Expression of EBV Encoded viral RNA 1, 2 and anti-inflammatory Cytokine (interleukin-10) in FFPE lymphoma specimens: a preliminary study for diagnostic implication in Pakistan

<p>Abstract</p> <p>Background</p> <p>Epstein Barr Virus (EBV) plays a significant role as a cofactor in the process of tumorigenesis and has consistently been associated with a variety of malignancies. EBV encoded RNAs (EBER1 and EBER2) are the most abundant viral transcripts in latently EBV-infected cells and their role in viral infection is still unclear. Formalin Fixed Paraffin Embedded (FFPE) tissues of surgically removed carcinoma biopsies are widely available form but have never been exploited for expressional studies previously in Pakistan. Immunohistochemistry (IHC) and <it>in situ </it>hybridization (ISH) in FFPE biopsy tissues remains the gold standard for proving EBV relationship in a histopathological lesion but their reagents associated limitations confines their reliability in some applications. Recently introduced targeted drug delivery systems induce viral lytic gene expression and therefore require more sensitive method to quantify viral as well as cellular gene expression.</p> <p>Methods</p> <p>Eight (8) lymphoma samples were screened to detect the EBV genome. Qualitative and quantitative expression of EBV Encoded RNAs (EBER1, EBER2) and anti-inflammatory cytokine (interleukin-10) in FFPE EBV positive lymphoma tissue samples were then analysed by using Reverse transcriptase Polymerase Chain Reaction (RT-PCR) and Real Time Polymerase Chain Reaction (qRT-PCR), respectively.</p> <p>Results</p> <p>In this study we have successfully quantified elevated expressional levels of both cellular and viral transcripts, namely EBER1, EBER2 and anti-inflammatory cytokine (IL-10) in the FFPE Burkitt's lymphoma (BL) specimens of Pakistani origin.</p> <p>Conclusions</p> <p>These results indicate that FFPE samples may retain viral as well as cellular RNA expression information at detectable level. To our knowledge, this is first study which represents elevated expressional levels of EBER1, EBER2 and IL-10 in FFPE tissue samples of Burkitt's lymphoma in Pakistan. These observations will potentially improve current lacunas in clinical as well as diagnostic practices in Pakistan and can be further exploited to develop new strategies for studying cellular and/or viral gene expression.</p

Immunophenotyping of mature T/NK cell neoplasm presenting as leukemia

Introduction : Mature T/NK cell lymphomas (MTNKL) presenting as leukemia are rare and show considerable overlapping of clinical, morphological and immunophenotypic features. AIM: Critical analysis of the morphology and immunophenotypic profile of MTNKL. Materials and Methods : We reviewed 380 consecutive cases of mature lymphoid neoplasm that presented as leukemia and were diagnosed on morphology and immunophenotyping of bone marrow and/or peripheral blood samples. Results : Peripheral blood and bone marrow involvement was seen in all cases. MTNKL constituted 4% (nine cases) of all mature lymphoid neoplasms presenting as leukemia. It included four cases of T-large granular leukemia (T-LGL), two of T-cell prolymphocytic leukemia small cell variant (T-PLL), two of adult T-cell leukemia/lymphoma (ATLL) and one of primary cutaneous gamma delta T-cell lymphoma (PCGDTCL). T-LGL revealed CD4-/CD8+ phenotype in three, and CD4+/CD8+ phenotype in one case. CD56 was absent in all the cases of T-LGL. One case of T- PLL small cell variant showed CD4+/CD8- phenotype, while the other revealed CD4-/CD8+ phenotype. Both cases of ATLL showed CD4+/CD8+/CD25+ phenotype. The single case of PCGDTCL showed CD4-/CD8- phenotype pattern. CD3 and CD5 were expressed in all MTNKL. CD7 was absent in three cases of T-LGL. TCR\u3b1/\u3b2 was performed in three cases of T-LGL and was positive in all. TCR\u3b1/\u3b2 was also seen in both the cases of T-PLL small variant. However, TCR\u3b1/\u3b2 was seen in the single case of PCGDTCL. Conclusion : Mature nodal T/NK cell neoplasms are rare and MTNKL presenting as leukemia are even rarer. There is an overlap between the immunophenotypic profiles of different MTNKL subtypes and elaborate T/NK cell panels are required for their evaluation

Role of RT-PCR and FISH in diagnosis and monitoring of acute promyelocytic leukemia

Background: Patients with a presence of Promyelocytic Leukemia-Retinoic Acid Receptor Alpha (PML-RARA) genes rearrangement predict a favorable response to all-trans retinoic acid (ATRA), and a significant improvement in survival. Therefore, establishing the presence of PML-RARA rearrangement is important for optimal patient management. Aim: The objective of this study is to compare and assess the role of fluorescent in situ hybridization (FISH) and reverse transcriptase polymerase chain reaction (RT-PCR) in the diagnosis and long-term monitoring of Acute Promyelocytic Leukemia (APL). Materials and Methods: We compared 145 samples received at different interval of times to analyze the sensitivity of RT-PCR and FISH. Results: The failure rate for RT-PCR was 4% at baseline, 13% at induction, and 0% at the end of consolidation. And for FISH it was 8% at baseline, 38% at induction, and 66% at the end of consolidation. The predictive values of relapse in the patients who were positive and negative by RT-PCR, at the end of induction, were 60 % and 3%, respectively, and at end of consolidation it was 67 % and 4%, respectively. On the other hand the predictive values of relapse in patients who were positive and negative by FISH at end of induction were 57 % and 6%, respectively; while at end of consolidation it was 14% who were negative by FISH. Conclusion: Both RT-PCR and FISH are important for the diagnosis of APL cases, as both techniques complement each other in the absence or failure of any one of them. However, RT-PCR is more sensitive than FISH for the detection of minimal residual disease in the long-term monitoring of these patients. The present study shows that the predictive value of relapse is more associated with minimal residual disease (MRD) results by RT-PCR than that by FISH

