Preetha Madhukumar

Genomic landscapes of breast fibroepithelial tumors

Breast fibroepithelial tumors comprise a heterogeneous spectrum of pathological entities, from benign fibroadenomas to malignant phyllodes tumors. Although MED12 mutations have been frequently found in fibroadenomas and phyllodes tumors, the landscapes of genetic alterations across the fibroepithelial tumor spectrum remain unclear. Here, by performing exome sequencing of 22 phyllodes tumors followed by targeted sequencing of 100 breast fibroepithelial tumors, we observed three distinct somatic mutation patterns. First, we frequently observed MED12 and RARA mutations in both fibroadenomas and phyllodes tumors, emphasizing the importance of these mutations in fibroepithelial tumorigenesis. Second, phyllodes tumors exhibited mutations in FLNA, SETD2 and KMT2D, suggesting a role in driving phyllodes tumor development. Third, borderline and malignant phyllodes tumors harbored additional mutations in cancer-associated genes. RARA mutations exhibited clustering in the portion of the gene encoding the ligand-binding domain, functionally suppressed RARA-mediated transcriptional activation and enhanced RARA interactions with transcriptional co-repressors. This study provides insights into the molecular pathogenesis of breast fibroepithelial tumors, with potential clinical implications.

Whole-exome sequencing of breast cancer, malignant peripheral nerve sheath tumor and neurofibroma from a patient with neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) is a genetic disorder characterized by the development of multiple neurofibromas, cafe‐au‐lait spots, and Lisch nodules. Individuals with NF1 are at increased risk of developing various tumors, such as malignant peripheral nerve sheath tumor (MPNST), pheochromocytoma, leukemia, glioma, rhabdomyosarcoma, and breast cancer. Here, we describe the exome sequencing of breast cancer, MPNST, and neurofibroma from a patient with NF1. We identified a germline mutation in the NF1 gene which resulted in conversion of leucine to proline at amino acid position 847. In addition, we showed independent somatic NF1 mutations in all the three tumors (frameshift insertion in breast cancer (p.A985fs), missense mutation in MPNST (p.G23R), and inframe deletion in dermal neurofibroma (p.L1876del‐Inf)), indicating that a second hit in NF1 resulting in the loss of function could be important for tumor formation. Each tumor had a distinct genomic profile with mutually exclusive mutations in different genes. Copy number analysis revealed multiple copy number alterations in the breast cancer and the MPNST, but not the benign neurofibroma. Germline loss of chromosome 6q22.33, which harbors two potential tumor suppressor genes, PTPRK and LAMA2, was also identified; this may increase tumor predisposition further. In the background of NF1 syndrome, although second‐hit NF1 mutation is critical in tumorigenesis, different additional mutations are required to drive the formation of different tumors.

Sentinel lymph nodes with isolated tumour cells and micrometastases in breast cancer: clinical relevance and prognostic significance

Aim We performed a retrospective review to determine the prognostic significance of isolated tumour cells (ITCs) and micrometastases to the sentinel lymph nodes of patients with breast cancer. Methods A total of 1044 patients with a diagnosis of invasive carcinoma of the breast who underwent surgical treatment including the sentinel lymph node biopsy procedure from July 2004 to October 2009 were included in the study. Results In 710 (68%) patients, no metastasis was seen to the sentinel lymph nodes. ITCs were detected in 22 (2.1%) patients, micrometastasis in 52 (5.0%) and macrometastases in 260 (24.9%). With a median follow-up of 28.8 months, disease recurrence was seen in 38 (3.6%) patients and 15 (1.5%) patients died of disease. No disease recurrence or deaths were recorded in women with ITCs in sentinel lymph nodes. In the micrometastasis group, 2 patients suffered disease recurrence and both died of disease. Conclusions We conclude that ITCs in the sentinel lymph nodes did not adversely impact disease free and overall survivals. Although only 2 recurrences with subsequent death occurred in the micrometastasis group, it may suggest a propensity for presence of micrometastases to augur a worse outcome, and justifies continued segregation of ITCs from micrometastasis.

