Building pharmacogenetics into a pharmacovigilance program in Singapore: using serious skin rash as a pilot study.

To study the possible genetic associations with adverse drug reactions (ADR), the Singapore Health Sciences Authority (HSA) has piloted a program to collect DNA and phenotype data of ADR cases as part of its pharmacovigilance program. Between 2009 and 2012, HSA screened 158 cases of Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). To assess the association between HLA-B*1502 and carbamazepine (CBZ)-induced SJS/TEN, 13 cases and 26 drug-tolerant controls were analyzed. All 13 CBZ-SJS/TEN cases and 3/26 controls were HLA-B*1502 positive (odds ratio 181, 95% confidence interval: 8.7-3785, P=6.9 × 10(-8)). Discussions of the finding with the Ministry of Health and an expert panel led to the decision to make HLA-B*1502 testing the standard of care prior to first use of CBZ in Asians and to subsidize the genotyping test at public hospitals. This program illustrates the role of a regulatory authority in advancing the use of pharmacogenetics for drug safety.

JAK2 mutations in Asian patients with essential thrombocythaemia.

BACKGROUND/AIM: JAK2V617F is an acquired mutation present in a considerable proportion of patients with chronic myeloproliferative disorders. Its reported prevalence in European and US studies of patients with essential thrombocythaemia (ET) is 23-57%. This study was conducted to determine the prevalence of the JAK2 mutation in Asian ET patients, and to examine their disease profile. METHODS: Asian patients with ET were either recruited to the study or registry data were analysed retrospectively. Blood samples were collected for analysis of JAK2 mutation status during routine patient follow up. Clinical data on these patients (including demographics and disease profiles) and complications at diagnosis were recorded. RESULTS: The JAK2 mutation was detected in 35/102 (34%) patients. Females were more likely than males to have JAK2 mutation (P = 0.031). At diagnosis, JAK2-mutated patients were found to be older (P = 0.012), have higher leucocyte counts (P = 0.036) and high-risk disease (P = 0.039). There were no other statistically significant differences between mutated and wild-type JAK2 ET patients. CONCLUSION: The prevalence of JAK2 mutations in this population of Asian ET patients was 34%. Patients with the JAK2 mutation were significantly more likely to have high-risk disease. Further studies are required to assess the role of JAK2 mutations in risk stratification in ET and compare the phenotype of Asian patients with other populations.

Development of quantitative-fluorescence polymerase chain reaction for the rapid prenatal diagnosis of common chromosomal aneuploidies in 1,000 samples in Singapore.

INTRODUCTION: We aimed to develop a rapid quantitative-fluorescence polymerase chain reaction (QF-PCR) to detect common foetal aneuploidies in the Singapore population within 48 hours of sample collection in order to alleviate parental anxiety. METHODS: DNA from 1,000 foetal samples (978 amniotic fluids, 14 chorion villi and eight foetal blood samples) was analysed using a QF-PCR of 19 microsatellite markers located on chromosomes 13, 18, 21, X and Y. A total of 523 samples were archived before the QF-PCR analysis (archived), while QF-PCR was performed and the results obtained within 48 hours of sample collection in the remaining 477 samples (live). The results were confirmed with their respective karyotypes. RESULTS: In total, 47 autosomal trisomies (T) were found: 30 among the archived (three T13, 12 T18, 15 T21) and 17 among the live (four T18, 13 T21) samples. The QF-PCR results were verified with their respective karyotypes. We achieved 100 percent sensitivity (lower 95 percent confidence interval [CI], 92.8 percent) and specificity (lower 95 percent CI, 99.5 percent), and the time taken from sample collection to the obtaining of results for the 477 live samples was less than 48 hours. CONCLUSION: Prenatal diagnostic results of common chromosomal abnormalities can be released within 48 hours of sample collection using QF-PCR. Parental anxiety is alleviated and clinical management is enhanced with this short waiting time.

How well do serum sTREM-1 measurements prognosticate in septic shock?

The soluble triggering receptor expressed on myeloid cells (sTREM)-1 has emerged as a potentially useful biomarker for the diagnosis of sepsis. This study aimed to evaluate the prognostic utility of serum sTREM-1 in septic shock, in comparison with that of procalcitonin measurements. Thirty-one consecutive patients in a tertiary medical intensive care unit with septic shock were studied. sTREM-1 levels in blood were measured using a modified immunoblot array technique on days one to three of intensive care unit admission. Serum procalcitonin and interleukin (IL)-1beta, IL-6, IL-IO and tumour necrosis factor-alpha levels were also measured. No significant difference was observed in the sTREM-1 levels on the first three days between survivors and nonsurvivors. sTREM-1 levels moderately correlated with the Acute Physiology and Chronic Health Evaluation II and Sequential Organ Failure Assessment scores on day three, but did not correlate with vasopressor requirements, cytokine levels and the presence of bacteraemia. In contrast, procalcitonin levels were significantly higher in nonsurvivors than in survivors on days two and three. A significant relationship also existed between procalcitonin levels and the other variables. In conclusion, this study found that the prognostic utility of serum sTREM-1 in septic shock is poor and that procalcitonin measurements perform better in this regard.

