Prevalence of high-risk human papillomavirus in oral squamous cell carcinoma with or without chewing habits.

Oral cancer (OC) is the most common cancer in Pakistani males and the second most common in females. Major risk factors include peculiar chewing habits, human papillomavirus (HPV) infection and molecular pathways. However, less data is available for this avertible cancer regarding its association with high-risk HPV (HR-HPV) and chewing habits in this region. Therefore, this study was done to determine the prevalence of HR-HPV in oral squamous cell carcinoma (OSCC) and its correlation with p16 and chewing habits. Formalin-fixed paraffin-embedded (FFPE) biopsy specimens of 186 samples were tested for HR-HPV type 16/18 by PCR, followed by p16 immunostaining (IHC) in a subset of cases (n = 50). Appropriate statistical tests were applied to find the association between HR-HPV/p16 and peculiar chewing habits with significance criteria of p<0.05 with 95% CI. HR-HPV (type 16 &18) was present in seven out of 186 cases (3.8%). Of these seven cases, five were positive for HPV16, whereas two were positive for HPV16/18. The overall expression of p16 protein in 50 samples was 38% (n = 19), and among these 19-IHC positive samples, 26% were positive for HR-HPV DNA. No significant association was found between HR-HPV positivity and p16 and chewing habits (p>0.05). It was concluded that HR-HPV prevalence in OSCC was very low in our population, with no statistically significant correlation with p16 and chewing habits. These results suggest the role of HR-HPV as an independent risk factor in OSCC in the local setting.

Spectrum of Cystic Fibrosis Conductance Regulator Gene Mutations Reported in Pakistani Descent Cystic Fibrosis Patients.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This study aims to determine the genotypic and phenotypic spectrum of the CFTR gene mutations reported in the literature for Pakistani-origin CF patients. Databases were searched for such studies from 1947-2019 for sample size, method of diagnosis, and CFTR gene mutations. The authors identified 12 studies reporting 33 CFTR gene mutations, both intronic as well as exonic in Pakistani origin patients. The most widely tested mutation was D508 with a frequency of 17%-60%. No hotspot zone was identified and not all reported mutations were causing disease. There is a need to identify common mutations in the Pakistani population to develop population-specific CFTR mutations panel. This will enable the researchers to perform phenotype-genotype correlation studies to improve the CF detection rate. Key Words: Cystic fibrosis, Pakistan, Mutations, CFTR.

Oral cancer: Clinicopathological features and associated risk factors in a high risk population presenting to a major tertiary care center in Pakistan.

Oral squamous cell carcinoma (OSCC) has the highest prevalence in head and neck cancers and is the first and second most common cancer in males and females of Pakistan respectively. Major risk factors include peculiar chewing habits like areca nut, betel quid, and tobacco. The majority of OSCC presents at an advanced stage with poor prognosis. On the face of such a high burden of this preventable cancer, there is a relative lack of recent robust data and its association with known risk factors from Pakistan. The aim of this study was to identify the socioeconomic factors and clinicopathological features that may contribute to the development of OSCC. A total of 186 patients diagnosed and treated at a tertiary care hospital, Karachi Pakistan were recruited. Clinicopathological and socioeconomic information was obtained on a structured questionnaire. Descriptive analysis was done for demographics and socioeconomic status (SES) while regression analysis was performed to evaluate the association between SES and chewing habits, tumor site, and tumor stage. The majority of patients were males and the mean age of OSCC patients was 47.62±12.18 years. Most of the patients belonged to low SES (68.3%) and 77.4% were habitual of chewing. Gender (male) and SES were significantly associated with chewing habits (p<0.05). Odds of developing buccal mucosa tumors in chewers (of any type of substance) and gutka users were 2 and 4 times higher than non-chewers respectively. Middle age, chewing habits, and occupation were significantly associated with late stage presentation of OSCC (p<0.05). In conclusion, male patients belonging to low SES in their forties who had chewing habits for years constituted the bulk of OSCC. Buccal mucosa was the most common site in chewers and the majority presented with late stage tumors.

Dedifferentiated Liposarcoma With Meningothelial-Like Whorls: Five Additional Cases and Review of the Literature.

