Microglandular adenosis: a prime suspect in triple‐negative breast cancer development

Microglandular adenosis (MGA) and atypical MGA (AMGA) are unusual lesions of the breast. They were once regarded as benign proliferative lesions and innocent bystanders. Several lines of evidence suggested that they could be neoplastic, clonal lesions and a non‐obligate precursor for triple‐negative breast cancers (TNBC). Recent work published in The Journal of Pathology by Guerini‐Rocco and colleagues provided further evidence regarding the precursor–product relationship between MGA/AMGA and TNBC. Using a massively parallel sequencing approach, they demonstrated that MGA/AMGA, particularly those associated with TNBC, could be clonal neoplastic lesions showing clonal non‐synonymous mutations, but none in pure MGA. Importantly, those alterations were observed in the associated TNBC. They were also able to identify recurrent alterations in TP53 in those MGA/AMGA cases as well as their associated TNBC. The findings, in conjunction with others, underscore the significance for MGA in clinical diagnosis. The potential of a benign lesion to progress into an aggressive malignant tumour implies that modification of the current management approach may be necessary. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

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.     

Frequent incidence of BARD1‐truncating mutations in germline DNA from triple‐negative breast cancer patients

Triple‐negative breast cancer (TNBC) accounts for 10–20% of all breast cancers (BCs), and conventional chemotherapy is the only effective systemic treatment. Germline BRCA1/2 mutations are found in approximately 15% of TNBC patients. In the past, we have documented pathogenic mutations in BARD1, a BRCA1 interacting protein, in families at high risk for BC. In this study, we have analyzed germline DNA from 61 estrogen receptor negative patients (of which 42 were TNBC) for the presence of mutations in the BRCA1, BRCA2 and BARD1 gene. BRCA1/2 mutations were found in 8 out of 42 (19%) TNBC patients, but not in the ER?/HER2+ cohort. We also found four good candidate pathogenic BARD1 mutations in the TNBC cohort, including two protein‐truncating mutations (p.Gln564Ter and p.Arg641Ter). Our data suggest that TNBC patients are enriched for pathogenic BARD1 germline mutations as compared to control samples and high BC risk families. Ten of the 42 investigated TNBC patients carry a BRCA pathway mutation (in BRCA1, BRCA2 or BARD1) rendering them susceptible to homologous recombination deficiency. These patients should become eligible for exploring the efficacy of poly (ADP‐ribose) polymerase (PARP) inhibitors.     

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.     

GABRP基因在基底样三阴乳腺癌中的差异表达

目的:探索GABRP基因在基底样三阴乳腺癌(basal-like Triple negative breast cancer,BLTNBC)中的表达,为临床提供预后诊断分子标志物.方法:通过生物信息学分析比较GABRP基因的表达;采用定量PCR方法检测GABRP基因在乳腺癌细胞系及104例乳腺癌石蜡样本中的差异表达,并采用相关性分析、卡方检验及Fisher精确概率法分析GABRP基因与细胞增殖、淋巴结转移、ER及HER-2基因表达的相关性.结果:生物信息学分析和定量PCR结果显示GABRP基因在三阴性乳腺癌(triple negative breast cancer,TNBC)及BLTNBC中显著性高表达(p＜0.05);在non-TNBC,TNBC,BLTNBC中,其表达与细胞增殖、HER-2表达无明显相关性(P＞0.05),而在BLTNBC中与淋巴结转移状态有明显相关性(P＜0.05),在non-TNBC巾与ER表达有明显相关性(P＜0.05).结论:GABRP基因可作为BLTNBC的有潜力的预后诊断分子标志物.     

MRI reveals increased tumorigenesis following high fat feeding in a mouse model of triple‐negative breast cancer

High animal fat consumption is associated with an increase in triple‐negative breast cancer (TNBC) risk. Based on previous MRI studies demonstrating the feasibility of detecting very early non‐palpable mammary cancers in simian virus 40 large T antigen (SV40TAg) mice, we examined the effect of dietary fat fed from weaning to young adulthood in this model of TNBC. Virgin female C3(1)SV40TAg mice (n = 16) were weaned at 3–4 weeks of age and then fed either a low fat diet (LFD) (n = 8, 3.7 kcal/g; 17.2% kcal from vegetable oil) or a high animal fat diet (HAFD) (n = 8, 5.3 kcal/g; 60% kcal from lard). After 8 weeks on the diet (12 weeks of age), fast spin echo MR images of inguinal mammary glands were acquired at 9.4 T. Following in vivo MRI, mice were sacrificed and inguinal mammary glands were excised and formalin fixed for ex vivo MRI. 3D volume‐rendered MR images were then correlated with mammary gland histology to assess the glandular parenchyma and tumor burden. Using in vivo MRI, an average of 3.88 ± 1.03 tumors were detected per HAFD‐fed mouse compared with an average of 1.25 ± 1.16 per LFD‐fed mouse (p < 0.007). Additionally, the average tumor volume was significantly higher following HAFD feeding (0.53 ± 0.45 mm³) compared with LFD feeding (0.20 ± 0.08 mm³, p < 0.02). Analysis of ex vivo MR and histology images demonstrated that HAFD mouse mammary glands had denser parenchyma, irregular and enlarged ducts, dilated blood vessels, increased white adipose tissue, and increased tumor invasion. MRI and histological studies of the SV40TAg mice demonstrated that HAFD feeding also resulted in higher cancer incidence and larger mammary tumors. Unlike other imaging methods for assessing environmental effects on mammary cancer growth, MRI allows routine serial measurements and reliable detection of small cancers as well as accurate tumor volume measurements and assessment of the three‐dimensional distribution of tumors over time.     