Soluble triggering receptor expressed on myeloid cells-1 in acute respiratory infections.

Levels of the soluble form of the triggering receptor expressed on myeloid cells (sTREM)-1 are elevated in severe sepsis. However, it is not known whether sTREM-1 measurements can distinguish milder bacterial infections from noninfectious inflammation. The present authors studied whether serum sTREM-1 levels differ in community-acquired pneumonia, exacerbations of chronic obstructive pulmonary disease (COPD), asthma and controls, and whether sTREM-1 may be used as a surrogate marker for the need for antibiotics. Serum sTREM-1 levels in 150 patients with pneumonia, COPD and asthma exacerbations and 62 healthy controls were measured. Serum sTREM-1 levels were significantly elevated in pneumonia (median 295.2 ng x mL(-1)), COPD (280.3 ng x mL(-1)) and asthma exacerbations (184.0 ng x mL(-1)) compared with controls (83.1 ng x mL(-1)). Levels were higher in pneumonia and Anthonisen type 1 COPD exacerbations than in type 2 and 3 COPD and asthma exacerbations. The area under the receiver operating characteristics curve for sTREM-1 as a surrogate marker for the need for antibiotics was 0.77. Serum levels of the soluble form of the triggering receptor expressed on myeloid cells-1 were elevated predominantly in pneumonia and Anthonisen type 1 COPD exacerbations versus type 2 and 3 chronic obstructive pulmonary disease exacerbations, asthma and controls. Serum levels of the soluble form of the triggering receptor expressed on myeloid cells-1 has moderate but insufficient accuracy as a surrogate marker for the need for antibiotics in lower respiratory tract infections.

Discordant quantitative detection of putative biomarkers in nodal micrometastases of colorectal cancer: biological and clinical implications.

AIMS: Nodal expression of the carcinoembryonic antigen (CEA), cytokeratin 20 (CK20), and guanylyl cyclase C (GCC) genes was measured in tandem in patients with colorectal cancer (CRC) to assess whether there would be sufficient agreement between these markers in their ability to detect micrometastasis to qualify one of them as a universal marker, and whether frozen and paraffin wax embedded tissues would yield similar results. METHODS: One hundred and seventy five frozen lymph nodes (FT) and 158 formalin fixed, paraffin wax embedded lymph nodes (PET) from 28 CRC cases were analysed using gene specific quantitative real time polymerase chain reaction, carried out on the LightCycler system with SYBR Green chemistry. RESULTS: There was significant disparity in positive detection of the three biomarkers in FT versus PET, with notable agreement achieved only for CEA (66.6%) in FT versus PET in Dukes' B disease, and between CK20 and GCC (44.6%) in FT, also in Dukes' B disease. One patient with full concordance in all three tumour markers with both tissue types suffered a relapse and died within two years of follow up. CONCLUSIONS: There was considerable discordance in the positive detection of the three tumour markers in both tissue types (FT versus PET). This brings into question whether using a single tumour marker to detect micrometastasis in one tissue type (FT or PET) is adequately representative, and challenges the concept of universal markers for molecular CRC metastatic detection. Multiple tumour markers would predict more accurately the metastatic potential of Dukes' B CRCs.

Lymphocytic gastritis-like T cell lymphoma: molecular evidence of an unusual recurrence.

This report describes a patient with a gastric biopsy specimen showing histomorphological and immunohistochemical appearances indistinguishable from those usually present in lymphocytic gastritis, a rare condition of unknown aetiology with a distinctive phenotype. The patient had a history of a biopsy confirmed T cell non-Hodgkin lymphoma at two anatomical sites (bladder and stomach), which was subsequently treated. Molecular analysis of the T cell receptor (TCR) gamma chain gene rearrangements showed a distinct monoclonal T cell population in the bladder and gastric biopsies. The same analysis in the lymphocytic gastritis-like biopsy sample showed a monoclonal population with identical base pair size to that identified in the other specimens. This report highlights the importance of TCR gene rearrangement analysis in the diagnosis of unusual gastric inflammation, and the use of capillary electrophoresis based polymerase chain reaction in the follow up of lymphoproliferative disorders.

Molecular diagnostic cytopathology: definitions, scope and clinical utility.

