A genetically engineered ovarian cancer mouse model based on fallopian tube transformation mimics human high‐grade serous carcinoma development

Recent evidence suggests that ovarian high‐grade serous carcinoma (HGSC) originates from the epithelium of the fallopian tube. However, most mouse models are based on the previous prevailing view that ovarian cancer develops from the transformation of the ovarian surface epithelium. Here, we report the extensive histological and molecular characterization of the mogp–TAg transgenic mouse, which expresses the SV40 large T‐antigen (TAg) under the control of the mouse müllerian‐specific Ovgp‐1 promoter. Histological analysis of the fallopian tubes of mogp–TAg mice identified a variety of neoplastic lesions analogous to those described as precursors to ovarian HGSC. We identified areas of normal‐appearing p53‐positive epithelium that are similar to ‘p53 signatures’ in the human fallopian tube. More advanced proliferative lesions with nuclear atypia and epithelial stratification were also identified that were morphologically and immunohistochemically reminiscent of human serous tubal intraepithelial carcinoma (STIC), a potential precursor of ovarian HGSC. Beside these non‐invasive precursor lesions, we also identified invasive adenocarcinoma in the ovaries of 56% of the mice. Microarray analysis revealed several genes differentially expressed between the fallopian tube of mogp–TAg and wild‐type (WT) C57BL/6. One of these genes, Top2a, which encodes topoisomerase IIα, was shown by immunohistochemistry to be concurrently expressed with elevated p53 and was specifically elevated in mouse STICs but not in the surrounding tissues. TOP2A protein was also found elevated in human STICs, low‐grade and high‐grade serous carcinoma. The mouse model reported here displays a progression from normal tubal epithelium to invasive HGSC in the ovary, and therefore closely simulates the current emerging model of human ovarian HGSC pathogenesis. This mouse therefore has the potential to be a very useful new model for elucidating the mechanisms of serous ovarian tumourigenesis, as well as for developing novel approaches for the prevention, diagnosis and therapy of this disease. Published 2014. This article has been contributed to by US Government employees and their work is in the public domain in the USA.     

High‐grade serous carcinoma of tubo‐ovarian origin: recent developments

Extra‐uterine high‐grade serous carcinoma (HGSC) accounts for most of the morbidity and mortality associated with ovarian carcinoma, and is one of the leading causes of cancer death in women. Until recently our understanding of HGSC was very limited compared to other common cancers, and it has only been during the last 15 years that we have learned how to diagnose this ovarian carcinoma histotype accurately. Since then, however, there has been rapid progress, with identification of a precursor lesion in the fallopian tube, development of prevention strategies for both those with inherited susceptibility (hereditary breast and ovarian cancer syndrome) and without the syndrome, and elucidation of the molecular events important in oncogenesis. This molecular understanding has led to new treatment strategies for HGSC, with the promise of more to come in the near future. In this review we focus on these recent changes, including diagnostic criteria/differential diagnosis, primary site assignment, precursor lesions and the molecular pathology of HGSC.     

One-tube real-time isothermal amplification assay to identify and distinguish human immunodeficiency virus type 1 subtypes A, B, and C and circulating recombinant forms AE and AG

