Genetic changes in bilateral breast cancer by comparative genomic hybridisation

Abstract The aim of this study is to find any specific genetic defect occurring frequently in bilateral breast cancer by examining the genetic changes of each chromosome using comparative genomic hybridisation (CGH). CGH was conducted for 36 breast cancer tissues taken from patients treated with surgery for bilateral breast cancer. Tumour and control DNAs were hybridised to metaphase chromosome with differential staining with fluorescein and rhodamine-dUTP. An average rate of green (DNA of tumour cell) against red (DNA of a normal peripheral blood lymphocyte) was calculated in these captured metaphase chromosomes and a ratio of more than 1.17 was defined as an acquisition, less than 0.85 as a loss and, finally, more than 2 as amplification. Twenty-six out of 36 cases (72.2%) showed a change in the number of DNA copies by CGH in one or more regions of gene. On average, 5.3 alterations for each chromosome (range, 1–14) were found, and gain was present more than loss at a ratio of 1.3:1. Loci that showed amplification were X, 17q, Xq, 8q, 14q11-21 and 17q22-qter. The locus showing the most gain was the X chromosome, which was observed in 15 (57.7%) out of 26 cases. Loss was most frequently observed in the short arm of chromosome 8. The concordance of genetic transformation of primary cancer and secondary cancer in bilateral breast cancer was an average of 18.7% in synchronous and 10.7% in metachronous cancer, showing higher similarity in synchronous breast cancer.     

Microglandular adenosis associated with triple‐negative breast cancer is a neoplastic lesion of triple‐negative phenotype harbouring TP53 somatic mutations

Microglandular adenosis (MGA) is a rare proliferative lesion of the breast composed of small glands lacking myoepithelial cells and lined by S100‐positive, oestrogen receptor (ER)‐negative, progesterone receptor (PR)‐negative, and HER2‐negative epithelial cells. There is evidence to suggest that MGA may constitute a non‐obligate precursor of triple‐negative breast cancer (TNBC). We sought to define the genomic landscape of pure MGA and of MGA, atypical MGA (AMGA) and associated TNBCs, and to determine whether synchronous MGA, AMGA, and TNBCs would be clonally related. Two pure MGAs and eight cases of MGA and/or AMGA associated with in situ or invasive TNBC were collected, microdissected, and subjected to massively parallel sequencing targeting all coding regions of 236 genes recurrently mutated in breast cancer or related to DNA repair. Pure MGAs lacked clonal non‐synonymous somatic mutations and displayed limited copy number alterations (CNAs); conversely, all MGAs (n = 7) and AMGAs (n = 3) associated with TNBC harboured at least one somatic non‐synonymous mutation (range 3–14 and 1–10, respectively). In all cases where TNBCs were analyzed, identical TP53 mutations and similar patterns of gene CNAs were found in the MGA and/or AMGA and in the associated TNBC. In the MGA/AMGA associated with TNBC lacking TP53 mutations, somatic mutations affecting PI3K pathway‐related genes (eg PTEN, PIK3CA, and INPP4B) and tyrosine kinase receptor signalling‐related genes (eg ERBB3 and FGFR2) were identified. At diagnosis, MGAs associated with TNBC were found to display subclonal populations, and clonal shifts in the progression from MGA to AMGA and/or to TNBC were observed. Our results demonstrate the heterogeneity of MGAs, and that MGAs associated with TNBC, but not necessarily pure MGAs, are genetically advanced, clonal, and neoplastic lesions harbouring recurrent mutations in TP53 and/or other cancer genes, supporting the notion that a subset of MGAs and AMGAs may constitute non‐obligate precursors of TNBCs. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Aneuploidy,TP53 mutation,and amplification of MYC correlate with increased intratumor heterogeneity and poor prognosis of breast cancer patients

