Sathiyamoorthy Selvarajan

Phyllodes tumors of the breast: the role of pathologic parameters.

We aimed to establish whether morphologic parameters were prognostically important in a large series of breast phyllodes tumors in Asian women. Of 335 phyllodes tumors diagnosed at the Department of Pathology, Singapore General Hospital, Singapore, between January 1992 and December 2002, 250 (74.6%) were benign, 54 (16.1%) borderline, and 31 (9.3%) malignant, based on histologic review of archival slides. Of the women, 43 (12.8%) experienced recurrences during the follow-up period. Recurrent disease was correlated with grade or classification (P = .028), stromal atypia (P = .016), stromal hypercellularity (P = .046), and permeative microscopic borders (P = .021). Multivariate analysis revealed that independent predictors of recurrence were pseudoangiomatous stromal hyperplasia (PASH) and margin status, whereby the presence of PASH and complete or negative margins reduced recurrence hazards by 51.3% and 51.7% respectively. The 7 women who died of disease during follow-up had malignant phyllodes tumor at the outset and experienced recurrences, and death was preceded by distant metastases.

Phyllodes Tumors of the Breast

We aimed to establish whether morphologic parameters were prognostically important in a large series of breast phyllodes tumors in Asian women. Of 335 phyllodes tumors diagnosed at the Department of Pathology, Singapore General Hospital, Singapore, between January 1992 and December 2002, 250 (74.6%) were benign, 54 (16.1%) borderline, and 31 (9.3%) malignant, based on histologic review of archival slides. Of the women, 43 (12.8%) experienced recurrences during the follow-up period. Recurrent disease was correlated with grade or classification ( P = .028), stromal atypia ( P = .016), stromal hypercellularity ( P = .046), and permeative microscopic borders ( P = .021). Multivariate analysis revealed that independent predictors of recurrence were pseudoangiomatous stromal hyperplasia (PASH) and margin status, whereby the presence of PASH and complete or negative margins reduced recurrence hazards by 51.3% and 51.7%, respectively. The 7 women who died of disease during follow-up had malignant phyllodes tumor at the outset and experienced recurrences, and death was preceded by distant metastases.

Immunohistochemical detection of Ki67 in breast cancer correlates with transcriptional regulation of genes related to apoptosis and cell death

Ki67 is a nuclear protein that is tightly linked to the cell cycle. It is a marker of cell proliferation and has been used to stratify good and poor prognostic categories in invasive breast cancer. Its correlation with gene expression patterns has not been fully elucidated. In this study, Ki67 immunohistochemistry using the MIB-1 antibody was performed on sections cut from 21 formalin-fixed, paraffin-embedded invasive breast cancers. Scoring was determined as nil (no immunostaining), low (10% or less immunopositivity) or high (>10% immunoreactive cells) respectively. The relationship of Ki67 immunohistochemical detection with clinicopathologic parameters was evaluated. Using Affymetrix U133A GeneChips, expression profiles for these tumors were generated and correlated with Ki67 immunohistochemical findings. Analysis of variance was used to define genes that were differentially regulated between the groups. Real-time polymerase chain reaction (PCR) was used to confirm the presence of a downregulated gene. Our results showed high, low and nil Ki67 immunostaining in nine (43%), six (28.5%) and six (28.5%) invasive breast cancers respectively, with increased Ki67 protein expression correlating with high histologic grade (P=0.02), mitotic score (P=0.001) and estrogen receptor immunonegativity (P=0.002). Expression profiling trends of the Ki67 gene mirrored the observed proportions of immunostained cells when the Ki67 immunoscore was >10%. Genes related to apoptosis and cell death (bcl2, MAP2K4, TNF10) were noted to be downregulated in tumors that disclosed >40% Ki67 immunostaining (P<0.001). Downregulation of the bcl2 gene was confirmed at the RNA level by real-time RT-PCR. Differential regulation of these genes, especially bcl2, may contribute to the biological nature of clinically more aggressive and highly proliferative breast cancers.

p53 and c-kit (CD117) protein expression as prognostic indicators in breast phyllodes tumors: a tissue microarray study