Triple negative breast cancer: adjuvant chemotherapy effect on survival

PurposeThe purpose of this study was to evaluate the overall survival of patients with triple negative breast cancer and the impact of different adjuvant chemotherapy regimens on survival.Material/MethodsThe study group consisted of 99 breast cancer patients with immunohistochemically confirmed triple negative breast cancer. The impact of various factors as well as the impact of different chemotherapy regimens on survival was evaluated.ResultsThe overall survival of breast cancer patients was 97.0% (95% CI 90.9–99.0), 84.9% (95% CI 76.1–90.6) and 66.5% (95% CI 55.5–75.3) 10, 30 and 60 months after diagnosis, respectively. Univariate analysis demonstrated that the following were significant risk factors for breast cancer patients survival: patient's age, stage of disease, tumour size, lymph node status, type of surgery and chemotherapy. Better survival was related to younger patients’ age, smaller tumour size, lower stage of disease, no lymph nodes involvement. Survival rates were higher among patients who received adjuvant chemotherapy and underwent quadrantectomy. In the multivariate statistical analysis the significant independent prognostic variables influencing survival were lymph node status and adjuvant chemotherapy. Survival rates of the patients, who received adjuvant anthracycline containing chemotherapy were higher, than those in non-anthracycline containing treatment group, but the difference was not statistically significant.ConclusionPatients who had lymph node status N2–3 and those who did not receive adjuvant chemotherapy showed worse prognosis and survival than other patients. The impact of chemotherapy type (anthracycline containing or non-anthracycline containing) on patients survival was not statistically significant.     

Molecular alterations in AKT1, AKT2 and AKT3 detected in breast and prostatic cancer by FISH

Kirkegaard T, Witton C J, Edwards J, Nielsen K V, Jensen L B, Campbell F M, Cooke T G & Bartlett J M S
(2010) Histopathology 56, 203–211 Molecular alterations in AKT1, AKT2 and AKT3 detected in breast and prostatic cancer by FISH Aims: The AKT family is implicated in cancer progression. There are three mammalian AKT isoforms located on chromosomes 14, 19 and 1, respectively. The aim of the study was to investigate genetic alterations of AKT in breast and prostatic cancers using fluorescence in situ hybridization (FISH). Methods and results: In oestrogen receptor(ER)‐positive breast carcinomas, AKT1 was deleted in five (4.8%) and amplified in one (1%) carcinoma. Deletions of AKT2 were seen in 19 (21.1%) cases. No AKT2 amplifications were identified. Ten (9.9%) AKT3 amplifications but no deletions were seen. In prostatic cancer, AKT1 was amplified in one carcinoma (2.6%). No genetic changes were observed for AKT2 and AKT3. High frequencies of aneusomy for all chromosomes were observed in breast and prostatic carcinomas. Conclusions: In breast cancer AKT3 amplifications and AKT1 and AKT2 deletions were seen, which, to our knowledge, have not been shown by FISH before. Although these two cohorts cannot be directly compared, only one AKT1 amplification was identified in prostatic carcinomas. This indicates differences in the genetic changes underlying development of breast and prostatic cancers. To evaluate further the role of genetic changes of AKT in breast cancer progression, a cohort of both ER+ and ER? patients should be evaluated.     

Gross cystic disease fluid protein‐15 and mammaglobin A expression determined by immunohistochemistry is of limited utility in triple‐negative breast cancer

Aims: In addition to oestrogen and progesterone receptors, gross cystic disease fluid protein‐15 (GCDFP‐15) and mammaglobin A (MAM) are the most common markers used to identify breast origin by immunohistochemistry. GCDFP‐15 expression has been reported in approximately 60% of breast carcinomas and MAM expression in approximately 80%. Data on their expression in triple‐negative breast cancer (TNBC) are very limited. The aim of this study was to examine the expression of these markers in TNBC to determine their utility in pathological diagnosis. Methods and results: We studied the immunohistochemical (IHC) expression of GCDFP‐15 and MAM in 63 primary and 118 metastatic TNBCs. GCDFP‐15 staining was present in 14% of primary and 21% of metastatic TNBCs. MAM staining was present in 25% of primary and 41% of metastatic TNBCs. The frequency of expression of GCDFP‐15 and/or MAM was 30% in primary and 43% in metastatic TNBCs, and many positive tumours had only focal staining. Conclusions: Staining for GCDFP‐15 and/or MAM in triple‐negative carcinomas helps to confirm breast origin, but most tumours in this subgroup of breast carcinomas lack expression of either marker.     