Background. Diagnosis of dedifferentiated liposarcoma (DDL) can sometimes be challenging due to a wide variety of histological features. "Meningothelial-like" whorl is an uncommon histological feature of DDL, which is also observed in neural tumors and follicular dendritic cell sarcoma. This feature is frequently associated with metaplastic bone formation. We conducted this study to describe the clinicopathological features of DDL with meningothelial-like whorls that would aid in establishing accurate diagnosis. Material and Methods. Microscopic glass slides of 5 cases of DDL with meningothelial-like whorls, diagnosed between January 2010 and December 2019, were reviewed. Results. Paratesticular region was the most common site. Whorls occupied 10% to 75% of tumor area and ranged in size from <0.1 cm to >2 cm. In 1 case, these whorls coalesced to form large areas of dedifferentiation. The cells forming whorls were spindle to epithelioid shaped and lacked significant nuclear pleomorphism and increased mitoses. Metaplastic bone formation was observed in 4 cases and cartilage formation in 3 cases. p16 and α-smooth muscle actin (α-SMA) immunohistochemical stains were positive in 2 cases, when performed. MDM2 gene amplification was observed in all cases by fluorescence in situ hybridization technique. These tumors showed aggressive behavior, similar to that of DDL without meningothelial-like whorls. Two patients died, 1 developed recurrence, 1 presented as recurrent tumor, and 1 developed metastasis. Conclusion. Meningothelial-like whorls in DDL most likely represent an early stage of dedifferentiation. Presence of well-differentiated liposarcoma areas, metaplastic bone formation, positive expressions for p16 and α-SMA immunohistochemical stains, and MDM2 gene amplification are useful diagnostic clues. These tumors have the potential to behave aggressively.

Distribution of Chromosomal Abnormalities Commonly Observed in Adult Acute Myeloid Leukemia in Pakistan as Predictors of Prognosis

Objectives: The heterogenous response to treatment in acute myeloid leukemia (AML) can be attributed largely to the difference in cytogenetic features identified in between cases. Cytogenetic analysis in acute leukemia is now routinely used to assist patient management, particularly in terms of diagnosis, disease monitoring, prognosis and risk stratification. Knowing about cytogenetic profile at the time of diagnosis is important in order to take critical decisions in management of these patients. The study was conducted to determine the distribution of cytogenetic abnormalities in Pakistani adult patients with AML in order to have insights regarding behavior of the disease. Methods: A retrospective analysis of all the cases of AML (≥15years old) diagnosed at Aga Khan University from January 2011 to December 2016 was performed. Cytogenetic analysis was made for all cases using the trypsin-Giemsa banding technique. Karyotypes were interpreted using the International System for Human Cytogenetic Nomenclature (ISCN) criteria. Results: A total of 321 patients were diagnosed with AML during the study period, of which 288 samples successfully yielded metaphase chromosomes. The male to female ratio was 1.7:1. A normal karyotype was present in 61% (n=176) of the cases whereas, 39% (n=112) had an abnormal karyotype. Of the abnormal cases, t (8;21) (q22;q22) and t (15;17) (q22;q12) were identified in 8.3% and 4.9% cases respectively. Adverse prognostic cytogenetic subgroups including complex karyotype, monosomy 7 and t(6;9)(p23;q34) were identified in 9%, 1% and 0.7% patients respectively. Conclusions: This largest cytogenetic data in adult AML from Pakistan showed comparable prevalence of favorable prognostic karyotype to international data. The prevalence of specific adverse prognostic karyotype was low.

