Spatial molecular profiling of mixed invasive ductal and lobular breast cancers reveals heterogeneity in intrinsic molecular subtypes, oncogenic signatures, and mutations.

Mixed invasive ductal and lobular carcinoma (MDLC) is a rare histologic subtype of breast cancer displaying both E-cadherin positive ductal and E-cadherin negative lobular morphologies within the same tumor, posing challenges with regard to anticipated clinical management. It remains unclear whether these distinct morphologies also have distinct biology and risk of recurrence. Our spatially resolved transcriptomic, genomic, and single-cell profiling revealed clinically significant differences between ductal and lobular tumor regions including distinct intrinsic subtype heterogeneity - e.g., MDLC with triple-negative breast cancer (TNBC) or basal ductal and estrogen receptor positive (ER+) luminal lobular regions, distinct enrichment of cell cycle arrest/senescence and oncogenic (ER and MYC) signatures, genetic and epigenetic CDH1 inactivation in lobular but not ductal regions, and single-cell ductal and lobular subpopulations with unique oncogenic signatures further highlighting intraregional heterogeneity. Altogether, we demonstrated that the intratumoral morphological/histological heterogeneity within MDLC is underpinned by intrinsic subtype and oncogenic heterogeneity which may result in prognostic uncertainty and therapeutic dilemma.

Decreased HER2 expression in endometrial cancer following anti-HER2 therapy.

Trastuzumab has demonstrated clinical efficacy in the treatment of HER2-positive serous endometrial cancer (EC), which led to its incorporation into standard-of-care management of this aggressive disease. Acquired resistance remains an important challenge, however, and its underlying mechanisms in EC are unknown. To define the molecular changes that occur in response to anti-HER2 therapy in EC, targeted next-generation sequencing (NGS), HER2 immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH) were performed on pre- and post-treatment tumour samples from 14 patients with EC treated with trastuzumab or trastuzumab emtansine. Recurrent tumours after anti-HER2 therapy acquired additional genetic alterations compared with matched pre-treatment ECs and frequently showed decreased HER2 protein expression by IHC (7/14, 50%). Complete/near-complete absence of HER2 protein expression (score 0/1+) observed post-treatment (4/14, 29%) was associated with retained HER2 gene amplification (n = 3) or copy number neutral status (n = 1). Whole-exome sequencing performed on primary and recurrent tumours from the latter case, which exhibited genetic heterogeneity of HER2 amplification in the primary tumour, revealed selection of an early HER2-non-amplified clone following therapy. Our findings demonstrate that loss of target expression, by selection of HER2-non-amplified clones or, more commonly, by downregulation of expression, may constitute a mechanism of resistance to anti-HER2 therapy in HER2-positive EC. © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Nonlobular Invasive Breast Carcinomas with Biallelic Pathogenic CDH1 Somatic Alterations: A Histologic, Immunophenotypic, and Genomic Characterization.

CDH1 encodes for E-cadherin, and its loss of function is the hallmark of invasive lobular carcinoma (ILC). Albeit vanishingly rare, biallelic CDH1 alterations may be found in nonlobular breast carcinomas (NL-BCs). We sought to determine the clinicopathologic characteristics and repertoire of genetic alterations of NL-BCs harboring CDH1 biallelic genetic alterations. Analysis of 5842 breast cancers (BCs) subjected to clinical tumor-normal sequencing with an FDA-cleared multigene panel was conducted to identify BCs with biallelic CDH1 pathogenic/likely pathogenic somatic mutations lacking lobular features. The genomic profiles of NL-BCs with CDH1 biallelic genetic alterations were compared with those of ILCs and invasive ductal carcinomas (IDCs), matched by clinicopathologic characteristics. Of the 896 CDH1-altered BCs, 889 samples were excluded based on the diagnosis of invasive mixed ductal/lobular carcinoma or ILC or the detection of monoallelic CDH1 alterations. Only 7 of the 5842 (0.11%) BCs harbored biallelic CDH1 alterations and lacked lobular features. Of these, 4/7 (57%) cases were ER-positive/HER2-negative, 1/7 (14%) was ER-positive/HER2-positive, and 2/7 (29%) were ER-negative/HER2-negative. In total, 5/7 (71%) were of Nottingham grade 2, and 2/7 (29%) were of grade 3. The NL-BCs with CDH1 biallelic genetic alterations included a mucinous carcinoma (n = 1), IDCs with focal nested growth (n = 2), IDC with solid papillary (n = 1) or apocrine (n = 2) features, and an IDC of no special type (NST; n = 1). E-cadherin expression, as detected by immunohistochemistry, was absent (3/5) or aberrant (discontinuous membranous/cytoplasmic/granular; 2/5). However, NL-BCs with CDH1 biallelic genetic alterations displayed recurrent genetic alterations, including TP53, PIK3CA (57%, 4/7; each), FGFR1, and NCOR1 (28%, 2/7, each) alterations. Compared with CDH1 wild-type IDC-NSTs, NL-BCs less frequently harbored GATA3 mutations (0% vs 47%, P = .03), but no significant differences were detected when compared with matched ILCs. Therefore, NL-BCs with CDH1 biallelic genetic alterations are vanishingly rare, predominantly comprise IDCs with special histologic features, and have genomic features akin to luminal B ER-positive BCs.

