The diagnostic utility of methylthioadenosine phosphorylase immunohistochemistry for pancreatic ductal adenocarcinoma in FNA and small biopsy specimens.

BACKGROUND: Accurate diagnosis of pancreatic lesions by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) or fine-needle biopsy can be challenging. Although surrogate immunohistochemical markers for genetic alterations associated with pancreatic ductal adenocarcinoma (PDAC) have been identified, they have modest sensitivity. Biallelic loss of CDKN2A occurs in up to 46% of PDACs, and methylthioadenosine phosphorylase (MTAP) immunohistochemistry (IHC) has been identified as a reliable surrogate marker for this alteration. The current study evaluates the utility of MTAP IHC for the diagnosis of PDAC. METHODS: In total, 136 cases of EUS-FNA cell block or core biopsy targeting solid pancreatic masses were identified. MTAP IHC was performed and evaluated for complete loss of expression in neoplastic cells. These results were correlated with available clinical next-generation sequencing that was performed on a subset of cases. RESULTS: Complete loss of MTAP expression was identified in 23 of 80 (29%) PDACs. A subset of cases classified as suspicious (4 of 21) and atypical (4 of 22) showed MTAP loss. All morphologically indeterminate cases with MTAP loss were confirmed as PDAC on resection/additional sampling. No benign samples (n = 13) showed loss of MTAP. In samples that had available clinical next-generation sequencing data (n = 13), copy number loss of CDKN2A was detected in all cases that had loss of MTAP expression (n = 4). CONCLUSIONS: Loss of MTAP was identified in approximately 30% of PDAC small biopsy specimens. As loss of MTAP expression is not expected in nonneoplastic cells, and these findings suggest that MTAP IHC can support a diagnosis of PDAC in small biopsy samples.

Angiomyxoma of the Breast: A Clinicopathologic Analysis of 40 Cases.

Superficial angiomyxoma is an uncommon benign mesenchymal neoplasm that usually arises in dermis/subcutis of the extremities or trunk. Some tumors are associated with Carney complex. When arising in breast, these tumors are not well-recognized, mainly due to a lack of uniform nomenclature in the literature. This study therefore aims to improve recognition of angiomyxomas of the breast region. Forty cases were identified: demographics, presence of Carney complex, imaging and histologic features, PRKAR1A expression, and outcomes were evaluated. There were 22 female and 18 male patients (median age 40 years, range: 14 to 72). Most tumors presented as slowly-growing masses (77%). All but one were solitary, and median size was 1.5 cm. Tumors were superficial (dermal/subcutaneous) in 52.5% and deep/parenchymal in 47.5%. Nine involved the nipple-areola complex. All showed characteristic features of superficial angiomyxoma: poorly circumscribed, hypocellular, myxoid neoplasms with lobulated (55%) or infiltrative (45%) architecture, bland spindled fibroblasts, prominent thin-walled vessels, and admixed neutrophils. Tumors involving the nipple-areola complex infiltrated through areolar smooth muscle, and deep/parenchymal tumors showed entrapment of lobules mimicking myxoid fibroadenoma. Mitoses were typically absent, as was significant atypia. Cystic change was common. Two-thirds showed loss of PRKAR1A expression by immunohistochemistry. Two patients had Carney complex (7%). Recurrence after incomplete excision occurred in 1 patient. Angiomyxoma of breast may arise at superficial, nipple-areola or deep/parenchymal locations, where it can be difficult to recognize classic histologic features. Loss of expression of PRKAR1A is not invariable, but may be a helpful diagnostic clue. Recognizing angiomyxoma is important for 2 reasons: first, the recurrence rate is low and therefore wide excision is not essential, and second, it may allow detection of Carney complex in some patients.

Melanoma arising in extracutaneous cellular blue naevus: report of two cases with comparison to cutaneous counterparts and uveal melanoma.