Insulin growth factor receptor‐1 expression and loss of PTEN protein predict early recurrence in triple‐negative breast cancer

Iqbal J, Thike A A, Cheok P Y, Tse G M‐K & Tan P H
(2012) Histopathology  61, 652–659 Insulin growth factor receptor‐1 expression and loss of PTEN protein predict early recurrence in triple‐negative breast cancer Aims: Insulin‐like growth factor receptor‐1 (IGFR‐1) and its signalling axis promote tumorigenesis, metastasis, and resistance to existing forms of cancer therapy, and have become a major focus for the development of anticancer drugs. As oncological management options for triple‐negative breast cancers (TNBCs) are limited, there is potential for the rapid development of novel selective anticancer agents specifically targeting components of the PTEN–phosphoinositide 3‐kinase–AKT pathway, including the phosphorylated form of AKT (pAKT) and the tumour suppressor molecule PTEN. The aim of this study was to conduct immunohistochemical analyses to examine the levels of PTEN, IGFR‐1 and pAKT expression in TNBCs, and determine whether these levels correlated with poor prognosis in this subset of aggressive breast cancers. Methods and results: Immunohistochemistry was performed on paraffin‐embedded tumour tissues from a consecutive cohort of 144 female patients diagnosed with TNBC. Associations of IGFR‐1, PTEN and pAKT expression with clinicopathological parameters, disease‐free survival (DFS) and overall survival (OS) were evaluated. There were significant increases in IGFR‐1 expression (99%) and pAKT expression (92%) with concomitant loss of PTEN expression in the majority of cases (63%). Increased IGFR‐1 expression and loss of PTEN expression were associated with reduced OS and DFS, respectively. pAKT expression showed a strong correlation with basal‐like expression. Combinatorial immunophenotypic analyses showed that loss of PTEN expression with concomitant IGFR‐1 expression correlated with poor DFS. Conclusions: A high percentage of PTEN loss with overexpression of IGFR‐1 and pAKT in TNBC indicates the potential of these molecules for predicting early recurrence and/or as targets in the formulation of effective alternative therapy regimens.     

Detection of numerical alterations of chromosome 1 in cytopathological specimens of breast tumors by chromogen in situ hybridization

To investigate the effectiveness of chromogen in situ hybridization (CISH) in the diagnosis of breast tumors, numerical alterations of chromosome 1 were examined by CISH and fluorescence in situ hybridization (FISH) methods, and the presence of der(16)t(1;16) was also examined by FISH in imprinted cytology specimens from resected tissues of 14 carcinomas and five non-malignant lesions. The modal signal counts of chromosome 1 were compared between the specimens processed by CISH and FISH for each case. Aneusomies of the long arm of chromosome 1 were detected in 10 (71%) carcinomas as the major clones by both methods. In addition, one atypical papilloma demonstrated tetrasomy of 1q12 as a major clone by CISH, but such a clone was at first overlooked by FISH. Four other benign lesions showed disomic 1q12 signals as a major clone by both CISH and FISH. As additional information from FISH, eight cancers showed structural or numerical alterations of chromosome 16, and four showed der(16)t(1;16). In total, 10 carcinomas showed chromosome 16 alterations, and all of these overlapped with the carcinomas with 1q12 aneusomies. The CISH method provided almost the same results as the FISH method, and both methods were considered applicable in supportive diagnosis of cytological specimens of breast tumors. In addition, the CISH method was superior in the detection of numerical alterations in carcinoma cells by referring to the morphology of cells and in the detection of significant clones which might be missed under dark-field microscopy.     