Frequency of ALK Rearrangement by FISH Testing and its Correlation with ALK-IHC in Adenocarcinoma of Primary Lung Origin

Anaplastic lymphoma kinase (ALK) gene can be oncogenic either by forming fusion with other genes, amplification of the gene or by having mutations. ALK rearrangement can either be detected by standard "fluorescence in situ hybridization (FISH)" or "immunohistochemistry (IHC)". Objective of this study was to record the prevalence of ALK rearrangement in adenocarcinoma of Primary Lung origin and compare it with ALK-IHC staining. Data of 64 patients of lung adenocarcinoma from 2015-2017 was analyzed. All of the FFPE biopsies were tested for EGFR (qPCR) followed by ALK rearrangement (by FISH and IHC) on EGFR negative samples. Out of 64 samples, 21.8% (14) showed EGFR mutations and 14% (7/50) were positive for ALK rearrangement when checked by FISH. In IHC testing for ALK (FISH positive) 8% (4/50) showed positivity. In conclusion ALK-FISH positive cases are higher than other studies likely due to the relatively small sample size. FISH testing was found to be more sensitive than IHC; one reason may be the low level of ALK. Our study warrants that currently FISH remains the gold standard for screening of ALK gene rearrangements.

Anaplastic lymphoma kinase protein positive diffuse large B cell lymphoma; A developing world experience.

Anaplastic lymphoma kinase (ALK) positive diffuse large B-cell lymphoma (ALK+DLBCL) is a rare, distinct and aggressive subtype of non-Hodgkin's lymphoma (NHL). These tumors are considered to be derived from post-germinal center B cells but peculiarly their distinction is based on the fact that they are ALK-positive neoplastic B cells but lack expression of B cell markers (CD19,CD20, CD79a), T cell markers (CD3, CD5) and CD30. Its broad differential diagnosis and similarities to plasmablastic lymphoma, immunoblastic DLBCL, Anaplastic large-cell lymphoma (ALCL) of T-null cell lineage, and poorly differentiated/anaplastic carcinoma pose a grave challenge to physicians with conventional costly treatment for DLBCL failing to yield any clinical or prognostic significance in ALK+DLBCL. In this article we present 7 cases which were reported at Aga Khan University Hospital, Department of Pathology and Laboratory Medicine from 2009 to 2015 and a review of literature on ALK+ DLBCL, which according to the best of our knowledge is the second largest reported series and the first from South Asian subcontinent.

Assessment of WT1 Expression as a Marker of Treatment Outcome in Karyotype Normal Acute Myeloid Leukemia Patients in Pakistan.

Currently, there is an effort to predict relapse by follow-up monitoring of MRD and subsequently to begin the treatment of the patients during their clinical and hematological remission prior to overt hematological relapse. Expression of WT1 in AML is known to be independently associated with significant inferior response to therapy and short survival outcome. Follow-up monitoring of WT1 gene expression during or after therapy would be a valuable predictive marker for early recurrence or relapse of AML disease. This pilot study evaluated newly diagnosed and post-induction or consolidation chemotherapy of AML patients who were registered with the Oncology Clinics of the Aga Khan University Hospital, Karachi. High WT1 burden (> 5000 copies/ml) in 2 patients was indicative of early recurrence of the disease along with shorter disease-free and overall survival. Low WT1 expression (< 200 copies/ml) in 2 patients after induction and consolidation therapy, respectively, was suggestive of better prognosis.

Cyclooxygenase-2 polymorphisms and breast cancer associated risk in Pakistani patients.

UNLABELLED: Prostaglandins produced by Cyclooxygenase-2 enzyme have been implicated to have a role in breast carcinogenesis. Several single nucleotide polymorphisms (SNPs) linked to COX-2 enzyme are reported to modulate its expression. The aim of the present study was to examine association of these SNPs to breast cancer risk in Pakistani patients. METHODS: In this case-control study, three sequence variants rs689465, rs689466, rs20417 in the promoter region of COX-2 were screened to evaluate the association with breast cancer risk. A total of 150 breast cancer patients and 101 healthy control genomic DNA were genotyped for rs689456, rs689466, rs20417 and their genotypes distribution in cases and control were compared using Pearson chi square test. Risk association was analyzed through odd ratio calculated by logistic regression. RESULTS: A screening analysis of COX-2 SNPs in 101 healthy controls showed distribution of Minor allelic frequency distribution of SNPs as follows : rs689465 (0.12), rs689466 (0.15), rs20417 (0.23). Further analyses revealed that their observed genotype frequencies were consistent with Hardy Weinberg equilibrium and strong linkage disequilibrium was identified between rs20417, rs689465 and rs689466. The Combined allele variants analysis showed that Haplotype rs68965G- 689466A-20417C (OR 2.909; CI 95 %1.3776.327; P = 0.007) was significantly associated with breast cancer. CONCLUSIONS: Our results indicate no strong association between three most frequent COX-2 SNPs rs689465 rs689466, rs20417 studied with breast cancer risk in the single locus analysis. However, our data suggested that combined COX-2 SNP haplotype have a role in breast cancer associated risk in Pakistani patients.