AIMS: Blue naevi are benign melanocytic lesions that typically occur in the dermis. Melanoma arising in blue naevus is rare, and shows a molecular profile distinct from conventional forms of cutaneous melanoma and more similar to uveal melanoma and central nervous system (CNS) melanocytomas. In contrast to conventional cutaneous melanoma, these tumour types typically show activating driver mutations in GNAQ or GNA11, a low mutational burden without evidence of a UV signature and a reproducible pattern of chromosomal copy number changes. Blue naevi can also occur at extracutaneous sites. Here we report two cases of melanoma arising in extracutaneous blue naevus and compare their molecular features to cohorts of melanoma arising in cutaneous blue naevus (five patients) and uveal melanoma (six patients). METHODS AND RESULTS: We describe the clinical, histomorphological, immunohistochemical and molecular findings in these two cases of melanoma arising in extracutaneous blue naevus. We compare their molecular profiles to melanomas arising in cutaneous blue naevus and uveal melanoma using a targeted next-generation DNA sequencing platform and find striking similarities between all three groups. CONCLUSIONS: The close relationship between blue naevus-associated melanomas, regardless of their anatomical site, supports and validates the concept of melanoma arising in extracutaneous blue naevus and suggests that the two groups share common pathogenic mechanisms. The similarity of both groups to uveal melanoma in turn supports the close relationship between blue naevus-associated melanoma, uveal melanoma and CNS melanocytoma, and their distinction from conventional UV-associated melanoma. These findings have important implications for prognosis and therapy.

Gastroesophageal Glomus Tumors: Clinicopathologic and Molecular Genetic Analysis of 26 Cases With a Proposal for Malignancy Criteria.

Although criteria for malignancy have been established for glomus tumors of soft tissue, there are no accepted criteria for gastroesophageal glomus tumors, the behavior of which is considered to be unpredictable. Recently, both benign and aggressive gastroesophageal glomus tumors have been shown to harbor CARMN :: NOTCH2 fusions, but, as yet, there are no described genetic features that predict clinical behavior. Here, we evaluated 26 gastroesophageal glomus tumors to investigate histologic and genetic features that might predict malignant behavior. Seventeen patients (65%) were male. The median age at presentation was 54.5 years (range: 16 to 81 y). Primary sites were stomach (25 tumors) and distal esophagus (1). The median tumor size was 4.05 cm (range: 0.8 to 19.5 cm). Tumors were composed of lobules of rounded cells with sharp borders, palely eosinophilic to clear cytoplasm, and round nuclei. All tumors involved the muscularis propria, and 12 also involved the serosal surface. Mitoses ranged from <1 to 53/10 HPF (median: 5/10 HPF). Sixteen tumors, including all 15 with mitoses ≥2/10 HPF, showed atypia (3 mild, 10 moderate, 3 severe), defined as spindle cell morphology, nuclear irregularity, nuclear size variability, enlarged nuclei, or coarse chromatin. Considering these histologic features and clinical behavior, tumors were classified as malignant (15 tumors) if they measured ≥5 cm or showed both atypia and mitoses ≥2/10 HPF, or benign (11 tumors) if these criteria were not met. Follow-up was available for 19 patients (73%; range: 1 to 15 y; median: 5.8 y), including 7 with benign tumors and 12 with malignant tumors. Two patients with malignant tumors had metastases at presentation, and 7 developed metastases subsequently. Follow-up was available for 8 of 9 patients with metastatic disease. Two were alive with disease at most recent follow-up. One underwent resection of a liver metastasis, with no subsequent metastases in 3 years of follow-up. Five patients died of metastatic disease. By immunohistochemistry, smooth muscle actin was diffusely positive in all evaluated tumors, and caldesmon and synaptophysin were positive in 94% and 73%, respectively. Sequencing identified NOTCH2 alterations in 4 benign tumors (80%) and 8 malignant tumors (80%), including CARMN :: NOTCH2 fusions in 2 benign and 5 malignant tumors. All 5 sequenced benign tumors lacked complex copy number alterations (CNAs), whereas all 10 sequenced malignant tumors showed complex CNAs, including recurrent loss of 9p21.3 (4/10, variably including CDKN2A / B and MTAP ) and ATRX inactivation (4/10). Complex CNAs were identified in all sequenced tumors that were ≥5 cm, exhibited both cytologic atypia and ≥2 mitoses/10 HPF, involved the serosa or metastasized. We propose that gastroesophageal glomus tumors ≥5 cm or with both atypia and mitoses ≥2/10 HPF should be considered malignant. Copy number analysis might be helpful in borderline cases.

Preclinical Modeling of Leiomyosarcoma Identifies Susceptibility to Transcriptional CDK Inhibitors through Antagonism of E2F-Driven Oncogenic Gene Expression.

