Triple-Negative Apocrine Carcinomas: Toward a Unified Group With Shared Molecular Features and Clinical Behavior.

Triple-negative apocrine carcinomas (TNACs) are rare breast tumors with limited studies evaluating their molecular characteristics and clinical behavior. We performed a histologic, immunohistochemical, genetic, and clinicopathologic assessment of 42 invasive TNACs (1 with a focal spindle cell component) from 41 patients, 2 pure apocrine ductal carcinomas in situ (A-DCIS), and 1 A-DCIS associated with spindle cell metaplastic carcinoma (SCMBC). All TNACs had characteristic apocrine morphology and expressed androgen receptor (42/42), gross cystic disease fluid protein 15 (24/24), and CK5/6 (16/16). GATA3 was positive in most cases (16/18, 89%), and SOX10 was negative (0/22). TRPS1 was weakly expressed in a minority of tumors (3/14, 21%). Most TNACs had low Ki67 proliferation (≤10% in 67%, 26/39), with a median index of 10%. Levels of tumor infiltrating lymphocytes were low (≤10% in 93%, 39/42, and 15% in 7%, 3/42). Eighteen percent of TNACs presented with axillary nodal metastasis (7/38). No patients treated with neoadjuvant chemotherapy achieved pathologic complete response (0%, 0/10). Nearly all patients with TNAC (97%, n = 32) were without evidence of disease at the time of study (mean follow-up of 62 months). Seventeen invasive TNACs and 10 A-DCIS (7 with paired invasive TNAC) were profiled by targeted capture-based next-generation DNA sequencing. Pathogenic mutations in phosphatidylinositol 3-kinase pathway genes PIK3CA (53%) and/or PIK3R1 (53%) were identified in all TNACs (100%), including 4 (24%) with comutated PTEN. Ras-MAPK pathway genes, including NF1 (24%), and TP53 were mutated in 6 tumors each (35%). All A-DCIS shared mutations, such as phosphatidylinositol 3-kinase aberrations and copy number alterations with paired invasive TNACs or SCMBC, and a subset of invasive carcinomas showed additional mutations in tumor suppressors (NF1, TP53, ARID2, and CDKN2A). Divergent genetic profiles between A-DCIS and invasive carcinoma were identified in 1 case. In summary, our findings support TNAC as a morphologically, immunohistochemically, and genetically homogeneous subgroup of triple-negative breast carcinomas and suggest overall favorable clinical behavior.

Integrated Genomic and Clinicopathologic Approach Distinguishes Pancreatic Grade 3 Neuroendocrine Tumor From Neuroendocrine Carcinoma and Identifies a Subset With Molecular Overlap.

Distinguishing grade 3 pancreatic neuroendocrine tumor (G3 PanNET) from neuroendocrine carcinoma (PanNEC) is a known diagnostic challenge, and accurate classification is critical because clinical behavior and therapies differ. Although current recommendations suggest that immunohistochemistry for p53, Rb, ATRX, and DAXX can distinguish most cases, some cases remain difficult to classify using this approach. In this study, we reviewed 47 high-grade neoplasms originally diagnosed as pancreatic neuroendocrine neoplasms. In addition to the currently recommended stains, we performed capture-based sequencing of approximately 500 cancer genes and immunohistochemistry for p16 and trypsin or chymotrypsin. Using an integrated molecular and clinicopathologic approach, 42 (89%) of 47 cases had a clear final diagnosis of either G3 PanNET (n = 17), PanNEC (n = 17), or mixed acinar-NEC (n = 8). The 17 G3 PanNETs demonstrated frequent alterations in MEN1 (71%), DAXX (47%), ATRX (24%), TSC2 (35%), SETD2 (42%), and CDKN2A (41%). Contrary to prior reports, TP53 alterations were also common in G3 PanNETs (35%) but were always mutually exclusive with CDKN2A alterations in this group. The 17 PanNECs demonstrated frequent alterations in TP53 (88%), cell cycle genes RB1 (47%), CCNE1/CCND1 (12%), CDKN2A (29%), and in KRAS (53%) and SMAD4 (41%); TP53 was coaltered with a cell cycle gene in 76% of PanNECs. Diffuse strong p16 staining was observed in 69% of PanNECs in contrast to 0% of G3 PanNETs. The 8 acinar-NECs had recurrent alterations in ATM (25%), APC (25%), and STK11 (25%). Five cases remained difficult to classify, 3 of which exhibited overlapping molecular features with alterations in MEN1 with or without ATRX, and RB1 with or without TP53, making it unclear whether to classify as PanNET or PanNEC. Our data demonstrate that molecular profiling and immunohistochemistry for p16 greatly improve the diagnostic accuracy of high-grade pancreatic neuroendocrine neoplasms and identify a subset of rare cases with overlapping features of both PanNET and PanNEC.

