sac tumour postpubertal-type.

AIMS: Yolk sac tumour postpubertal-type (YSTpt) shows a wide range of histological patterns and is challenging to diagnose. Recently, forkhead box transcription factor A2 (FoxA2) emerged as a driver of YSTpt formation and a promising marker for diagnosing YSTpt. However, FoxA2 has not been tested in the different patterns of YSTpt. This study aimed to assess the staining pattern of FoxA2 in te different patterns of YSTpt and other germ cell tumours of the testis (GCTT), comparing it with glypican-3 (GPC3) and α-fetoprotein (AFP). METHODS AND RESULTS: FOXA2, GPC3 and AFP immunohistochemistry was performed on 24 YSTpt (24 microcystic/reticular, 10 myxoid, two macrocystic, five glandular/alveolar, two endodermal sinus/perivascular, four solid, two polyembryoma/embryoid body and two polyvesicular vitelline) and 81 other GCTT. The percentage of positive cells (0, 1+, 2+, 3+) and the intensity (0, 1, 2, 3) were evaluated regardless of and within each YSTpt pattern. FoxA2 was positive in all YSTpt (24 of 24) and all but one (23 of 24) exhibited 2+/3+ stain, with higher intensity [median value (mv): 2.6] than AFP (1.8) and GPC3 (2.5). Both FoxA2 and GPC3 were positive in all microcystic/reticular (24 of 24), myxoid (10 of 10), macrocystic (two of two), endodermal sinus/perivascular (four of four) and polyembryoma/embryoid body (two of two) patterns. Nevertheless, only FoxA2 was positive in all glandular/alveolar (five of five), solid (four of four) and polyvesicular vitelline (two of two) patterns. The intensity of FoxA2 was higher than AFP and GPC3 in almost all YST patterns. In the other GCTT, FoxA2 was positive only in teratoma postpubertal-type (Tpt) [13 of 20 (65%)], with staining almost exclusively confined to the mature gastrointestinal/respiratory tract epithelium. CONCLUSIONS: FoxA2 is a highly sensitive and specific biomarker that supports the diagnosis of YSTpt. FoxA2 is superior to GPC3 and AFP, especially in rare and difficult-to-diagnose histological patterns of YSTpt, but mature glands of Tpt could represent a potential diagnostic pitfall.

Analysis of GATA3 and FOXA2 expression suggests that downregulation of genes involved in the maintenance of a mature yolk sac tumor phenotype may underlie sarcomatoid transformation.

In the post-chemotherapy setting, germ cell tumors of the testis (GCTT) that resemble non-specific sarcomas and co-express cytokeratins and glypican-3 (GPC3) are diagnosed as "sarcomatoid yolk sac tumor postpubertal-type (YSTpt)". The diagnosis of sarcomatoid YSTpt is clinically relevant but challenging due to its rarity, non-specific histology, and negative α-fetoprotein (AFP) staining. Recently, FOXA2 has emerged as a key-gene in the reprogramming of GCTT (activating the transcription of several genes, among which GATA3), and immunohistochemical studies showed that GATA3 and FOXA2 have a higher sensitivity for non-sarcomatoid YSTpt than GPC3 and AFP. We found that sarcomatoid YSTpt did not express FOXA2 [0: 14/14 (100%)] and showed focal expression of GATA3 [0: 12/14 (85.7%), 1 + : 2/14 (14.3%)], thus suggesting that these markers are not useful in diagnosing this tumor. Furthermore, we proposed a potential mechanism of sarcomatoid transformation in the post-chemotherapy setting of GCTT, mediated by the downregulation of FOXA2 and GATA3.

Yolk sac tumor of postpubertal-type does not exhibit immunohistochemical loss of SMARCB1/INI1 and SMARCA4/BRG1 but choriocarcinoma?

The recently described SWI/SNF complex-deficient sinonasal carcinoma (SMARCB1 & SMARCA4) may exhibit a yolk sac-like morphology. Tumors with similar features (yolk sac-like histology combined with the immunohistochemical loss of SMARCB1/INI1 and/or SMARCA4/BRG1) have also been described in other sites, such as the female genital tract. In this study, we immunohistochemically assessed SMARCB1/INI1 and SMARCA4/BRG1 expression to evaluate if these proteins could be involved in the pathogenesis of testicular yolk sac tumors of postpubertal type (YSTpt). Specifically, we analyzed a retrospective case series comprising pure YSTpt and mixed germ cell tumors of the testis (GCTT) with YSTpt components. In the present study, no testicular YSTpt showed loss of SMARCB1/INI1 (0/24, 0%) or SMARCA4/BRG1 (0/24, 0%). However, testicular choriocarcinoma (CHC) and isolated syncytiotrophoblast cells (iSTCs) demonstrated abnormal staining patterns for SMARCA4/BRG1 [CHC: 4/4 (100%); iSTCs: 12/12 (100%), respectively], including focal or diffuse loss of expression in a subset of cases. The results of our study suggest that functional loss of SMARCA4/BRG1 represents a recurrent event that may be relevant for the pathogenesis of a subset of testicular CHC.