PURPOSE: Leiomyosarcoma (LMS) is a neoplasm characterized by smooth muscle differentiation, complex copy-number alterations, tumor suppressor loss, and the absence of recurrent driver mutations. Clinical management for advanced disease relies on the use of empiric cytotoxic chemotherapy with limited activity, and novel targeted therapies supported by preclinical research on LMS biology are urgently needed. A lack of fidelity of established LMS cell lines to their mesenchymal neoplasm of origin has limited translational understanding of this disease, and few other preclinical models have been established. Here, we characterize patient-derived xenograft (PDX) models of LMS, assessing fidelity to their tumors of origin and performing preclinical evaluation of candidate therapies. EXPERIMENTAL DESIGN: We implanted 49 LMS surgical samples into immunocompromised mice. Engrafting tumors were characterized by histology, targeted next-generation sequencing, RNA sequencing, and ultra-low passage whole-genome sequencing. Candidate therapies were selected based on prior evidence of pathway activation or high-throughput dynamic BH3 profiling. RESULTS: We show that LMS PDX maintain the histologic appearance, copy-number alterations, and transcriptional program of their parental tumors across multiple xenograft passages. Transcriptionally, LMS PDX cocluster with paired LMS patient-derived samples and differ primarily in host-related immunologic and microenvironment signatures. We identify susceptibility of LMS PDX to transcriptional cyclin-dependent kinase (CDK) inhibition, which disrupts an E2F-driven oncogenic transcriptional program and inhibits tumor growth. CONCLUSIONS: Our results establish LMS PDX as valuable preclinical models and identify strategies to discover novel vulnerabilities in this disease. These data support the clinical assessment of transcriptional CDK inhibitors as a therapeutic strategy for patients with LMS.

Updates from the 2020 World Health Organization Classification of Soft Tissue and Bone Tumours.

The fifth edition of the World Health Organization (WHO) classification of soft tissue and bone tumours was published in May 2020. This 'Blue Book', which is also available digitally for the first time, incorporates an array of new information on these tumours, amassed in the 7 years since the previous edition. Major advances in molecular characterisation have driven further refinements in classification and the development of ancillary diagnostic tests, and have improved our understanding of disease pathogenesis. Several new entities are also included. This review summarises the main changes introduced in the 2020 WHO classification for each subcategory of soft tissue and bone tumours.

Primary intra-abdominal melanoma arising in association with extracutaneous blue naevus: a report of two cases.

AIMS: Blue naevi are uncommon dermal melanocytic neoplasms characterised by GNAQ/GNA11 mutations, which very rarely progress to melanoma. Such melanomas also often have BAP1 mutations, and lack genetic events associated with conventional melanoma. Exceptionally, blue naevi arise in extracutaneous locations; one melanoma arising in this setting has been reported. We report the clinicopathological, immunohistochemical and molecular genetic features of two cases of melanoma arising in extracutaneous blue naevus. METHODS AND RESULTS: Both arose in males, aged 25 and 63 years, with no history of other melanocytic lesions, and presented as large, painful intra-abdominal masses. The tumours were dark-brown/black, multilobulated, involved small intestinal mesentery and consisted of a predominantly fascicular and spindled, but occasionally nested and epithelioid, proliferation of variably pigmented, relatively monotonous cells with pale cytoplasm and ovoid nuclei with mild to moderate atypia. Mitotic activity was variable but generally low. Both cases showed areas of conventional and cellular blue naevus. Recurrent tumour in one case showed predominantly epithelioid morphology and greater cytological atypia and mitotic activity. One case expressed Melan-A, SOX10 and CD117, with absent expression of S100 protein and DOG1; the other expressed Melan-A, HMB45 and S100 protein. Next-generation sequencing identified GNAQ and BAP1 mutations in one case and GNA11 mutation in the other. Both patients developed widespread metastatic disease. CONCLUSION: Exceptionally rare, aggressive melanomas arising in extracutaneous blue naevi should be distinguished from metastatic melanoma, gastrointestinal stromal tumour and malignant melanotic nerve sheath tumour, especially given the significant therapeutic and prognostic differences between these different entities.

Radiation-associated sarcomas other than malignant peripheral nerve sheath tumours demonstrate loss of histone H3K27 trimethylation†.

