Detection of numerical chromosome 17 abnormalities in fine-needle aspirates of breast cancer using a novel in situ hybridization signal amplification method

We describe a method of in situ hybridization (ISH) to assess numerical chromosome abnormalities on alcohol-fixed smears obtained by fine-needle aspiration from breast cancer patients, using a commercially available amplification kit to demonstrate numerical chromosome alterations of chromosome 17. In this staining procedure after detection of the biotin-labeled α-satellite probe for chromosome 17 with avidin-biotin-peroxidase, we incorporated a signal amplification based on the peroxidase-catalyzed deposition of a biotinylated phenolic compound followed by a secondary reaction with peroxidase. The reactions are revealed by deposition of diaminobenzidine and can be analyzed in an optical microscope, with total preservation of the morphology, allowing a direct morphologic-cytogenetic correlation. A series of 25 cases of aspirates from breast cancer were analyzed with this methodology. Aneusomy was found in 14 cases (56%), whereas 11 (44%) had a normal number of chromosome 17 copies. Polysomy occurred in all aneusome cases except one. We did not find concordance between numerical chromosome abnormalities of chromosome 17 and nuclear grading as well as with the immunoexpression of p53 and c-erbB2 studied in the smears. We conclude that the application of the ISH signal amplification method on alcohol-fixed smears will eliminate the need for fresh material and will provide several advantages, such as improvement of morphological concomitant analysis without the need for a fluorescence microscope; utilization, whenever malignancy is found, without necessity to reaspirate the patient; and adequacy of archival material. Diagn. Cytopathol. 1998;19:141–146. © 1998 Wiley-Liss, Inc.     

1148例乳腺肿物细针吸取活检的分析

分析１１４８例乳腺肿物细针吸取活检（ＦＮＡＢ）的细胞学诊断结果，与３４８例经手术获组织病理学诊断的结果比较，ＦＮＡＢ对恶性肿瘤诊断的敏感性为９７．２％，特异性为９６．８％。细胞学诊断为阳性者（Ⅳ，Ⅴ级）手术后证实全部为恶性。文中对细胞学诊断的标准、误导诊断的因素如不典型的细胞学改变、出现异型细胞的良性病变及易于漏诊的乳腺癌等进行了简要讨论。     

Nuclear grading and flow cytometric DNA pattern in fine-needle aspirates of primary breast cancer

Fine-needle aspiration (FNA) biopsy is increasingly used in the diagnosis and biological characterization of breast carcinomas in patients who receive preoperative chemotherapy. In this context, nuclear cytologic grade supplemented by DNA content could play an important role in the morphologic assessment of breast cancer. In this study, DNA ploidy pattern, analyzed by flow cytometry on FNAs from 92 primary breast carcinomas, was related to cytologic nuclear grade. Twenty-seven samples were cytologic grade 1, 33 were grade 2, and 32 were grade 3. Ploidy correlated with cytologic nuclear grade (P = 0.0001). Thirty percent of grade 1, 55% of grade 2, and 84% of grade 3 tumors were DNA aneuploid. For 30 of the 92 FNAs, it was possible to compare nuclear cytologic grade with the corresponding histologic grade using the Scarff, Bloom, and Richardson system. A high concordance (80%) between nuclear grade on FNAs and histologic grade was found. DNA flow cytometry in combination with nuclear cytologic grade might represent additional information for the characterization of breast cancer diagnosed by FNA. Diagn Cytopathol 1996;15:116–120. © 1996 Wiley-Liss, Inc.     

Expression of Ki-67 and p27(Kip1) in fine-needle aspirates from breast carcinoma and benign breast diseases