Clinico-Hematological Profile in Biphenotypic Acute Leukemia

Background : We present a clinico-hematological profile and treatment outcome of Biphenotypic Acute Leukemia (BAL). Aim : Study incidence and subtypes of BAL, correlate with age, morphology, and cytogenetic findings and correlate the clinico-hematological data with the treatment response. St Jude\u2032s and the EGIL\u2032s criteria have been compared for their diagnostic and clinical relevance. Material and Methods : Diagnosis was based on WHO classification, including clinical details, morphology, cytochemistry, immunophenotyping, and molecular genetics. We included those cases, which fulfilled the European Group for the Immunological Characterization of Acute Leukemia\u2032s (EGIL\u2032s) scoring system criteria for the diagnosis of BAL, as per recommendation of the WHO classification. Results : There were 32 patients diagnosed with BAL, based on EGIL\u2032s criteria. Incidence of BAL was 1.2%. B-Myeloid (14 cases) followed by T-Myeloid BAL (13 cases) were the commonest subtypes. Polymorphous population of blasts (16 cases) was commonly associated with T-Myeloid BAL (10 cases). BCR ABL fusion positivity was a common cytogenetic abnormality (seven cases). Fifteen patients received chemotherapy; eight achieved complete remission (CR) at the end of the induction period. Conclusions : Pediatric BAL and T-B lymphoid BAL have a better prognosis. A comprehensive panel of reagents is required, including cytoplasmic markers; to diagnose BAL. St Jude\u2032s criteria is a simple, easy, and cost-effective method to diagnose BAL. The outcome-related prognostic factors include age, HLA-DR, CD34 negativity, and subtype of BAL. BCR-ABL expression is an important prognostic factor, as these cases will be labeled as Chronic myeloid leukemia (CML) in blast crisis with biphenotypic expression and treated accordingly

Clinico-Hematological Profile in Biphenotypic Acute Leukemia

Background : We present a clinico-hematological profile and treatment outcome of Biphenotypic Acute Leukemia (BAL). Aim : Study incidence and subtypes of BAL, correlate with age, morphology, and cytogenetic findings and correlate the clinico-hematological data with the treatment response. St Jude′s and the EGIL′s criteria have been compared for their diagnostic and clinical relevance. Material and Methods : Diagnosis was based on WHO classification, including clinical details, morphology, cytochemistry, immunophenotyping, and molecular genetics. We included those cases, which fulfilled the European Group for the Immunological Characterization of Acute Leukemia′s (EGIL′s) scoring system criteria for the diagnosis of BAL, as per recommendation of the WHO classification. Results : There were 32 patients diagnosed with BAL, based on EGIL′s criteria. Incidence of BAL was 1.2%. B-Myeloid (14 cases) followed by T-Myeloid BAL (13 cases) were the commonest subtypes. Polymorphous population of blasts (16 cases) was commonly associated with T-Myeloid BAL (10 cases). BCR ABL fusion positivity was a common cytogenetic abnormality (seven cases). Fifteen patients received chemotherapy; eight achieved complete remission (CR) at the end of the induction period. Conclusions : Pediatric BAL and T-B lymphoid BAL have a better prognosis. A comprehensive panel of reagents is required, including cytoplasmic markers; to diagnose BAL. St Jude′s criteria is a simple, easy, and cost-effective method to diagnose BAL. The outcome-related prognostic factors include age, HLA-DR, CD34 negativity, and subtype of BAL. BCR-ABL expression is an important prognostic factor, as these cases will be labeled as Chronic myeloid leukemia (CML) in blast crisis with biphenotypic expression and treated accordingly

Immunophenotyping of mature T/NK cell neoplasm presenting as leukemia

Introduction : Mature T/NK cell lymphomas (MTNKL) presenting as leukemia are rare and show considerable overlapping of clinical, morphological and immunophenotypic features. AIM: Critical analysis of the morphology and immunophenotypic profile of MTNKL. Materials and Methods : We reviewed 380 consecutive cases of mature lymphoid neoplasm that presented as leukemia and were diagnosed on morphology and immunophenotyping of bone marrow and/or peripheral blood samples. Results : Peripheral blood and bone marrow involvement was seen in all cases. MTNKL constituted 4% (nine cases) of all mature lymphoid neoplasms presenting as leukemia. It included four cases of T-large granular leukemia (T-LGL), two of T-cell prolymphocytic leukemia small cell variant (T-PLL), two of adult T-cell leukemia/lymphoma (ATLL) and one of primary cutaneous gamma delta T-cell lymphoma (PCGDTCL). T-LGL revealed CD4-/CD8+ phenotype in three, and CD4+/CD8+ phenotype in one case. CD56 was absent in all the cases of T-LGL. One case of T- PLL small cell variant showed CD4+/CD8- phenotype, while the other revealed CD4-/CD8+ phenotype. Both cases of ATLL showed CD4+/CD8+/CD25+ phenotype. The single case of PCGDTCL showed CD4-/CD8- phenotype pattern. CD3 and CD5 were expressed in all MTNKL. CD7 was absent in three cases of T-LGL. TCRα/β was performed in three cases of T-LGL and was positive in all. TCRα/β was also seen in both the cases of T-PLL small variant. However, TCRα/β was seen in the single case of PCGDTCL. Conclusion : Mature nodal T/NK cell neoplasms are rare and MTNKL presenting as leukemia are even rarer. There is an overlap between the immunophenotypic profiles of different MTNKL subtypes and elaborate T/NK cell panels are required for their evaluation