The clinical course of breast cancer varies from one patient to another. Currently, the choice of therapy relies on clinical parameters and histological and molecular tumor features. Alas, these markers are informative in only a subset of patients. Therefore, additional predictors of disease outcome would be valuable for treatment stratification. Extensive studies showed that the degree of variation of the nuclear DNA content, i.e., aneuploidy, determines prognosis. Our aim was to further elucidate the molecular basis of aneuploidy. We analyzed five diploid and six aneuploid tumors with more than 20 years of follow‐up. By performing FISH with a multiplexed panel of 10 probes to enumerate copy numbers in individual cells, and by sequencing 563 cancer‐related genes, we analyzed how aneuploidy is linked to intratumor heterogeneity. In our cohort, none of the patients with diploid tumors died of breast cancer during follow‐up in contrast to four of six patients with aneuploid tumors (mean survival 86.4 months). The FISH analysis showed markedly increased genomic instability and intratumor heterogeneity in aneuploid tumors. MYC gain was observed in only 20% of the diploid cancers, while all aneuploid cases showed a gain. The mutation burden was similar in diploid and aneuploid tumors, however, TP53 mutations were not observed in diploid tumors, but in all aneuploid tumors in our collective. We conclude that quantitative measurements of intratumor heterogeneity by multiplex FISH, detection of MYC amplification and TP53 mutation could augment prognostication in breast cancer patients.     

Intratumoral heterogeneity in breast carcinoma revealed by laser-microdissection and comparative genomic hybridization

To evaluate the potential cytogenetic heterogeneity in breast carcinoma, several small cell groups (each consisting of 20 to 50 cells) were investigated within paraffin sections. By laser-microdissection, three to seven cell groups were taken per case. The DNA was amplified by degenerate oligonucleotide primed PCR (DOP-PCR), and the samples were analyzed by CGH for chromosomal gains and losses. Two ductal invasive breast carcinomas, one of them with two lymphnode metastases, were investigated. To compare the results from the small samples, CGH was also performed on DNA isolated from the tumorous regions of three to five serial sections (10⁷ to 10⁶ cells). The aberrations observed in the microdissected tumor samples were multiple and involved up to 14 different chromosomal or subchromosomal regions. The most frequent changes were gains on chromosomes 12q (14/20) and 20q (16/20), and loss on 13q (12/20). Some aberrations have rarely been detected (e.g., loss on 2p, gain on 8q). Comparing chromosomal imbalances in primary tumors and lymph node metastases, more consistent changes were found between the primary tumor and its corresponding metastases than between both primary tumors. The laser-microdissected samples in general showed more chromosomal aberrations than DNA isolated from several tumor sections. Our CGH results were confirmed by fluorescence in situ hybridization (FISH) for the chromosomal regions of centromere 1 and 20, and 20q13. In addition, microsatellite analyses on 31 samples confirmed our CGH findings for selected chromosome regions 2p and 11q. It can be concluded that there is a distinct intratumoral heterogeneity in primary breast tumors as well as in the corresponding lymph node metastases. The combination of microdissection and CGH enabled us to detect cytogenetic aberrations from important clones which are missed when analyzing DNA extracted from large cell numbers.     

Impact of intratumoral heterogeneity on the metabolic profiling of breast cancer tissue using high-resolution magic angle spinning magnetic resonance spectroscopy

High-resolution magic angle spinning (HR-MAS) magnetic resonance spectroscopy (MRS) is a useful metabolic profiling technique for human tissue. However, the impact of intratumoral heterogeneity on the metabolite levels of breast cancers is not yet established. The purpose of this prospective study was to investigate whether the tumor cell fraction of core needle biopsy (CNB) specimens of breast cancers affect metabolic profiles assessed with HR-MAS MRS. From June 2015 to December 2016, 46 patients with 47 breast cancers were enrolled. HR-MAS MRS was used for the metabolic profiling of 285 CNB specimens from the 47 cancers. Multiple CNB samples (range 2–8) for the HR-MAS MRS experiment were obtained from surgical specimens under ultrasound guidance following surgical removal of the tumor. Tumor cell fraction was expressed as a percentage of the tumor cell volume relative to the total tumor volume contained in each CNB sample. Metabolite quantification levels were compared according to primary tumor characteristics using the t-test. Multivariate analyses were performed including primary tumor characteristics and tumor cell percentages as variables. Correlations between tumor cell percentage and metabolite levels in the CNB specimens were assessed according to the immunohistochemical status of the primary tumor. In univariate analysis, levels of choline-containing compounds, glutamate, glutamine, glycine, serine, and taurine were correlated with primary tumor characteristics. In multivariate analysis, most metabolite levels were not affected by tumor cell percentage. Tumor cell percentage showed poor correlation with metabolite levels in hormone receptor-positive cancer and triple-negative cancer, and poor to fair correlation with metabolite levels in HER2-positive cancer. This study showed that differences in the tumor cell fraction of CNB samples do not affect predictions on the primary cancer from which the samples are obtained.     