Genomic profiling of combined hepatocellular-cholangiocarcinoma reveals similar genetics to hepatocellular carcinoma.

Combined hepatocellular-cholangiocarcinomas (CHC) are mixed tumours with both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) components. CHC prognosis is similar to intrahepatic CC (ICC) and worse than HCC; staging and treatment generally follow ICC algorithms. However, the molecular biology of CHC remains poorly characterised. We performed capture-based next-generation sequencing of 20 CHC and, for comparison, 10 ICC arising in cirrhosis. Intratumour heterogeneity was assessed by separately sequencing the HCC and CC components of nine CHC. CHC demonstrated molecular profiles similar to HCC, even in the CC component. CHC harboured recurrent alterations in TERT (80%), TP53 (80%), cell cycle genes (40%; CCND1, CCNE1, CDKN2A), receptor tyrosine kinase/Ras/PI3-kinase pathway genes (55%; MET, ERBB2, KRAS, PTEN), chromatin regulators (20%; ARID1A, ARID2) and Wnt pathway genes (20%; CTNNB1, AXIN, APC). No CHC had alterations in IDH1, IDH2, FGFR2 or BAP1, genes typically mutated in ICC. TERT promoter mutations were consistently identified in both HCC and CC components, supporting TERT alteration as an early event in CHC evolution. TP53 mutations were present in both components in slightly over half the TP53-altered cases. By contrast, focal amplifications of CCND1, MET and ERRB2, as well as Wnt pathway alterations, were most often exclusive to one component, suggesting that these are late events in CHC evolution. ICC in cirrhosis demonstrated alterations similar to ICC in non-cirrhotic liver, including in IDH1 or IDH2 (30%), CDKN2A (40%), FGFR2 (20%), PBRM1 (20%), ARID1A (10%) and BAP1 (10%). TERT promoter and TP53 mutation were present in only one ICC each. Our data demonstrate that CHC genetics are distinct from ICC (even in cirrhosis) and similar to HCC, which has diagnostic utility and implications for treatment. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Genomic profiling of well-differentiated hepatocellular neoplasms with diffuse glutamine synthetase staining reveals similar genetics across the adenoma to carcinoma spectrum.

Well-differentiated hepatocellular neoplasms are currently classified in the World Health Organization scheme as hepatocellular adenoma or hepatocellular carcinoma. There is no recognized diagnostic category for atypical cases with borderline features, and we have designated these as atypical hepatocellular neoplasms. Diffuse glutamine synthetase staining is used as a surrogate marker to detect ß-catenin activation, a well-recognized high risk feature in hepatocellular tumors. This study examined 27 well-differentiated hepatocellular neoplasms with diffuse glutamine synthetase staining, including 7 atypical hepatocellular neoplasms with no cytoarchitectural atypia, 6 atypical hepatocellular neoplasms with focal cytoarchitectural atypia, and 14 well-differentiated hepatocellular carcinomas. Capture-based next-generation sequencing was performed, and alterations in WNT pathway genes (CTNNB1, APC, AXIN1) were seen in 81% of cases (10/13 atypical hepatocellular neoplasms and 12/14 of hepatocellular carcinomas), while the molecular basis of diffuse glutamine synthetase staining was unclear in the remaining 19% of cases. Additional non-WNT pathway mutations (TP53, TSC1, DNMT3A, CREBBP) or copy number alterations were present in 56% of atypical hepatocellular neoplasms, with no significant difference in cases with or without focal cytoarchitectural atypia, supporting that all cases with ß-catenin activation should be classified as atypical irrespective of atypia. Atypical hepatocellular neoplasm and hepatocellular carcinoma also demonstrated largely similar genomic profiles, but TERT promoter mutations were restricted to hepatocellular carcinoma (21%) and copy number alterations were more common in hepatocellular carcinoma (64 vs 31%). Mutational and copy number analysis may be helpful in characterization and risk stratification of atypical hepatocellular neoplasms when morphology and glutamine synthetase staining yield ambiguous results.

Immunohistochemical and molecular features of cholangiolocellular carcinoma are similar to well-differentiated intrahepatic cholangiocarcinoma.