Distribution of EGFR mutations commonly observed in primary lung adenocarcinomas in Pakistan as predictors for targeted therapy.

BACKGROUND: Acquired genetic alterations and presence of sensitizing mutations in the tyrosine kinase domain of EGFR and other signaling molecules have been found in different subsets of primary lung adenocarcinoma. The commonest EGFR mutations are small in frame deletions of exon 19 and a point mutation (L858R) in exon 21, having a combined occurrence of around 90%. The objective of this study was to determine the frequency and types of EGFR mutations in primary lung adenocarcinomas in Pakistan. MATERIALS AND METHODS: EGFR mutations in tumor samples were screened by multiplex real time PCR. Briefly, DNA from formalin fixed paraffin-embedded tissue was amplified with primers and probes specific to 43 different EGFR mutations in a Cobas z 480 instrument. The assay detects mutations in four exons (18-21) of the EGFR gene. RESULTS: Out of 94 patients, 65 were males and 29 females with a M:F ratio of 2.2: 1. The median age was 62 years (range, 28 - 85 years). In our biopsy samples 70 (74%) cases were of primary lung adenocarcinoma, whereas 24 (26%) were confirmed metastatic adenocarcinoma of primary lung origin. EGFR mutation was positive in 29% of the patients. The highest frequency of L858R was observed in 48% of these, followed by deletion in exon 19 (44%). In addition, other rare mutations such as compound G718X:S768I and insertions in exon 20 insertion were detected in approximately 4% of the patients. CONCLUSIONS: This study showed that Del 19 and L858R are the most frequent mutations in Pakistani lung adenocarcinoma patients and around 29% of the patients were found eligible for erlotinib therapy.

Estimating window period blood donations for human immunodeficiency virus Type 1, hepatitis C virus, and hepatitis B virus by nucleic acid amplification testing in Southern Pakistan.

BACKGROUND: Recently, strategic planning was initiated by the National Blood Transfusion Services Pakistan to improve its blood bank facilities. Emphasis has been placed on appropriate screening of blood products. Located in the southern region, Aga Khan University Hospital is a 700-bed tertiary care academic institute with comprehensive blood banking. Screening of blood donors has been based on verbal screening and serologic testing to date. Additionally, the need of implementing nucleic acid testing (NAT) was considered in 2011 because of an upsurge in hepatitis epidemiology. The aim of this study was to analyze the efficacy of this additional donor screening program and to evaluate the impact of NAT on the yield and residual risk of transfusion-transmissible viral infections. STUDY DESIGN AND METHODS: A total of 42,830 blood donations collected between 2011 and 2012 were screened for routine serologic assays. Only serologically negative donors (n=41,304) were tested for NAT. The frequency of viral infections was evaluated through serologic techniques and NAT yield for viral agents was estimated for computing window period donors. Residual risk per million donors was computed for viral infections in seronegative blood donors. RESULTS: Serologic work-up showed 1571 abnormal screening results in 1526 blood donors with the following results: hepatitis C virus antibodies (anti-HCV; n=708), hepatitis B surface antigen (n=555), human immunodeficiency virus antibodies (anti-HIV; n=29), malaria (n=30), VDRL (n=249), and coinfection (n=45). Thirty-five NAT-reactive samples were identified: HIV-1, one; HCV, 27; and hepatitis B virus (HBV), seven. Incident rates per 10(5) donors were highest for HCV (453.3) followed by HBV (171.5) and HIV (72.2). Calculated residual risk per million donors was highest at 1 in 10,900 for HBV, intermediate at 1 in 13,900 for HCV, and least at 1 in 62,600 for HIV. CONCLUSION: Incidence rates and estimated residual risk indicate that the current risk of transfusion-transmitted viral infections attributable to blood donation is relatively high in this country. The study recommends the parallel use of both serology and NAT screening of donated blood in countries that have high seroprevalence of these viral infections.

