Alvin Soon Tiong Lim

Estimates of sperm sex chromosome disomy and diploidy rates in a 47,XXY/46,XY mosaic Klinefelter patient

Abstract A 47,XXY/46,XY male was investigated for the incidence of aneuploidy in sperm sex chromosomes using a three-colour X/Y/18 fluorescence in situ hybridisation (FISH) protocol. A total of 1701 sperm nuclei were analysed. The ratio of X-bearing to Y-bearing sperm did not differ from the expected 1 : 1 ratio although there were more 23,Y sperm than 23,X sperm (844 vs 795). There was a significantly increased proportion of disomy XY and XX sperm compared with normal controls (0.41% vs 0.10%, P < 0.001 and 0.29% vs 0.04%, P < 0.01). However, the incidence of YY sperm was similar to the controls (0.06% vs 0.02%). The diploidy rate was also significantly increased (1.7% vs 0.13%, P < 0.0001), as was disomy 18 (0.71% vs 0.01%) and 25,XXY (0.47% vs 0%). The results support the hypothesis that some 47,XXY cells are able to undergo meiosis and produce mature spermatozoa. Patients with mosaic Klinefelter syndrome with severe oligozoospermia have significantly elevated incidences of disomy XY and XX sperm and may be at a slightly increased risk of producing 47,XXX and 47,XXY offspring. Additionally, they may be at risk of producing offspring with autosomal trisomies. Hence, patients with Klinefelter mosaicism scheduled for intracytoplasmic sperm injection intervention should first undergo FISH analysis of their sperm to determine their risk.

Dasatinib Induces a Response in Malignant Thymoma

TO THE EDITOR: Dasatinib is a novel, oral, multitargeted kinase inhibitor of Bcr-Abl and Src family kinases, as well as ephrin receptor kinases, platelet-derived growth factor receptor, and c-Kit. It was recently shown to be effective in inducing hematologic and cytogenetic responses in imatinib-resistant BCR-ABL-positive leukemias. Preclinical studies have also revealed that dasatinib has activity in head and neck, lung, pancreatic, and prostate cancer cells, raising the possibility that dasatinib may have a wider spectrum for other clinical diseases. We report, in this letter, the first clinical case of a dasatinibresponsive thymoma. The patient was diagnosed with chronic myeloid leukemia in 1994 and developed lymphoid blast crisis in 2003 after interferon-alfa therapy. He was treated with imatinib and conventional chemotherapy and achieved a complete cytogenetic remission (CCR). He was maintained on imatinib and remained in CCR. In October 2005, he developed another lymphoid blast crisis with white cell counts of 17,840/mm, blasts 86%, hemoglobin 5.9 g/dL, and platelet counts of 5,000/mm. Direct sequencing of the Abl kinase revealed the Y253H mutation. A chest x-ray showed a left hilar mass and left pleural effusion. Computer tomography (CT) of the thorax confirmed the presence of the anterior mediastinal mass with a volumetric measurement of 69.3 cm. Cytologic examination of the effusion revealed a predominant lymphocytic yield. Severe thrombocytopenia precluded a biopsy of the mass. He was enrolled onto the local internal review board approved phase II dasatinib clinical trial and started on 140 mg daily in November 2005. Two months after dasatinib, he achieved a complete hematologic and cytogenetic remission. CT of the thorax showed a partial resolution in the size of the mediastinal mass (40.6 cm) but worsening of the pleural effusion, a possible dasatinib-related adverse effect (Fig 1). His thrombocytopenia had resolved and a thoracotomy was performed to drain the pleural fluid and the mass was completely resected. The thymic tumor (Fig 2A) featured lobular, organotypic proliferation of neoplastic epithelial cells, with large vesicular nuclei and prominent nucleoli, admixed with a background population of small lymphocytes. There were occasional perivascular spaces and scattered macrophages. Cystic degeneration accompanied by hemorrhage and siderophages was prominently displayed. Although there were lymphocyte poor areas where the tumor cells appeared to form more confluent sheets, the nuclear features remained those of B2-type. The tumor had infiltrated into but not through the capsule (modified Masaoka stage I). Leukemic infiltrates were not seen. The tumor cells were evaluated for their cell membrane reactivity with epidermal growth factor receptor (EGFR) using the anti-EGFR mouse monoclonal antibody (clone E30; DakoCytomation, Glostrup, Denmark). The guidelines of the College of American Pathologists for HER2/cerb-B2 were used to grade EGFR membrane expression. EGFR cell membrane staining of a score of 1 (faint incomplete) to 2 (moderate but complete) was detected in 20% of the tumor cells (Fig 2B). The

