Long-molecule scars of backup DNA repair in BRCA1- and BRCA2-deficient cancers.

Homologous recombination (HR) deficiency is associated with DNA rearrangements and cytogenetic aberrations1. Paradoxically, the types of DNA rearrangements that are specifically associated with HR-deficient cancers only minimally affect chromosomal structure2. Here, to address this apparent contradiction, we combined genome-graph analysis of short-read whole-genome sequencing (WGS) profiles across thousands of tumours with deep linked-read WGS of 46 BRCA1- or BRCA2-mutant breast cancers. These data revealed a distinct class of HR-deficiency-enriched rearrangements called reciprocal pairs. Linked-read WGS showed that reciprocal pairs with identical rearrangement orientations gave rise to one of two distinct chromosomal outcomes, distinguishable only with long-molecule data. Whereas one (cis) outcome corresponded to the copying and pasting of a small segment to a distant site, a second (trans) outcome was a quasi-balanced translocation or multi-megabase inversion with substantial (10 kb) duplications at each junction. We propose an HR-independent replication-restart repair mechanism to explain the full spectrum of reciprocal pair outcomes. Linked-read WGS also identified single-strand annealing as a repair pathway that is specific to BRCA2 deficiency in human cancers. Integrating these features in a classifier improved discrimination between BRCA1- and BRCA2-deficient genomes. In conclusion, our data reveal classes of rearrangements that are specific to BRCA1 or BRCA2 deficiency as a source of cytogenetic aberrations in HR-deficient cells.

Ovarian cancer mutational processes drive site-specific immune evasion.

High-grade serous ovarian cancer (HGSOC) is an archetypal cancer of genomic instability1-4 patterned by distinct mutational processes5,6, tumour heterogeneity7-9 and intraperitoneal spread7,8,10. Immunotherapies have had limited efficacy in HGSOC11-13, highlighting an unmet need to assess how mutational processes and the anatomical sites of tumour foci determine the immunological states of the tumour microenvironment. Here we carried out an integrative analysis of whole-genome sequencing, single-cell RNA sequencing, digital histopathology and multiplexed immunofluorescence of 160 tumour sites from 42 treatment-naive patients with HGSOC. Homologous recombination-deficient HRD-Dup (BRCA1 mutant-like) and HRD-Del (BRCA2 mutant-like) tumours harboured inflammatory signalling and ongoing immunoediting, reflected in loss of HLA diversity and tumour infiltration with highly differentiated dysfunctional CD8+ T cells. By contrast, foldback-inversion-bearing tumours exhibited elevated immunosuppressive TGFß signalling and immune exclusion, with predominantly naive/stem-like and memory T cells. Phenotypic state associations were specific to anatomical sites, highlighting compositional, topological and functional differences between adnexal tumours and distal peritoneal foci. Our findings implicate anatomical sites and mutational processes as determinants of evolutionary phenotypic divergence and immune resistance mechanisms in HGSOC. Our study provides a multi-omic cellular phenotype data substrate from which to develop and interpret future personalized immunotherapeutic approaches and early detection research.

Single-cell genomic variation induced by mutational processes in cancer.

How cell-to-cell copy number alterations that underpin genomic instability1 in human cancers drive genomic and phenotypic variation, and consequently the evolution of cancer2, remains understudied. Here, by applying scaled single-cell whole-genome sequencing3 to wild-type, TP53-deficient and TP53-deficient;BRCA1-deficient or TP53-deficient;BRCA2-deficient mammary epithelial cells (13,818 genomes), and to primary triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSC) cells (22,057 genomes), we identify three distinct 'foreground' mutational patterns that are defined by cell-to-cell structural variation. Cell- and clone-specific high-level amplifications, parallel haplotype-specific copy number alterations and copy number segment length variation (serrate structural variations) had measurable phenotypic and evolutionary consequences. In TNBC and HGSC, clone-specific high-level amplifications in known oncogenes were highly prevalent in tumours bearing fold-back inversions, relative to tumours with homologous recombination deficiency, and were associated with increased clone-to-clone phenotypic variation. Parallel haplotype-specific alterations were also commonly observed, leading to phylogenetic evolutionary diversity and clone-specific mono-allelic expression. Serrate variants were increased in tumours with fold-back inversions and were highly correlated with increased genomic diversity of cellular populations. Together, our findings show that cell-to-cell structural variation contributes to the origins of phenotypic and evolutionary diversity in TNBC and HGSC, and provide insight into the genomic and mutational states of individual cancer cells.

Diverse alterations associated with resistance to KRAS(G12C) inhibition.