To halt the human immunodeficiency virus type 1 (HIV-1) epidemic requires interventions that can prevent transmission of numerous HIV-1 subtypes. The most frequently transmitted viruses belong to the subtypes A, B, and C and the circulating recombinant forms (CRFs) AE and AG. A fast one-tube assay that identifies and distinguishes among subtypes A, B, and C and CRFs AE and AG of HIV-1 was developed. The assay amplifies a part of the gag gene sequence of the genome of all currently known HIV-1 subtypes and can identify and distinguish among the targeted subtypes as the reaction proceeds, because of the addition of subtype-specific molecular beacons with multiple fluorophores. The combination of isothermal nucleic acid sequence-based amplification and molecular beacons is a new approach in the design of real-time assays. To obtain a sufficiently specific assay, we developed a new strategy in the design of molecular beacons, purposely introducing mismatches in the molecular beacons. The subtype A and CRF AG isolates reacted with the same molecular beacon. We tested the specificity and sensitivity of the assay on a panel of the culture supernatant of 34 viruses encompassing all HIV-1 subtypes: subtypes A through G, CRF AE and AG, a group O isolate, and a group N isolate. Assay sensitivity on this panel was 92%, with 89% correct subtype identification relative to sequence analysis. A linear relationship was found between the amount of input RNA in the reaction mixture and the time that the reaction became positive. The lower detection level of the assay was approximately 10(3) copies of HIV-1 RNA per reaction. In 38% of 50 serum samples from HIV-1-infected individuals with a detectable amount of virus, we could identify subtype sequences with a specificity of 94% by using sequencing and phylogenetic analysis as the "gold standard." In conclusion, we showed the feasibility of the approach of using multiple molecular beacons labeled with different fluorophores in combination with isothermal amplification to identify and distinguish subtypes A, B, and C and CRFs AE and AG of HIV-1. Because of the low sensitivity, the assay in this format would not be suited for clinical use but can possibly be used for epidemiological monitoring as well as vaccine research studies.     

Distinct evolutionary trajectories of primary high‐grade serous ovarian cancers revealed through spatial mutational profiling

High‐grade serous ovarian cancer (HGSC) is characterized by poor outcome, often attributed to the emergence of treatment‐resistant subclones. We sought to measure the degree of genomic diversity within primary, untreated HGSCs to examine the natural state of tumour evolution prior to therapy. We performed exome sequencing, copy number analysis, targeted amplicon deep sequencing and gene expression profiling on 31 spatially and temporally separated HGSC tumour specimens (six patients), including ovarian masses, distant metastases and fallopian tube lesions. We found widespread intratumoural variation in mutation, copy number and gene expression profiles, with key driver alterations in genes present in only a subset of samples (eg PIK3CA, CTNNB1, NF1). On average, only 51.5% of mutations were present in every sample of a given case (range 10.2–91.4%), with TP53 as the only somatic mutation consistently present in all samples. Complex segmental aneuploidies, such as whole‐genome doubling, were present in a subset of samples from the same individual, with divergent copy number changes segregating independently of point mutation acquisition. Reconstruction of evolutionary histories showed one patient with mixed HGSC and endometrioid histology, with common aetiologic origin in the fallopian tube and subsequent selection of different driver mutations in the histologically distinct samples. In this patient, we observed mixed cell populations in the early fallopian tube lesion, indicating that diversity arises at early stages of tumourigenesis. Our results revealed that HGSCs exhibit highly individual evolutionary trajectories and diverse genomic tapestries prior to therapy, exposing an essential biological characteristic to inform future design of personalized therapeutic solutions and investigation of drug‐resistance mechanisms. © 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Measurement of HIV RNA in patients infected by subtype C by assays optimized for subtype B results in an underestimation of the viral load

Quantitation assays of HIV-1 RNA used currently were designed and optimized for subtype B viruses. However, infection with non-B HIV viruses has become more common worldwide. Unfortunately, little information is available regarding the suitability of these assays for measurement of viral load in specific non-B subtypes. The performance of two commercial HIV-1 RNA quantitation assays was evaluated in 82 HIV subtype C-infected patients and in 43 HIV-1 subtype B-infected patients. Blood samples were tested by the Amplicor HIV-1 Monitor Assay, Version 1.5, and by the nucleic acid sequence-based amplification HIV-1 assay (NucliSens). The results were compared by using a paired, two-tailed Student's t-test; the difference between the assays was found to be significant only for subtype C. Discordant results (>0.5 log difference) between the two assays were detected in 39% of subtype C samples, compared to 23.2% of subtype B samples. In all cases in which a discordant result was detected, the lower results were obtained by the NucliSens assay. Discordant results between CD4 and viral load (CD4 < 200 cells/ml with a viral load <5,000 copies/ml) were observed in eight of the subtype C-infected patients when a viral load was measured by NucliSens (9.7%), compared to three patients (3.6%) when measured by the Amplicor assay. In conclusion, in patients with HIV subtype C infection, measurement of HIV RNA by the NucliSens assay resulted in a significant underestimation of the viral load as compared to the Amplicor assay. As a consequence, such an underestimation may result in sub-optimal care of patients infected with HIV subtype C.     

