Digital pathology implementation in cancer diagnostics: towards informed decision-making.

Digital pathology (DP) has become a part of the cancer healthcare system, creating additional value for cancer patients. DP implementation in clinical practice provides plenty of benefits but also harbors hidden ethical challenges affecting physician-patient relationships. This paper addresses the ethical obligation to transform the physician-patient relationship for informed and responsible decision-making when using artificial intelligence (AI)-based tools for cancer diagnostics. DP application allows to improve the performance of the Human-AI Team shifting focus from AI challenges towards the Augmented Human Intelligence (AHI) benefits. AHI enhances analytical sensitivity and empowers pathologists to deliver accurate diagnoses and assess predictive biomarkers for further personalized treatment of cancer patients. At the same time, patients' right to know about using AI tools, their accuracy, strengths and limitations, measures for privacy protection, acceptance of privacy concerns and legal protection defines the duty of physicians to provide the relevant information about AHI-based solutions to patients and the community for building transparency, understanding and trust, respecting patients' autonomy and empowering informed decision-making in oncology.

Comprehensive clinicopathological, molecular, and methylation analysis of mesenchymal tumors with NTRK and other kinase gene aberrations.

Alterations in kinase genes such as NTRK1/2/3, RET, and BRAF underlie infantile fibrosarcoma (IFS), the emerging entity 'NTRK-rearranged spindle cell neoplasms' included in the latest WHO classification, and a growing set of tumors with overlapping clinical and pathological features. In this study, we conducted a comprehensive clinicopathological and molecular analysis of 22 cases of IFS and other kinase gene-altered spindle cell neoplasms affecting both pediatric and adult patients. Follow-up periods for 16 patients ranged in length from 10 to 130 months (mean 38 months). Six patients were treated with targeted therapy, achieving a partial or complete response in five cases. Overall, three cases recurred and one metastasized. Eight patients were free of disease, five were alive with disease, and two patients died. All cases showed previously reported morphological patterns. Based on the cellularity and level of atypia, cases were divided into three morphological grade groups. S100 protein and CD34 were at least focally positive in 12/22 and 14/22 cases, respectively. Novel PWWP2A::RET, NUMA1::RET, ITSN1::RAF1, and CAPZA2::MET fusions, which we report herein in mesenchymal tumors for the first time, were detected by RNA sequencing. Additionally, the first uterine case with BRAF and EGFR mutations and CD34 and S100 co-expression is described. DNA sequencing performed in 13 cases uncovered very rare additional genetic aberrations. The CNV profiles showed that high-grade tumors demonstrate a significantly higher percentage of copy number gains and losses across the genome compared with low- and intermediate-grade tumors. Unsupervised clustering of the tumors' methylation profiles revealed that in 8/9 cases, the methylation profiles clustered with the IFS methylation class, irrespective of their clinicopathological or molecular features. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Utility of Immunohistochemistry With Antibodies to SS18-SSX Chimeric Proteins and C-Terminus of SSX Protein for Synovial Sarcoma Differential Diagnosis.

Synovial sarcoma is a relatively common soft tissue tumor characterized by highly specific t(X;18)(p11;q11) translocation resulting in the fusion of SS18 with members of SSX gene family. Typically, detection of SS18 locus rearrangement by fluorescence in situ hybridization or SS18 :: SSX fusion transcripts confirms the diagnosis. More recently, immunohistochemistry (IHC) for SS18-SSX chimeric protein (E9X9V) and C-terminus of SSX (E5A2C) showed high specificity and sensitivity for synovial sarcoma. This study screened a cohort of >1000 soft tissue and melanocytic tumors using IHC and E9X9V and E5A2C antibodies. Three percent (6/212) of synovial sarcomas were either negative for SS18-SSX or had scattered positive tumor cells (n=1). In these cases, targeted RNA next-generation sequencing detected variants of SS18 :: SSX chimeric transcripts. DNA methylation profiles of 2 such tumors matched with synovial sarcoma. A few nonsynovial sarcoma tumors (n=6) revealed either focal SS18-SSX positivity (n=1) or scattered positive tumor cells. However, targeted RNA next-generation sequencing failed to detect SS18 :: SSX transcripts in these cases. The nature of this immunopositivity remains elusive and may require single cell sequencing studies. All synovial sarcomas showed positive SSX IHC. However, a mosaic staining pattern or focal loss of expression was noticed in a few cases. Strong and diffuse SSX immunoreactivity was also seen in epithelioid sclerosing osteosarcoma harboring EWSR1 :: SSX1 fusion, while several sarcomas and melanocytic tumors including cellular blue nevus (5/7, 71%) revealed focal to diffuse, mostly weak to intermediate SSX staining. The SS18-SSX and SSX IHC is a useful tool for synovial sarcoma differential diagnosis, but unusual immunophenotype should trigger molecular genetic testing.