TAMs PD-L1(+) in the reprogramming of germ cell tumors of the testis.

BACKGROUND: In recent years, several studies focused on the process of reprogramming of seminoma (S) cells, which regulates the transition from pure S (P-S) to S component (S-C) of mixed germ cell tumors of the testis (GCTT) and finally to embryonal carcinoma (EC) and other nonseminomatous GCTT (NS-GCTT). The accepted pathogenetic model is driven and regulated by cells (macrophages, B- and T-lymphocytes) and molecules of the tumor microenvironment (TME). Herein, we tested a series of GCTT with double staining (DS) for CD68-PD-L1 to evaluate tumor-associated macrophages (TAMs) expressing programmed death-ligand 1 (PD-L1) [TAMs PD-L1(+)] and clarify if these cells may be involved in establishing the fate of GCTT. METHODS: We collected 45 GCTT (comprising a total of 62 different components of GCTT). TAMs PD-L1(+) were evaluated with three different scoring systems [TAMs PD-L1(+)/mm2, TAMs PD-L1(+)/mm2H-score, TAMs PD-L1(+) %], and compared using pertinent statistic tests (Student's t-test and Mann-Whitney U test). RESULTS: We found that TAMs PD-L1(+) values were higher in S rather than EC (p = 0.001, p = 0.015, p = 0.022) and NS-GCTT (p < 0.001). P-S showed statistically significant differences in TAMs PD-L1(+) values compared to S-C (p < 0.001, p = 0.006, p = 0.015), but there were no differences between S-C and EC (p = 0.107, p = 0.408, p = 0.800). Finally, we found statistically significant differences also in TAMs PD-L1(+) values between EC and other NS-GCTT (p < 0.001). CONCLUSIONS: TAMs PD-L1(+) levels gradually decrease during the reprogramming of S cells {P-S [(high values of TAMs PD-L1(+)] → S-C and EC [(intermediate values of TAMs PD-L1(+)] → other NS-GCTT [(low values of TAMs PD-L1(+)], supporting a complex pathogenetic model where the interactions between tumor cells and TME components [and specifically TAMs PD-L1(+)] play a key role in determining the fate of GCTT.

H&E and OCT4/CD34 for the assessment of lympho-vascular invasion in seminoma and embryonal carcinoma.

BACKGROUND: Lymphovascular invasion (LVI) is a relevant prognostic factor in germ cell tumors of the testis (GCTT), and it is included in the pT stage. However, its detection on hematoxylin and eosin (H&E) slides is very challenging, and previous studies reported fair to moderate inter-observer agreement among dedicated uropathologists. In the present study, we tested H&E and a recently developed in-house double staining for OCT4/CD34 to detect LVI in GCTT. METHODS: Nine authors [5 non-uropathologists and 4 uropathologists] independently evaluated 34 consecutive and retrospectively enrolled cases of GCTT. We assessed the inter-observer agreement (Fleiss's Kappa) with both H&E and OCT4/CD34. Besides, we compared the consensus diagnosis on both H&E and OCT4/CD34-stained sections with the original diagnosis to evaluate the pT re-staging (McNemar test) and identify the sources of disagreement. RESULTS: The inter-observer agreement among uropathologists plus non-uropathologists was fair with both H&E (KF=0.398; p < 0.001) and OCT4/CD34 (KF=0.312; p < 0.001). OCT4/CD34 (KF=0.290; p < 0.001) slightly reduces the inter-observer agreement compared to H&E (KF=0.321; p < 0.001) for non-uropathologists; in contrast, OCT4/CD34 (KF=0.293; p < 0.001) significantly reduces the inter-observer agreement compared to H&E (KF=0.529; p < 0.001) for uropathologists, changing it from moderate to fair. Consensus diagnosis with H&E modified the LVI status of the original diagnosis in 8/34 (23.5 %) cases (p: 0.070), with pT re-staging in 2/34 (5.9 %) cases (p: 0.500). Consensus diagnosis with OCT4/CD34 modified the LVI status of the original diagnosis in 8/34 (23.5 %) cases (p: 0.289), with pT re-staging in 3/34 (8.8 %) cases (p: 0.250). The consensus diagnosis with OCT4/CD34 modified the consensus diagnosis with H&E in 8/34 (23.5 %) cases (p: 0.727), and these findings resulted in pT-restaging in 3/34 (8.8 %) cases (p: 0.500). The sources of disagreement among uropathologists were: H&E [artefactual clefts misinterpreted as LVI in 4/6 (66.7 %) cases and true foci of LVI misinterpreted as clusters of histiocytes within the vessels in 2/6 (33.3 %) cases], OCT4/CD34 [artefactual clefts misinterpreted as LVI in 2/8 (25 %) cases, true LVI misinterpreted as artefactual clefts in 2/8 (25 %) cases or floaters in 4/8 (50 %) cases]. CONCLUSIONS: OCT4/CD34 does not improve the inter-observer agreement for the assessment of LVI in OCT4(+) GCTT. Consensus diagnosis with H&E modifies the LVI status in a significant number of cases, resulting in changes of the pT stage in a relatively small subgroup. Consensus diagnosis with OCT4/CD34 provides little additional benefit since it cannot exclude mimickers of LVI such as floaters and artefactual clefts. These results argue against the adoption of this diagnostic tool for the routine assessment of OCT4(+) GCTT.