Cholangiolocellular carcinoma is characterized by low grade cytologic atypia, and anastomosing cords and glands resembling cholangioles or canals of Hering. Cholangiolocellular carcinoma has been variously regarded as a subtype of intrahepatic cholangiocarcinoma (World Health Organization 2000), combined hepatocellular-cholangiocarcinoma of stem cell subtype (World Health Organization 2010) and a distinct type of primary liver carcinoma. Capture-based next generation sequencing targeting the coding regions of 479 cancer genes and select introns was performed on 17 cases (5 cholangiolocellular carcinomas, 7 intrahepatic cholangiocarcinomas, 5 mixed cholangiolocellular-intrahepatic cholangiocarcinomas) along with immunohistochemistry for CK19, SALL4, CD56, CD117, and EMA. For 5 mixed cholangiolocellular-intrahepatic cholangiocarcinoma, the individual areas were micro-dissected prior to sequencing. CK19 and EMA were positive in all cases; both luminal and cytoplasmic EMA was seen in 3/5 cholangiolocellular carcinoma and 3/6 intrahepatic cholangiocarcinomas. CD117 and SALL4 were negative in all cases. CD56 was positive in 2/5 cholangiolocellular carcinoma, 4/6 intrahepatic cholangiocarcinoma and 2/5 mixed cases. Mutations typical of intrahepatic cholangiocarcinoma (IDH1/2, PBRM1, FGFR2) were present in 90% of cases with cholangiolocellular carcinoma component. The genomic profile (IDH1/2 mutations, FGFR2 fusions, chromatin-remodeling gene mutations such as ARID1A, PBRM1) and copy number alterations were similar in cholangiolocellular carcinoma, intrahepatic cholangiocarcinoma and mixed cholangiolocellular-intrahepatic cholangiocarcinoma. In all mixed cases, the immunohistochemistry results, mutational profile and copy number alterations in both components were similar. Cholangiolocellular carcinoma should be categorized as a histologic subtype of well-differentiated intrahepatic cholangiocarcinoma, and should not be considered a distinct entity, or combined hepatocellular-cholangiocarcinoma unless a distinct hepatocellular component is also present.

Identification of high-risk human papillomavirus and Rb/E2F pathway genomic alterations in mutually exclusive subsets of colorectal neuroendocrine carcinoma.

Colorectal neuroendocrine carcinomas, both small cell and large cell types, are highly aggressive tumors with poor prognosis compared with colorectal adenocarcinoma. The molecular drivers of neuroendocrine carcinoma are best defined in small cell lung cancer, which shows near-universal genomic alterations in TP53 and RB1. The genetics of colorectal neuroendocrine carcinoma remain poorly understood; recent studies demonstrated infrequent RB1 alterations and genetics closely resembling colorectal adenocarcinoma. To better define the molecular pathogenesis of colorectal neuroendocrine carcinoma, we performed capture-based next-generation sequencing on 25 cases and evaluated for expression of p53, Rb, p16, and high-risk human papillomavirus (HR-HPV) subtypes using immunohistochemistry, in situ hybridization, and polymerase chain reaction. Rb/E2F pathway dysregulation was identified in nearly all cases (23/25, 92%) and occurred via three distinct mechanisms. First, RB1 genomic alteration was present in 56% (14/25) of cases and was accompanied by Rb protein loss, high p16 expression, and absence of HR-HPV; these cases also had frequent genomic alterations in TP53, the PI3K/Ras and Wnt pathways, as well as in DNA repair genes, with 4/14 cases being hypermutated. Second, 16% (4/25) of cases, all left-sided, had TP53 alteration without RB1 alteration; half of these harbored high-level amplifications in CCNE1 and MYC or MYCN and arose in patients with ulcerative colitis. Finally, 28% (7/25) of cases, all rectal or anal, lacked genomic alterations in RB1 or TP53 but were positive for HR-HPV. Our data demonstrate that Rb/E2F pathway dysregulation is essential in the pathogenesis of colorectal neuroendocrine carcinoma, akin to neuroendocrine carcinomas in other anatomic sites. Moreover, colorectal neuroendocrine carcinomas stratify into three distinct molecular subgroups, which can be differentiated based on Rb protein and HR-HPV status. HR-HPV infection represents a distinct mechanism for Rb and p53 inactivation in cases lacking genomic alterations in either gene. Differential treatment strategies for hypermutated and HPV-driven cases could improve patient outcomes.

DNA flow cytometric and interobserver study of crypt cell atypia in inflammatory bowel disease.