乳腺癌HER2基因遗传异质性与其临床病理特征的相关性研究

目的 介绍乳腺癌患者HER2基因扩增的瘤内遗传异质性(genetic heterogeneity,GH)现象,并探讨HER2基因GH与临床病理学参数间的相关性,寻找与HER2基因GH现象密切相关的病理学因素.方法 整理100例浸润性乳腺癌患者分期、组织学分型、分子分型等临床数据,应用免疫组化技术(immunohistochemistry,IHC)检测HER2、雌激素受体α(estrogen peceptor α,ERα)和孕激素受体(progesterone receptor,PR)蛋白表达,用荧光原位杂交(fluorescence in situ hybridization,FISH)技术检测HER2基因扩增情况,并分析HER2基因异质性与临床病理参数间的关系.结果 100例乳腺癌患者中有20%的病例(20/100)存在HER2基因GH现象.在瘤内异质性样本中,扩增细胞所占比例≥25%的样本组其FISH结果阳性率明显高于扩增细胞＜25%的样本组(P=0.012).HER2蛋白的表达程度(P=0.004)和ER蛋白表达(P=0.002)与HER2基因GH现象显著相关.结论 HER2基因GH现象最易出现于HER2蛋白轻到中度表达、ER蛋白表达的乳腺癌组织,故而对于具有上述病理学特征的患者进行FISH检测时应特别注意GH现象,以免遗漏阳性细胞或者只计数阳性细胞,而给出不准确的FISH结果.     

Identification of novel BRCA large genomic rearrangements in Singapore Asian breast and ovarian patients with cancer

Large genomic rearrangements have been reported to account for about 10-15% of BRCA1 gene mutations. Approximately, 90 BRCA rearrangements have been described to date, all of which but one have been reported in Caucasian populations of predominantly Western European descent. Knowledge of BRCA genomic rearrangements in Asian populations is still largely unknown. In this study, we have investigated for the presence of BRCA rearrangements among Asian patients with early onset or familial history of breast or ovarian cancer. Using multiplex ligation-dependent probe amplification (MLPA), we have analyzed 100 Singapore patients who previously tested negative for deleterious BRCA mutations by the conventional polymerase chain reaction-based mutation detection methods. Three novel BRCA rearrangements were detected, two of which were characterized. The patients with the rearrangements, a BRCA1 exon 13 duplication, a BRCA1 exon 13-15 deletion and a BRCA2 exon 4-11 duplication, comprise 3% of those previously tested negative for BRCA mutations. Of the BRCA1 and BRCA2 pathogenic mutations identified in our studies on Asian high-risk breast and ovarian patients with cancer to date, these rearrangements constitute 2/19 and 1/2 of the BRCA1 and BRCA2 pathogenic mutations, respectively. Given the increasing number of rearrangements reported in recent years and their contribution to the BRCA mutation spectrum, the presence of BRCA large exon rearrangements in Asian populations should be investigated where clinical, diagnostic service is recommended.     