Inactive state-selective KRAS(G12C) inhibitors1-8 demonstrate a 30-40% response rate and result in approximately 6-month median progression-free survival in patients with lung cancer9. The genetic basis for resistance to these first-in-class mutant GTPase inhibitors remains under investigation. Here we evaluated matched pre-treatment and post-treatment specimens from 43 patients treated with the KRAS(G12C) inhibitor sotorasib. Multiple treatment-emergent alterations were observed across 27 patients, including alterations in KRAS, NRAS, BRAF, EGFR, FGFR2, MYC and other genes. In preclinical patient-derived xenograft and cell line models, resistance to KRAS(G12C) inhibition was associated with low allele frequency hotspot mutations in KRAS(G12V or G13D), NRAS(Q61K or G13R), MRAS(Q71R) and/or BRAF(G596R), mirroring observations in patients. Single-cell sequencing in an isogenic lineage identified secondary RAS and/or BRAF mutations in the same cells as KRAS(G12C), where they bypassed inhibition without affecting target inactivation. Genetic or pharmacological targeting of ERK signalling intermediates enhanced the antiproliferative effect of G12C inhibitor treatment in models with acquired RAS or BRAF mutations. Our study thus suggests a heterogenous pattern of resistance with multiple subclonal events emerging during G12C inhibitor treatment. A subset of patients in our cohort acquired oncogenic KRAS, NRAS or BRAF mutations, and resistance in this setting may be delayed by co-targeting of ERK signalling intermediates. These findings merit broader evaluation in prospective clinical trials.

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.

HER2 Genetic Intratumor Heterogeneity Is Associated With Resistance to Trastuzumab and Trastuzumab Emtansine Therapy in Recurrent High-Grade Endometrial Cancer.

Anti-HER2 targeted therapies have recently demonstrated clinical activity in the treatment of high-grade endometrial carcinomas (ECs), particularly serous carcinomas with HER2 amplification and/or overexpression. Intratumor heterogeneity of HER2 amplification or HER2 genetic intratumor heterogeneity (G-ITH) has been associated with resistance to anti-HER2 therapies in breast and gastroesophageal cancers; however, its clinical relevance in EC is unknown. To characterize HER2 G-ITH in EC, archival specimens from a clinically annotated cohort of 57 ECs treated with trastuzumab or trasutuzmab emtansine in the recurrent (n = 38) or adjuvant (n = 19) setting were subjected to central pathology review, HER2 assessment by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), and next-generation sequencing. HER2 G-ITH, defined as HER2 amplification in 5% to 50% of tumor cells examined by FISH, was identified in 36% (19/53) of ECs and was associated with lower HER2 copy number and levels of protein expression. HER2 IHC revealed spatially distinct areas of strong expression juxtaposed with areas of low/absent expression in tumors with the "cluster" pattern of G-ITH, whereas the "mosaic" pattern was typically associated with a diffuse admixture of cells with variable levels of HER2 expression. HER2 G-ITH was frequently observed in cases with IHC/FISH or FISH/next-generation sequencing discrepancies and/or with an equivocal/negative FISH result (9/13, 69%). Although the objective response rate to anti-HER2 therapy in recurrent ECs was 52% (13/25) for tumors lacking HER2 G-ITH, none (0%, 0/10) of the patients with HER2 G-ITH achieved a complete or partial response (P = .005). HER2 G-ITH was significantly associated with worse progression-free survival (hazard ratio, 2.88; 95% CI, 1.33-6.27; P = .005) but not overall survival. HER2 IHC score, HER2/CEP17 ratio, HER2 copy number, histologic subtype, and other genetic alterations, including PIK3CA hotspot mutations, were not significantly associated with therapeutic response or survival outcomes. Treatment responses were not restricted to serous carcinomas, supporting consideration of anti-HER2 therapy in patients with HER2-positive high-grade ECs of non-serous histology. Our results demonstrate that HER2 G-ITH is an important determinant of response to trastuzumab and trastuzumab emtansine in EC, providing a rationale for the development of novel therapeutic strategies to target HER2-nonamplified resistant tumor subpopulations, such as HER2 antibody-drug conjugates with bystander effects.

Ovarian RASoma With Mesonephric-like Adenocarcinoma and Mixed Mullerian Components: A Case Report With Molecular Analysis Demonstrating Multidirectional Mullerian Differentiation.