Ewing's sarcoma arising from the adrenal gland in a young male: a case report.

BACKGROUND: Ewing's sarcoma uncommonly arises from extraosseous soft tissue or parenchymal organs. Primary adrenal Ewing's Sarcoma, although very rare, is extremely aggressive and commonly fatal. CASE PRESENTATION: A 17 year old Pakistani male was referred to the outpatient oncology clinic at our center with a three month history of concomitant pain, swelling and dragging sensation in the right hypochondrium. Abdominal examination revealed a large, firm mass in the right hypochondrium extending into the right lumbar region and epigastrium. His genital exam was unremarkable and there were no stigmata of hepatic or adrenal disease.Computed tomography scans revealed a large peripherally enhancing mass in the hepatorenal area, biopsy of which showed a neoplastic lesion composed of small round blue cells which exhibited abundance of glycogen and stained diffusely positive for CD99 (MIC2 antigen). Fluorescence in situ hybridization demonstrated gene rearrangement at chromosome 22q12 which confirmed the diagnosis of Ewing's sarcoma. Staging scans revealed pulmonary metastasis and hence he was commenced on systemic chemotherapy. CONCLUSION: This case report highlights the importance of keeping Ewing's sarcoma in mind when a young patient presents with a large non-functional adrenal mass.

Prenatal screening for ß-thalassemia major reveals new and rare mutations in the Pakistani population.

ß-Thalassemia is the most common genetic disorder in Pakistan, where more than 6000 affected children are born annually, and the carrier population is around 10 million. The objective was to study ß-globin gene mutations in chorionic villous biopsy samples. Prenatal screening of 383 pregnant women between 2003 and 2010 was carried out using a panel of 13 mutation primers and amplification refractory mutations system (ARMS)-PCR. In addition, DNA sequencing was used to confirm uncharacterized mutations and in some cases fetal disease status was confirmed by linkage analysis. Families enrolled in this study represented major ethnic groups in Pakistan. Of the 13 mutations tested, three mutations accounted 71% of the total, including IVS1-5(G-C)[HBB:c.92+5G>C], codon 8/9(+G) [HBB:c.27_28insG] and del 619[NG_000007.3:g71609-72227del619]. Mutations in four uncharacterized samples were later confirmed by DNA sequencing as -88(C-T)[HBB:c.-138C>G], -90(C-T)[HBB:c.-140C>T] and codon 59(+T)[HBB:c.178_179insT]. To our knowledge, this is the first report of these mutations in Pakistan. Moreover, 19.2% fetal samples were normal and 52.3% heterozygous, whereas 26.4% were affected with thalassemia major. IVS1-5:IVS1-5 was the most common genotype in fetal samples. Prenatal diagnosis of ß-thalassemia using ARMS PCR is an efficient approach for reducing the burden of this disease in Pakistan. In addition, rare mutations reported in this study should be incorporated in the diagnostic strategy.

BRCA1 status in Pakistani breast cancer patients with moderate family history.

OBJECTIVE: To determine BRCA1 status in breast carcinoma patients of Pakistani origin. STUDY DESIGN: Observational study. PLACE AND DURATION OF STUDY: The Oncology Clinics of the Aga Khan University Hospital, Karachi, between May 2005 and December 2009. METHODOLOGY: Fifty three breast cancer patients based on clinical and laboratory diagnosis were recruited for this study. Moderate family history was defined as having a close relative (mother, daughter, sister) diagnosed with breast cancer under 45 years. Peripheral blood samples were collected from each patient in a 5 ml tube containing EDTA as anticoagulant. Subsequent to DNA extraction, mutational analysis of BRCA1 exons 2, 5, 6, 16, 20 and 22 was carried out using single strand conformation polymorphism (SSCP) assay while protein truncation test (PTT) was used to examine mutations in exon 11. All BRCA1 sequence variants were confirmed by DNA sequencing. RESULTS: Twenty-three patients were diagnosed with early onset breast cancer, 30 patients had moderate family history. At the time of diagnosis, the median age of enrolled patients was 39 years (range 24-65 years). Out of 53 patients, analyzed by SSCP assay, mobility shift was detected in exon 6, 16 and 20 of three patients, whereas one patient was tested positive for mutation in exon 11 by PTT assays. All patients with BRCA1 mutations were further confirmed by DNA sequencing analysis. In exon 16 c.4837A > G was confirmed, which is a common polymorphism reported in several populations including Asians. Moreover, mutations in exon 6 (c.271T > G), exon 20 (c.5231 delG) and exon 11 (c.1123 T > G) were reported first time in the Pakistani population. CONCLUSION: Several BRCA1 mutations were observed in Pakistani breast cancer patients with moderate family history. Therefore, mutation-based genetic counselling for patients with moderate family history can facilitate management, if one first or second degree relative or early onset disease is apparent.