Prevalence of BRCA1/2 large genomic rearrangements in Chinese women with sporadic triple‐negative or familial breast cancer

The prevalence of BRCA1/2 large genomic rearrangements (LGRs) and their underlying mechanisms have not been fully evaluated in Chinese women with breast cancer. In this study, we determined the prevalence of BRCA1/2 LGRs in 834 patients with familial breast cancer (FBC) and 660 patients with sporadic triple‐negative breast cancer (TNBC) who were negative for BRCA1/2 small‐range mutations using the multiplex ligation‐dependent probe amplification method. We found that 20 index patients (2.4%) in the FBC group carried a BRCA1 or BRCA2 LGR, and the frequencies of BRCA1 and BRCA2 LGRs were 1.6% and 0.8%, respectively. Seven index patients (1.1%) carried a BRCA1 LGR in 660 sporadic TNBC patients, whereas no BRCA2 LGRs were found in these patients. Among the BRCA1/2 LGRs, 48.1% (13/27) were novel, and the breakpoints of the majority of the LGRs were identified. ΨBRCA1‐mediated homologous recombination (HR) and Alu‐mediated HR/non‐homologous end‐joining (NHEJ) accounted for 40% and 30% of the BRCA1 LGRs, respectively. Alu‐mediated HR accounted for 71.4% of the BRCA2 LGRs, and the remaining one‐third was generated through Long interspersed nuclear elements (LINE)‐mediated NHEJ. Our findings suggest that both FBC patients and sporadic TNBC patients should be tested for BRCA1/2 LGRs.     

TP53 mutations are associated with a particular pattern of genomic imbalances in breast carcinomas

TP53 mutations play an important role in the development of several cancers and are present in 20-40% of all breast carcinomas, contributing to increased genomic instability. In order to address the relationship of mutated TP53 to genomic complexity, the present study analysed 61 breast carcinomas for TP53 mutations and compared mutation status with the pattern of genomic imbalances as assessed by comparative genomic hybridization (CGH). Twenty per cent of the present series of breast carcinomas harboured TP53 mutations. An increasing number of abnormalities, as identified by CGH (higher genomic complexity), correlated significantly with mutant TP53. Among the chromosome arms most commonly altered (in more than 20% of the tumours), loss of 8p and gain of 8q were associated with TP53 mutations, whereas loss of 16q was associated with wild-type TP53. By performing supervised hierarchical clustering analysis of the CGH data, a cluster of chromosome imbalances was observed that showed differences between wild-type and mutant TP53 cases. Among these, loss of chromosome arm 5q revealed the strongest correlation with altered TP53. To investigate further the most commonly deleted region of 5q, gene expression patterns from two publicly available microarray data sets of breast carcinomas were evaluated statistically. The expression data sets identified potential target genes, including genes involved in ubiquitination and the known TP53 target CSPG2. The genomic complexity of breast carcinomas as assessed by CGH is associated with TP53 mutation status; breast cancers with TP53 mutations display more complex genomes than do those with wild-type TP53. The pattern of genomic imbalances associated with mutant TP53 is non-random, with loss of chromosome arm 5q being particularly closely associated with TP53 mutations.     

Immunophenotypic and genomic characterization of papillary carcinomas of the breast

Papillary carcinomas are a special histological type of breast cancer and have a relatively good outcome. We characterized the genomic and phenotypic characteristics of papillary carcinomas to determine whether they would constitute an entity distinct from grade- and oestrogen receptor (ER)-matched invasive ductal carcinomas of no special type (IDC-NSTs). The phenotype of 63 papillary carcinomas of the breast and grade- and ER-matched IDC-NSTs was determined by immunohistochemistry. DNA of sufficient quality was extracted from 49 microdissected papillary carcinomas and 49 microdissected grade- and ER-matched IDC-NSTs. These samples were subjected to high-resolution microarray-based comparative genomic hybridization (aCGH) and Sequenom MassARRAY sequencing analysis of 19 known oncogenes. Papillary carcinomas were predominantly of low histological grade, expressed immunohistochemical markers consistent with a luminal phenotype, and a lower rate of lymph node metastasis and p53 expression than grade- and ER-matched IDC-NSTs. Papillary carcinomas displayed less genomic aberrations than grade- and ER-matched IDC-NSTs; however, the patterns of gene copy number aberrations found in papillary carcinomas were similar to those of ER- and grade-matched IDC-NSTs, including 16q losses. Furthermore, PIK3CA mutations were found in 43% and 29% of papillary carcinomas and grade- and ER-matched IDC-NSTs, respectively. The genomic profiles of encapsulated, solid and invasive papillary carcinomas, the three morphological subtypes, were remarkably similar. Our results demonstrate that papillary carcinomas are a homogeneous special histological type of breast cancer. The similarities in the genomic profiles of papillary carcinomas and grade- and ER-matched IDC-NSTs suggest that papillary carcinomas may be best positioned as part of the spectrum of ER-positive breast cancers, rather than as a distinct entity. Furthermore, the good prognosis of papillary carcinomas may stem from the low rates of lymph node metastasis and p53 expression, low number of gene copy number aberrations and high prevalence of PIK3CA mutations.     