Gynecologic carcinomas with RAS mutations may show a wide spectrum of histologic types, including mixed types. We present the case of a 63-yr-old patient diagnosed with an ovarian tumor harboring a mesonephric-like adenocarcinoma in a background of mixed mesonephric-like, mucinous, and endometrioid components. Molecular analysis revealed that all 3 components shared the same clonal KRAS mutation (p.G12A) and chromosome 1q gain. Based on shifts in clonality, copy number gains, and acquisition of an additional mutation, our data suggest that the mucinous component likely constituted the substrate from which the mesonephric-like and endometrioid components arose.

Cervical Pleuropulmonary Blastoma-like Tumor Associated With DICER1 and TP53 Mutations.

We describe a very unusual cervical tumor in a 12-yr-old patient with a clinical history indicative of DICER1 syndrome. Morphologic, immunohistochemical, and molecular genetic analysis together helped to diagnose this lesion as a cervical pleuropulmonary blastoma-like tumor, associated with TP53 and DICER1 mutations. The tumor displayed usual histologic features including mixtures of embryonal rhabdomyosarcoma, sarcomatous cartilage, compact blastema, primitive spindle cells and anaplasia, akin to type III pleuropulmonary blastoma, and trabecular and retiform patterns. In addition to expanding the phenotypic spectrum of DICER1 -associated conditions, we draw attention to genotype-phenotype correlations in DICER1 -associated tumors, particularly as they relate to the discovery of a heritable tumor predisposition syndrome.

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.

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.

Hyperthermic intraperitoneal chemotherapy (HIPEC) with carboplatin induces distinct transcriptomic changes in ovarian tumor and normal tissues.

OBJECTIVE: To determine the effect of hyperthermic intraperitoneal chemotherapy (HIPEC) with carboplatin on the transcriptomic profiles of normal and ovarian cancer (OC) tissues. METHODS: Normal and tumor samples from four OCs were prospectively collected pre- and immediately post-HIPEC treatment and subjected to RNA-sequencing. Differential gene expression, gene ontology enrichment and pathway analyses were performed. Heat shock protein and immune-response protein expression was assessed using protein arrays and western blotting. RESULTS: RNA-sequencing revealed 4231 and 322 genes significantly differentially expressed between pre- and post-treatment normal and OC tissues, respectively (both adjusted p-value <0.05). Gene enrichment analyses demonstrated that the most significantly upregulated genes in normal tissues played a role in immune as well as heat shock response (both adjusted p < 0.001). In contrast, HIPEC induced an increased expression of primarily heat shock response and protein folding-related genes in tumor tissues (both adjusted p < 0.001). HIPEC-induced heat shock protein (HSP) expression changes, including in HSP90, HSP40, HSP60, and HSP70, were also observed at the protein level in both normal and tumor tissues. CONCLUSIONS: HIPEC with carboplatin resulted in an upregulation of heat shock-related genes in both normal and tumor tissue, with an additional immune response gene induction in normal and protein folding in tumor tissue. The findings of our exploratory study provide evidence to suggest that HIPEC administration may suffice to induce gene expression changes in residual tumor cells and raises a biological basis for the consideration of combinatorial treatments with HSP inhibitors.

Clonal relationship and directionality of progression of synchronous endometrial and ovarian carcinomas in patients with DNA mismatch repair-deficiency associated syndromes.

Sporadic synchronous endometrial (ECs) and ovarian cancers (OCs), although clinically considered to be independent primaries, have been shown to be clonally related and likely constitute metastases from each other. We sought to define whether synchronous ECs/OCs in patients with DNA mismatch repair (MMR)-deficiency syndromes would be clonally related. We subjected synchronous ECs/OCs from four patients (LS3-LS6) with clinically confirmed Lynch syndrome (LS) and one patient with constitutional mismatch repair-deficiency syndrome (CMMRD) to massively parallel sequencing targeting 468 cancer-related genes. Somatic mutations, copy number alterations (CNAs), clonal relatedness and clonal decomposition analyses were performed using previously described bioinformatics methods. All synchronous ECs/OCs analyzed were considered independent primaries based on clinicopathologic criteria. Sequencing analysis revealed that the ECs/OCs of three cases (LS2-CMMRD, L3, L4) harbored similar repertoires of somatic mutations and CNAs and were clonally related. In these three cases, a subset of subclonal mutations in the EC became clonal in the OC, suggesting that the EC was likely the substrate from which the OC developed. LS5's EC/OC had distinct mutational profiles but shared specific CNAs. In contrast, LS6's EC/OC harbored distinct somatic mutations and lacked CNAs, consistent with each tumor constituting an independent primary lesion. In LS5 and LS6, PTEN mutations and PTEN loss of protein expression were found to be restricted to the EC. Finally, re-analysis of sequencing data of sporadic synchronous ECs/OCs supported the observations made in the current study that the directionality of progression is likely from the endometrium to the ovary. In conclusion, contrary to sporadic synchronous ECs/OCs, which are almost invariably clonally related, ECs/OCs simultaneously involving the uterus and ovary in LS patients may represent distinct primary tumors. A subset of MMR-deficiency syndrome-related synchronous ECs/OCs, however, may originate from a single primary tumor at variance with their clinical diagnosis, with the endometrium being the likeliest site of origin.