Adenoid cystic carcinoma of the Bartholin's gland is underpinned by MYB- and MYBL1- rearrangements.

OBJECTIVE: Adenoid cystic carcinoma (AdCC) of the Bartholin's gland (AdCC-BG) is a very rare gynecologic vulvar malignancy. AdCC-BGs are slow-growing but locally aggressive and are associated with high recurrence rates. Here we sought to characterize the molecular underpinning of AdCC-BGs. METHODS: AdCC-BGs (n = 6) were subjected to a combination of RNA-sequencing, targeted DNA-sequencing, reverse-transcription PCR, fluorescence in situ hybridization (FISH) and MYB immunohistochemistry (IHC). Clinicopathologic variables, somatic mutations, copy number alterations and chimeric transcripts were assessed. RESULTS: All six AdCC-BGs were biphasic, composed of ductal and myoepithelial cells. Akin to salivary gland and breast AdCCs, three AdCC-BGs had the MYB::NFIB fusion gene with varying breakpoints, all of which were associated with MYB overexpression by IHC. Two AdCC-BGs were underpinned by MYBL1 fusion genes with different gene partners, including MYBL1::RAD51B and MYBL1::EWSR1 gene fusions, and showed MYB protein expression. Although the final AdCC-BG studied had MYB protein overexpression, no gene fusion was identified. AdCC-BGs harbored few additional somatic genetic alterations, and only few mutations in cancer-related genes were identified, including GNAQ, GNAS, KDM6A, AKT1 and BCL2, none of which were recurrent. Two AdCC-BGs, both with a MYB::NFIB fusion gene, developed metastatic disease. CONCLUSIONS: AdCC-BGs constitute a convergent phenotype, whereby activation of MYB or MYBL1 can be driven by the MYB::NFIB fusion gene or MYBL1 rearrangements. Our observations further support the notion that AdCCs, irrespective of organ site, constitute a genotypic-phenotypic correlation. Assessment of MYB or MYBL1 rearrangements may be used as an ancillary marker for the diagnosis of AdCC-BGs.

Recurrent WWTR1 S89W mutations and Hippo pathway deregulation in clear cell carcinomas of the cervix.

Clear cell carcinoma (CCC) of the cervix (cCCC) is a rare and aggressive type of human papillomavirus (HPV)-negative cervical cancer with limited effective treatment options for recurrent or metastatic disease. Historically, CCCs of the lower genital tract were associated with in utero diethylstilbestrol exposure; however, the genetic landscape of sporadic cCCCs remains unknown. Here we sought to define the molecular underpinning of cCCCs. Using a combination of whole-exome, targeted capture, and RNA-sequencing, we identified pathogenic genetic alterations in the Hippo signaling pathway in 50% (10/20) of cCCCs, including recurrent WWTR1 S89W somatic mutations in 40% (4/10) of the cases harboring mutations in the Hippo pathway. Irrespective of the presence or absence of Hippo pathway genetic alterations, however, all primary cCCCs analyzed in this study (n = 20) harbored features of Hippo pathway deregulation at the transcriptomic and protein levels. In vitro functional analysis revealed that expression of the WWTR1 S89W mutation leads to reduced binding of TAZ to 14-3-3, promoting constitutive nuclear translocation of TAZ and Hippo pathway repression. WWTR1 S89W expression was found to lead to acquisition of oncogenic behavior, including increased proliferation, migration, and colony formation in vitro as well as increased tumorigenesis in vivo, which could be reversed by targeted inhibition of the TAZ/YAP1 complex with verteporfin. Finally, xenografts expressing WWTR1 S89W displayed a shift in tumor phenotype, becoming more infiltrative as well as less differentiated, and were found to be composed of cells with conspicuous cytoplasmic clearing as compared to controls. Our results demonstrate that Hippo pathway alterations are likely drivers of cCCCs and likely contribute to the clear cell phenotype. Therapies targeting this pathway may constitute a new class of treatment for these rare, aggressive tumors. © 2022 The Pathological Society of Great Britain and Ireland.

Mullerian adenosarcoma: clinicopathologic and molecular characterization highlighting recurrent BAP1 loss and distinctive features of high-grade tumors.