BACKGROUND AND AIMS: Complete loss of histone H3 lysine 27 trimethylation (H3K27me3) has recently emerged as a biomarker for malignant peripheral nerve sheath tumours (MPNST). Loss of H3K27me3 staining has also been reported in post-radiation MPNST; however, it has not been evaluated in a large series of radiation-associated sarcomas of different histological subtypes. The aim of this study was to assess H3K27me3 labelling by immunohistochemistry in radiation-associated sarcomas and to determine the prevalence of H3K27me3 loss in these tumours. METHODS AND RESULTS: Radiation-associated sarcomas (n = 119) from two tertiary care referral centres were evaluated for loss of H3K27me3, defined as complete loss of staining within tumour cells in the presence of a positive internal control. Twenty-three cases (19%) showed H3K27me3 loss, including nine of 10 (90%) MPNST, seven of 77 (9%) undifferentiated spindle cell/pleomorphic sarcomas, five of 25 (20%) angiosarcomas, one of five (20%) leiomyosarcomas and one of two (50%) osteosarcomas. CONCLUSIONS: Complete H3K27me3 loss was present in 19% of radiation-associated sarcomas in our series. Our findings demonstrate that loss of H3K27me3 is not specific for radiation-associated MPNST and may also occur in other histological subtypes of RAS, including radiation-associated undifferentiated spindle cell/pleomorphic sarcoma, angiosarcoma, leiomyosarcoma and osteosarcoma.

MYC expression has limited utility in the distinction of undifferentiated radiation-associated sarcomas from sporadic sarcomas and sarcomatoid carcinoma.

AIMS: MYC is a proto-oncogene that is frequently dysregulated in various malignancies, through translocation or amplification. Radiation-associated angiosarcoma frequently shows MYC amplification, and immunohistochemical expression has been shown to be a reliable surrogate marker for amplification, but less is known about MYC expression in other sarcoma types, despite reports of MYC amplification in some undifferentiated/unclassified radiation-associated sarcomas (RASs). Distinguishing putative RAS from non-radiation-associated sarcoma or sarcomatoid carcinoma can be difficult. The aim of this study was to determine the prevalence and potential diagnostic utility of MYC in this context, by evaluating MYC expression in a cohort of RASs, non-radiation-associated sarcomas, and sarcomatoid carcinomas. METHODS AND RESULTS: Three hundred and eighty-five neoplasms were evaluated, including 81 RASs (18 angiosarcomas; 57 undifferentiated sarcomas; three leiomyosarcomas; and three malignant peripheral nerve sheath tumours), 267 non-radiation-associated sarcomas, and 37 sarcomatoid carcinomas. Immunohistochemistry was performed with a monoclonal anti-MYC antibody. Staining in tumour cells was scored on the basis of extent (focal, 1-4%; multifocal, 5-49%; and diffuse, ≥50%) and intensity (strong, moderate, and weak). One hundred percent of radiation-associated angiosarcomas expressed MYC diffusely. Expression was infrequent among other types of RAS (9.5%), and the frequency was similar to that in non-radiation-associated sarcomas (9.7%). MYC expression was more common in sarcomatoid carcinomas, occurring in 43%. The extent and intensity of staining were variable in all groups. CONCLUSION: MYC expression is infrequent among RASs other than angiosarcoma, and has a similar prevalence in sporadic sarcomas. Given the frequency of expression in sarcomatoid carcinomas, MYC expression outside the context of radiation-associated angiosarcoma is of limited diagnostic utility, and should be interpreted with caution after exclusion of sarcomatoid carcinoma where relevant.

Secondary Sarcomas: Biology, Presentation, and Clinical Care.

Secondary sarcomas are a subset of sarcomas that occur in patients with prior cancer diagnoses and are associated with environmental or genetic factors. Although secondary sarcomas are rare in general, there are predisposing factors that can substantially increase this risk in certain populations. Herein, we review the environmental factors with the strongest association of sarcoma risk, including chemical exposure, certain viruses, cytotoxic and immunosuppressive agents, chronic edema, and radiation exposure. Additionally, the most common genetic disorders that carry a predisposition for sarcoma development will be discussed, including hereditary retinoblastoma (RB), Li-Fraumeni syndrome (LFS), neurofibromatosis type 1 (NF1), and DICER1 syndrome. Although treatment does not generally differ for sporadic versus secondary sarcomas, awareness of the risk factors can alter therapeutic strategies to minimize risk, aid prompt diagnosis by increasing clinical suspicion, and allow for appropriate surveillance and genetic counseling for those patients with cancer predisposition syndromes.

Smooth muscle tumors of the gastrointestinal tract: an analysis of prognostic features in 407 cases.