Breast phyllodes tumors are fibroepithelial neoplasms whose clinical behavior is difficult to predict on histology. There is relatively scant data on the role of biological markers. In this study, we determined if p53 and CD117 (c-kit) protein expression was predictive of behavior in a series of 335 phyllodes tumors diagnosed at the Singapore General Hospital, using immunohistochemistry on tissue microarrays. Representative areas from 250 (75%) benign, 54 (16%) borderline and 31 (9%) malignant phyllodes tumors were selected for construction of tissue microarrays using the 2 mm punch. Immunohistochemistry for p53 and CD117 was carried out using the streptavidin–biotin method. Staining proportion and intensity of both epithelial and stromal elements were analyzed. p53 immunostaining was observed in the epithelium of 28 (10%) of 278 microarrays; myoepithelium of 53 (21%) of 251 microarrays; and stromal cells in 105 (36%) of 289 microarrays. CD117 immunohistochemical reactivity was noted in epithelial and stromal components of 175 (of 267, 66%) and 17 (of 273, 6%) microarrays, respectively. Stromal p53 and CD117 protein expression was associated with tumor grade (P<0.05). Of 43 (13%) women who suffered recurrences during the follow-up period, CD117 stromal staining predicted recurrent disease (P<0.05), but p53 was not correlative. We conclude that tissue microarrays are a convenient method for evaluating immunostaining results of large numbers of phyllodes tumors. Although positive p53 stromal immunohistochemical detection may corroborate histologic malignancy, it is CD117 protein expression in phyllodes tumor stromal cells that may be of potential utility in predicting recurrent disease.

Effect of Fixation Period on HER2/neu Gene Amplification Detected by Fluorescence In Situ Hybridization in Invasive Breast Carcinoma

This study investigated if formalin fixation duration affects HER2/neu gene amplification detection by fluorescence in situ hybridization (FISH) in breast cancer. Tumor tissues from 35 cases were divided into three groups and subjected to two formalin fixation protocols per group (12 hr, 27 hr in the first; 2 hr, 17.5 hr in the second; 28.5 hr, 541 hr in the third) before FISH analysis. There was no significant difference in FISH signal detection between the two different fixation protocols in the first two groups. In the third, no signal was detected in 4/6 cases fixed for an extended duration.

Parafibromin expression in breast cancer: a novel marker for prognostication?

Background: Parafibromin is a novel protein product of HRPT2, a recently identified tumour suppressor gene. Mutations of the HRPT2 gene are common in parathyroid carcinomas, and these exhibit reduced protein expression. Parafibromin expression in breast cancer has not been previously studied. Aims: To determine the distribution of parafibromin in breast cancer tissues, and correlate its expression with conventional pathological parameters. Methods: Tissue microarrays were constructed from archival paraffin embedded breast cancer samples. Sections cut from tissue microarray blocks were subjected to immunohistochemistry. Immunopositivity for parafibromin and intensity-percentage scores were derived by blinded evaluation. Findings were correlated with clinicopathological parameters. Results: 163 breast cancers were assessed. Larger tumours were less likely to express parafibromin than smaller ones, with the association approaching statistical significance (p = 0.05). Staining intensity correlated inversely with tumour size (p = 0.016) and pathological stage (p = 0.008); as did parafibromin intensity-percentage score with pathological stage (p = 0.03), lymphovascular invasion (p = 0.03) and cerbB2 intensity-percentage score (p = 0.04). Conclusion: Parafibromin in breast cancer, as in parathyroid tumours, appears to have tumour suppressor functions, with loss of protein expression associated with adverse pathological parameters. These findings may indicate a potential role of parafibromin as a prognostic marker in breast cancer.

c-erbB-2 (HER-2/neu) immunohistochemistry in invasive breast cancer: is there concordance between standard sections and tissue microarrays?

Aims: Immunohistochemical detection of the 185‐kDa transmembrane glycoprotein product of the proto‐oncogene c‐erbB‐2 (also known as HER‐2/neu), located on chromosome 17q21, is a well established method of evaluation in invasive breast cancer. This investigation is currently performed on standard sections cut from the tumour containing paraffin block. It is uncertain if concordant results can be obtained on tissue microarray (TMA) sections, a high throughput technique that is particularly advantageous in research and validation protocols. Our aim in this study was to compare the results of c‐erbB‐2 immunoexpression in standard sections of invasive breast cancers with those of TMAs. Methods: Standard sections and TMAs constructed from archival paraffin‐embedded breast cancers of 184 patients who had surgery in Singapore General Hospital during the period 1998–2002 were subjected to immunohistochemistry using the commercial antibody (A0485, Dako). c‐erbB‐2 over‐expression was evaluated according to cytoplasmic membrane staining intensity, which was defined as 2+ and 3+ staining. Results: Over‐expression of c‐erbB‐2 protein was found in 21.2% (39/184) and 18.6% (34/183) of cases on standard sections and TMAs, respectively. There was substantial agreement between these two types of sections (k = 0.724) when positive and negative staining was considered. Conclusion: Immunohistochemistry on TMAs for c‐erbB‐2 expression in breast cancer is a reliable alternative to that performed on routine standard sections, as it is both cost effective and time efficient, especially in a research setting.