Evaluation of an institutional series of low-grade oncocytic tumor (LOT) of the kidney and review of the mutational landscape of LOT.

The 2022 WHO classification of urinary and male genital tumors introduced several novel kidney entities exhibiting eosinophilic/oncocytic features with specific mutational backgrounds. Thus, molecular techniques, such as next-generation sequencing (NGS), became more commonly used for their evaluation. We studied 12 low-grade oncocytic tumors (LOT) of the kidney (from 11 patients), identified in a cohort of 210 eosinophilic/oncocytic renal tumors, diagnosed in our institution between October 2019 and May 2023, which represented 5.7% (12/210) of all eosinophilic/oncocytic renal tumors during this period. We reviewed their clinicopathologic, histologic, and immunohistochemical features, as well as their mutational profiles. We also reviewed the literature on NGS-derived data of LOT, by selecting papers in which LOT diagnosis was rendered according to the criteria proposed initially. Median age was 65 years (mean: 63.5; range 43-79) and median tumor size was 2.0 cm (mean: 2.2; range: 0.9-3.1). All tumors were positive for PAX8, CK7, and GATA3, and negative or focally positive for CD117/KIT. We found the following gene mutations: MTOR ((6/11), 54.5%)), TSC1 ((2/11), 18.2%)), and 1 had both NOTCH1 and NOTCH4 ((1/11), 9.1%)). Wild-type status was found in 2/11 (18.2%) patients and one tumor was not analyzable. A review of 8 previous studies that included 79 LOTs revealed frequent mutations in the genes that regulate the mammalian target of rapamycin (mTOR) pathway: MTOR (32/79 (40.5%)), TSC1 (21/79 (26.6%)), and TSC2 (9/79 (11.4%)). Other mutated genes included PIK3CA, NF2, and PTEN, not typically known to affect the mTOR pathway, but potentially acting as upstream and downstream effectors. Our study shows that LOT is increasingly diagnosed in routine practice when applying the appropriate diagnostic criteria. We also confirm that the mTOR pathway is strongly implicated in the pathogenesis of this tumor mainly through MTOR, TCS1, and TSC2 mutations, but other genes could also be involved in the pathway activation, especially in LOTs without "canonical" mutations.

The role of next-generation sequencing in the differential diagnosis of composite neoplasms.

Composite neoplasms (CNs) are rare and diagnostically challenging lesions that require differentiating between mixed clonal tumors with divergent phenotypes (MT), collision of 2 independent tumors adjacent to each other (CT), and tumor-to-tumor metastasis (TTM). To that end, pathologists have traditionally used immunohistochemistry and limited molecular studies, such as Sanger sequencing. Herein we evaluate the potential application of NGS in the differential diagnosis of these rare neoplasms. Four CNs were included in the study. Two were diagnosed as MT (mixed adenoneuroendocrine carcinoma of the gallbladder and metastatic papillary thyroid carcinoma with squamous dedifferentiation) and 2 were interpreted as TTM (esophageal adenocarcinoma to lung adenocarcinoma and small cell carcinoma of the lung to meningeal melanoma). Diagnoses were made using clinical, histologic, and immunophenotypic information, with the aid of limited molecular studies in 2 cases. Formalin-fixed, paraffin-embedded tissue was dissected for DNA and RNA extraction, and NGS was performed using the Oncomine Comprehensive Panel. The 2 tumors initially interpreted as MT showed shared genetic aberrations in the different neoplastic components, supporting the pathologic diagnosis. NGS results for the lesion diagnosed as esophageal adenocarcinoma metastatic to lung adenocarcinoma did not support the histopathologic interpretation and were deemed inconclusive. However, the identification of an identical CDKN2A mutation in all components and in the adjacent benign lung parenchyma suggests a possible germline aberration. Sequencing results in the last case were clearly supportive of TTM. This study illustrates the role of NGS in the diagnostic workup of CNs, as an adjunct to light microscopy and immunohistochemistry.