AIMS: The pathological features and diagnostic reliability of crypt cell atypia (CCA) arising in inflammatory bowel disease (IBD) and its clinical significance are unknown. METHODS AND RESULTS: DNA flow cytometry (FCM) was performed on 14 colon biopsies of CCA from seven IBD patients (male-to-female ratio, 5:2; mean age, 53 years; mean IBD duration, 15 years) using paraffin-embedded tissue. Seven gastrointestinal pathologists were asked to diagnose each biopsy as negative for dysplasia (NEG), indefinite for dysplasia (IND), low-grade dysplasia (LGD) or high-grade dysplasia (HGD) by morphology alone, then again with knowledge of FCM results. Aneuploidy was detected in all 14 biopsies, and five of eight biopsies (63%) also showed strong and diffuse nuclear staining for p53 in the areas of CCA. Six (86%) patients developed HGD (n = 5) or adenocarcinoma (n = 1) in the same colonic segment where CCA had been diagnosed within a mean follow-up time of 27 months. No follow-up information was available in the remaining one patient. When diagnoses were grouped as NEG or 'atypical' (including IND, LGD or HGD), the overall agreement rate of 76% (kappa = 0.51) based on morphology alone improved to 90% (kappa = 0.81) with knowledge of FCM results. Even when categorised as NEG or dysplasia (LGD or HGD) with each of the IND diagnoses reclassified into three categories (NEG, LGD or HGD) based on the degree of suspicion for dysplasia, the overall agreement rate of 63% (kappa = 0.25) based on morphology alone improved to 73% (kappa = 0.46) with knowledge of FCM results. However, when grouped as NEG, LGD or HGD, the overall agreement rate was less than 40% (kappa < 0.09) regardless of knowledge of FCM results. CONCLUSIONS: The presence of aneuploidy, p53 positivity and development of HGD or adenocarcinoma on follow-up indicate that CCA likely represents a dysplastic lesion (at least LGD) and is a histological marker of neoplastic progression. Although the grading of CCA, largely based on cytological abnormalities, is subject to significant interobserver variability, CCA can be histologically identified and should lead to a recommendation of increased endoscopic surveillance, especially if aneuploidy is detected.

Adenomatoid tumors of the male and female genital tract are defined by TRAF7 mutations that drive aberrant NF-kB pathway activation.

Adenomatoid tumors are the most common neoplasm of the epididymis, and histologically similar adenomatoid tumors also commonly arise in the uterus and fallopian tube. To investigate the molecular pathogenesis of these tumors, we performed genomic profiling on a cohort of 31 adenomatoid tumors of the male and female genital tracts. We identified that all tumors harbored somatic missense mutations in the TRAF7 gene, which encodes an E3 ubiquitin ligase belonging to the family of tumor necrosis factor receptor-associated factors (TRAFs). These mutations all clustered into one of five recurrent hotspots within the WD40 repeat domains at the C-terminus of the protein. Functional studies in vitro revealed that expression of mutant but not wild-type TRAF7 led to increased phosphorylation of nuclear factor-kappa B (NF-kB) and increased expression of L1 cell adhesion molecule (L1CAM), a marker of NF-kB pathway activation. Immunohistochemistry demonstrated robust L1CAM expression in adenomatoid tumors that was absent in normal mesothelial cells, malignant peritoneal mesotheliomas and multilocular peritoneal inclusion cysts. Together, these studies demonstrate that adenomatoid tumors of the male and female genital tract are genetically defined by TRAF7 mutation that drives aberrant NF-kB pathway activation.

Hepatic angiomyolipoma: mutation analysis and immunohistochemical pitfalls in diagnosis.

AIMS: Hepatic angiomyolipoma (AML) often shows epithelioid morphology with inconspicuous fat. Epithelioid component can mimic hepatocellular adenoma (HCA) or carcinoma (HCC). The aims of this study were to examine the expression of commonly used markers for HCA or HCC in hepatic AML and highlight pitfalls in diagnosis. METHODS AND RESULTS: Resected hepatic AMLs (n = 16) were reviewed; reticulin stain, immunohistochemistry for glutamine synthetase (GS), ß-catenin and liver fatty acid binding protein (LFABP) were performed along with Sanger sequencing of exon 3 of CTNNB1 and next-generation sequencing (NGS). Predominant epithelioid component (≥50%) was seen in 80% of cases. Foamy macrophage was present in 33% of cases. High-risk histological features were often present in tumours with benign outcome: marked atypia (19%), mitoses (20%) and necrosis (33%). GS staining (≥10% of tumour) was seen in epithelioid components in 13 (87%) cases, and was diffuse (>50% of tumour) in six (40%) cases. LFABP staining or nuclear ß-catenin staining was not seen in any case. Sanger sequencing and NGS did not reveal CTNNB1 mutation in any tested case. NGS demonstrated TSC2 mutations in all five cases tested. CONCLUSIONS: The predominance of epithelioid component resembling HCA or HCC is common in hepatic AML. Absence of LFABP and presence of fat can be mistaken for HNF1α-inactivated HCA. Diffuse GS staining can be mistaken for ß-catenin-activated HCA or HCC. Diffuse GS expression is not related to CTNNB1 mutation. All tested cases showed TSC2 mutation, supporting this as the driving genetic event for hepatic AML.