Analysis of the sex chromosome constitution of sperm in men with a 47,XYY mosaic karyotype by fluorescence in situ hybridization

OBJECTIVE To determine the incidence of sex chromosome aneuploidy in the sperm of two men with a 47,XYY/46,XY karyotype. DESIGN Case report. SETTING Infertility clinic in a teaching hospital. PATIENT(S) One patient with near normal semen parameters whose wife had a history of miscarriages and one patient with primary infertility and severe oligoasthenozoospermia. INTERVENTION(S) Cytogenetic analysis of peripheral lymphocytes and three-color X/Y/18 fluorescence in situ hybridization analysis of sperm. MAIN OUTCOME MEASURE(S) Analysis of sex chromosome disomy and diploidy rates in sperm. RESULT(S) Both patients had a 47,XYY/46,XY karyotype. The hyperdiploidy rate of patient 1 was 19% and that of patient 2 was 90%. The incidence of disomy XY was significantly elevated in both patients compared with the controls (0.23% and 1.02%, respectively, versus 0.10%). The incidence of disomy YY (0.44% versus 0.10%) was increased only in patient 2, as was the incidence of disomy 18 (0.49% versus 0.09%) and the rate of diploidy (0.83% versus 0.13%). The rate of 24,XX sperm in both patients was not different from that in the controls. CONCLUSION(S) Patients with a 47,XYY mosaic karyotype may be at risk of producing offspring with a hyperdiploid sex constitution. These patients should have their sperm investigated by fluorescence in situ hybridization to determine their particular risks before they undergo intracytoplasmic sperm injection.

Implications of the Updated 2013 American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations on Human Epidermal Growth Factor Receptor 2 Gene Testing Using Immunohistochemistry and Fluorescence In Situ Hybridization for Breast Cancer

CONTEXT Human epidermal growth factor receptor 2 (HER2/neu) amplification is used as a predictive marker for trastuzumab treatment in breast cancer. Both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) testing algorithms have been based on the 2007 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. In late 2013, the guidelines were updated with new scoring criteria. OBJECTIVE To assess the impact of the revised ASCO/CAP recommendations on both IHC and FISH results by using the dual-color HER2/neu and centromeric FISH probes. DESIGN Retrospective analysis of 590 invasive carcinomas with concurrent IHC and dual-color HER2/neu and centromeric 17 (CEP17) FISH results, based on 2007 ASCO/CAP guidelines, was conducted from July 2011 to June 2013. With the revised guidelines, patients were recategorized and concordance rates between the 2 assays were recalculated. RESULTS Overall concordance rates for FISH and IHC decreased from 94.9% to 93.8% with reclassification. Negative FISH cases decreased from 79.1% to 69.3%. However, equivocal FISH cases were significantly increased from 0.7% to 9.5%, leading to more retesting. Both positive IHC and FISH cases were also noted to be increased, leading to more patients being eligible for trastuzumab treatment, especially those patients with concurrent HER2/neu and CEP17 polysomy. Approximately 1% of patients with initial FISH negative results were reclassified as having positive results when both the ratios and average copy number of HER2/neu were considered under the revised guidelines. CONCLUSIONS The revised 2013 ASCO/CAP guidelines can potentially lead to more patients being eligible for trastuzumab therapy but additional retesting is to be expected owing to an increased number of equivocal FISH cases.

Gefitinib, cisplatin, and concurrent radiotherapy for locally advanced head and neck cancer: EGFR FISH, protein expression, and mutational status are not predictive biomarkers

BACKGROUND Gefitinib was demonstrated to be synergistic with cisplatin and radiotherapy (RT) in in vitro studies. Biomarkers predictive of response to gefitinib in squamous cell head and neck cancer is still lacking. METHODS Thirty-one patients with locally advanced and easily accessible primary tumor sites for biopsies were recruited. Gefitinib was started 3 weeks before the start of cisplatin/concurrent radiotherapy (CTRT) and continued during the CTRT phase and thereafter for 4 months as consolidation phase. Two baselines and a repeat tumor sample were taken after 2 weeks of gefitinib alone to study its impact on tumor gene expression. Epidermal growth factor receptor (EGFR) protein expression, FISH and mutational status, and matrix metallopeptidase 11 (MMP11) protein expression were correlated with response and survival outcome. RESULTS The overall response rate to gefitinib alone was 9.7%. The survival outcome is as follows: median disease free 1.3 years, median survival time 2.4 years, 3-year disease free 42.9%, and 3-year overall survival 48.4%. EGFR FISH, protein expression, and mutational status did not predict for response nor survival outcome of patients. Although MMP11 overexpression did not predict for response, it predicted significantly for a poorer survival outcome. CONCLUSIONS Gefitinib can be combined safely with cisplatin/RT. More studies are needed to uncover predictive biomarkers of benefit to gefitinib.