Status of HER2 amplification, polysomy 17 and histopathological features of 425 Pakistani breast cancer patients.

HER2 gene amplification in invasive breast cancer is a robust predictive marker for response to transtuzumab therapy. This study was undertaken to measure concordance between immunohistochemistry (IHC) and FISH for HER2 gene amplification in invasive breast tumors, as well as the presence of polysomy 17 and possible correlation with demographics and histopathological variables, including ER and PR positivity. A total of 425 cases of infiltrating carcinoma of breast (99% IDC-NOS) were studied. HER2 over expression was tested by IHC and FISH methods. Association between IHC and FISH in both subsets was calculated by amplification ratio including polysomy 17. Out of 425 specimens, 128 (30%) were positive for HER2 amplification by FISH test, whereas only 78 (24%) tumors with 2+ expression showed amplification. In contrast, 39 (74%) demonstrated 3+ IHC score and HER2 gene amplification. The histological variables including tumor size, tumor type, and lymph node involvement did not influence the outcome of FISH analysis. The ER and PR status showed significantly greater positivity in patients negative for HER2 amplification. Polysomy 17 was detected in 23.7% patients and was positively associated with ER and PR expression (P= <0.05). Our study showed a concordance of 24% between 2+ IHC and FISH amplification, while in 3+ IHC cases the concordance was 74%. Significant links of HER2 amplification was seen with ER andPR negativity and higher tumor grade. In addition, non-significant correlations were noted with other variables like tumor type, size and lymph node status.

Cystic fibrosis: defining a disease under-diagnosed in Pakistan.

OBJECTIVE: Cystic fibrosis is frequently missed in the Pakistani population due to lack of appropriate diagnostic tools. Thus our aim was to define unknown disease-causing mutations to help create suitable diagnostic tests and improve understanding of what appears to be an aggressive and under-diagnosed disease in this population. METHODS: Patients with elevated sweat chloride values and clinically suspected CF were recruited from Aga Khan University, Pakistan. Mutations DF508, S549R, S549N, Y569D, 296 + 12(T>C), G553X, G551D and G551X were screened for by allele specific polymerase chain reactions. CFTR exons 10, 11 and 12 were sequenced by direct DNA sequencing. RESULTS: Of 150 patients tested by PCR, 26 (17.3%) were positive for DeltaF508. One patient was a F508/S549N compound heterozygote. Eighty-three of 87 patients sequenced for mutations in exon 10 were normal; 42/43 for exon 11 and 29 for exon 12 were normal. CONCLUSION: This first step in defining mutations involved in Pakistani CF suggests that DeltaF508 is uncommon and S549 was the only additional mutation identified in CFTR exons 10, 11 and 12. Identification of the remaining mutations and their frequency is required to design appropriate tests and improve understanding and management of the disease.

Characterization of genetic lesions in apoptosis-regulating and proliferation control genes in diffuse large B-cell non-Hodgkin's lymphoma.