DNA Genotyping of Nonmolar Donor Egg Pregnancies With Abnormal Villous Morphology: Allele Zygosity Patterns Prevent Misinterpretation as Complete Hydatidiform Mole.

DNA genotyping is the gold standard diagnostic test to distinguish hydatidiform moles from nonmolar but morphologically abnormal products of conception (POC). The test is based on comparison of alleles at 15 short tandem repeat loci in the chorionic villi of the POC to those in the maternal decidual tissue. If alleles in the POC are not present in the decidua, then the most concerning interpretation is that the POC has a paternal uniparental genome diagnostic of a complete hydatidiform mole (CHM). However, a nonmolar pregnancy from a donated egg would also appear the same because the maternal genome of the POC would match that of the maternal donor, not that of the decidua of the individual carrying the pregnancy. Not surprisingly, 2 cases of potential misclassification of the genotype of a donor egg POC as CHM have been reported in the literature. We hypothesize that the ratio of heterozygous loci to homozygous loci (so-called allele zygosity ratio) distinguishes the genotype of a donor egg POC from CHM. We compared the allele zygosity ratio in 11 nonmolar donor egg POC, 5 dispermic (heterozygous) CHM and 31 monospermic (homozygous) CHM, without knowledge of the use of a donor egg, the histologic findings, or results of p57 immunohistochemical staining. In all 47 cases, the alleles from the chorionic villi did not match those in the decidua. The average ratio of heterozygous to homozygous loci was 4:1 in donor egg POC and 1:3 in dispermic CHM (P<0.0001). Monospermic CHM contained 100% homozygous loci. p57 staining was intact in all donor egg POC. We conclude that the allele zygosity ratio is important to evaluate when interpreting the genotype of morphologically abnormal POC that does not match the genotype of the decidua. A high heterozygous:homozygous ratio should raise concern for a nonmolar donor egg pregnancy. Correlation of this variable along with review of the histologic findings and p57 immunohistochemistry may prevent misclassification of the genotype of a donor egg POC with abnormal villous morphology as a dispermic (heterozygous) CHM.

An Ovarian Adenocarcinoma With Combined Low-grade Serous and Mesonephric Morphologies Suggests a Müllerian Origin for Some Mesonephric Carcinomas.

Mesonephric carcinomas are rare adenocarcinomas of the female genital tract that occur most commonly in the uterine cervix. They are classically thought to arise from benign mesonephric remnants, and are rarely reported at other sites in the gynecologic tract. Here we present an interesting biphenotypic ovarian adenocarcinoma with intimately associated but distinct components of both low-grade serous carcinoma and mesonephric-like carcinoma. A serous borderline tumor was present adjacent to the invasive carcinoma, and no benign mesonephric precursors were identified. Numerous invasive peritoneal metastases were present, including multiple metastases with both low-grade serous and mesonephric-like elements. Consistent with recent reports, foci of mesonephric-like carcinoma were morphologically and immunohistochemically identical to classic mesonephric carcinoma of the cervix. On molecular analysis, the serous borderline tumor, primary and metastatic low-grade serous carcinoma, and primary and metastatic mesonephric-like carcinoma each harbored a shared NRAS p.Q61R hotspot mutation, shared gains in chromosome 1q and 18p, and shared losses in chromosomes 1p, 18q, and 22. These shared molecular features indicate a clonal relationship between all morphologic elements of this ovarian adenocarcinoma, suggesting that at least some mesonephric carcinomas may arise from Müllerian precursors.

Recurrent GNAQ mutations in anastomosing hemangiomas.

Anastomosing hemangiomas are recently described benign vascular lesions that occur chiefly in the genitourinary tract and paravertebral soft tissues. Owing to their rarity and unusual cytoarchitectural features, anastomosing hemangiomas are frequently confused with low-grade angiosarcomas. The specific genetic alterations underlying these lesions are currently unknown. We performed capture-based next-generation DNA sequencing analysis on 13 anastomosing hemangiomas and identified frequent somatic mutations in the heterotrimeric G-protein alpha-subunit, GNAQ. Nine of 13 cases (69%) harbored a somatic mutation at GNAQ codon 209, a known hotspot that is commonly mutated in uveal melanoma and blue nevi, as well as various congenital vascular proliferations. No other pathogenic or likely pathogenic mutations were identified in these genetically simple lesions. The finding of a recurrent driver mutation in the G-protein signal transduction pathway provides strong evidence that anastomosing hemangiomas are indeed clonal vascular neoplasms.