Cell atypia in breast fine needle aspiration (FNA) can introduce some diagnostic difficulties. Molecules reflecting proliferative cell potential, such as Ki‐67 and p27^Kip1, can help in recognizing the true biological nature of a cell. Thus, the objective of the study was to analyze the difference in Ki‐67 and p27^Kip1 cell immunoexpression in breast FNA specimens between fibroadenomas, fibrocystic changes (FCC) with atypia, and breast carcinoma. Microscopic analyses of cell cytomorphology and Ki‐67 and p27^Kip1 breast cell immunoexpression were done after standard Pappenheim and immunocytochemical staining (labeled streptavidin‐biotin, LSAB) method in autostainer DakoCytomation TechMate?. The study included 50 patients with breast carcinoma, 20 patients with fibroadenoma, and 20 patients with FCC with atypia. High Ki‐67 and low or absent p27^Kip1 were found in most patients with breast carcinoma, while majority of FCC with atypia were characterized by low Ki‐67 and moderate to high p27^Kip1 cell immunoexpression. Majority of fibroadenomas were associated with low Ki‐67 and low to moderate p27^Kip1 cell immunoexpression indicating progressive decrease in cell cycle inhibition, but still not so high proliferative activity as in carcinoma. However, although statistically significant difference for Ki‐67 and p27^Kip1 was found between breast lesions in our study, the large ranges observed for each marker make them essentially useless for better cytological diagnosis in a single case. Regarding their opposite role in cell cycle, inverse correlation of Ki‐67 and p27^Kip1 was noticed. Poorly differentiated carcinoma cells had mostly high Ki‐67 andlow p27^Kip1 cell immunoexpression. Diagn. Cytopathol. 2011;39:333–340. © 2010 Wiley‐Liss, Inc.     

Cytologic diagnosis of papillary carcinoma of the breast in needle aspirates

Eleven cases of rare papillary carcinoma of the breast diagnosed by fine-needle aspiration cytology (FNAC) are reported. Five of these were pure papillary carcinomas and six were mixed papillary and ductal, lobular, or mucinous carcinomas. In each case, cytological material was collected by washing the needle and syringe contents into 30% alcohol in saline, and the Gelman cytosieve method was used for the cytological preparations. In this article, the cytological features of these tumors are described, including the presence of single papillae and papillary clusters, tall columnar cells, diathesis of blood with hemosiderin-laden macrophages, naked nuclei, and high cell recovery.     

The cancer genetics and pathology of male breast cancer

Male breast cancer (MBC) is an uncommon and poorly understood disease. Recent molecular studies have shown important differences from female breast cancer which are likely to influence treatment strategies from the current female‐based management towards a more tailored approach. Significantly more MBCs than female breast cancers arise with an underlying germline cancer predisposition, and display a vastly different penetrance compared with females. Furthermore, the genophenotypical association of basal‐like cancer with BRCA1 present in female breast cancer is not observed in male breast cancer. Differences in somatic changes between male and female breast cancer have also been reported, with particular enrichment of PIK3CA mutations and a paucity of TP53 mutations. In general, chromosomal‐based changes, in particular regions of gains, are seen more frequently in male than female breast cancer and methylation is seen less frequently. Clinically, several molecular subtypes with prognostic relevance have been described, including chromosomal complex high and methylation high groups, and subgroups with profiling signatures pertaining to epithelial mesenchymal transition and hormonal therapy insensitivity. As with female breast cancer, attention to male specific multicentre trials based on the individual characteristics are needed, together with establishment of reliable preclinical models to understand more clearly the pathogenesis of male breast cancer and improve the general poor outcome of this disease.     

Quantitative assessment of HER2 gene amplification of breast cancer using droplet digital PCR

We previously reported the usefulness of droplet digital polymerase chain reaction (ddPCR) for the assessment of Human epithelial growth factor receptor 2 (HER2) gene amplification in breast cancer using formalin-fixed and paraffin-embedded sections. In our previous study, we combined HER2/CEP17 ratio (HER2 gene signals to chromosome 17 signals) with ddPCR and tumor content ratio (TCR) of each sample and determined the HER2 status by adopting a two-dimensional chart. This “ddPCR-TCR method” showed a high concordance with conventional HER2 status. In this study, we updated our method to assess the HER2 status of breast cancer in a more quantitative manner. We combined obtained data of the ddPCR ratio [R_x] and TCR [x]; we calculated “(R_x − 1)/x + 1” for 41 samples with primary breast cancer and named the value led by this formula as “eHER2 (estimated HER2/CEP17 ratio of a tumor cell)”. eHER2 was equivalent to conventional in situ hybridization (ISH) HER2/CEP17 ratio in most cases. eHER2 and ISH ratio showed a strong correlation (Spearman rank correlation ρ = 0.70, p < 0.0001). The obtained results indicated that eHER2 is a potential tool for HER2 status diagnosis in breast cancer.     