Mullerian adenosarcoma is an uncommon mesenchymal tumor of the gynecologic tract. Most cases are low-grade, while high-grade adenosarcomas are rare and not well studied. Herein, we characterize the clinicopathologic and molecular features of 27 adenosarcomas of gynecologic origin, enriched for high-grade tumors subjected to targeted panel sequencing. Sarcomatous overgrowth was more frequently seen in high-grade compared to low-grade tumors (12/17, 71%, vs 1/10, 10%, p = 0.004) and heterologous elements were exclusive to high-grade cases (n = 7, p = 0.03). All deaths were from high-grade disease (advanced primary, n = 2, or recurrence, n = 5). Genetic alterations specific to high-grade adenosarcomas have known associations with chromosome instability, including TP53 mutations (n = 4) and amplifications of MDM2 (n = 2) and CCNE1 (n = 2). Somatic ATRX frameshift mutations were found in 2 patients with high-grade recurrences following a primary low-grade adenosarcoma and ATRX deletion in 1 high-grade adenosarcoma with an adjacent low-grade component. The fraction of genome altered by copy number alterations was significantly higher in high-grade compared to low-grade adenosarcomas (P = 0.001). Other recurrent genetic alterations across the entire cohort included BAP1 homozygous deletions (n = 4), DICER1 mutations (n = 4), ARID1A mutations (n = 3), TERT promoter mutations (n = 2) and amplification (n = 1), as well as alterations involving members of the PI3K and MAPK signaling pathways. One tumor harbored an ESR1-NCOA3 fusion and another had an MLH1 homozygous deletion. Immunohistochemical analysis for BAP1 revealed loss of nuclear expression in 6/24 (25%) cases, including all four tumors with BAP1 deletions. Notably, out of 196 mesenchymal neoplasms of gynecologic origin, BAP1 homozygous deletion was only found in adenosarcomas (P = 0.0003). This study demonstrates that high-grade adenosarcomas are heterogeneous at the molecular level and are characterized by genomic instability and TP53 mutations; ATRX loss may be involved in high-grade transformation of low-grade adenosarcoma; and BAP1 inactivation appears to be a specific pathogenic driver in a subset of adenosarcomas.

ESR1 hotspot mutations in endometrial stromal sarcoma with high-grade transformation and endocrine treatment.

High-grade endometrial stromal sarcomas (HGESSs) are more aggressive and have higher rates of resistance to endocrine therapy than low-grade endometrial stromal sarcomas (LGESSs). The pathogenesis of hormonal resistance in these lesions has yet to be defined. Here we sought to histologically and genetically characterize 3 LGESSs and their recurrences that underwent histologic high-grade transformation following endocrine therapy. For this, DNA from primary tumors and select subsequent recurrences were subject to massively parallel sequencing targeting 468 cancer-related genes. Somatic mutation analyses were performed using validated bioinformatics methods. In addition, RNA from each case was evaluated for the presence of gene fusions using targeted RNA-sequencing. All patients initially presented with LGESS, developed HGESS recurrences, and received at least 2 lines of hormonal suppressive therapy. Gene fusions classically described as associated with LGESS were identified in all 3 cases, including JAZF1-PHF1, EPC1-PHF1 and JAZF1-SUZ12 fusions for Cases 1, 2 and 3, respectively. Targeted sequencing analysis revealed that none of the primary LGESS, however the HGESS recurrences of Cases 1 and 3, and the LGESS and HGESS recurrences of Case 2 post endocrine treatment harbored ESR1 p.Y537S hotspot mutations. These ESR1 ligand-binding domain mutations have been found as a mechanism of acquired endocrine resistance in breast cancer. Also, a reduction in estrogen receptor (ER) expression was observed in recurrences. Our findings suggest that the ESR1 p.Y537S hotspot mutation in LGESS with histologic high-grade transformation may be associated with endocrine resistance in these lesions. Furthermore, our data suggest that genetic analyses may be performed in recurrent LGESS following hormonal therapy, development of high-grade morphology, and/or altered/diminished ER expression.

The clinical behavior and genomic features of the so-called adenoid cystic carcinomas of the solid variant with basaloid features.

Classic adenoid cystic carcinomas (C-AdCCs) of the breast are rare, relatively indolent forms of triple negative cancers, characterized by recurrent MYB or MYBL1 genetic alterations. Solid and basaloid adenoid cystic carcinoma (SB-AdCC) is considered a rare variant of AdCC yet to be fully characterized. Here, we sought to determine the clinical behavior and repertoire of genetic alterations of SB-AdCCs. Clinicopathologic data were collected on a cohort of 104 breast AdCCs (75 C-AdCCs and 29 SB-AdCCs). MYB expression was assessed by immunohistochemistry and MYB-NFIB and MYBL1 gene rearrangements were investigated by fluorescent in-situ hybridization. AdCCs lacking MYB/MYBL1 rearrangements were subjected to RNA-sequencing. Targeted sequencing data were available for 9 cases. The invasive disease-free survival (IDFS) and overall survival (OS) were assessed in C-AdCC and SB-AdCC. SB-AdCCs have higher histologic grade, and more frequent nodal and distant metastases than C-AdCCs. MYB/MYBL1 rearrangements were significantly less frequent in SB-AdCC than C-AdCC (3/14, 21% vs 17/20, 85% P < 0.05), despite the frequent MYB expression (9/14, 64%). In SB-AdCCs lacking MYB rearrangements, CREBBP, KMT2C, and NOTCH1 alterations were observed in 2 of 4 cases. SB-AdCCs displayed a shorter IDFS than C-AdCCs (46.5 vs 151.8 months, respectively, P < 0.001), independent of stage. In summary, SB-AdCCs are a molecularly heterogeneous but clinically aggressive group of tumors. Less than 25% of SB-AdCCs display the genomic features of C-AdCC. Defining whether these tumors represent a single entity or a collection of different cancer types with a similar basaloid histologic appearance is warranted.