False negative rate for intraoperative sentinel lymph node frozen section in patients with breast cancer: a retrospective analysis of patients in a single Asian institution

Background and objective Intraoperative frozen section of the sentinel lymph node (SLN) in clinically node negative breast cancer patients detects metastatic disease and enables axillary lymph node dissection to be performed in the same operative setting. Internationally, the false negative rate (FNR) for SLN biopsy ranges from 5.5% to 43%. The size of SLN metastasis has been identified as a key factor affecting FNR. We review our institutional experience on the accuracy of intraoperative SLN biopsy. Methods Data were collected retrospectively from patients undergoing SLN biopsy performed at Singapore General Hospital. The SLN was identified using blue dye, radioisotope or both. Frozen section was performed intraoperatively. When SLN was positive for metastasis on frozen section, completion axillary clearance was performed. False negative cases were defined as patients in whom a negative frozen section result was obtained, whose final permanent paraffin section was positive. We determined the FNR of SLN frozen section and evaluated the factors associated with it. Results A total of 2202 SLN biopsies were performed between January 2005 and June 2012. There were 89 false negative cases, of which there were 23 (25.8%) cases of isolated tumour cells (ITCs), 49 (55.1%) cases of micrometastasis, and 17 (19.1%) cases of macrometastasis. The overall FNR was 13.5%. FNR was 79.3% in ITCs, 59.8% in micrometastasis, and 3.1% in macrometastatic disease. Non-ductal histological subtype, absence of lymphovascular invasion and the size of SLN metastasis were identified as significant independent factors associated with a higher FNR. Conclusions FNRin our institution is acceptable when compared to other large centres. Failure to detect metastasis in frozen section in more than half of our patients was due to ITCs and micrometastasis.

Predicting the likelihood of additional lymph node metastasis in sentinel lymph node positive breast cancer: validation of the Memorial Sloan-Kettering Cancer Centre (MSKCC) nomogram

Aim To identify important clinicopathological parameters that are most helpful in predicting additional non-sentinel lymph node (SLN) metastasis among patients with a positive SLN biopsy in the Singapore breast cancer population. Methods A total of 1409 patients who underwent SLN biopsy were reviewed over a 5 year period from July 2004 to October 2009. A Singapore General Hospital (SGH) nomogram was developed from predictors in the Memorial Sloan-Kettering Cancer Centre (MSKCC) nomogram using 266 patients with primary invasive breast cancer and a positive SLN biopsy who subsequently had an axillary lymph node dissection. The SGH nomogram was calibrated using bootstrapped data, while the MSKCC nomogram was calibrated using SGH data. The performance of these two nomograms was compared with the calculation of the area under the receiver–operator characteristics curve and adequacy indices. Results The MSKCC nomogram achieved an area under the curve (AUC) of 0.716 (range 0.653–0.779) in our study population, while the SGH nomogram, which used only three pathological parameters, lymphovascular invasion, number of positive and negative SLN biopsies, achieved an AUC of 0.750 (range 0.691–0.808). The SGH nomogram with a higher adequacy index (0.969) provided better estimates compared with the MSKCC nomogram (0.689). Conclusions The use of the MSKCC nomogram was validated in our local patient population. The SGH nomogram showed promise to be equally, if not, more predictive as a model in our own population, while using only three pathological parameters.