Molecular diagnosis is the application of molecular biology techniques and knowledge of the molecular mechanisms of disease to diagnosis, prognostication and treatment of diseases. Although it is not widely used in routine molecular cytological practice, some examples are presented here of the application of molecular techniques to the routine cytopathological diagnosis of solid tumours and lymphoreticular malignancies. The term 'molecular diagnostic cytopathology' is proposed to define the application of molecular diagnosis to cytopathology, and the challenges of the introduction of molecular diagnosis into routine diagnostic histopathology and cytopathology are discussed. Finally, the importance of a combined morphological, immunophenotypic and molecular approach to maintain the diagnostic pathologist at the heart of the clinical decision-making process is emphasized.

Lactacystin-induced apoptosis of cultured mouse cortical neurons is associated with accumulation of PTEN in the detergent-resistant membrane fraction.

The tumor suppressor function of PTEN is attributed to its phospholipid phosphatase activity that dephosphorylates the plasma membrane phosphatidylinositol-(3,4,5)-triphosphate [PtdIns(3,4,5)P3]. Implicit in this notion is that PTEN needs to be targeted to the plasma membrane to dephosphorylate PtdIns(3,4,5)P3. However, the recruitment of PTEN to the plasma membrane is not fully understood. Here, we demonstrate PTEN accumulation in the detergent-insoluble fraction of neuronal cells in response to treatment by the proteasome inhibitor lactacystin. First, lactacystin induces apoptosis and the activation of caspase-3 in cultured cortical neurons. Second, PTEN undergoes proteolysis to form a truncated 50-kDa form that lacks parts of its C-terminal tail. Third, the truncated PTEN is stably associated with the detergent-insoluble fraction in which the plasma membrane marker protein flotillin-1 resides. Taken together, our results suggest that truncation and accumulation of PTEN to the detergent-insoluble membrane fraction are two events associated with the apoptotic signals of the proteasome inhibitor in cortical neurons.

Sequence confirmation of the EWS-WT1 fusion gene transcript in the peritoneal effusion of a patient with desmoplastic small round cell tumor.

Desmoplastic small round cell tumor (DSRCT) is a rare undifferentiated neoplasm. The prognosis is poor, even if therapy is instituted promptly, and thus it is important to differentiate it from other histologically and cytologically similar-looking malignancies of the young adult. We present a case of DSRCT in a 17-yr-old male with disseminated peritoneal disease and peritoneal effusion. The cytology sample showed a malignant small round cell tumor, the classical cytological features of DSRCT, and immunohistochemistry performed in the prepared cell block exhibited an antibody expression profile in keeping with DSRCT. Further material from the effusion was prepared for RNA extraction, following which a reverse-transcriptase polymerase chain reaction (RT-PCR) and sequencing of the t(11;22)(p13;q11 or q12) were carried out. The result showed the presence of the reciprocal translocation and thus confirmed the diagnosis of DSRCT. This case shows how molecular techniques (including sequencing) can be applied to cytology in clarifying and confirming certain difficult diagnosis of undifferentiated neoplasms, DSRCT in this particular case.

Immunocytochemistry versus molecular fingerprinting of metastases.

Examination of cytological samples of cancer to suggest a possible primary site of origin is one of the commonest and most difficult tasks of diagnostic cytopathologists. Currently, both cytomorphology and immunocytochemistry are the main approaches to this diagnostic dilemma. We report the application of microsatellite analysis in cytological samples in a patient with a primary colonic tumour and two subsequent lung nodules, which were suspected on CT scans of the chest, and compared the findings with those obtained with conventional immunocytochemistry. The molecular results were in agreement with the radiological impression and conflicted with the immunocytochemistry. We conclude that immunocytochemical and molecular biology approaches to the diagnosis of tumours may give rise to contradictory results.

Intrinsic variability in the detection of micrometastases in lymph nodes for re-staging of colorectal cancer. effect of individual markers and tissue samples.

In this study, we investigated whether (a) carcinoembryonic antigen (CEA), cytokeratin-20 (CK-20) and guanylyl cyclase C (GCC) are clinically useful markers for the molecular detection of submicroscopic metastases in colorectal cancer (CRC) and (b) whether overexpression of CEA, CK-20 and GCC can be reliably detected in formalin-fixed, paraffin-embedded tissues as well as frozen lymph nodes. We studied 175 frozen lymph nodes and 158 formalin-fixed, paraffin-embedded lymph nodes from 28 cases of CRC. CEA or CK-20 or GCC-specific polymerase chain reaction (PCR) was carried out on mRNA transcripts extracted from the nodal tissues. Ten out of 11 Dukes' B CRC cases had detectable CEA and CK-20 while 6 out of 11 Dukes' B CRC cases had detectable GCC. In general, the difference of re-staged cases when comparing frozen and paraffin-embedded samples was marked; the only statistically significant correlation between frozen and paraffin tissue was for the CEA marker. Our results indicated a high incidence (>50%) of detecting micrometastases in histologically-negative lymph nodes at the molecular level.