Molecular alterations differentiate microinvasive carcinoma from ductal carcinoma in situ and invasive breast carcinoma: retrospective analysis of a large single-center series

Microinvasive carcinoma (MIC) of the breast is a rare lesion. The clinicopathologic features and biologic behavior of MIC are unclear. Whether MIC is a distinct entity or an interim stage in the progression from ductal carcinoma in situ (DCIS) to invasive breast carcinoma (IBC) remains to be determined. A retrospective review of clinicopathologic features and analysis of the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), and Ki-67 in patients with MIC (90 cases), DCIS (268 cases) and IBC (1504 cases) was performed. Most MICs (93.3%) exhibited an intermediate to high nuclear grade, and this proportion was larger than that of DCIS (62.7%, P < 0.001) or IBC (85.4%, P = 0.036). The incidence of sentinel lymph node metastasis in MIC (12.5%) was higher than that of DCIS (1.6%, P < 0.001), but much lower than that of IBC (39.7%, P < 0.001). MICs had higher expression of HER-2 and lower expression of ER and PR compared to DCIS and IBC; and MIC was more likely to present with a HER-2+ subtype. Furthermore, DCIS exhibited greater HER-2 overexpression or gene amplification (P < 0.001) levels and lower proliferation index of Ki-67 (P < 0.001) compared to IBC. Our results suggest that the clinicopathologic and molecular phenotype of MIC are different from DCIS and IBC. Thus, MIC may be a distinct entity rather than an interim stage in the progression from DCIS to IBC. The prognosis of MIC and the biologic behavior of this uncommon subset need to be further explored.     

Diagnostic significance of signet ring cells in fine-needle aspirates of the breast

Fine-needle aspiration (FNA) is a reliable and cost-effective procedure in the evaluation and management of breast lesions. One diagnostic dilemma that may sometimes arise is the finding of signet ring cells. The isolated finding of such cells in aspirate smears may be particularly problematic in cases of low cellularity or those with otherwise benign features. Although it is generally held that such cells are almost exclusively associated with carcinoma (particularly the lobular subtype), their significance in FNA smears has never been systematically evaluated. To establish their diagnostic utility, we evaluated aspirate smears from 150 cases of histologically proven benign (77) and malignant (73) breast lesions for the presence of signet ring cells, defined as those containing a prominent intracytoplasmic vacuole with nuclear displacement. Signet ring cells were identified in 71% of malignant cases (75% of ductal carcinomas and 71% of lobular carcinomas), mostly as single cells or within small, loosely cohesive tissue fragments. Such cells were also present in 6% of histologically proven benign lesions, most commonly within large tissue fragments. Many of these cells were proven to be vacuolated myoepithelial cells, based on histologic correlation and immunostaining results using anti-muscle-specific actin. On the basis of these findings, we conclude that (1) the presence of signet ring cells within small loose tissue fragments or as single cells in FNA smears should prompt close clinical follow-up (including repeat FNA and perhaps surgical biopsy), regardless of smear cellularity, (2) the presence of signet ring cells in cases of adenocarcinoma does not predict a particular tumor subtype, and (3) rare benign breast lesions may contain signet ring cells, particularly within large tissue fragments, and do not, in isolation, warrant surgical biopsy to exclude malignancy. Diagn. Cytopathol. 16:117–121, 1997. © 1997 Wiley-Liss, Inc.     

The effects of chemotherapy on breast cancer tissue in locally advanced breast cancer

It is well known that chemotherapy induces cytomorphological changes in neoplastic and non-neoplastic tissue. Thirty-one stage III breast-carcinoma patients, treated with both pre-operative chemotherapy and mastectomy, were evaluated to define the effects of systemic chemotherapeutic agents in tumours, non-neoplastic breast tissue, and lymph nodes. Histological changes were compared with those observed in patients who had been treated by surgery alone. Cytoplasmic vacuolization was the most striking change in the tumour cells (59%). Chemotherapy was especially effective in the terminal duct lobular unit in non-neoplastic breast tissue. Lobular atrophy was observed in 20 (65%) cases, and lobular cellular atypia was seen in 16 (52%). The rate of ductal cellular atypia (42%) was not different from the control group. The most important changes seen in the non-neoplastic stromal component were fibrosis and hyalinization. These were found in 31 out of 727 evaluated lymph nodes. In serial sections, metastatic deposits were seen in or around these fibrotic or hyalinized areas. Chemotherapy is widely used in the treatment of early and locally advanced breast carcinomas. Familaritiy with chemotherapy induced changes in breast tissue and lymph nodes have considerable importance in the accurate interpretation of these specimens.     