输卵管在卵巢癌中的遗传学作用的研究进展

卵巢癌在三种妇科恶性肿瘤中恶性度最高,是妇科恶性肿瘤患者死亡的主要病因.高级别浆液性卵巢癌(HGSC)是最常见、恶性度最高的上皮性卵巢癌,在发现时一般已为晚期.HGSC可能与BRCA1以及BRCA2基因的突变有直接关系.长久以来,我们一直认为HGSC起源于卵巢上皮细胞,但是,最近越来越多的证据表明,无论是在高风险的妇女中,还是在一般人群中,绝大多数卵巢癌均起源于输卵管上皮细胞.浆液性上皮内癌(STIC)可能最终发展成卵巢癌或腹膜癌.目前,预防性保留卵巢的输卵管切除术越来越被人们所接受,成为高风险妇女在绝经前降低患卵巢癌风险的做法.     

Use of mutation profiles to refine the classification of endometrial carcinomas

The classification of endometrial carcinomas is based on pathological assessment of tumour cell type; the different cell types (endometrioid, serous, carcinosarcoma, mixed, undifferentiated, and clear cell) are associated with distinct molecular alterations. This current classification system for high-grade subtypes, in particular the distinction between high-grade endometrioid (EEC-3) and serous carcinomas (ESC), is limited in its reproducibility and prognostic abilities. Therefore, a search for specific molecular classifiers to improve endometrial carcinoma subclassification is warranted. We performed target enrichment sequencing on 393 endometrial carcinomas from two large cohorts, sequencing exons from the following nine genes: ARID1A, PPP2R1A, PTEN, PIK3CA, KRAS, CTNNB1, TP53, BRAF, and PPP2R5C. Based on this gene panel, each endometrial carcinoma subtype shows a distinct mutation profile. EEC-3s have significantly different frequencies of PTEN and TP53 mutations when compared to low-grade endometrioid carcinomas. ESCs and EEC-3s are distinct subtypes with significantly different frequencies of mutations in PTEN, ARID1A, PPP2R1A, TP53, and CTNNB1. From the mutation profiles, we were able to identify subtype outliers, ie cases diagnosed morphologically as one subtype but with a mutation profile suggestive of a different subtype. Careful review of these diagnostically challenging cases suggested that the original morphological classification was incorrect in most instances. The molecular profile of carcinosarcomas suggests two distinct mutation profiles for these tumours: endometrioid-type (PTEN, PIK3CA, ARID1A, KRAS mutations) and serous-type (TP53 and PPP2R1A mutations). While this nine-gene panel does not allow for a purely molecularly based classification of endometrial carcinoma, it may prove useful as an adjunct to morphological classification and serve as an aid in the classification of problematic cases. If used in practice, it may lead to improved diagnostic reproducibility and may also serve to stratify patients for targeted therapeutics.     