Spindle cell rhabdomyosarcomas: With TFCP2 rearrangements, and novel EWSR1::ZBTB41 and PLOD2::RBM6 gene fusions. A study of five cases and review of the literature.

AIMS: Spindle-cell/sclerosing rhabdomyosarcomas (SS-RMS) are clinically and genetically heterogeneous. They include three well-defined molecular subtypes, of which those with EWSR1/FUS::TFCP2 rearrangements were described only recently. This study aimed to evaluate five new cases of SS-RMS and to perform a clinicopathological and statistical analysis of all TFCP2-rearranged SS-RMS described in the English literature to more comprehensively characterize this rare tumour type. METHODS AND RESULTS: Cases were retrospectively selected and studied by immunohistochemistry, fluorescence in situ hybridization with EWSR1/FUS and TFCP2 break-apart probes, next-generation sequencing (Archer FusionPlex Sarcoma kit and TruSight RNA Pan-Cancer Panel). The PubMed database was searched for relevant peer-reviewed English reports. Five cases of SS-RMS were found. Three cases were TFCP2 rearranged SS-RMS, having FUSex6::TFCP2ex2 gene fusion in two cases and triple gene fusion EWSR1ex5::TFCP2ex2, VAX2ex2::ALKex2 and VAX2intron2::ALKex2 in one case. Two cases showed rhabdomyoblastic differentiation and spindle-round cell/sclerosing morphology, but were characterized by novel genetic fusions including EWSR1ex8::ZBTB41ex7 and PLOD2ex8::RBM6ex7, respectively. In the statistical analysis of all published cases, CDKN2A or ALK alterations, the use of standard chemotherapy and age at presentation in the range of 18-24 years were negatively correlated to overall survival. CONCLUSION: EWSR1/FUS::TFCP2-rearranged SS-RMS is a rare rhabdomyosarcoma subtype, affecting predominantly young adults with average age at presentation 34 years (median 29.5 years; age range 7-86 years), with a predilection for craniofacial bones, rapid clinical course with frequent bone and lung metastases, and poor prognosis (3-year overall survival rate 28%).

EWSR1::POU2AF3(COLCA2) Sarcoma: An Aggressive, Polyphenotypic Sarcoma With a Head and Neck Predilection.