Smooth muscle tumors represent the second most common mural mesenchymal neoplasm in the gastrointestinal tract, but established criteria for prognostic assessment of these tumors are lacking. A large cohort of surgically resected intramural gastrointestinal smooth muscle tumors from 31 institutions was analyzed to identify potential prognostic features. Pathologic features were assessed by expert gastrointestinal and/or soft tissue pathologists at each center. Immunohistochemical confirmation was required. A total of 407 cases from the esophagus (n = 97, 24%), stomach (n = 180, 44%), small bowel (n = 74, 18%), and colorectum (n = 56, 14%) were identified. Patients ranged in age from 19 to 92 years (mean 55 years), with a slight female predominance (57%). Mean tumor size was 5.4 cm, with the largest tumor measuring 29 cm. Disease progression following surgery, defined as local recurrence, metastasis, or disease-related death, occurred in 56 patients (14%). Colorectal tumors were most likely to progress, followed by small bowel and gastric tumors. None of the esophageal tumors in this series progressed. Receiver operator characteristic analysis identified optimal cutoffs of 9.8 cm and 3 mitoses/5 mm2 for discriminating between progressive and non-progressive tumors. Histologic features strongly associated with progression by univariate analysis included moderate-to-severe atypia, high cellularity, abnormal differentiation (defined as differentiation not closely resembling that of normal smooth muscle), tumor necrosis, mucosal ulceration, lamina propria involvement, and serosal involvement (P < 0.0001 for all features). Age, sex, and margin status were not significantly associated with progression (P = 0.23, 0.82, and 0.07, respectively). A risk assessment table was created based on tumor site, size, and mitotic count, and Kaplan-Meier plots of progression-free survival for each subgroup revealed progression-based tiers. Based on our findings, it appears that nonesophageal gastrointestinal smooth muscle tumors measuring >10 cm and/or showing ≥3 mitoses/5 mm2 may behave aggressively, and therefore close clinical follow-up is recommended in these cases.

Sclerosing epithelioid mesenchymal neoplasm of the pancreas - a proposed new entity.

We have encountered pancreatic tumors with unique histologic features, which do not conform to any of the known tumors of the pancreas or other anatomical sites. We aimed to define their clinicopathologic features and whether they are characterized by recurrent molecular signatures. Eight cases were identified; studied histologically and by immunohistochemistry. Selected cases were also subjected to whole-exome sequencing (WES; n = 4), RNA-sequencing (n = 6), Archer FusionPlex assay (n = 5), methylation profiling using the Illumina MethylationEPIC (850k) array platform (n = 6), and TERT promoter sequencing (n = 5). Six neoplasms occurred in females. The mean age was 43 years (range: 26-75). Five occurred in the head/neck of the pancreas. All patients were treated surgically; none received neoadjuvant/adjuvant therapy. All patients are free of disease after 53 months of median follow-up (range: 8-94). The tumors were well-circumscribed, and the median size was 1.8 cm (range: 1.3-5.8). Microscopically, the unencapsulated tumors had a geographic pattern of epithelioid cell nests alternating with spindle cell fascicles. Some areas showed dense fibrosis, in which enmeshed tumor cells imparted a slit-like pattern. The predominant epithelioid cells had scant cytoplasm and round-oval nuclei with open chromatin. The spindle cells displayed irregular, hyperchromatic nuclei. Mitoses were rare. No lymph node metastases were identified. All tumors were positive for vimentin, CD99 and cytokeratin (patchy), while negative for markers of solid pseudopapillary neoplasm, neuroendocrine, acinar, myogenic/rhabdoid, vascular, melanocytic, or lymphoid differentiation, gastrointestinal stromal tumor as well as MUC4. Whole-exome sequencing revealed no recurrent somatic mutations or amplifications/homozygous deletions in any known oncogenes or tumor suppressor genes. RNA-sequencing and the Archer FusionPlex assay did not detect any recurrent likely pathogenic gene fusions. Single sample gene set enrichment analysis revealed that these tumors display a likely mesenchymal transcriptomic program. Unsupervised analysis (t-SNE) of their methylation profiles against a set of different mesenchymal neoplasms demonstrated a distinct methylation pattern. Here, we describe pancreatic neoplasms with unique morphologic/immunophenotypic features and a distinct methylation pattern, along with a lack of abnormalities in any of key genetic drivers, supporting that these neoplasms represent a novel entity with an indolent clinical course. Given their mesenchymal transcriptomic features, we propose the designation of "sclerosing epithelioid mesenchymal neoplasm" of the pancreas.