超声靶向微泡破坏技术及其在乳腺癌治疗方面的应用

超声靶向微泡破坏(UTMD)技术利用超声造影剂微泡(UCAs)作为载体,联合基因或药物治疗多种疾病,目前已表现出强大潜力。在乳腺癌治疗方面,低频超声辐照下微泡破裂产生的声孔效应及空化效应有效增加靶细胞内基因或药物浓度,增强对癌细胞杀伤力,具多重优点。但是,UTMD技术在实际应用中仍然面临许多问题。     

Identification of cellular and genetic drivers of breast cancer heterogeneity in genetically engineered mouse tumour models

The heterogeneous nature of mammary tumours may arise from different initiating genetic lesions occurring in distinct cells of origin. Here, we generated mice in which Brca2, Pten and p53 were depleted in either basal mammary epithelial cells or luminal oestrogen receptor (ER)‐negative cells. Basal cell‐origin tumours displayed similar histological phenotypes, regardless of the depleted gene. In contrast, luminal ER‐negative cells gave rise to diverse phenotypes, depending on the initiating lesions, including both ER‐negative and, strikingly, ER‐positive invasive ductal carcinomas. Molecular profiling demonstrated that luminal ER‐negative cell‐origin tumours resembled a range of the molecular subtypes of human breast cancer, including basal‐like, luminal B and ‘normal‐like’. Furthermore, a subset of these tumours resembled the ‘claudin‐low’ tumour subtype. These findings demonstrate that not only do mammary tumour phenotypes depend on the interactions between cell of origin and driver genetic aberrations, but also multiple mammary tumour subtypes, including both ER‐positive and ‐negative disease, can originate from a single epithelial cell type. This is a fundamental advance in our understanding of tumour aetiology. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

The Japanese Society of Pathology Practical Guidelines on the handling of pathological tissue samples for cancer genomic medicine

Clinical cancer genomic testing based on next-generation sequencing can help select genotype-matched therapy and provide diagnostic and prognostic information. Pathological tissue from malignant tumors obtained during routine practice are frequently used for genomic testing. This article is aimed to standardize the proper handling of pathological specimens in practice for genomic medicine based on the findings established in “Guidelines on the handling of pathological tissue samples for genomic medicine (in Japanese)” published by The Japanese Society of Pathology (JSP) in 2018. The two-part practical guidelines are based on empirical data analyses; Part 1 describes the standard preanalytic operating procedures for tissue collection, processing, and storage of formalin-fixed paraffin-embedded (FFPE) samples, while Part 2 describes the assessment and selection of FFPE samples appropriate for genomic testing, typically conducted by a pathologist. The guidelines recommend that FFPE sample blocks be used within 3 years from preparation, and the tumor content should be ≥30% (minimum 20%). The empirical data were obtained from clinical studies performed by the JSP in collaboration with leading Japanese cancer genome research projects. The Japanese Ministry of Health, Labour, and Welfare (MHLW) recommended to comply with the JSP practical guidelines in implementing cancer genomic testing under the national health insurance system in over 200 MHLW-designated core and cooperative cancer genome medicine hospitals in Japan.     