TSC2-mutant uterine sarcomas with JAZF1-SUZ12 fusions demonstrate hybrid features of endometrial stromal sarcoma and PEComa and are responsive to mTOR inhibition.

Uterine perivascular epithelioid cell tumor (PEComa) is a rare mesenchymal neoplasm that occasionally shares morphologic and immunohistochemical overlap with low- and high-grade endometrial stromal sarcoma (LGESS and HGESS). In this study, we sought to characterize the clinical, morphologic, genetic, and epigenetic features of five uterine sarcomas that display histologic features of LGESS, HGESS, and PEComa. All tumors demonstrated epithelioid cells often associated with a low-grade spindled component resembling LGESS, with both regions expressing CD10, ER, PR, variable HMB45, and Melan-A immunoreactivity, and strong cathepsin K and pS6 expression. Targeted massively parallel sequencing analysis revealed the presence of somatic TSC2 mutations in all five cases, of which four harbored concurrent or consecutive JAZF1-SUZ12 gene fusions. Unsupervised hierarchical clustering analysis of methylation profiles of TSC2-mutant uterine sarcomas (n = 4), LGESS (n = 10), and HGESS (n = 12) demonstrated two clusters consisting of (1) all LGESS and TSC2-mutant uterine sarcomas and (2) all HGESS. KEGG pathway analysis detected methylation differences in genes involved in PI3K/AKT, calcium, and Rap1 signaling. TSC2-mutant uterine sarcomas were responsive to hormone suppression, and mTOR inhibition demonstrated clinical benefit in four patients with these neoplasms. Our results suggest that these tumors represent histologically distinctive LGESS with TSC2 mutations. TSC2 mutations and JAZF1-SUZ12 fusion may help diagnose these tumors and possibly direct effective treatment.

Histologic and genomic features of breast cancers with alterations affecting the SWI/SNF (SMARC) genes.

The SWI/SNF family of proteins is a multisubunit ATPase complex frequently altered in human cancer. Inactivating mutations in SWI/SNF-related matrix-associated actin-dependent regulator of chromatin (SMARCs) underpin a subset of tumors such as the malignant rhabdoid tumor and small cell carcinoma of the ovary, hypercalcemic type. Here, we investigated the genotypic and phenotypic characteristics of breast cancers harboring somatic genetic alterations affecting genes of the SMARC family. We analyzed a series of 6026 primary and metastatic breast cancers subjected to targeted-capture sequencing. SMARC core subunit (SMARCA4, SMARCB1, and SMARCA2) alterations were identified in <1% of all breast cancers, consisting of 27 primary and 30 recurrent/metastatic tumors. The majority of SMARC alterations were monoallelic mutations (47/57, 82%) and thus categorized into two groups: Class 1 alterations consisting of potentially pathogenic mutations and rearrangements and Class 2 alterations consisting of missense mutations and small in-frame deletions of unknown significance. Biallelic events in a SMARC gene were present in a minority of cases (10/57, 18%). Histologic patterns in the form of rhabdoid, composite rhabdoid, sarcomatoid or anaplastic features were observed in a subset of Class 1 primary and metastatic tumors (7/57, 12%). SMARC protein was preserved in nearly all tumors analyzed with immunohistochemistry (26/30, 87%). Four Class 1 tumors demonstrated altered SMARC protein expression in the form of loss (1/30, 3%) or mosaic pattern (3/30, 10%). Complete loss of SMARCA2 (BRM) was observed in a sole tumor with composite rhabdoid morphology, and biallelic hits in the SMARCA2 gene. The genomic landscape of both primary Class 1 and 2 breast cancers did not reveal any characteristic findings. In summary, SMARC alterations likely contribute to the biology of a rare subset of breast cancers in the form of biallelic or pathogenic alterations in SMARC, as evidenced by SMARC-deficient phenotype or altered expression of SMARC protein.

Mesonephric and mesonephric-like carcinomas of the female genital tract: molecular characterization including cases with mixed histology and matched metastases.