Association of C-MYC amplification with progression from the in situ to the invasive stage in C-MYC-amplified breast carcinomas

Human carcinoma in situ of the breast already demonstrates genomic changes found in invasive lesions. However, no specific genetic alterations have previously been identified that are associated with progression from the in situ to the invasive stage. By comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) analysis of an invasive breast carcinoma with a large associated in situ component, high-level amplification of C-MYC was found in the invasive component only. To determine the frequency of this correlation in a panel of 188 invasive breast carcinomas, 18 additional cases with C-MYC amplification were identified. Nine of these cases had a detectable adjacent in situ component. FISH analysis demonstrated increased (>5) C-MYC signals per nucleus in seven invasive components and increased (>4) C-MYC/centromere 8 signal ratios in five of these. None of the associated in situ components demonstrated these increases. The minimal amplified region was defined at 8q24.13-8qter. C-MYC amplification was correlated with overexpression of C-MYC and two of its target genes, TERT and FBL. Thus, C-MYC amplification is the first identified genetic alteration that is associated with progression from the in situ to the invasive stage of breast carcinoma.     

乳腺癌Her2基因荧光原位杂交及其临床病理关系

目的探讨Her2基因过表达的比例及其与临床病理之间的联系。方法收集乳腺浸润型导管癌标本50例,总结其临床及病理资料,应用石蜡包埋组织切片,以EnVision两步法进行免疫组织化学染色标记ER、PR及HER2,以金菩嘉DNA探针试剂盒行荧光原位杂交检测HER2基因。结果患者平均年龄55.5岁,病理组织分级Ⅰ级11例,Ⅱ级30例,Ⅲ级9例。术后分期Ⅰ期13例,ⅡA期15例,ⅡB期13例,ⅢA期6例,ⅢB期2例,ⅢC期1例。淋巴结中位转移率6.91%。ER阳性33例,PR阳性32例,IHC法HER2阳性40例,FISH阳性33例,阴性17例。FISH法检测HER2基因与HER2蛋白过表达之间相关(P0.05),其与病理分级、术后分期、淋巴结转移率及ER、PR的表达无相关(P0.05),3例脉管内瘤栓及3例原发瘤数大于1者,FISH均为阳性。结论FISH检测HER2基因扩增和免疫组化检测HER2蛋白一致性较好,HER2基因与脉管内瘤栓及多个原发瘤有一定关系,但其作为间接预测预后的指标有待于进一步研究。     

Altered glutamine metabolism in breast cancer; subtype dependencies and alternative adaptations

Cancer cells must alter their metabolism in order to satisfy the demands of necessary energy and cellular building blocks. These metabolic alterations are mediated by many oncogenic changes that affect cellular signalling pathways, which result in sustained cell growth and proliferation. Recently, metabolomics has received great attention in the field of cancer research, and as the essential metabolic pathways that drive tumour growth and progression are determined the possibilities of new targets for therapeutic intervention are opened. More specifically, as breast cancer is a heterogeneous disease, there is growing evidence that differences in metabolic changes exist between molecular subtypes. In this review, the most recent findings in breast cancer cell metabolism are discussed, with particular emphasis on glutamine and its transporters, which is considered one of the key amino acids fuelling cancer growth. Furthermore, the metabolic differences between the molecular subtypes of breast cancer are examined, highlighting the clinical utility for breast cancer diagnosis and treatment.     