Magnetic resonance angiography reveals increased arterial blood supply and tumorigenesis following high fat feeding in a mouse model of triple‐negative breast cancer

Breast cancer is the second most commonly diagnosed malignancy among women globally. Past MRI studies have linked a high animal fat diet (HAFD) to increased mammary cancer risk in the SV40Tag mouse model of triple‐negative breast cancer. Here, serial MRI examines tumor progression and measures the arterial blood volume feeding mammary glands in low fat diet (LFD) or HAFD fed mice. Virgin female C3(1)SV40Tag mice (n = 8), weaned at 3 weeks old, were assigned to an LFD (n = 4, 3.7 kcal/g, 17.2% kcal from vegetable oil) or an HAFD (n = 4, 5.3 kcal/g, 60% kcal from lard) group. From ages 8 to 12 weeks, weekly fast spin echo MR images and time‐of‐flight (TOF) MR angiography of inguinal mammary glands were acquired at 9.4 T. Following in vivo MRI, mice were sacrificed. Inguinal mammary glands were excised and fixed for ex vivo MRI and histology. Tumor, blood, and mammary gland volumes for each time point were measured from manually traced regions of interest; tumors were classified as invasive by histopathology‐blinded observers. Our analysis confirmed a strong correlation between total tumor volume and blood volume in the mammary gland. Tumor growth rates from weeks 8‐12 were twice as high in HAFD‐fed mice (0.42 ± 0.14/week) as in LFD‐fed mice (0.21 ± 0.03/week), p < 0.004. Mammary gland blood volume growth rate was 2.2 times higher in HAFD mice (0.29 ± 0.11/week) compared with LFD mice (0.13 ± 0.06/week), p < 0.02. The mammary gland growth rate of HAFD‐fed mice (0.071 ± 0.011/week) was 2.7 times larger than that of LFD‐fed mice (0.026 ± 0.009/week), p < 0.01. This is the first non‐invasive, in vivo MRI study to demonstrate a strong correlation between an HAFD and increased cancer burden and blood volume in mammary cancer without using contrast agents, strengthening the evidence supporting the adverse effects of an HAFD on mammary cancer. These results support the potential future use of TOF angiography to evaluate vasculature of suspicious lesions.     

The use of fine‐needle aspiration biopsy samples for the assessment of basal phenotype in triple negative breast cancer patients: A correlative study

Molecular profiling of breast cancer has gained popularity due to the possibility of studying the biological spectrum of the disease and to evaluate prognostic and predictive characteristics that can lead to more accurate therapeutic decisions. Although studies have been carried out on tissue using tissue microarray technology (TMA), to our knowledge, this technology has not been applied to samples obtained by fine‐needle aspiration biopsy (FNAB). This study was designed to correlate FNAB samples with corresponding surgical specimens for the assessment of basal phenotype in patients with triple negative breast cancer (TNBC). A total of 198 cases of TNBC with matching FNAB and surgical specimens were identified. Forty‐six cases with sufficient tissue in both FNAB cell blocks and surgical specimens were selected. Tissue microarray blocks were prepared and stained with six biomarkers (CK5/6, p63, SMA, EGFR, C‐Kit, and p53). For statistical analysis, we used the observed (Gross) percentage of agreement and also calculated the Cohen's Kappa for each biomarker. We found a high agreement between the two groups as shown by the values of Observed (Gross) percentage of agreement (mostly 90% or higher except for the C‐Kit which was 78%). In addition, Cohen's Kappa point estimate showed substantial agreement (0.61–0.80) for CK 5/6, p63, EGFR, p53, moderate agreement (0.41–0.60) for C‐Kit, and, fair agreement (0.21–0.40) for SMA. Our study shows that the advantages of FNAB are not restricted to cost‐effectiveness, but also attested that cytology samples are suitable for the evaluation of biomarkers that have important implications on patient's therapy and prognosis. Diagn. Cytopathol. 2011;39:1–7. © 2010 Wiley‐Liss, Inc.     

Fine‐needle aspiration cytology of triple‐negative basal‐like breast cancer

Invasive breast cancer is divided into luminal A, luminal B, HER2 overexpression, basal‐like (BL) and normal‐like subtypes, among which the BL subtype has the worst prognosis. The purpose of this study was to determine the clinicopathological and cytological characteristics of BL breast cancer (BLBC). Fine‐needle aspiration cytology samples from 17 patients with consecutive BLBC were investigated, and the findings were compared with those of other subtypes (10 cases each) for the following cytomorphological features: necrosis; lymphocyte infiltration; mitotic index; apoptosis; naked nuclei; nuclear/cytoplasmic ratio; nuclear margin, size and pleomorphism; chromatin granularity and density; and nucleolar appearance. Histologically, the BLBCs were heterogeneous, and included medullary carcinoma and metaplastic carcinoma, in addition to invasive ductal carcinoma. Cytologically, high mitotic index, naked nuclei, and irregular nuclear margin were significantly observed when compared with both the luminal A and B subtypes. Large nuclei with nucleoli and lymphocyte infiltration were frequently seen compared with the luminal A and B subtypes, respectively. Squamous nodules were seen in all metaplastic cases, but not in the HER2 overexpression subtype. Lymphocyte infiltration, squamous metaplasia, and nuclear findings such as a high mitotic index, naked or large nuclei, an irregular nuclear margin and the presence of nucleoli, may be clues indicating BLBC. Diagn. Cytopathol. 2013;41:283–287. © 2011 Wiley Periodicals, Inc.