HER2/neu revisited: quality and interpretive issues

Background Immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) are the only tests currently approved by the US Food and Drug Administration for classifying which patients will benefit from trastuzumab therapy. The accuracy of these two testing methods can be adversely affected by a variety of pre-analytical, analytical and post-analytical factors. According to the latest published recommendations of the panel of the Joint Committee of the American Society of Clinical Oncology and College of American Pathologists for HER2/neu testing, laboratories performing IHC and FISH for HER2/neu status in breast cancer are now required to have a high concordance of at least 95% between IHC and FISH, significantly higher than that in the published literature. Aim To perform a retrospective analysis of the concordance of IHC and FISH analysis for HER2/neu at Singapore General Hospital and review potential causes of disparity between these two methods. Method A retrospective review of a total of 106 invasive ductal carcinomas evaluated for HER2/neu at the Department of Pathology, Singapore General Hospital between 2007 and 2008 were included in the study. The initial HER2/neu immunostained slides were reviewed independently without knowledge of FISH results, and concordance between IHC and FISH was determined. Results Concordance between IHC and FISH assay was excellent and within the published range (104/106=98.1%). The discordant cases represent a well-recognised subset of genetic heterogeneity in HER2/neu, which is known to contribute to positive IHC and negative FISH tests.

Concordance of anaplastic lymphoma kinase ( ALK ) gene rearrangements between circulating tumor cells and tumor in non-small cell lung cancer

Anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung cancer (NSCLC) is routinely evaluated by fluorescent in-situ hybridization (FISH) testing on biopsy tissues. Testing can be challenging however, when suitable tissue samples are unavailable. We examined the relevance of circulating tumor cells (CTC) as a surrogate for biopsy-based FISH testing. We assessed paired tumor and CTC samples from patients with ALK rearranged lung cancer (n = 14), ALK-negative lung cancer (n = 12), and healthy controls (n = 5) to derive discriminant CTC counts, and to compare ALK rearrangement patterns. Blood samples were enriched for CTCs to be used for ALK FISH testing. ALK-positive CTCs counts were higher in ALK-positive NSCLC patients (3–15 cells/1.88 mL of blood) compared with ALK-negative NSCLC patients and healthy donors (0–2 cells/1.88 mL of blood). The latter range was validated as the ‘false positive’ cutoff for ALK FISH testing of CTCs. ALK FISH signal patterns observed on tumor biopsies were recapitulated in CTCs in all cases. Sequential CTC counts in an index case of lung cancer with no evaluable tumor tissue treated with crizotinib showed six, three and eleven ALK-positive CTCs per 1.88 mL blood at baseline, partial response and post-progression time points, respectively. Furthermore, ALK FISH rearrangement suggestive of gene copy number increase was observed in CTCs following progression. Recapitulation of ALK rearrangement patterns in the tumor on CTCs, suggested that CTCs might be used to complement tissue-based ALK testing in NSCLC to guide ALK-targeted therapy when suitable tissue biopsy samples are unavailable for testing.

ETV6 disruption does not predict indolent clinical behavior in secretory breast carcinoma.

Mabel Wong, MRCP,* Ana Richelia Jara-Lazaro, MD, Raymond Chee Hui Ng, FRACP,* Alvin Soon Tiong Lim, PhD, Poh Yian Cheok, BSc, Tse-Hui Lim, MSc, Puay Hoon Tan, FRCPA, and Nan Soon Wong, FAMS *Department of Medical Oncology, National Cancer Centre, Singapore; Department of Pathology, Singapore General Hospital, Singapore; Oncocare Cancer Centre, Mt Elizabeth Medical Centre, Singapore; and Department of Clinical Sciences, Duke-NUS, Singapore

Primary nodal follicular lymphoma with spindle cell features: a potential diagnostic pitfall

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Clear cell sarcomas of kidney are characterized by BCOR gene abnormalities including exon 15 internal tandem duplications and BCOR-CCNB3 gene fusion

Clear cell sarcoma of the kidney (CCSK) is a rare paediatric renal malignant tumour. The majority of CCSKs have internal tandem duplications (ITDs) of the BCOR gene, whereas a minority have the YWHAE–NUTM2 gene fusion. A third ‘double‐negative’ (DN) category comprises CCSKs with neither BCOR ITDs nor YWHAE–NUTM2 fusion. The aim of this study was to characterise 11 histologically diagnosed CCSKs immunohistochemically (with CCND1, BCOR and CCNB3 stains) and genetically.