Multilayer spectrum of intratumoral heterogeneity in basal bladder cancer

Basal/squamous (Ba/Sq) subtype represents an intrinsic and robust group in the consensus molecular classification of muscle-invasive bladder cancer (MIBC), with poor outcome and controversial chemosensitivity. We aimed to investigate the spectrum of intratumor heterogeneity (ITH) in the Ba/Sq subtype. First, we validated a 29-gene NanoString CodeSet to predict the Ba/Sq subtype for FFPE samples. We identified heterogeneous Ba/Sq tumors in a series of 331 MIBC FFPE samples using dual GATA3/KRT5/6 immunohistochemistry (IHC). Heterogeneous regions with distinct immunostaining patterns were studied separately for gene expression using the 29-gene CodeSet, for mutations by targeted next-generation sequencing, and for copy number alteration (CNA) by microarray hybridization. Among 83 Ba/Sq tumors identified by GATA3/KRT5/6 dual staining, 19 tumors showed heterogeneity at the IHC level. In one third of the 19 cases, regions from the same tumor were classified in different distinct molecular subtypes. The mutational and CNA profiles confirmed the same clonal origin for IHC heterogeneous regions with possible subclonal evolution. Overall, two patterns of intratumoral heterogeneity (ITH) were observed in Ba/Sq tumors: low ITH (regions with distinct immunostaining, but common molecular subtype and shared CNA) or high ITH (regions with distinct immunostaining, molecular subtype, and CNA). These results showed multilayer heterogeneity in Ba/Sq MIBC. In view of personalized medicine, this heterogeneity adds complexity and should be taken into account for sampling procedures used for diagnosis and treatment choice. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Ovarian carcinoma. Do the subtypes reflect different diseases?

Lack of therapeutic options and poor reproducibility of histopathological subtypes have been the reasons that ovarian carcinomas are currently treated as monolithic entity. Histopathological grading is used to identify those patients who can be spared adjuvant therapy. With slight modifications of the WHO based subtype classification we have shown that subtypes (i.e. serous, endometrioid, clear cell, mucinous) can be reproducibly used to stratify patients according to disease-specific survival. As these pathologically identifiable subtypes have different epidemiologic and genetic risk factors, precursor lesions, molecular abnormalities and clinical behaviour, screening and management strategies have to be subtype-specific.     

Key features of extrauterine pelvic serous tumours (fallopian tube, ovary, and peritoneum)

Key features of extrauterine pelvic serous tumours (fallopian tube, ovary, and peritoneum) Ovarian serous carcinoma (OSC) is the most common of the ovarian epithelial malignancies, and accounts for most of the mortality. Traditionally, ovarian cancer has been considered to be one disease; however, it is now apparent that it actually consists of many different entities. OSC can be further segregated into two processes: high-grade serous carcinoma (HGSC) and low-grade serous carcinoma. This classification is supported by molecular changes, morphological appearance, clinical behaviour, and distinct precursor lesions. This review will describe in detail these features of OSC, differential diagnosis, prognosis, and recent challenges to the existing hypotheses regarding the origin of these tumours. Special attention will be paid to HGSC and its relationship with BRCA abnormalities, especially those seen in hereditary breast and ovarian cancer syndrome. Finally, treatment options based on specific molecular targets will be discussed.     

Increasing heterosexual transmission of HIV‐1 subtype C in Inland Central Western Brazil

The molecular epidemiology of HIV‐1 in Brazil is complex and heterogeneous because several subtypes co‐circulate with some important regional differences. This study evaluated HIV‐1 subtypes amongst pregnant women living in the metropolitan area and in the interior cities from central western Brazil. From June 2008 to June 2010, 86.9% of confirmed cases of HIV‐1 infection amongst pregnant women (172 out of 198 cases) were recruited in Goiania/Goias state. The HIV‐1 pol gene was sequenced after nested‐PCR. HIV‐1 subtypes were assigned by REGA, phylogenetic, and bootscan analyses. The median age of participants was 26 years (15–41 years range); 58.7% of participants were diagnosed during prenatal care and 51.7% of participants came from >50 interior cities within Goias state. Amongst the 131 HIV‐1 pol sequences, 64.9% were subtype B, 13.0% were BF1 recombinant, 11.4% were subtype C, 7.6% were subtype F1, and 2.3% were BC recombinant. According to the HIV‐1 diagnosis date (1994–2010), a significant increase in subtype C and a decrease of BF1 mosaics were observed over time. All subtype C patients lived in interior cities where the highest prevalence of subtype C outside southern Brazil was observed (18.4%). Phylogenetic analysis revealed multiple independent introductions of the Brazilian subtype C clade from the southern/southeastern regions of Brazil. The HIV‐1 epidemic in women from central western Brazil infected by the heterosexual route is characterized by an unexpectedly high prevalence of subtype C viruses highly related to those circulating in southern/southeastern Brazil. These findings highlight the importance of molecular surveillance programs outside large metropolitan regions in Brazil. J. Med. Virol. 85:396–404, 2013. © 2012 Wiley Periodicals, Inc.     