Predicting the Behavior of Perivascular Epithelioid Cell Tumors of the Uterine Corpus.

Perivascular epithelioid cell tumors (PEComas) are rare neoplasms that share phenotypic features with angiomyolipomas, clear cell sugar tumors, and lymphangioleiomyomatosis. They presumably represent the neoplastic counterpart of a yet-unidentified perivascular epithelioid cell that expresses smooth muscle and melanocytic immunomarkers. The uterus is the second most common site of origin for perivascular epithelioid cell tumors, after the retroperitoneum. Although most uterine perivascular epithelioid cell tumors are clinically benign and can be cured by a complete surgical excision, there is a subset characterized by both local and distant dissemination. Unfortunately, no single histopathologic or immunohistochemical parameter can accurately predict the clinical behavior of these tumors, which is why the 2012 World Health Organization classification of tumors of the female reproductive organs suggests the use of several criteria to predict the risk of aggressive clinical behavior. Here we review those perivascular epithelioid cell tumors of the uterine corpus with aggressive clinical behavior reported in the literature, and we discuss their most relevant clinical and histopathologic features.

Mitogen-Activated Protein Kinase Signaling Pathway in Cutaneous Melanoma: An Updated Review.

The mitogen-activated protein kinase (MAPK) signaling pathway is a cascade of protein kinases that act in a sequential and predominantly linear fashion, albeit displaying some cross talk with other signaling cascades. Mutations in proteins integral to the MAPK signaling pathway are present in more than 50% of cutaneous melanomas. The most frequently mutated protein is v-raf murine sarcoma viral oncogene homolog B (BRAF), followed by neuroblastoma Ras viral oncogene homolog (NRAS). Recently, the development of targeted drugs for the treatment of BRAF-mutant melanoma has led to the widespread implementation of molecular assays for the detection of specific BRAF mutations. There have been some attempts to standardize testing of BRAF mutations, but this has not been achieved so far. Here we provide an updated review on the role of the MAPK signaling pathway in the pathogenesis of cutaneous melanoma, focusing on several different BRAF mutations and their diagnostic and therapeutic implications.

Primary Ureteral Thyroid Transcription Factor 1-Positive Small Cell Neuroendocrine Carcinoma: Case Report and Review of the Literature.

Upper urinary tract neoplasms comprise only 5% of urinary malignancies overall, the overwhelming majority showing an urothelial histology. Nonurothelial lesions represent 1.9% of upper tract neoplasms, and small cell carcinomas (SmCCs) only account for a minute fraction of them, with few cases reported in the literature to date. In the genitourinary tract, these lesions are most frequently found in the urinary bladder, where they tend to show a mixed histology with a characteristic small cell neuroendocrine component that is positive for chromogranin A, synaptophysin, and CD56 immunostains. Urinary SmCCs are almost invariably aggressive, with a propensity to infiltrate surrounding tissues and vascular structures. Unfortunately, clinical stage at presentation is often advanced, with early dissemination to lymph nodes or distant organs in a significant proportion of patients. For the most part, prognosis is dismal and can be predicted by the pathologic stage at diagnosis. Recently, new chemotherapeutic regimens have shown some potential benefit for the treatment of urinary SmCC, albeit not achieving curative results. Here we present the case of a 71-year-old male patient with a history of treated bladder urothelial carcinoma that presented with subacute flank pain and was diagnosed with a primary pure SmCC of the ureter. Besides the usual neuroendocrine immunomarkers, this tumor showed intense positivity for TTF-1 (thyroid transcription factor-1), a phenomenon that has been described for extrapulmonary SmCC of other locations.

Primary Mixed Adenoneuroendocrine Carcinoma of the Gallbladder in a 55-Year-Old Female Patient: A Case Report and Review of the Literature.

Mixed adenoneuroendocrine carcinomas (MANECs) of the biliary tract are rare tumors, and to date only a few cases arising in the gallbladder have been reported. Their histogenesis is a matter of debate, since the biliary tract normally lacks neuroendocrine cells. However, the immunohistochemical identification of nonneoplastic neuroendocrine cells in both biliary adenocarcinomas and intestinal metaplasia has been documented. Here we report a case of a 55-year-old female patient presenting with right upper quadrant pain, cholelithiasis, and a gallbladder mass identified after cholecystectomy. The histopathologic examination showed a MANEC, composed of an intestinal-type adenocarcinoma and a large cell neuroendocrine carcinoma, arising in a background of enteric metaplasia with extensive high-grade dysplasia. Moreover, we report the presence of focal pancreatic intraepithelial neoplasia-like epithelial lesions, which has not been described for these tumors yet. The histopathologic features of this case provide further support for the theory that MANECs arise following a metaplasia-dysplasia-carcinoma pathway.