BACKGROUND: This study was conducted to analyze the frequency, expression patterns, and the impact of individual proteins BCL2, BCL6, and p53 on overall survival (OS) in adult, diffuse large B-cell lymphoma (DLBCL) patients. BCL2 gene was further investigated for potential alterations at the DNA level and correlated with OS. MATERIALS AND METHODS: A total of 117 adult well-characterized DLBCL cases were included. The panel of antibodies comprised CD45, CD20, CD79a, CD3, BCL2, BCL6, and p53. PCR was also employed to correlate the events at the DNA level in BCL2. RESULTS: The mean and median ages were 47.74 and 49 with a M:F ratio of 2.07:1. The incidence of BCL2, BCL6, and p53 expression was observed in 64.10%, 37.60%, and 52.13% of cases, respectively. Amplifiable quality DNA was available from 90 cases. BCL2/IGH translocation was found in 35/90 patients (38.88%) with 24 cases showing BCL2 (MBR)/IGH and 11 cases BCL2 (mcr)/IGH translocation. No association between BCL2 overexpression and BCL2 /IGH translocation was seen. Clinical data were available for 52 patients treated by CHOP therapy. It was found that patients with p53 overexpression had decreased overall survival (P = 0.0004) whereas BCL2, BCL6 expression, and BCL2/IGH translocation had no impact on overall survival. CONCLUSION: Our data suggest that simple p53 protein expression by IHC at the time of diagnosis may help to identify high-risk patients, who may benefit with more aggressive and newer treatments in addition to standard CHOP.

Phylogenetic analysis of hepatitis B virus in Pakistan.

OBJECTIVE: To identify the distribution pattern of Hepatitis B Virus (HBV) genotype in a group of patients and to study its phylogenetic divergence. STUDY DESIGN: An observational study. PLACE AND DURATION OF STUDY: The clinics of Gastroenterology Unit, Ziauddin University, from January to December 2006. METHODOLOGY: Two hundred and one HBV infected patients were genotyped for this study. All HbsAg positive individuals, either healthy carriers or suffering from conditions such as acute or chronic hepatitis, cirrhosis and hepatocellular carcinoma were included. Hepatitis B patients co-infected with other hepatic viruses were excluded. Hepatitis B virus DNA was extracted from serum, and subjected to a nested PCR, using the primers type-specific for genotype detection. Phylogenetic analysis was performed in the pre-S1 through S genes of HBV. The divergence was studied through 15 sequences of 967 bp submitted to the DBJ/EMBL/GenBank databases accessible under accession number EF584640 through EF584654. RESULTS: Out of 201 patients tested, 156 were males and 45 were females. Genotype D was the predominant type found in 128 (64%) patients followed by A in 47 (23%) and mixed A/D in 26 (13%). Phylogenetic analysis confirmed the dominance of genotype D and subtype ayw2. CONCLUSION: There was dominance of genotype D subtype ayw2. It had a close resemblance with HBV strains that circulate in Iran, India and Japan.

Identification of hemoglobin Q India (alpha 1-64 Asp-His) through ARMS-PCR. First report from Pakistan.

Various hemoglobinopathies have been reported from Pakistan excepting the rare ones like hemoglobin Q India. Our purpose of study was to identify the mutation (alpha 1 64 aspartate to histidine) through amplification restriction mutation system-polymerase chain reaction (ARMS-PCR) in patients where hemoglobin Q has been detected via high performance liquid chromatography (HPLC) and also to evaluate the cost effectiveness of the two technologies. All patients irrespective of age and gender who underwent HPLC for identification of their hemoglobin variant during January 1, 2006 to January 30, 2007 were studied. The blood samples with unknown peak at a retention time of 4.7 min were evaluated at the molecular level. Analysis of HPLC tracings of 11,008 subjects over a thirteen-month period identified ten individuals with hemoglobin Q. Male to female ratio was 1:1.5 and their age was variable ranging from 1 to 49 (mean 22.8) years. The mean hemoglobin level was 11.3 g/dl while MCV (fl) and MCH (pg) were 73.0 and 20.8 respectively. HPLC showed an unknown peak of 17.7% which was detected as Hb Q. ARMS based PCR showed Hb Q specific product of 370 bp and also an amplified product of 766 bp as the control fragment in these samples. This is the first ever report that documents the presence of Hb Q India (alpha 64 Asp to His) in Pakistani population. We recommend that HPLC be used as a useful screening tool especially in developing countries where PCR facilities may not be accessible.