EWSR1::POU2AF3 (COLCA2) sarcomas are a recently identified group of undifferentiated round/spindle cell neoplasms with a predilection for the head and neck region. Herein, we report our experience with 8 cases, occurring in 5 men and 3 women (age range, 37-74 years; median, 60 years). Tumors involved the head/neck (4 cases), and one each the thigh, thoracic wall, fibula, and lung. Seven patients received multimodal therapy; 1 patient was treated only with surgery. Clinical follow-up (8 patients; range, 4-122 months; median, 32 months) showed 5 patients with metastases (often multifocal, with a latency ranging from 7 to 119 months), and 3 of them also with local recurrence. The median local recurrence-free and metastasis-free survival rates were 24 months and 29 months, respectively. Of the 8 patients, 1 died of an unknown cause, 4 were alive with metastatic disease, 1 was alive with unresectable local disease, and 2 were without disease. The tumors were composed of 2 morphologic subgroups: (1) relatively bland tumors consisting of spindled to stellate cells with varying cellularity and fibromyxoid stroma (2 cases) and (2) overtly malignant tumors composed of nests of "neuroendocrine-appearing" round cells surrounded by spindled cells (6 cases). Individual cases in the second group showed glandular, osteogenic, or rhabdomyoblastic differentiation. Immunohistochemical results included CD56 (4/4 cases), GFAP (5/8), SATB2 (4/6), keratin (AE1/AE3) (5/8), and S100 protein (4/7). RNA sequencing identified EWSR1::POU2AF3 gene fusion in all cases. EWSR1 gene rearrangement was confirmed by fluorescence in situ hybridization in 5 cases. Our findings confirm the head/neck predilection and aggressive clinical behavior of EWSR1::POU2AF3 sarcomas and widen the morphologic spectrum of these rare lesions to include relatively bland spindle cell tumors and tumors with divergent differentiation.

Biphenotypic sinonasal sarcoma with PAX3::MAML3 fusion transforming into high-grade rhabdomyosarcoma: report of an emerging rare phenomenon.

We report a case of a 67-year-old male patient with a sinonasal tumor that showed areas of classic biphenotypic sinonasal sarcoma (BSNS) which in some sections sharply transitioned into high-grade rhabdomyosarcoma. Immunohistochemically, the conventional BSNS parts showed S100 protein, SMA, PAX7, and focal MyoD1 expression, whereas desmin and myogenin were negative. In contrast, the cells in high-grade areas expressed desmin, MyoD1, myogenin, and PAX7, while being negative for S100 protein and SMA. Using the Archer FusionPlex assay, the classical PAX3::MAML3 gene fusion was detected. FISH for PAX3 and MAML3 confirmed a break of these genes in both components. Despite aggressive therapy, the tumor progression resulted in the patient's death. The herein presented case, together with 2 previously published cases of BSNS with high-grade transformation, helps to better understand this novel phenomenon. Although the risk for such transformation appears low, it has important clinical and diagnostic implications which are discussed.

A minority of cases of acinic cell carcinoma of the salivary glands are driven by an NR4A2 rearrangement: the diagnostic utility of the assessment of NR4A2 and NR4A3 alterations in salivary gland tumors.

Acinic cell carcinoma (AciCC) is a common salivary gland malignancy, typically composed of neoplastic acinic cells with zymogen granules. The vast majority of cases are driven by a t(4;9)(q13;q31) leading to enhancer hijacking and upregulation of the NR4A3 gene. However, a minority of cases do not display NR4A3 overexpression on immunohistochemical examination and are negative for the rearrangement involving the NR4A3 gene when tested by FISH. Such cases overexpress NR4A2, and the protein product is detectable by immunohistochemistry. In this study, we aimed to assess the utility of NR4A2 and NR4A3 immunohistochemistry in the differential diagnosis of salivary gland tumors. Eighty-five cases of classic low-grade ACiCC, as well as 36 cases with high-grade transformation (HGT) and 7 high-grade AciCC cases were included in the analysis. NR4A3 was at least focally positive in 105/128 (82%) cases. Out of the 23 cases that were immunohistochemically negative for NR4A3, 6 displayed nuclear immunopositivity with the NR4A2 antibody. The NR4A3 rearrangement was confirmed by FISH in 38/52 (73%) cases. In addition, this is the first report of an NR4A2 rearrangement being detected by FISH in 2 AciCC cases that were negative for the NR4A3 rearrangement. Our analysis confirms that the majority of AciCC, including high-grade cases and cases with HGT, are immunopositive for NR4A3, and suggests that NR4A3 immunohistochemistry is a powerful tool in the differential diagnosis of salivary gland tumors. However, its utility is limited in sub-optimally fixed samples which often display weaker and focal positivity. Our study also indicates that in a minority of cases, AciCC might be negative for NR4A3 immunostaining, because the pathogenic genetic event in these cases is instead a rearrangement involving the NR4A2 gene.