Mesonephric carcinoma of the cervix is a rare tumor derived from Wolffian remnants. Mesonephric-like carcinomas of the ovary and endometrium, while morphologically similar, do not have obvious Wolffian derivation. Here, we sought to characterize the repertoire of genetic alterations in primary mesonephric and mesonephric-like carcinomas, in the distinct histologic components of mixed cases, as well as in matched primary tumors and metastases. DNA from microdissected tumor and normal tissue from mesonephric carcinomas (cervix, n = 8) and mesonephric-like carcinomas (ovarian n = 15, endometrial n = 13) were subjected to sequencing targeting 468 cancer-related genes. The histologically distinct components of four cases with mixed histology and four primary tumors and their matched metastases were microdissected and analyzed separately. Mesonephric-like carcinomas were underpinned by somatic KRAS mutations (25/28, 89%) akin to mesonephric carcinomas (8/8, 100%), but also harbored genetic alterations more frequently reported in Müllerian tumors. Mesonephric-like carcinomas that lacked KRAS mutations harbored NRAS (n = 2, ovary) or BRAF (n = 1, endometrium) hotspot mutations. PIK3CA mutations were identified in both mesonephric-like (8/28, 28%) and mesonephric carcinomas (2/8, 25%). Only mesonephric-like tumors harbored CTNNB1 hotspot (4/28, 14%) and PTEN (3/13, 23%) mutations. Copy number analysis revealed frequent gains of chromosomes 1q and 10 in both mesonephric (87% 1q; 50% chromosome 10) and mesonephric-like tumors (89% 1q; 43% chromosome 10). Chromosome 12 gains were more frequent in ovarian mesonephric-like carcinomas, and losses of chromosome 9 were more frequent in mesonephric than in mesonephric-like carcinomas (both p = 0.01, Fisher's exact test). The histologically distinct components of four mixed cases were molecularly related and shared similar patterns of genetic alterations. The progression from primary to metastatic lesions involved the acquisition of additional mutations, and/or shifts from subclonal to clonal mutations. Our findings suggest that mesonephric-like carcinomas are derived from a Müllerian substrate with differentiation along Wolffian/mesonephric lines.

Oncogenic properties and signaling basis of the PAX8-GLIS3 fusion gene.

Hyalinizing trabecular tumors of the thyroid are rare and mostly benign epithelial neoplasms of follicular cell origin, which have recently been shown to be underpinned by the PAX8-GLIS3 fusion gene. In our study, we sought to investigate the potential oncogenic mechanisms of the PAX8-GLIS3 fusion gene. Forced expression of PAX8-GLIS3 was found to increase proliferation, clonogenic potential and migration of human nonmalignant thyroid (Nthy-ori 3-1) and embryonic kidney (HEK-293) cells. Moreover, in xenografts, Nthy-ori 3-1 PAX8-GLIS3 expressing cells generated significantly larger and more proliferative tumors compared to controls. These oncogenic effects were found to be mediated through activation of the Sonic Hedgehog (SHH) pathway. Targeting of smoothened (SMO), a key protein in the SHH pathway, using the small molecule inhibitor Cyclopamine partially reversed the increased proliferation, colony formation and migration in PAX8-GLIS3 expressing cells. Our data demonstrate that the oncogenic effects of the PAX8-GLIS3 fusion gene are, at least in part, due to an increased activation of the SHH pathway.

Histologic spectrum of polymorphous adenocarcinoma of the salivary gland harbor genetic alterations affecting PRKD genes.

Polymorphous adenocarcinoma (PAC) and cribriform adenocarcinoma of (minor) salivary gland (CASG) are salivary gland tumors with overlapping spectrum of morphology. Whether these represent distinct entities or a histologic spectrum of the same tumor remains contentious. PACs harbor recurrent PRKD1 E710D hotspot mutations in >70% of cases, whereas 80% of CASGs display rearrangements involving PRKD1, PRKD2, or PRKD3 (PRKD1/2/3). We studied the molecular and morphologic features of 37 PACs/CASGs, seeking to identify the associations among genotype, histologic phenotype, and classification. DNA was subjected to Sanger sequencing analysis of the PRKD1 hotspot locus. Fluorescence in situ hybridization (FISH) analysis for PRKD1/2/3 was performed using dual-color break-apart probes. Tumors were classified into four categories as described previously: PAC, CASG, tumor with indeterminate features (TIF), and tumor with a predominant papillary pattern (TPPP). PRKD1 E710D hotspot mutations were identified in 56%, 20%, 43% and 0% of PACs, CASGs, TIFs, and TPPPs, respectively. FISH demonstrated PRKD1/2/3 rearrangements in 13%, 78%, 36%, and 75% of PACs, CASGs, TIFs, and TPPPs, respectively. Histologically, fusion-positive tumors were associated with a high percentage of papillary growth, low percentage of single filing arrangement, a propensity of base of tongue location, and frequent (50%) lymph node metastasis, compared with the mutation-related tumors which had negligible nodal metastasis risk. Our results demonstrated that (1) PACs/CASGs are underpinned by genetic alterations affecting PRKD genes; (2) despite the associations between PAC and PRKD1 hotspot mutations and CASG and PRKD1/2/3 fusion, such distinction is not absolute; and (3) there is of a novel genotypic-phenotypic association whereby fusion-positive tumors are usually located in the base of the tongue, show papillary architecture and have a high risk of nodal metastasis. Genetic analysis of PRKD genes appears to be useful characterizing this spectrum of tumors, not only histologically but also clinically identifying those tumors with high risk of nodal metastasis.