Assessment of suitability of the one step nucleic acid amplification (OSNA) assay as an intraoperative procedure for detection of metastasis in sentinel lymph nodes of breast cancer

Aim We aimed to assess the one step nucleic acid amplification (OSNA) assay as an intraoperative method in comparison with frozen sections (FS) for detection of metastasis in sentinel lymph nodes (SLNs) of breast cancer. Method 100 SLNs from patients with breast carcinoma were enrolled within a 3-month period. Alternate 2 mm node slices were subjected to routine FS, and later to permanent histology, and the rest for automated molecular detection of CK19 mRNA using OSNA. FS and OSNA findings were compared with permanent histology results. Difference in turnaround time was also noted. Results With permanent histology as gold standard, OSNA was discrepant in 8 of 98 (3 false negative, 5 false positive) included SLNs whereas FS had 2 false negative cases. FS had higher sensitivity (89%, p=<0.001), specificity (100%, p=0.001) and concordance rate (98%) than OSNA (83%, 94% and 92%, respectively). FS showed almost perfect agreement (κ=0.929) whereas OSNA showed substantial agreement (κ=0.740) when compared with permanent histology. OSNA turnaround time was twice longer (mean of 47.7 min) than FS. Conclusions Automation of SLN assessment using OSNA is a potentially useful intraoperative diagnostic tool with acceptable accuracy. Discordant findings in this study may be due to sampling allocation. Since OSNA is more time-consuming, its practical advantage over routine FS requires further study in view of current technical workflow considerations.

Trends in Post‐Mastectomy Reconstruction in an Asian Population: A 12‐Year Institutional Review

Post‐mastectomy breast reconstruction is an integral component of breast cancer treatment. It is often perceived that women in Asian countries have a lower rate of post‐mastectomy reconstruction than Western populations. This study describes trends in timing and types of breast reconstruction performed in the largest healthcare provider in Singapore, over a period of 12 years. It also reports on the oncological outcomes and surgical safety. A retrospective review of all patients who underwent post‐mastectomy reconstruction from January 2001 to December 2012 at the National Cancer Centre Singapore and Singapore General Hospital was performed. Six hundred and twenty post‐mastectomy reconstructions were performed in 579 patients. The proportion of reconstructions increased from 4% in 2001 to 18% in 2012. Younger patients (<50 years old) and those with early stage cancer were more likely to undergo reconstruction. Immediate breast reconstruction was favored by more than 90% of patients. Postoperatively, 9% developed acute surgical complications that were treated surgically; 6% had additional surgery for late complications. Only 4% had delay of adjuvant chemotherapy. At median follow‐up of 63 months (range 3–166), loco‐regional recurrence was 4%, and distant metastases 8%. Post‐mastectomy reconstruction for breast cancer is increasingly performed in our institution. Both younger age and lower stage disease were associated with choice for reconstruction in our study. Low rates of delay to adjuvant therapy were noted, and it may safely be offered to suitable women undergoing mastectomy.

Unexplained Small-Bowel Obstruction in a Patient with Presumptive Achalasia: Need for Early Recognition of Chronic Intestinal Pseudo-obstruction (CIPO)

Chronic intestinal pseudo-obstruction (CIPO) is a rare condition characterized by impaired gastrointestinal propulsion associated with features of intestinal obstruction (IO) in the absence of any mechanical cause [1–5]. Although gut involvement may be isolated or diffuse [1], the clinical picture is dominated by small-bowel involvement [2]. In contrast, esophageal involvement is usually clinically silent despite achalasia-like features evident on barium studies [3] or on esophageal manometry [5–8]. We report a patient who was treated for achalasia 10 years prior to his first presentation for small-bowel IO, and emphasize the need to consider CIPO early in the differential diagnosis of a patient with underlying achalasia presenting with unexplained small-bowel IO. In July 1998, a 38-year-old Chinese male was referred for worsening dysphagia and regurgitation. Barium swallow and gastroscopy revealed a dilated esophagus with smooth tapering at the gastroesophageal junction. A presumptive diagnosis of achalasia was made. He responded well to serial esophageal dilatations between July 1998 and January 2003 and defaulted follow-up. He represented in January 2009 with severe abdominal distension, vomiting, and constipation. In the preceding 6 months, he had experienced intermittent episodes of abdominal bloating, weight loss, and reduced frequency of bowel movements. He did not complain of dysphagia. Clinical and radiological investigations established a diagnosis of small-bowel IO without a mechanical cause. He was treated conservatively. A colonoscopy was normal. His chest X-ray was clear. A laparotomy was performed 2 weeks later for recurrent IO. Intraoperative findings revealed multiple dilated smallbowel loops without any structural lesion. Enterotomy, evacuation of small intestinal bezoar, and appendicectomy were performed. The diagnosis of CIPO was entertained following another three admissions for subacute IO. Failure of conservative management on the third occasion led to a second laparotomy, revealing dilated small-bowel loops. Full-thickness intestinal biopsies were performed. Histology revealed a complete absence of ganglion cells and neural hypoplasia but intact smooth muscles (Fig. 1a, b), supporting a diagnosis of a neuropathic degenerative variant [9] of CIPO. Eight weeks later, a third laparotomy was required, whereby resection of a contained small-bowel perforation, adhesiolysis, and a venting ileostomy were performed. The case patient had no prior history of metabolic, neurological, cardiovascular, or pulmonary disease or history of travel to any Chaga-prone endemic areas. No The authors do not have any conflict of interests.