Sclerosing Polycystic Adenoma of Salivary Glands: A Novel Neoplasm Characterized by PI3K-AKT Pathway Alterations-New Insights Into a Challenging Entity.

Sclerosing polycystic adenoma (SPA) is a rare salivary gland neoplasm originally thought to represent a non-neoplastic lesion. Recently we have encountered an index case of apocrine intraductal carcinoma of parotid gland of 62-year-old man with invasive salivary duct carcinoma component arising from SPA, a combination of tumor entities that has never been published so far. Here, we further explore the nature of SPA by evaluating 36 cases that were identified from the authors' consultation files. The patients were 25 females and 11 males aged 11 to 79 years (mean, 47.8 y). All tumors originated from the parotid gland. Their size ranged from 11 to 70 mm (mean, 28 mm). Histologically, all cases revealed characteristic features of SPA, such as lobulated well-circumscribed growth, focal hyalinized sclerosis, presence of large acinar cells with abundant brightly eosinophilic intracytoplasmic granules, and ductal components with variable cytomorphologic characteristics, including foamy, vacuolated, apocrine, mucous, clear/ballooned, squamous, columnar and oncocyte-like cells. In all cases, there were foci of intraluminal solid and cribriform intercalated duct-like epithelial proliferations with variable dysplasia which were positive for S100 protein and SOX10, and fully enveloped by an intact layer of myoepithelial cells. In addition, 14/36 cases (39%) had focal intraductal cribriform and micropapillary apocrine-type dysplastic epithelial structures composed of cells positive for androgen receptors and negative for S100/SOX10. The intraductal proliferations of both types showed focal mild to severe dysplasia in 17 cases (17/36; 47%). Two cases showed overt malignant morphology ranging from high-grade intraductal carcinoma to invasive carcinoma with an apocrine ductal phenotype. Next generation sequencing using ArcherDX panel targeting RNA of 36 pan-cancer-related genes and/or a TruSight Oncology 170/500 Kit targeting a selection of DNA from 523 genes and RNA from 55 genes was performed. Tumor tissue was available for molecular analysis in 11 cases, and 9 (9/11; 82%) of them harbored genetic alterations in the PI3K pathway. Targeted sequencing revealed HRAS mutations c.37G>C, p.(Gly13Arg) (2 cases) and c.182A>G, p.(Gln61Arg) (2 cases), and PIK3CA mutations c.3140A>G, p.(His1047Arg) (3 cases), c.1633G>A, p.(Glu545Lys) (1 case), and c.1624G>A, p.(Glu542Lys) (1 case). Moreover, mutations in AKT1 c.49G>A, p.(Glu17Lys) and c.51dup, p.(Tyr18ValfsTer15); c.49_50delinsAG, p.(Glu17Arg) (as a double hit) were found (2 cases). In addition, germinal and somatic mutation of PTEN c.1003C>T, p.(Arg335Ter); c.445C>T, p.(Gln149Ter), respectively, were detected. Gene fusions were absent in all cases. These prevalent molecular alterations converging on one major cancer-related pathway support the notion that SPA is a true neoplasm with a significant potential to develop intraluminal epithelial proliferation with apocrine and/or intercalated duct-like phenotype. The name SPA more correctly reflects the true neoplastic nature of this enigmatic lesion.

EWSR1-PATZ1-rearranged sarcoma: a report of nine cases of spindle and round cell neoplasms with predilection for thoracoabdominal soft tissues and frequent expression of neural and skeletal muscle markers.