Intraindividual genomic heterogeneity of high‐grade serous carcinoma of the ovary and clinical utility of ascitic cancer cells for mutation profiling

Intraindividual tumoural heterogeneity (ITH) is a hallmark of solid tumours and impedes accurate genomic diagnosis and selection of proper therapy. The aim of this study was to identify ITH of ovarian high‐grade serous carcinomas (OSCs) and to determine the utility of ascitic cancer cells as a resource for mutation profiling in spite of ITH. We performed whole‐exome sequencing, copy number profiling and DNA methylation profiling of four OSC genomes by using multiregional biopsies from 13 intraovarian lesions, 12 extraovarian tumour lesions (omentum/peritoneum), and ascitic cells. We observed substantial levels of heterogeneity in mutations and copy number alterations (CNAs) of the OSCs. We categorized the mutations into ‘common’, ‘shared’ and ‘private’ according to the regional distribution. Six common, eight shared and 24 private mutations were observed in known cancer‐related genes. Common mutations had a higher mutant allele frequency, and included TP53 mutations in all four OSCs. Region‐specific chromosomal amplifications and deletions involving BRCA1, PIK3CA and RB1 were also identified. It is of note that the mutations detected in ascitic cancer cells represented 92.3–100% of overall somatic mutations in the given case. Phylogenetic analyses of ascitic genomes predicted a polyseeding origin of somatic mutations in ascitic cells. Our results demonstrate that, despite ITH, somatic mutations, CNAs and DNA methylations in both ‘common’ category and cancer‐related genes were highly conserved in ascitic cells of OSCs, highlighting the clinical relevance of genome analysis of ascitic cells. Ascitic tumour cells may serve as a potential resource for discovering somatic mutations of primary OSC with diagnostic and therapeutic relevance. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Comparative study of the histopathology of breast cancer in a screened and unscreened population investigated by mammography

The histopathology of 360 surgical resections for breast cancer in a consecutive series of patients aged 45-69 years from 1979-1983 is described. Two hundred and seventeen patients who were offered screening (screened patients) were compared with 143 patients who were referred as out-patients with breast problems and were not offered screening (unscreened patients). Both groups were investigated by mammography. Comparisons between tumour staging, grading and quadrant involvement are reported. In the screened patients 31% were in the in-situ stage or had an invasive carcinoma less than 1.0 cm in maximum diameter, compared with 7% in the unscreened patients. Conversely 26% of the screened patients had cancers greater than 2.0 cm compared with 52% in the unscreened group. The percentage of cancer patients with lymph node metastases was comparable in both 1.0-2.0 cm and greater than 2.0 cm groups of invasive carcinoma. There were more multiquadrant cancers in the unscreened patients (32%) than screened patients (17%) and this was mainly due to differences in the incidence of carcinomas 1.0-2.0 cm in diameter. This suggests that invasive tumours of comparable size are more likely to produce symptoms leading to detection if multiquadrant. Multicentric cancers were more common in unscreened patients. Differences in the histological grading related to tumour size were found within the screened and unscreened groups but not between the two groups.     

Frequency of three BRCA1 gene founder mutations in breast/ovarian cancer families from the Pomerania-Kujawy region of Poland

A group of 63 families from the Pomerania-Kujawy region were analyzed for three BRCA1 gene Polish founder mutations, 5382insC, 300T>G, and 4153delA, because of breast (BrCa) and/or ovarian cancer (OvCa) history. The analysis was carried out by multiplex polymerase chain reaction method. BRCA1 mutation was revealed in nine (14%) families: in three (33%) of hereditary BrCa and OvCa families, in three (8%) of hereditary BrCa families, and in three (21%) of hereditary OvCa families. According to risk criteria, it was revealed in 45% of high-risk families with more than three cancers, 13% of moderate-risk families with two cancers, and 8% of families with sporadic OvCa. In six families, the mutation was found in a proband with BrCa or OvCa and in three families, the mutation was found in a healthy proband, first-degree relative of a patient deceased of BrCa or OvCa. 5382insC frameshift mutation accounted for 67% and 300T>G missense mutation for 33% of all identified familial mutations. 4153delA frameshift mutation was not found in analyzed sample of families. 5382insC mutation was found in 9% and 300T>G in 5% of all investigated families, and in 27 and 18%, respectively, of high-risk families. This underlines the importance of applying strict inclusion criteria to analyze mutation frequency in hereditary BrCa/OvCa families.