Atypical lipomatous tumour/well-differentiated liposarcoma and de-differentiated liposarcoma in patients aged ≤ 40 years: a study of 116 patients.

AIMS: Limited data exist on atypical lipomatous tumour (ALT)/well-differentiated liposarcoma (WDL) and de-differentiated liposarcoma (DDLPS) in children and young adults. METHODS AND RESULTS: Cases of ALT/WDL/DDLPS arising in patients aged ≤ 40 years were collected from multiple institutional and consultation archives. A total of 116 cases of ALT/WDL (75) and DDLPS (41) were identified, representing fewer than 5% of these tumours seen at our institutions during this time-period. The patients (59 male/57 female) ranged in age from 8 to 40 years. Sites included deep central (abdomen/retroperitoneum/pelvis/groin) (n = 60), extremity (n = 42), trunk (n = 5), head/neck (n = 8) and mediastinum (n = 1). De-differentiated patterns included: high-grade pleomorphic sarcoma, myxofibrosarcoma-like, heterologous rhabdomyoblastic differentiation, low-grade spindle cell sarcoma and homologous lipoblastic differentiation. Forty-one patients experienced a local recurrence and 11 patients with DDLPS developed metastasis. ALT arising in the extremities had lower recurrence rates than deep central WDL (5-year recurrence-free survival 88.9% versus 59.0%; P = 0.002), while patients with deep central DDLPS experienced significantly more adverse events compared to WDL at this site (5-year event-free survival 11.9% versus 59.0%) (P < 0.0001). Seven (of eight) head/neck tumours had follow-up available; five recurred, and one patient (DDLPS) with recurrence also experienced a metastasis. The single mediastinal tumour (DDLPS) recurred and metastasised. CONCLUSION: ALT/WDL and DDLPS occurring in patients aged ≤ 40 years is rare, but exhibits similar morphological features to its counterparts in older adults, including potential for heterologous and homologous de-differentiation in the latter. Although case numbers are limited, tumours arising in the head and neck exhibit high rates of adverse events, suggesting that classification as WDL rather than ALT is more appropriate.

Novel Methylated DNA Markers Discriminate Advanced Neoplasia in Pancreatic Cysts: Marker Discovery, Tissue Validation, and Cyst Fluid Testing.

OBJECTIVES: Pancreatic cystic lesions (PCLs) may be precancerous. Those likely to harbor high-grade dysplasia (HGD) or pancreatic cancer (PC) are targets for surgical resection. Current algorithms to predict advanced neoplasia (HGD/PC) in PCLs lack diagnostic accuracy. In pancreatic tissue and cyst fluid (CF) from PCLs, we sought to identify and validate novel methylated DNA markers (MDMs) that discriminate HGD/PC from low-grade dysplasia (LGD) or no dysplasia (ND). METHODS: From an unbiased whole-methylome discovery approach using predefined selection criteria followed by multistep validation on case (HGD or PC) and control (ND or LGD) tissues, we identified discriminant MDMs. Top candidate MDMs were then assayed by quantitative methylation-specific polymerase chain reaction on archival CF from surgically resected PCLs. RESULTS: Of 25 discriminant MDMs identified in tissue, 13 were selected for validation in 134 CF samples (21 cases [8 HGD, 13 PC], 113 controls [45 ND, 68 LGD]). A tree-based algorithm using 2 CF-MDMs (TBX15, BMP3) achieved sensitivity and specificity above 90%. Discrimination was significantly better by this CF-MDM panel than by mutant KRAS or carcinoembryonic antigen, with areas under the receiver operating characteristic curve of 0.93 (95% confidence interval: 0.86-0.99), 0.71 (0.57-0.85), and 0.72 (0.60-0.84), respectively. Cutoffs for the MDM panel applied to an independent CF validation set (31 cases, 56 controls) yielded similarly high discrimination, areas under the receiver operating characteristic curve = 0.86 (95% confidence interval: 0.77-0.94, P = 0.2). DISCUSSION: Novel MDMs discovered and validated in tissue accurately identify PCLs harboring HGD/PC. A panel of 2 MDMs assayed in CF yielded results with potential to enhance current risk prediction algorithms. Prospective studies are indicated to optimize and further evaluate CF-MDMs for clinical use.

Loss of SMAD4 protein expression in gastrointestinal and extra-gastrointestinal carcinomas.