The knowledge of clinical features and, particularly, histopathological spectrum of EWSR1-PATZ1-rearranged spindle and round cell sarcomas (EPS) remains limited. For this reason, we report the largest clinicopathological study of EPS to date. Nine cases were collected, consisting of four males and five females ranging in age from 10 to 81 years (average: 49 years). Five tumors occurred in abdominal wall soft tissues, three in the thorax, and one in the back of the neck. Tumor sizes ranged from 2.5 to 18 cm (average 6.6 cm). Five patients had follow-up with an average of 38 months (range: 18-60 months). Two patients had no recurrence or metastasis 19 months after diagnosis. Four patients developed multifocal pleural or pulmonary metastasis and were treated variably by surgery, radiotherapy, and chemotherapy. The latter seemed to have little to no clinical benefit. One of the four patients was free of disease 60 months after diagnosis, two patients were alive with disease at 18 and 60 months, respectively. Morphologically, low, intermediate, and high-grade sarcomas composed of a variable mixture of spindled, ovoid, epithelioid, and round cells were seen. The architectural and stromal features also varied, resulting in a broad morphologic spectrum. Immunohistochemically, the following markers were most consistently expressed: S100-protein (7/9 cases), GFAP (7/8), MyoD1 (8/9), Pax-7 (4/5), desmin (7/9), and AE1/3 (4/9). By next-generation sequencing, all cases revealed EWSR1-PATZ1 gene fusion. In addition, 3/6 cases tested harbored CDKN2A deletion, while CDKN2B deletion and TP53 mutation were detected in one case each. Our findings confirm that EPS is a clinicopathologic entity, albeit with a broad morphologic spectrum. The uneventful outcome in some of our cases indicates that a subset of EPS might follow a more indolent clinical course than previously appreciated. Additional studies are needed to validate whether any morphological and/or molecular attributes have a prognostic impact.

Molecular Profiling of Clear Cell Myoepithelial Carcinoma of Salivary Glands With EWSR1 Rearrangement Identifies Frequent PLAG1 Gene Fusions But No EWSR1 Fusion Transcripts.

Myoepithelial carcinoma of salivary glands is an underrecognized and challenging entity with a broad morphologic spectrum, including an EWSR1-rearranged clear cell variant. Myoepithelial carcinoma is generally aggressive with largely unknown genetic features. A retrospective review of Salivary Gland Tumor Registry in Pilsen searching for the key words "clear cell myoepithelial carcinoma," "hyalinizing clear cell," and "clear cell malignant myoepithelioma" yielded 94 clear cell myoepithelial carcinomas (CCMCs) for molecular analysis of EWSR1 rearrangement using fluorescence in situ hybridization (FISH). Tumors positive for EWSR1 gene rearrangement were tested by next-generation sequencing (NGS) using fusion-detecting panels. NGS results were confirmed by reverse-transcription polymerase chain reaction or by FISH. Twenty-six tumors originally diagnosed as CCMC (26/94, 27.6%) revealed split signals for EWSR1 by FISH. Six of these tumors (6/26, 23%) displayed amplification of the EWSR1 locus. Fifteen cases were analyzable by NGS, whereas 9 were not, and tissue was not available in 2 cases. None of the CCMCs with EWSR1 rearrangements detected by FISH had an EWSR1 fusion transcript. Fusion transcripts were detected in 6 cases (6/15, 40%), including LIFR-PLAG1 and CTNNB1-PLAG1, in 2 cases each, and CHCHD7-PLAG1 and EWSR1-ATF1 fusions were identified in 1 case each. Seven cases, including those with PLAG1 fusion, were positive for PLAG1 rearrangement by FISH, with notable exception of CHCHD7-PLAG1, which is an inversion not detectable by FISH. One single case with EWSR1-ATF1 fusion in NGS showed ATF1 gene rearrangement by FISH and was reclassified as clear cell carcinoma (CCC). In addition, another 4 cases revealed ATF1 rearrangement by FISH and were reclassified as CCC as well. Moreover, 12/68 (17%) CCMCs with intact EWSR1 gene were selected randomly and analyzed by NGS. PLAG1 fusions were found in 5 cases (5/12, 41.6%) with LIFR (2 cases), FGFR1 (2 cases), and CTNNB1 (1 case) as partner genes. Overall, PLAG1 gene rearrangements were detected in 10/38 (26%) tested cases. None of the tumors had SMARCB1 loss by immunohistochemistry as a possible explanation for the EWSR1 abnormalities in FISH. Novel findings in our NGS study suggest that EWSR1-FISH positive CCMC is a gene fusion-driven disease with frequent oncogenic PLAG1 fusions, including LIFR-PLAG1 and CTNNB1-PLAG1 in most cases. Productive EWSR1 fusions are found only in a minority of EWSR1-ATF1-rearranged cases, which were in part reclassifiable as CCCs. Detectable EWSR1-FISH abnormality in CCMCs without gene fusion perhaps represents a passenger mutation with minor or no oncologic effect.