Genomic profiling of malignant peritoneal mesothelioma reveals recurrent alterations in epigenetic regulatory genes BAP1, SETD2, and DDX3X.

Malignant mesothelioma is a rare cancer that arises from the mesothelial cells that line the pleural cavity and less commonly from the peritoneal lining of the abdomen and pelvis. Most pleural mesotheliomas arise in patients with a history of asbestos exposure, whereas the association of peritoneal mesotheliomas with exposure to asbestos and other potential carcinogens is less clear, suggesting that the genetic alterations that drive malignant peritoneal mesothelioma may be unique from those in pleural mesothelioma. Treatment options for all malignant mesotheliomas are currently limited, with no known targeted therapies available. To better understand the molecular pathogenesis of malignant peritoneal mesothelioma, we sequenced 510 cancer-related genes in 13 patients with malignant mesothelioma arising in the peritoneal cavity. The most frequent genetic alteration was biallelic inactivation of the BAP1 gene, which occurred in 9/13 cases, with an additional two cases demonstrating monoallelic loss of BAP1. All 11 of these cases demonstrated loss of BAP1 nuclear staining by immunohistochemistry, whereas two tumors without BAP1 alteration and all 42 cases of histologic mimics in peritoneum (8 multilocular peritoneal inclusion cyst, 6 well-differentiated papillary mesothelioma of the peritoneum, 16 adenomatoid tumor, and 12 low-grade serous carcinoma of the ovary) demonstrated intact BAP1 nuclear staining. Additional recurrently mutated genes in this cohort of malignant peritoneal mesotheliomas included NF2 (3/13), SETD2 (2/13), and DDX3X (2/13). While these genes are known to be recurrently mutated in pleural mesotheliomas, the frequencies are distinct in peritoneal mesotheliomas, with nearly 85% of peritoneal tumors harboring BAP1 alterations versus only 20-30% of pleural tumors. Together, these findings demonstrate the importance of epigenetic modifiers including BAP1, SETD2, and DDX3X in mesothelial tumorigenesis and suggest opportunities for targeted therapies.

Genomic profiling of breast secretory carcinomas reveals distinct genetics from other breast cancers and similarity to mammary analog secretory carcinomas.

Secretory carcinomas of the breast are rare tumors with distinct histologic features, recurrent t(12;15)(p13;q25) translocation resulting in ETV6-NTRK3 gene fusion and indolent clinical behavior. Mammary analog secretory carcinomas arising in other sites are histopathologically similar to the breast tumors and also harbor ETV6-NTRK3 fusions. Breast secretory carcinomas are often triple (estrogen and progesterone receptor, HER2) negative with a basal-like immunophenotype. However, genomic studies are lacking, and whether these tumors share genetic features with other basal and/or triple negative breast cancers is unknown. Aside from shared ETV6-NTRK3 fusions, the genetic relatedness of secretory carcinomas arising in different sites is also uncertain. We immunoprofiled and sequenced 510 cancer-related genes in nine breast secretory carcinomas and six salivary gland mammary analog secretory carcinomas. Immunoprofiles of breast and salivary gland secretory carcinomas were similar. All the tumors showed strong diffuse MUC4 expression (n=15), and SOX10 was positive in all nine breast and in five out of six salivary gland tumors. All breast secretory carcinomas were triple negative or weakly ER-positive, and all tumors at both the sites expressed CK5/6 and/or EGFR, consistent with a basal-like phenotype. Sequencing revealed classic ETV6-NTRK3 fusion genes in all cases, including in carcinoma in situ of one breast tumor. Translocations were reciprocal and balanced in six out of nine breast and three out of six salivary gland tumors and were complex in three others. In contrast to most breast basal carcinomas, the mutational burden of secretory carcinomas was very low, and no additional pathogenic aberrations were identified in genes typically mutated in breast cancer. Five (56%) breast and two (33%) salivary gland tumors had simple genomes without copy number changes; the remainder had very few changes, averaging 1.3 per tumor. The ETV6-NTRK3 derivative chromosome was duplicated in one breast and one salivary gland tumor, and was the only copy number change in the latter. The findings highlight breast secretory carcinoma as a subtype more closely related to mammary analog secretory carcinoma than to basal/triple negative breast cancers of no special type. Lack of pathogenic mutations in common cancer-related genes suggests that ETV6-NTRK3 alone may suffice to drive these tumors and likely helps explain their indolent behavior.

FOXL2 Mutation Status in Granulosa Theca Cell Tumors of the Ovary.