Genomic profiling of primary and recurrent adult granulosa cell tumors of the ovary.

Adult-type granulosa cell tumor (aGCT) is a rare malignant ovarian sex cord-stromal tumor, harboring recurrent FOXL2 c.C402G/p.C134W hotspot mutations in 97% of cases. These tumors are considered to have a favorable prognosis, however aGCTs have a tendency for local spread and late recurrences, which are associated with poor survival rates. We sought to determine the genetic alterations associated with aGCT disease progression. We subjected primary non-recurrent aGCTs (n = 7), primary aGCTs that subsequently recurred (n = 9) and their matched recurrences (n = 9), and aGCT recurrences without matched primary tumors (n = 10) to targeted massively parallel sequencing of ≥410 cancer-related genes. In addition, three primary non-recurrent aGCTs and nine aGCT recurrences were subjected to FOXL2 and TERT promoter Sanger sequencing analysis. All aGCTs harbored the FOXL2 C134W hotspot mutation. TERT promoter mutations were found to be significantly more frequent in recurrent (18/28, 64%) than primary aGCTs (5/19, 26%, p = 0.017). In addition, mutations affecting TP53, MED12, and TET2 were restricted to aGCT recurrences. Pathway annotation of altered genes demonstrated that aGCT recurrences displayed an enrichment for genetic alterations affecting cell cycle pathway-related genes. Analysis of paired primary and recurrent aGCTs revealed that TERT promoter mutations were either present in both primary tumors and matched recurrences or were restricted to the recurrence and absent in the respective primary aGCT. Clonal composition analysis of these paired samples further revealed that aGCTs display intra-tumor genetic heterogeneity and harbor multiple clones at diagnosis and relapse. We observed that in a subset of cases, recurrences acquired additional genetic alterations not present in primary aGCTs, including TERT, MED12, and TP53 mutations and CDKN2A/B homozygous deletions. Albeit harboring relatively simple genomes, our data provide evidence to suggest that aGCTs are genetically heterogeneous tumors and that TERT promoter mutations and/or genetic alterations affecting other cell cycle-related genes may be associated with disease progression and recurrences.

Immunohistochemical analysis of IDH2 R172 hotspot mutations in breast papillary neoplasms: applications in the diagnosis of tall cell carcinoma with reverse polarity.

Tall cell carcinoma with reverse polarity is a rare subtype of breast carcinoma with solid and papillary growth and nuclear features reminiscent of those of the tall cell variant of papillary thyroid carcinoma. These tumors harbor recurrent IDH2 R172 hotspot mutations or TET2 mutations, co-occurring with mutations in PI3K pathway genes. Diagnosis of tall cell carcinomas with reverse polarity is challenging in view of their rarity and the range of differential diagnosis. We sought to determine the sensitivity and specificity of IDH2 R172 immunohistochemistry for the detection of IDH2 R172 hotspot mutations in this entity. We evaluated 14 tall cell carcinomas with reverse polarity (ten excision and five core needle biopsy specimens), 13 intraductal papillomas, 16 solid papillary carcinomas, and 5 encapsulated papillary carcinomas by Sanger sequencing of the IDH2 R172 hotspot locus and of exons 9 and 20 of PIK3CA, and by immunohistochemistry using monoclonal antibodies (11C8B1) to the IDH2 R172S mutation. The 14 tall cell carcinomas with reverse polarity studied harbored IDH2 R172 hotspot mutations, which co-occurred with PIK3CA hotspot mutations in 50% of cases. None of the other papillary neoplasms analyzed displayed IDH2 R172 mutations, however PIK3CA hotspot mutations were detected in 54% of intraductal papillomas, 6% of solid papillary carcinomas, and 20% of encapsulated papillary carcinomas tested. Immunohistochemical analysis with anti-IDH2 R172S antibodies (11C8B1) detected IDH2 R172 mutated protein in 93% (14/15) of tall cell carcinomas with reverse polarity samples including excision (n = 9/10) and core needle biopsy specimens (n = 5), whereas the remaining papillary neoplasms (n = 34) were negative. Our findings demonstrate that immunohistochemical analysis of IDH2 R172 is highly sensitive and specific for the detection of IDH2 R172 hotspot mutations, and likely suitable as a diagnostic tool in the evaluation of excision and core needle biopsy material of tall cell carcinomas with reverse polarity.

Immunohistochemical assessment of HRAS Q61R mutations in breast adenomyoepitheliomas.