AIMS: SMAD4 (DPC4) is a tumour suppressor gene that is dysregulated in various tumour types, particularly pancreaticobiliary and gastrointestinal carcinomas. Corresponding loss of protein expression has been reported in approximately 50% of pancreatic and 25% of colonic adenocarcinomas. In the evaluation of carcinoma of unknown primary site, immunohistochemical loss of SMAD4 expression is often used to suggest pancreaticobiliary origin, but there are limited data on the spectrum of SMAD4 expression in carcinomas of other sites. This study evaluates the frequency of SMAD4 loss in a large cohort of carcinomas from diverse anatomical sites. METHODS AND RESULTS: Immunohistochemistry for SMAD4 was performed on tissue microarrays or whole tissue sections of 1210 carcinomas from various organs: gastrointestinal tract, liver, pancreas/biliary tract, lung, breast, thyroid, kidney, ovary and uterus. Expression was considered lost when there was complete absence of staining in tumour cell nuclei, in the presence of intact staining in non-neoplastic cells. SMAD4 loss was seen in 58% of pancreatic adenocarcinomas, 27% of appendiceal adenocarcinomas, 19% of colorectal adenocarcinomas, 16% of cholangiocarcinomas, 10% of lung adenocarcinomas and <5% of oesophageal, breast, gastric and mucinous ovarian adenocarcinomas. All papillary thyroid, hepatocellular, non-mucinous ovarian, endometrial and renal cell carcinomas showed intact SMAD4 nuclear expression. CONCLUSION: In addition to pancreaticobiliary, appendiceal and colonic tumours, SMAD4 loss is also seen in a small subset of other carcinomas, specifically breast, lung, oesophageal and gastric adenocarcinomas, all of which are typically CK7-positive, similar to pancreaticobiliary carcinoma. Awareness of SMAD4 loss in these other carcinoma types is helpful in the evaluation of carcinomas of unknown or uncertain primary site.

Characteristics of mismatch repair deficiency in sarcomas.

Due to the efficacy of immune checkpoint inhibitor therapy in tumors with deficient mismatch repair, there has been a surge in demand for mismatch repair deficiency testing in various tumor types. Mismatch repair deficiency is not known to play a significant role in the pathogenesis of sarcomas, and the utility of testing these tumor types is not established. This study aimed to determine the frequency, pattern, and clinicopathologic correlates of mismatch repair deficiency in sarcomas. Three hundred and four sarcomas were profiled using a genomic platform that employs massively parallel sequencing to interrogate 447 cancer-associated genes. Mismatch repair status was evaluated by determining the number of small insertion/deletion events occurring in homopolymer regions per megabase of exonic sequence data across all genes.  Molecular characteristics of mismatch repair-deficient sarcomas were compared to mismatch repair-deficient carcinomas (n = 70) also identified using the sequencing panel. Seven sarcomas (2.3%) were classified as mismatch repair-deficient: four unclassified sarcomas, and one each of pleomorphic rhabdomyosarcoma, epithelioid leiomyosarcoma and malignant PEComa. One patient had an established diagnosis of Lynch syndrome. In the remaining patients, the mismatch repair gene mutation was confirmed or suspected to be somatic. Mismatch repair immunohistochemistry confirmed the mismatch repair-deficiency status of all cases with alterations in the tested proteins. As expected, mismatch repair-deficient sarcomas showed a significantly elevated tumor mutation burden relative to mismatch repair-proficient sarcomas (median 16 versus 4.6, p < 0.001). However, in comparison to mismatch repair-deficient carcinomas, mismatch repair-deficient sarcomas showed a lower tumor mutation burden (median 28 versus 16, p = 0.006) and a significantly greater degree of chromosomal instability. Among mismatch repair-deficient sarcomas, PD-L1 was variably expressed on tumor-associated macrophages but not on tumor cells. Three patients received pembrolizumab: two progressed and one has stable disease with five months follow-up. Mismatch repair deficiency in histologically classifiable sarcomas is rare (1%) and is more common in unclassified sarcomas (10%). Additional study is required to determine the predictive role of mismatch repair-deficiency in sarcomas for immunotherapy.

Radiation-Associated Sarcomas: An Update on Clinical, Histologic, and Molecular Features.

Approximately half of all cancer patients receive radiation therapy as part of their oncologic treatment. Radiation-associated sarcomas occur in fewer than 1% of patients who receive radiation therapy but account for up to 5% of all sarcomas. As the use of radiation has increased in the past few decades and overall oncologic outcomes are improving, the incidence of radiation-associated sarcomas is also expected to increase. Historically, radiation-associated sarcomas have been associated with poor outcomes but recent data suggest the prognosis is improving. Distinguishing the sarcoma from the primary malignancy is a major diagnostic criterion.