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.

Genetic characterisation of adult primary pleomorphic uterine rhabdomyosarcoma and comparison with uterine carcinosarcoma.

AIMS: To characterise the genetic alterations in adult primary uterine rhabdomyosarcomas (uRMSs) and to investigate whether these tumours are genetically distinct from uterine carcinosarcomas (UCSs). METHODS AND RESULTS: Three tumours originally diagnosed as primary adult pleomorphic uRMS were subjected to massively parallel sequencing targeting 468 cancer-related genes and RNA-sequencing. Mutational profiles were compared with those of UCSs (n = 57) obtained from The Cancer Genome Atlas. Sequencing data analyses were performed using validated bioinformatic approaches. Pathogenic TP53 mutations and high levels of genomic instability were detected in the three cases. uRMS1 harboured a likely pathogenic YTHDF2-FOXR1 fusion. uRMS2 harboured a PPP2R1A hotspot mutation and amplification of multiple genes, including WHSC1L1, FGFR1, MDM2, and CCNE1, whereas uRMS3 harboured an FBXW7 hotspot mutation and an ANKRD11 homozygous deletion. Hierarchical clustering of somatic mutations and copy number alterations revealed that these tumours initially diagnosed as pleomorphic uRMSs and UCSs were similar. Subsequent comprehensive pathological re-review of the three uRMSs revealed previously unidentified minute pan-cytokeratin-positive atypical glands in one case (uRMS3), favouring its reclassification as UCS with extensive rhabdomyosarcomatous overgrowth. CONCLUSIONS: Adult pleomorphic uRMSs harbour TP53 mutations and high levels of copy number alterations. Our findings underscore the challenge in discriminating between uRMS and UCS with rhabdomyosarcomatous differentiation.

Diagnosis and management of an endometrial cancer patient with Cowden syndrome.

Somatic PTEN alterations are common in endometrial carcinoma (EC), but in rare cases PTEN mutations are associated with inherited syndromes. Here, we present a case of Cowden syndrome-associated EC. We discuss clinical, pathologic and molecular features of her tumor and PTEN-mutated EC, inherited syndromes predisposing to EC and PTEN-targeted therapies.

Genetic and molecular subtype heterogeneity in newly diagnosed early- and advanced-stage endometrial cancer.

OBJECTIVE: To characterize and compare the molecular subtypes and profiles of prospectively-accrued newly-diagnosed early- and advanced-stage endometrial cancers (ECs). METHODS: EC patients consented to an IRB-approved protocol of massively parallel sequencing of 410-468 cancer-related genes; 175 ECs of 7 histologic types (n = 135 FIGO stages I/II, n = 40 FIGO stages III/IV) were included. Previously reported sequencing data from 99 additional advanced-stage ECs were retrieved for comparisons. RESULTS: Irrespective of histologic type, all 175 ECs could be stratified into the molecular subtypes, with 75 (43%) being of p53 wild-type, 49 (28%) MMR-deficient, 39 (22%) p53 abnormal and 12 (7%) of POLE molecular subtypes. Subtype distribution, mutational and copy number profiles varied according to histologic type. In endometrioid ECs, genetic alterations varied according to histologic grade. Potential therapeutic targets, including high tumor mutational burden, ERBB2 amplification and PIK3CA hotspot mutations, were found across histologic types in 63% (n = 110) of all ECs. Compared to their early-stage counterparts, advanced-stage endometrioid ECs had a significantly higher fraction of genome altered (median 0.1% vs 12%, p < 0.001) and ARID1B mutations (0% vs 11%, p = 0.01), and advanced-stage serous ECs harbored more frequent ERBB2 amplification (18% vs 8%, p > 0.05) and PIK3CA mutations (46% vs 27%, p > 0.05). Whole-genome doubling was found in advanced- but not early-stage carcinosarcomas and clear cell carcinomas. CONCLUSIONS: Our findings demonstrate the molecular heterogeneity within and across histologic types of EC and the increased genomic complexity of advanced-stage ECs. Molecular subtypes are present across EC histologic types and may help stratify EC patients for prognostic and therapeutic purposes.