Genomic Analysis Reveals Distinct Subtypes in Two Rare Cases of Primary Ovarian Lymphoma

Primary (localized) non-Hodgkin lymphoma (NHL) of the ovary is extremely rare; only a few cases have been reported in the literature. We report two cases of primary ovarian lymphoma (POL), one involving bilateral ovaries in a 15-year-old girl and other involving one ovary in a 5-year-old girl. This report describes detailed clinical, histopathological, and imaging findings, along with the review of literature of primary diffuse large B-cell lymphoma (DLBCL) arising from an ovary. In addition, we describe findings of targeted capture panel sequencing on both tumors and identify the major genetic mutations that are recurrently mutated in pan-cancers. Compared to the genomic mutation features of major subtypes of DLBCL, we distinguish that each POL belongs to distinctive subtypes, GCB (germinal center B-cell subtype) DLBCL and ABC (activated B-cell subtype) DLBCL, respectively. The findings from the genomic analysis may help to understand the pathogenesis of POL and to guide potential targeted therapy in the future.     

Molecular classification of urothelial carcinoma: global mRNA classification versus tumour‐cell phenotype classification

Global mRNA expression analysis is efficient for phenotypic profiling of tumours, and has been used to define molecular subtypes for almost every major tumour type. A key limitation is that most tumours are communities of both tumour and non‐tumour cells. This problem is particularly pertinent for analysis of advanced invasive tumours, which are known to induce major changes and responses in both the tumour and the surrounding tissue. To identify bladder cancer tumour‐cell phenotypes and compare classification by tumour‐cell phenotype with classification by global gene expression analysis, we analysed 307 advanced bladder cancers (cystectomized) both by genome gene expression analysis and by immunohistochemistry with antibodies for 28 proteins. According to systematic analysis of gene and protein expression data, focusing on key molecular processes, we describe five tumour‐cell phenotypes of advanced urothelial carcinoma: urothelial‐like, genomically unstable, basal/SCC ‐like, mesenchymal‐like, and small‐cell/neuroendocrine‐like. We provide molecular pathological definitions for each subtype. Tumours expressing urothelial differentiation factors show inconsistent and abnormal protein expression of terminal differentiation markers, suggesting pseudo‐differentiation. Cancers with different tumour‐cell phenotypes may co‐cluster (converge), and cases with identical tumour‐cell phenotypes may cluster apart (diverge), in global mRNA analyses. This divergence/convergence suggests that broad global commonalities related to the invasive process may exist between muscle‐invasive tumours regardless of specific tumour‐cell phenotype. Hence, there is a systematic disagreement in subtype classification determined by global mRNA profiling and by immunohistochemical profiling at the tumour‐cell level. We suggest that a combination of molecular pathology (tumour‐cell phenotype) and global mRNA profiling (context) is required for adequate subtype classification of muscle‐invasive bladder cancer. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Salivary duct carcinomas can be classified into luminal androgen receptor‐positive,HER2 and basal‐like phenotypes*