A Comparison of Outcomes and Prognostic Features for Radiation-Associated Angiosarcoma of the Breast and Other Radiation-Associated Sarcomas.

PURPOSE: Radiation-associated sarcomas (RAS) are considered to have a poor prognosis. Although the incidence is anticipated to rise, contemporary data regarding predictors of outcomes are few. We performed a retrospective analysis to identify RAS prognostic factors and subset analyses for radiation-associated angiosarcoma arising after treatment for breast cancer (RAAB) and other RAS subtypes (other-RAS). METHODS AND MATERIALS: Patients with localized RAS evaluated at an institutional multidisciplinary sarcoma clinic were identified. Clinical and histologic review was performed, and outcomes were assessed to identify prognostic features. A subset of cases underwent molecular analysis by next-generation sequencing. RESULTS: Among 176 patients, histologic subtypes of RAS included angiosarcoma (41%), undifferentiated/unclassified sarcoma (40%), leiomyosarcoma (8%), malignant peripheral nerve sheath tumor (6%), and osteosarcoma (2%). Sixty-seven patients (38%) had RAAB, and 109 (62%) had other-RAS. RAAB had significantly shorter latency from time of initial radiation compared with other-RAS (8 vs. 15 years; P < .001). Treatment approaches included surgery (91%), chemotherapy (44%), and radiation therapy (27%). Median follow-up was 3.2 years; 3-year overall survival (OS) was 74%. On multivariate analysis, positive margins (P < .0001), deep tumor location (intrathoracic/intra-abdominal, P = .002), and high grade (P < .0001) were associated with worse OS. In particular, 3-year OS with negative versus positive margins was 90% versus 66%. Patients with RAAB versus other-RAS showed a trend for higher 3-year OS (84% vs 68%; P = .09), significantly higher 3-year metastasis-free survival (82% vs 67%; P = .001), but similar 3-year local recurrence-free survival (54% vs 61%; P = .28). Next-generation sequencing identified overall low tumor mutational burden, recurrent MYC amplification in RAAB, and few clinically actionable mutations. CONCLUSIONS: Margin negative excision, superficial tumor location, and low tumor grade are determinants of improved OS for RAS, suggesting that complete surgical excision, when possible, is an optimal component of treatment. RAAB is a clinicopathologically distinct type of RAS with shorter latency from initial RT, different recurrence patterns, and when aggressively managed has potentially better outcomes compared with other-RAS.

Germline cancer susceptibility gene variants, somatic second hits, and survival outcomes in patients with resected pancreatic cancer.

PURPOSE: Germline variants in double-strand DNA damage repair (dsDDR) genes (e.g., BRCA1/2) predispose to pancreatic adenocarcinoma (PDAC) and may predict sensitivity to platinum-based chemotherapy and poly(ADP) ribose polymerase (PARP) inhibitors. We sought to determine the prevalence and significance of germline cancer susceptibility gene variants in PDAC with paired somatic and survival analyses. METHODS: Using a customized next-generation sequencing panel, germline/somatic DNA was analyzed from 289 patients with resected PDAC ascertained without preselection for high-risk features (e.g., young age, personal/family history). All identified variants were assessed for pathogenicity. Outcomes were analyzed using multivariable-adjusted Cox proportional hazards regression. RESULTS: We found that 28/289 (9.7%; 95% confidence interval [CI] 6.5-13.7%) patients carried pathogenic/likely pathogenic germline variants, including 21 (7.3%) dsDDR gene variants (3 BRCA1, 4 BRCA2, 14 other dsDDR genes [ATM, BRIP1, CHEK2, NBN, PALB2, RAD50, RAD51C]), 3 Lynch syndrome, and 4 other genes (APC p.I1307K, CDKN2A, TP53). Somatic sequencing and immunohistochemistry identified second hits in the tumor in 12/27 (44.4%) patients with germline variants (1 failed sequencing). Compared with noncarriers, patients with germline dsDDR gene variants had superior overall survival (hazard ratio [HR] 0.54; 95% CI 0.30-0.99; P = 0.05). CONCLUSION: Nearly 10% of PDAC patients harbor germline variants, although the majority lack somatic second hits, the therapeutic significance of which warrants further study.