Ovarian sex-cord stromal tumors that have between 10% and 50% granulosa cells in a prominent fibrothecomatous background have been referred to as granulosa theca cell tumors or mixed granulosa theca cell tumors. The classification and prognosis of these tumors is not clear. Most adult granulosa cell tumors of the ovary harbor a mutation in the FOXL2 gene, whereas fibromas and thecomas lack this mutation. The aim of our study was to assess the FOXL2 mutation status of ovarian granulosa theca cell tumors and to correlate the mutation status with morphologic and clinical characteristics. A FOXL2 mutation was detected in 6 of 12 (50%) granulosa theca cell tumors. Tumors with higher cellularity of granulosa cells were more likely to harbor a FOXL2 mutation as were tumors in which the granulosa cells formed large lobules. No conclusions could be drawn regarding the clinical and prognostic significance of the presence of a mutation given the small number of cases and limited clinical follow-up. Our study shows that half of granulosa theca cell tumors harbor the same FOXL2 mutation that characterizes adult granulosa cell tumors but there is no outcome evidence to guide whether mutation status should alter the classification of the tumor or the management of the patient.

Genomic profiling of malignant phyllodes tumors reveals aberrations in FGFR1 and PI-3 kinase/RAS signaling pathways and provides insights into intratumoral heterogeneity.

Malignant phyllodes tumors of the breast are poorly understood rare neoplasms with potential for aggressive behavior. Few efficacious treatment options exist for progressed or metastatic disease. The molecular features of malignant phyllodes tumors are poorly defined, and a deeper understanding of the genetics of these tumors may shed light on pathogenesis and progression and potentially identify novel treatment approaches. We sequenced 510 cancer-related genes in 10 malignant phyllodes tumors, including 5 tumors with liposarcomatous differentiation and 1 with myxoid chondrosarcoma-like differentiation. Intratumoral heterogeneity was assessed by sequencing two separate areas in 7 tumors, including non-heterologous and heterologous components of tumors with heterologous differentiation. Activating hotspot mutations in FGFR1 were identified in 2 tumors. Additional recurrently mutated genes included TERT promoter (6/10), TP53 (4/10), PIK3CA (3/10), MED12 (3/10), SETD2 (2/10) and KMT2D (2/10). Together, genomic aberrations in FGFR/EGFR PI-3 kinase and RAS pathways were identified in 8 (80%) tumors and included mutually exclusive and potentially actionable activating FGFR1, PIK3CA and BRAF V600E mutations, inactivating TSC2 mutation, EGFR amplification and PTEN loss. Seven (70%) malignant phyllodes tumors harbored TERT aberrations (six promoter mutations, one amplification). For comparison, TERT promoter mutations were identified by Sanger sequencing in 33% borderline (n=12) and no (0%, n=8) benign phyllodes tumors (P=0.391 and P=0.013 vs malignant tumors, respectively). Genetic features specific to liposarcoma, including CDK4/MDM2 amplification, were not identified. Copy number analysis revealed intratumoral heterogeneity and evidence for divergent tumor evolution in malignant phyllodes tumors with and without heterologous differentiation. Tumors with liposarcomatous differentiation revealed more chromosomal aberrations in non-heterologous components compared with liposarcomatous components. EGFR amplification was heterogeneous and present only in the non-heterologous component of one tumor with liposarcomatous differentiation. The results identify novel pathways involved in the pathogenesis of malignant phyllodes tumors, which significantly increase our understanding of tumor biology and have potential clinical impact.

The loss of Nf1 transiently promotes self-renewal but not tumorigenesis by neural crest stem cells.

Neurofibromatosis is caused by the loss of neurofibromin (Nf1), leading to peripheral nervous system (PNS) tumors, including neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs). A long-standing question has been whether these tumors arise from neural crest stem cells (NCSCs) or differentiated glia. Germline or conditional Nf1 deficiency caused a transient increase in NCSC frequency and self-renewal in most regions of the fetal PNS. However, Nf1-deficient NCSCs did not persist postnatally in regions of the PNS that developed tumors and could not form tumors upon transplantation into adult nerves. Adult P0a-Cre+Nf1(fl/-) mice developed neurofibromas, and Nf1(+/-)Ink4a/Arf(-/-) and Nf1/p53(+/-) mice developed MPNSTs, but NCSCs did not persist postnatally in affected locations in these mice. Tumors appeared to arise from differentiated glia, not NCSCs.

Gliosarcomas lack BRAFV600E mutation, but a subset exhibit ß-catenin nuclear localization.