Over-expression of c-erbB-2 correlates with nuclear morphometry and prognosis in breast carcinoma in Asian women

Aim: We aimed to investigate the immunohistochemical expression of c‐erbB‐2 in invasive breast carcinoma in Asian women and its correlations with clinicopathological parameters and nuclear morphometry. Patients were followed up for disease relapse and overall survival, and the data were reviewed in conjunction with c‐erbB‐2 over‐expression. Methods: Paraffin sections from 321 invasive breast cancers were immunohistochemically stained with anti‐human c‐erbB‐2 antibody using the streptavidin–biotin technique. Results: c‐erbB‐2 was over‐expressed in 110 (34.3%) cases, with an inverse correlation with oestrogen receptor (ER) and progesterone receptor (PR) status (p = 0.0001) and a positive correlation with histological grade (p = 0.017). Nuclear morphometry in 96 cases revealed rounder nuclei in c‐erbB‐2 negative tumours (p = 0.0322) when compared with c‐erbB‐2 positive tumours. Among c‐erbB‐2 positive cases, malignant cells of histological grade 3 tumours revealed larger nuclear area and perimeter than grade 1 and 2 cases (p = 0.0095, p = 0.03, respectively) while increasing tumour size correlated with greater nuclear perimeter (p = 0.046). c‐erbB‐2 positivity was significantly associated with poor survival when all patients were included in the analysis (p = 0.0166) and for subsets of node positive, histological grade 1 and 2, and ER positive tumours, and in women aged over 50 years (p = 0.0047, p = 0.0367, p = 0.0092, p = 0.0096, respectively). Conclusions: c‐erbB‐2 was independently prognostic when histological grade, nodal and ER status were considered. Our results show that c‐erbB‐2 over‐expression correlates with poor histological grade and negative ER/PR status, and predicts poor overall survival in Asian women with breast cancer.

Sarcoma of the breast: an update on a rare entity

Breast sarcoma is a rare condition. It consists of a heterogeneous group of non-epithelial tumours arising from the mesenchymal tissue of the breast. It has a distinctly different natural history, treatment response and prognosis as compared with carcinoma of the breast. A different diagnostic approach and treatment strategy have to be defined for this group of tumours. Due to its rarity, the current understanding on breast sarcoma is limited and is mostly based on small retrospective case series or case reports. Hence, the management generally follows the algorithms derived from randomised control trials of soft tissue sarcomas in the extremities and chest wall. Through this review, we discuss the results of major retrospective studies on breast sarcomas including data on epidemiology, aetiology, diagnostic approach, treatment strategies and outcomes of this challenging and potentially aggressive condition.

SETD2 histone modifier loss in aggressive GI stromal tumours

Background GI stromal tumours (GISTs) are clinically heterogenous exhibiting varying degrees of disease aggressiveness in individual patients. Objectives We sought to identify genetic alterations associated with high-risk GIST, explore their molecular consequences, and test their utility as prognostic markers. Designs Exome sequencing of 18 GISTs was performed (9 patients with high-risk/metastatic and 5 patients with low/intermediate-risk), corresponding to 11 primary and 7 metastatic tumours. Candidate alterations were validated by prevalence screening in an independent patient cohort (n=120). Functional consequences of SETD2 mutations were investigated in primary tissues and cell lines. Transcriptomic profiles for 8 GISTs (4 SETD2 mutated, 4 SETD2 wild type) and DNA methylation profiles for 22 GISTs (10 SETD2 mutated, 12 SETD2 wild type) were analysed. Statistical associations between molecular, clinicopathological factors, and relapse-free survival were determined. Results High-risk GISTs harboured increased numbers of somatic mutations compared with low-risk GISTs (25.2 mutations/high-risk cases vs 6.8 mutations/low-risk cases; two sample t test p=3.1×10−5). Somatic alterations in the SETD2 histone modifier gene occurred in 3 out of 9 high-risk/metastatic cases but no low/intermediate-risk cases. Prevalence screening identified additional SETD2 mutations in 7 out of 80 high-risk/metastatic cases but no low/intermediate-risk cases (n=29). Combined, the frequency of SETD2 mutations was 11.2% (10/89) and 0% (0/34) in high-risk and low-risk GISTs respectively. SETD2 mutant GISTs exhibited decreased H3K36me3 expression while SETD2 silencing promoted DNA damage in GIST-T1 cells. In gastric GISTs, SETD2 mutations were associated with overexpression of HOXC cluster genes and a DNA methylation signature of hypomethylated heterochromatin. Gastric GISTs with SETD2 mutations, or GISTs with hypomethylated heterochromatin, showed significantly shorter relapse-free survival on univariate analysis (log rank p=4.1×10−5). Conclusions Our data suggest that SETD2 is a novel GIST tumour suppressor gene associated with disease progression. Assessing SETD2 genetic status and SETD2-associated epigenomic phenotypes may guide risk stratification and provide insights into mechanisms of GIST clinical aggressiveness.