Evaluation of three polygenic risk score models for the prediction of breast cancer risk in Singapore Chinese

Genome-wide association studies (GWAS) have proven highly successful in identifying single nucleotide polymorphisms (SNPs) associated with breast cancer (BC) risk. The majority of these studies are on European populations, with limited SNP association data in other populations. We genotyped 51 GWAS-identified SNPs in two independent cohorts of Singaporean Chinese. Cohort 1 comprised 1294 BC cases and 885 controls and was used to determine odds ratios (ORs); Cohort 2 had 301 BC cases and 243 controls for deriving polygenic risk scores (PRS). After age-adjustment, 11 SNPs were found to be significantly associated with BC risk. Five SNPs were present in <1% of Cohort 1 and were excluded from further PRS analysis. To assess the cumulative effect of the remaining 46 SNPs on BC risk, we generated three PRS models: Model-1 included 46 SNPs; Model-2 included 11 statistically significant SNPs; and Model-3 included the SNPs in Model-2 but excluded SNPs that were in strong linkage disequilibrium with the others. Across Models-1, -2 and -3, women in the highest PRS quartile had the greatest ORs of 1.894 (95% CI = 1.157–3.100), 2.013 (95% CI = 1.227–3.302) and 1.751 (95% CI = 1.073–2.856) respectively, suggesting a direct correlation between PRS and BC risk. Given the potential of PRS in BC risk stratification, our findings suggest the need to tailor the selection of SNPs to be included in an ethnic-specific PRS model.

Breast carcinoma and phyllodes tumour: a case series

Malignant transformation of the epithelial component of phyllodes tumours (PT) is rare and only reported in literature as sporadic cases of carcinoma associated with PTs. We report the clinicopathological characteristics of in situ and invasive carcinoma coexisting with PT in 10 patients treated in our institution over an 11-year period from 1992 to 2012. Ten patients with coexisting PT and in situ or invasive carcinoma were identified from our records. Six had carcinoma found within the PT. All were female with a median age of 47 (43–72) years. One patient had a history of PT in the same breast while another had a history of PT in the same breast as well as invasive ductal carcinoma in the contralateral breast. The rest did not have any risk factors of breast cancer. Five patients had a preoperative core needle biopsy performed with the report of a fibroepithelial lesion. The rest of the patients had surgery upfront for their breast masses. Two patients who had ER/PR positive invasive carcinoma received adjuvant hormonal therapy. Patients were followed up for a mean of 3.6 years (9 months–10 years) and all patients were alive and recurrence free. PT associated with carcinoma is rare, and we present a series of cases that add to the limited current literature. It is often difficult to detect the presence of the carcinomatous component preoperatively. Hence, close examination of resected PT specimens must be carried out to allow prompt detection of any associated carcinomas, however rare, such that adequate treatment can be given.