Di Palma S, Simpson R H W, Marchiò C, Skálová A, Ungari M, Sandison A, Whitaker S, Parry S & Reis‐Filho J S
(2012) Histopathology  61, 629–643 Salivary duct carcinomas can be classified into luminal androgen receptor‐positive, HER2 and basal‐like phenotypes Aims: The aim of this study was to devise a molecular classification for salivary duct carcinomas (SDCs) based on the similarities between SDCs and breast carcinomas and on characteristics of the microarray‐based gene expression profiling‐defined molecular subtypes of breast cancer. Methods and results: Forty‐two pure salivary duct carcinomas, 35 of which contained an in‐situ component as defined by histological review and/or immunohistochemical analysis, were stained with antibodies for oestrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR) and cytokeratin (CK) 5/6. Based on these markers, tumours were classified into HER2, luminal androgen receptor‐positive, basal‐like, luminal and indeterminate phenotype. Analysis revealed that 16.7%, 69%, 4.8%, 9.5% and 0% were of HER2, luminal androgen receptor‐positive, basal‐like, indeterminate and luminal phenotype, respectively. The in‐situ and invasive components displayed the same molecular subtype in all but one case. Conclusions: Salivary duct carcinomas can be classified into molecular subgroups approximately equivalent to those in the breast. We also report on the existence of a subgroup of bona fide pure salivary duct carcinomas that have a ‘basal‐like’ phenotype. Understanding the phenotypic complexity of SDCs may help to expedite the identification of novel therapeutic targets for these aggressive tumours.     

Molecular abnormalities in ovarian carcinoma: clinical,morphological and therapeutic correlates

The histopathological classification of ovarian surface epithelial carcinomas (referred to hereafter as ‘ovarian carcinoma’) has shifted over the past 10 years to reflect more clearly our understanding of molecular events during carcinogenesis. Ovarian carcinoma is no longer viewed as a single entity but as multiple disease processes, with each having different molecular pathways altered during oncogenesis, resulting in differences in clinical and pathological features, such as biomarker expression, pattern of spread and response to chemotherapy. There are five subtypes of ovarian carcinoma that are sufficiently distinct and well‐characterized that they should be considered to be different diseases, i.e. high‐grade serous, clear cell, endometrioid, mucinous and low‐grade serous, from most to least common, respectively. This review summarizes the molecular abnormalities of these five ovarian carcinoma subtypes, relating them to clinical and pathological features.     

Adaptation of the ultrasensitive HIV-1 p24 antigen assay to dried blood spot testing

Implementation of molecular tests for the assessment of pediatric HIV-1 infection in resource-limited countries is difficult because of technical complexity and costs. Alternatives like the ultrasensitive HIV-1 p24 antigen enzyme-linked immunosorbent assay have therefore been proposed. We have now adapted this test to dried blood spot (DBS) plasma p24 antigen (p24). High background activity was recognized as originating from endogenous peroxidase and eliminated by H2O2 quenching. The assay was evaluated with 72 pediatric specimens from Tanzania and with 210 pediatric or adult specimens from Switzerland. A real-time polymerase chain reaction assay for DBS DNA and/or plasma RNA identified HIV-1 infection in 38 Tanzanian children. HIV-1 subtypes included 18 C, 9 A1, 8 D, 1 AC, 1 J-like, and 1 unidentified. The detection rates for the different assays were as follows: DBS-p24, 32 (84%) of 38 samples; DBS DNA, 30 (79%) of 38 samples; plasma-p24, 23 (85%) of 27 samples; and plasma RNA, 30 (100%) of 30 samples. False-negative DBS-p24 was associated with subtype D (P < 0.01). DBS-p24 detection for non-D subtypes was 93% (95% confidence interval: 81% to 99%), and for subtype C, it was 94% (95% confidence interval: 76% to 99%). Specificity among 193 HIV-negative DBS samples was 100%. Correlation of DBS-p24 and plasma-p24 concentrations was excellent (R = 0.83, P < 0.0001). DBS-p24 is thus a promising alternative to molecular tests for HIV-1 in subtype C regions. It should now be evaluated in large studies of children for accurate assessment of diagnostic sensitivity.