AIMS: Breast adenomyoepitheliomas (AMEs) are uncommon tumours. Most oestrogen receptor (ER)-positive AMEs have mutations in phosphoinositide 3-kinase (PI3K) pathway genes, whereas ER-negative AMEs usually harbour concurrent mutations affecting the HRAS Q61 hotspot and PI3K pathway genes. Here, we sought to determine the sensitivity and specificity of RAS Q61R immunohistochemical (IHC) analysis for detection of HRAS Q61R mutations in AMEs. METHODS AND RESULTS: Twenty-six AMEs (14 ER-positive; 12 ER-negative) previously subjected to massively parallel sequencing (n = 21) or Sanger sequencing (n = 5) of the HRAS Q61 hotspot locus were included in this study. All AMEs were subjected to IHC analysis with a monoclonal (SP174) RAS Q61R-specific antibody, in addition to detailed histopathological analysis. Nine ER-negative AMEs harboured HRAS mutations, including Q61R (n = 7) and Q61K (n = 2) mutations. Five of seven (71%) AMEs with HRAS Q61R mutations were immunohistochemically positive, whereas none of the AMEs lacking HRAS Q61R mutations (n = 17) were immunoreactive. RAS Q61R immunoreactivity was restricted to the myoepithelium in 80% (4/5) of cases, whereas one case showed immunoreactivity in both the epithelial component and the myoepithelial component. RAS Q61R immunohistochemically positive AMEs were associated with infiltrative borders (P < 0.001), necrosis (P < 0.01) and mitotic index in the epithelial (P < 0.05) and myoepithelial (P < 0.01) components. RAS Q61R IHC assessment did not reveal Q61K mutations (0/2). CONCLUSIONS: IHC analysis of RAS Q61R shows high specificity (100%) and moderate sensitivity (71%) for detection of HRAS Q61R mutations in breast AMEs, and appears not to detect HRAS Q61K mutations. IHC analysis of RAS Q61R may constitute a useful technique in the diagnostic workup of ER-negative AMEs.

Clinical patterns and genomic profiling of recurrent 'ultra-low risk' endometrial cancer.

OBJECTIVE: Despite good prognosis for patients with low-risk endometrial cancer, a small subset of women with low-grade/low-stage endometrial cancer experience disease recurrence and death. The aim of this study was to characterize clinical features and mutational profiles of recurrent, low-grade, non-myoinvasive, 'ultra-low risk' endometrioid endometrial adenocarcinomas. METHODS: We retrospectively identified patients with International Federation of Gynecology and Obstetrics (FIGO) stage IA endometrioid endometrial cancers who underwent primary surgery at our institution, between January 2009 and February 2017, with follow-up of ≥12 months. 'Ultra-low risk' was defined as FIGO tumor grade 1, non-myoinvasive, and lacking lymphovascular space invasion. Tumor-normal profiling using massively parallel sequencing targeting 468 genes was performed. Microsatellite instability was assessed using MSIsensor. DNA mismatch repair (MMR) protein proficiency was determined by immunohistochemistry. RESULTS: A total of 486 patients with ultra-low risk endometrioid endometrial cancers were identified: 14 (2.9%) of 486 patients developed a recurrence. Median follow-up for non-recurrent endometrioid endometrial cancers: 34 (range 12-116) months; for recurrent endometrioid endometrial cancers: 50.5 (range 20-116) months. Patients with recurrent disease were older, had lower body mass index, and were most commonly non-White (p=0.025, p<0.001, and p<0.001, respectively). Other clinical characteristics did not differ. MMR immunohistochemistry was obtained for 211 (43%) tumors: 158 (75%) MMR-proficient and 53 (25%) MMR-deficient. Primary tumors of 9 recurrent and 27 non-recurrent endometrioid endometrial cancers underwent mutational profiling. Most were microsatellite stable (6/9, 67% recurrent; 25/27, 93% non-recurrent). Recurrent PTEN and PIK3CA mutations were present in both groups. Exon 3 CTNNB1 hotspot mutations were found in 4/9 (44%) recurrent and 8/27 (30%) non-recurrent (p=0.44). CONCLUSIONS: Patients diagnosed with ultra-low risk endometrioid endometrial cancers have an overall excellent prognosis. However, in our study, 2.9% of patients with no identifiable clinical or pathologic risk factors developed recurrence. Further work is warranted to elucidate the mechanism for recurrence in this population.

PAX8-GLIS3 gene fusion is a pathognomonic genetic alteration of hyalinizing trabecular tumors of the thyroid.