Over‐expression of metallothionein predicts chemoresistance in breast cancer

Metallothionein (MT) plays a role in fundamental cellular processes such as proliferation, apoptosis and differentiation. We examined MT expression in women with invasive breast ductal carcinoma who underwent mastectomy/lumpectomy without neo‐adjuvant treatment. We showed that MT was over‐expressed in 87.9% of breast cancer tissues examined, with the mean percentage of positive cells at 30%. There were two patterns of MT expression: predominantly cytoplasmic in 75.9% and nuclear in 24.1% of MT‐positive cases. Higher MT scores were associated with poorer histological grade (p = 0.009) but were independent of age, tumour size and oestrogen receptor status. For patients who were treated with adjuvant chemotherapy (cyclophosphamide/methotrexate/5 fluorouracil‐ or doxorubicin‐based regimes), those with high MT expression had a significantly lower recurrence‐free survival (p = 0.048), suggesting a role of MT in predicting disease recurrence. Down‐regulation of MT in MCF‐7 cells by silencing the MT‐2A gene (the most abundantly expressed of the 10 known functional MT isoforms) increased chemosensitivity of the cells to doxorubicin. To examine the mechanisms underlying these clinical data, we used siRNAs to decrease MT‐2A mRNA expression and protein expression. In MT down‐regulated cells challenged with the IC₅₀ concentration of doxorubicin, we observed a significant reduction in cell viability. Cell cycle analysis also revealed a corresponding increase in apoptosis in the MT down‐regulated cells following doxorubicin exposure, showing that down‐regulation of MT increased susceptibility to doxorubicin cytotoxicity. The data suggest that MT could be a potential marker of chemoresistance and a molecular therapeutic target. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Immunocytochemical expression of E-cadherin on fine-needle aspirates from breast carcinomas correlate with the cell dissociation pattern seen on smears.

Altered E-cadherin expression has been suggested to be of prognostic significance in breast cancers and to correlate with tumor subtype and grade. A dysfunctional, intercellular adhesion system may be responsible for the tumor cell dissociation pattern seen on fine-needle aspirate cytology (FNAC). The aim of our study was to determine E-cadherin expression on direct FNAC smears from breast carcinomas and compare the results with the dyscohesion grade of the tumor cells and the cytological grading. The material consisted of FNAs from 56 breast carcinomas. The degree of cellular cohesion was estimated semiquantitatively. Full expression of E-cadherin was defined as a complete and strong membrane staining of virtually all tumor cells. All nonductal as well as 85% of the invasive ductal carcinomas revealed reduced expression or negativity for E-cadherin. In all, 25% of carcinomas with dyscohesion Grade 1 (mainly in groups) revealed full expression of E-cadherin, in contrast to 12.5% of tumors with dyscohesion Grade 3 (mainly dispersed cells). Nuclear positivity was seen in 21% of the tumors (12 cases) and seven of these were G3 ductal carcinomas. In conclusion, E-cadherin expression correlated with the cell dissociation pattern seen on direct smears from FNAC of breast carcinomas, but is only one of several markers that modulate this pattern. High-grade carcinomas rarely revealed full expression and had a high incidence of aberrant nuclear localization of E-cadherin.     

Ploidy analysis by in situ hybridization of interphase cell nuclei in fine-needle aspirates from breast carcinomas: Correlation with cytologic grading

Fine-needle aspirates from 54 breast cancer patients were investigated for numeric aberrations in chromosomes 6, 7, 12, and 17 by in situ hybridization (ISH) of interphase cell nuclei. Ploidy findings were compared with cytologic grading of tumors. Aneuploidy was found in 73% of cases. Chromosomes 6 and 7 showed numeric abnormalities in 63% and 62% of cases, respectively, whereas chromosome 17 retained a disome pattern in 2/3 of the tumors. Thirteen cancers (28% of 47 with four analyzed probes) had a normal signal number in all four chromosomes. In 17 (36%), all four had signal gain. Another 17 showed a mixed disome/aneusome pattern. They presented a continuum of increasing numeric abnormalities, 82% disomy for chromosome 17, and 13 of them were grade 2, indicating intermediate biologic properties. Correlation between grading and ploidy was good, with 10 of 11 grade 1 carcinomas showing diploidy, whereas 33 of 36 grade 2 and 3 tumors had numeric aberrations. Diagn. Cytopathol. 1997; 17:267–271. © 1997 Wiley-Liss, Inc.     

The transition from hyperplasia to invasive carcinoma of the breast

The multistep model of carcinogenesis in the breast suggests a transition from normal epithelium to invasive carcinoma via non-atypical and atypical hyperplasia and in situ carcinoma. Within the breast, these proliferations are heterogeneous in their cytological and architectural characteristics. This review considers the evidence supporting a precursor role for these preinvasive lesions. Copyright © 1999 John Wiley & Sons, Ltd.