Molecular Profiling of Salivary Oncocytic Mucoepidermoid Carcinomas Helps to Resolve Differential Diagnostic Dilemma With Low-grade Oncocytic Lesions.

Oncocytic mucoepidermoid carcinoma (OMEC) is a rare but diagnostically challenging variant of mucoepidermoid carcinoma (MEC). OMEC is notable for differential diagnostic considerations that are raised as a result of overlap with other benign and low-grade oncocytic salivary gland tumors. Diffuse and strong immunoreactivity of p63 protein may be useful in distinguishing OMEC from its mimics. However, focal p63 staining can be present in benign oncytomas. Presence of mucin-containing cells, mucinous cystic formation, and foci of extravasated mucin are considered a hallmark of MEC. True mucocytes may be, however, very few and hardly discernable in OMECs. Recent evidence has shown that most MECs harbor gene fusions involving MAML2. A retrospective review of archived pathology files and the authors' own files was conducted to search for "low-grade/uncertain oncocytic tumor," "oncocytoma," and "oncocytic carcinoma" in the period from 1996 to 2019. The tumors with IHC positivity for p63 and/or p40, and S100 negativity, irrespective of mucicarmine staining, were tested by next-generation sequencing using fusion-detecting panels to detect MAML2 gene rearrangements. Two index cases from consultation practice (A.S. and A.A.) of purely oncocytic low-grade neoplasms without discernible mucinous cells showed a CRTC1-MAML2 fusion using next-generation sequencing, and were reclassified as OMEC. In total, 22 cases of oncocytic tumors, retrieved from the authors' files, and from the Salivary Gland Tumor Registry, harbored the MAML2 gene rearrangements. Presence of mucocytes, the patterns of p63 and SOX10 immunopositivity, and mucicarmine staining were inconsistent findings. Distinguishing OMEC devoid of true mucinous cells from oncocytoma can be very challenging, but it is critical for proper clinical management. Diffuse and strong positivity for p63 and visualization of hidden mucocytes by mucicarmine staining may be misleading and does not always suffice for correct diagnosis. Our experience suggests that ancillary studies for the detection of MAML2 rearrangement may provide useful evidence in difficult cases.

Predictive factors of pathological response to neoadjuvant chemotherapy in patients with breast cancer.

PURPOSE: To identify predictive factors connected with pathologic response in patients with breast cancer (BC) having received neoadjuvant chemotherapy (NACT). METHODS: 49 patients with BC were investigated before and after treatment in this prospective research. Different chemotherapy regimes were administered. The Miller-Payne scoring system was used to assess the tumour response. The nuclear proliferation markers Ki67 and the expression of topoisomerase IIα (Topo IIα) were evaluated. RESULTS: Six patients (12.2 %) achieved pathological complete response (pCR). Noticeable decrease of tumor cellularity was detected in all BC subtypes and pCR in the triple-negative BC (TNBC) group (p=0.007) was observed. Poorly differentiated tumors could be considered as predictive factors of pCR (p=0.07). Ki67 appeared to be a predictive marker of achieving pCR (p<0.001) with a threshold of 28% (AUC=0.89, 95% CI 0.75-0.96). The additional factor of reaching pCR was operable BC (p=0.04). The expression level of Topo IIα (p=0.50) and using different regimens of NACT (p=0.97) did not influence pCR achievement. CONCLUSION: To sum it up, poorly differentiated carcinomas with high cellularity in the primary tumor, TNBC, Ki 67 with a threshold above 28% and operable BC can be considered as early predictors of reaching pCR.