The hyalinizing trabecular adenoma/tumor is a rare and poorly characterized follicular-derived thyroid neoplasm recently shown to harbor recurrent PAX8-GLIS1 or PAX8-GLIS3 gene fusions. Here we sought to define the repertoire of genetic alterations of hyalinizing trabecular tumors, and whether PAX8-GLIS3 fusions are pathognomonic for hyalinizing trabecular tumors. A discovery series of eight hyalinizing trabecular tumors was subjected to RNA-sequencing (n = 8), whole-exome sequencing (n = 3) or targeted massively parallel sequencing (n = 5). No recurrent somatic mutations or copy number alterations were identified in hyalinizing trabecular tumor, whereas RNA-sequencing revealed the presence of a recurrent genetic rearrangement involving PAX8 (2q14.1) and GLIS3 (9p24.2) genes in all cases. In this in-frame fusion gene, which comprised exons 1-2 of PAX8 and exons 3-11 of GLIS3, GLIS3 is likely placed under the regulation of PAX8. Reverse transcription RT-PCR and/or fluorescence in situ hybridization analyses of a validation series of 26 hyalinizing trabecular tumors revealed that the PAX8-GLIS3 gene fusion was present in all hyalinizing trabecular tumors (100%). No GLIS1 rearrangements were identified. Conversely, no PAX8-GLIS3 gene fusions were detected in a cohort of 237 control thyroid neoplasms, including 15 trabecular thyroid lesions highly resembling hyalinizing trabecular tumor from a morphological standpoint, as well as trabecular/solid follicular adenomas, solid/trabecular variants of papillary carcinoma, and Hurthle cell adenomas or carcinomas. Our data provide evidence to suggest that the PAX8-GLIS3 fusion is pathognomonic for hyalinizing trabecular tumors, and that the presence of the PAX8-GLIS3 fusion in thyroid neoplasms may be used as an ancillary marker for the diagnosis of hyalinizing trabecular tumor, thereby avoiding overtreatment in case of misdiagnoses with apparently similar malignant tumors.

Genomic analysis of recurrences and high-grade forms of polymorphous adenocarcinoma.

AIMS: Polymorphous adenocarcinoma (PAC) usually follows an indolent course, but some cases may show recurrences and high-grade features. The genetic events associated with recurrences and high-grade versions are yet to be defined. Our aim was to determine the genetic underpinning of recurrent PACs of the salivary gland and the repertoire of somatic genetic alterations in cases with high-grade histology. METHODS AND RESULTS: Four PACs from three patients, including one case with matching primary and recurrent tumours, one de-novo high-grade PAC, and a PAC that transformed to a high-grade tumour following multiple recurrences, were subjected to targeted sequencing (Memorial Sloan Kettering Mutation Profiling of Actionable Cancer Targets assay) or whole-exome sequencing. Both matching primary and recurrent tumours, and the de-novo high-grade PAC, harboured clonal PRKD1 E710D hotspot mutations, whereas the PAC that underwent high-grade transformation upon recurrence, which was wild-type for PRKD1, harboured a PRKD2 rearrangement. The PACs analysed here also harboured mutations targeting cancer genes such as PIK3CA, SETD2, ARID1A, and NOTCH2. A clonal decomposition analysis of the matching primary and recurrent PACs revealed that a minor subclone from the primary tumour became dominant in the recurrent tumour following a clonal selection evolutionary pattern. CONCLUSIONS: Our findings demonstrate that recurrent and high-grade PACs are underpinned by PRKD1 E710D hotspot mutations or PRKD2 rearrangements, and that recurrences of PACs may stem from the selection of pre-existing subclones in the primary tumour.

Micropapillary variant of mucinous carcinoma of the breast shows genetic alterations intermediate between those of mucinous carcinoma and micropapillary carcinoma.

AIMS: Micropapillary variant of mucinous carcinoma of the breast (MPMC) is a rare histological form of oestrogen receptor (ER)-positive invasive carcinoma that is characterised by micropapillary clusters of tumour cells in lakes of extracellular mucin. The aims of this study were to determine the genetic alterations underpinning MPMCs, and to determine whether they overlap with those of mucinous carcinomas and/or invasive micropapillary carcinomas. METHODS AND RESULTS: DNA from five MPMCs was subjected to whole-exome sequencing. Somatic mutations, copy number alterations and mutational signatures were determined with state-of-the-art bioinformatics methods. No mutations in genes significantly mutated in breast cancer, including TP53, PIK3CA, GATA3, and MAP3K1, were detected. We identified copy number alterations that have been reported in invasive micropapillary carcinomas, such as recurrent gains in 1q, 6p, 8q, and 10q, and recurrent losses in 16q, 11q, and 13q, as well as a recurrent 8p12-8p11.2 amplification encompassing FGFR1. Like mucinous carcinomas, three of the five MPMCs analysed lacked PIK3CA mutations, 1q gains, and 16q losses, which are the hallmark genetic alterations of ER-positive breast cancers, whereas two MPMCs harboured 16q losses and/or a complex pattern of copy number alterations similar to those found in breast-invasive micropapillary carcinomas. CONCLUSIONS: MPMCs are heterogeneous at the genetic level; some tumours show a pattern of somatic genetic alterations similar to those of mucinous carcinomas, whereas others resemble invasive micropapillary carcinomas at the genetic level. These findings suggest that MPMCs may not constitute one histological subtype, but rather a convergent phenotype that can stem from mucinous carcinomas or invasive micropapillary carcinomas.