Gliosarcoma (GS) is a rare subtype of glioblastoma (GBM) characterized by both glial and mesenchymal components. Unlike GBM, there are no specific prognostic markers, and optimized treatments for patients with GS do not exist. Recent reports describe BRAFV600E mutation in malignant peripheral nerve sheath tumors, and aberrant Wnt signaling and CTNNB1 (ß-catenin gene) mutations have been described in GBM. We sought to determine whether GS tumors harbor BRAFV600E mutations or aberrant Wnt signaling, as indicated by nuclear localization of ß-catenin, by immunohistochemical detection. Forty-eight (48) cases of primary and secondary adult GS (including recurrent ones) were evaluated by immunohistochemical techniques for the presence of nuclear ß-catenin and the BRAFV600E mutation. A small subset (6/46, 13%) showed nuclear localization of ß-catenin. None of the cases harbored BRAFV600E mutations (0/48). These results are the first to describe the presence of Wnt signaling pathway abnormalities, manifested by nuclear ß-catenin, in a subset, as well as the lack of BRAFV600E mutation in GS. We propose a potential role for Wnt pathway alterations in the pathogenesis of a subset of GS.

Diagnostic utility and limitations of glutamine synthetase and serum amyloid-associated protein immunohistochemistry in the distinction of focal nodular hyperplasia and inflammatory hepatocellular adenoma.

Inflammatory hepatocellular adenoma can show overlapping histological features with focal nodular hyperplasia, including inflammation, fibrous stroma, and ductular reaction. Expression of serum amyloid-associated protein in inflammatory hepatocellular adenoma and map-like pattern of glutamine synthetase in focal nodular hyperplasia can be helpful in this distinction, but the pitfalls and limitations of these markers have not been established. Morphology and immunohistochemistry were analyzed in 54 inflammatory hepatocellular adenomas, 40 focal nodular hyperplasia, and 3 indeterminate lesions. Morphological analysis demonstrated that nodularity, fibrous stroma, dystrophic blood vessels, and ductular reaction were more common in focal nodular hyperplasia, while telangiectasia, hemorrhage, and steatosis were more common in inflammatory hepatocellular adenoma, but there was frequent overlap of morphological features. The majority of inflammatory hepatocellular adenomas demonstrated perivascular and/or patchy glutamine synthetase staining (73.6%), while the remaining cases had diffuse (7.5%), negative (3.8%), or patchy pattern of staining (15%) that showed subtle differences from the classic map-like staining pattern and was designated as pseudo map-like staining. Positive staining for serum amyloid-associated protein was seen in the majority of inflammatory hepatocellular adenomas (92.6%) and in the minority of focal nodular hyperplasia (17.5%). The glutamine synthetase staining pattern was map-like in 90% of focal nodular hyperplasia cases, with the remaining 10% of cases showing pseudo map-like staining. Three cases were labeled as indeterminate and showed focal nodular hyperplasia-like morphology but lacked map-like glutamine synthetase staining pattern; these cases demonstrated a patchy pseudo map-like glutamine synthetase pattern along with the expression of serum amyloid-associated protein. Our results highlight the diagnostic errors that can be caused by variant patterns of staining with glutamine synthetase and serum amyloid-associated protein in inflammatory hepatocellular adenoma and focal nodular hyperplasia.

Apparent diffusion coefficient in glioblastoma with PNET-like components, a GBM variant.

Glioblastoma (GBM) with primitive neuroectodermal tumor (PNET)-like (GBM-PNET) components is a rare variant of GBM. Recent studies describe PNET-like clinical behavior in these patients-with significantly increased propensity for CSF dissemination and a benefit of "PNET-like" chemotherapy. The imaging appearance of GBM-PNET is not well-described and given areas of marked cellularity in the PNET components one might expect significantly reduced diffusion on MRI. The purpose of this study is to quantitatively evaluate the diffusion characteristics in GBM-PNET and compare them with conventional GBMs. Nine patients with surgical specimens yielding GBM-PNET were identified from the UCSF Pathology files. MR images of these patients were reviewed retrospectively. DWI (diffusion-weighted imaging) sequences were analyzed with multiple regions of interests placed within the tumor, and ADC (apparent diffusion coefficient) values were measured. Results were compared to previously published ADC values in pathology-proven conventional GBM cases from our institution. Reduced ADC was seen in GBM-PNET (mean 581 × 10(-6) mm(2)/s, range 338-817) compared to previously published mean of 1,030 × 10(-6) mm(2)/s in the enhancing components of conventional GBMs. We report substantially reduced ADC values in GBM-PNETs compared to conventional GBMs. If demonstrated in a larger sample, when areas of marked reduced diffusion are seen in a suspected GBM, MRI may appropriately direct tissue sampling and can advocate a thorough search for PNET-like components on histopathology. These patients may have a higher chance of developing CSF dissemination and may benefit from "PNET-like" platinum-based chemotherapy.