Domnita Crisan

Angiotensin I-Converting Enzyme: Genotype and Disease Associations

Cardiovascular diseases (CVD) represent a paradigm for the complex interplay of environmental risk factors and multiple genetic risk factors. Genetic abnormalities that are disease-causing, however, are less frequent than genetic factors that confer increased risk for CVD. Genetic predisposition for CVD appears to be the end result of cumulative effects of common genetic polymorphisms, which would confer only modestly increased risk when present as a single genetic risk factor. 1 Such cumulative effects may be further modulated by environmental factors. Furthermore, in patients with already diagnosed CVD, the genetic risk factors may influence the patients’ response to therapy and diet. 1, 2 Polymorphisms of the genes encoding components of the renin-angiotensin system (RAS) represent an area of intense research for cardiovascular disease associations, with promising, although sometimes contradictory reported findings.

Hepatosplenic gamma/delta T-Cell Lymphoma in Immunocompromised Patients Report of Two Cases and Review of Literature

We describe 2 male patients in whom hepatosplenic gamma/delta T-cell lymphoma (HSTL) developed 6 and 10 years after renal transplantation. The onset was abrupt with systemic symptoms, cytopenia, and hepatosplenomegaly. The histologic examination of the spleen (case 1), liver, and bone marrow revealed sinusoidal infiltrates of markedly abnormal lymphocytes. The neoplastic cells in these cases were CD2+, CD3+, CD4-, CD5-, CD7+, CD8+, CD16+, CD56+, beta F1-negative, and TIA-1-negative. Both cases displayed clonal rearrangement of the T-cell receptor (TCR) delta gene and the TCR beta gene. The spleen in case 1 was positive for Epstein-Barr virus genome and showed TCR-gamma gene rearrangement by polymerase chain reaction. Isochromosome 7 [i(7)(q10)] was found in each case. Both patients died within 4 months of diagnosis. HSTL has been reported in only 5 renal transplant recipients. HSTL may be relatively more frequent in immunocompromised patients compared with the general population.

Specimen Stability for Dna-based Diagnostic Testing

The use of molecular diagnostic testing is increasing in the clinical setting; therefore, data regarding DNA stability in clinical specimens are essential for correct test performance and interpretation. This study was designed to determine DNA stability in peripheral blood and solid tissue under different storage conditions. DNA quality and yield were assayed by spectrophotometric absorbance, gel electrophoresis, and suitability for Southern hybridization and polymerase chain reaction (PCR), the most widely employed clinical DNA analyses. A second goal of the study was to evaluate DNA stability during storage at 4°C for 1 month to 3 years. The data show that freezing or refrigeration of separated leukocytes is preferable for short- to intermediate-term storage and freezing is preferable for solid tissue. DNA degradation varying from slight to severe is seen inconsistently with such specimens, probably due to sampling of unevenly frozen-tissue areas. Depending on the degree of DNA degradation, analysis may still be possible by PCR and in some cases even by Southern hybridization. Once isolated, DNA was stable at 4°C for at least 3 years. These results suggest a more flexible approach to specimen requirements for molecular pathology, as some samples that would routinely be rejected gave interpretable results.

Separate clones in concomitant multiple myeloma and a second B-cell neoplasm demonstrated by molecular and immunophenotypic analysis

Abstract: The occurrence of multiple myeloma (MM) and a second B‐cell neoplasm in the same patient is a rare event. We present 2 such patients, and provide evidence to support the presence of separate clones in these coexisting neoplasms. In the first case, MM became evident 14 months after the diagnosis of chronic lymphocytic leukemia (CLL). In past reports, most occurrences of this association, when investigated, have been regarded to be biclonal disease processes; however, with few exceptions, most were documented by immunologic studies alone. To establish the clonality in our case of CLL with MM, we examined both immunophenotypic data obtained by standard two‐color flow cytometric analysis, and patterns of immunoglobulin gene rearrangement, using standard Southern analysis and hybridization with 32P‐labelled JH and JK probes. This provided evidence for the presence in this patient of two separate monoclonal populations of B cells, manifested as light chain restrictions and gene rearrangements which differed in blood (CLL) and bone marrow (MM) samples. In the second case, MM presented simultaneously with bone marrow lymphocytosis and abnormal peripheral lymphocytes. Clonality studies on blood were not done. Bone marrow B‐cell gene rearrangement studies, however, revealed the presence of three bands in the JK blot of significantly different intensities, suggestive of two monoclonal populations. A monoclonal population of small cells with surface B markers and surface IgM was demonstrated by flow cytometry, while a second population of larger cells with intracytoplasmic IgG matching the patients serum monoclonal protein was detected by immunofluorescence microscopy. The results in these 2 cases expand previous findings of the rare association of MM with a second B‐cell neoplasm, and demonstrate the usefulness of molecular diagnostic investigation.

Detection of Circulating Epithelial Cells After Surgery for Benign Breast Disease

AbstractBackground: Cytokeratins are predominantly expressed in epithelial cells and their malignant counterparts. Ultrasensitive methods for cytokeratin messenger RNAs (mRNAs) can detect rare circulating tumor cells consistent with hematogenous dissemination in epithelial-derived malignancies, including breast carcinomas. Intraoperative tumor-cell shedding may contribute to this process; this hypothesis is based on the assumption that only tumor cells can be mobilized during surgical manipulation. Methods and Results: The present study addresses this issue by using cytokeratin 19 mRNA detection by reverse transcription-polymerase chain reaction (RT-PCR) in preoperative and postoperative blood samples from 54 patients undergoing excisional biopsy for benign breast disease; 22 healthy volunteers represented the control group. No cytokeratin RT-PCR positivity was found in the control or preoperative samples. Cytokeratin RT-PCR positivity was found in 21 postoperative samples (39%). Conclusions: This finding shows that benign epithelial cells can be mobilized during breast surgery; this effect of surgical manipulation warrants caution in the interpretation of RT-PCR positivity for cytokeratin mRNA in the peripheral blood of patients undergoing surgery for breast cancer.

Amplification of intermediate-size DNA sequences from formalin and B-5 fixed tissue by polymerase chain reaction

Retrospective analysis of DNA from paraffin-embedded fixed bone marrow biopsy specimens is possible if preceded by amplification of the DNA sequences of interest by the polymerase chain reaction (PCR). These fixed specimens yield degraded DNA that may not be suitable for direct analysis by conventional digestion and hybridization methods. This limitation is circumvented by PCR amplification and subsequent analysis of the amplified products. The model used in this study is the amplification of a 725 base-pair (bp) beta-globin gene sequence encompassing the sickle-cell anemia point mutation, followed by Cvn I digestion. The beta A beta A, beta A beta S, and beta S beta S genotypes are derived from analysis of the allele-specific digestion patterns. Two fixatives were compared: neutral-buffered formalin and a mercury-based fixative (B-5) routinely used for bone marrow biopsies. DNA extracted from B-5-fixed bone marrow specimens was found to be more degraded than DNA from neutral-buffered, formalin-fixed bone marrow aliquots from the same specimens. PCR amplification of the 725 bp beta-globin gene sequence was successful with DNA from formalin-fixed bone marrow specimens, but not with DNA from B-5-fixed identical specimens. Analysis of the amplified product by Cvn I digestion resulted in correct genotype derivation for all patients, normal controls and positive controls (patients diagnosed with sickle-cell anemia or trait). These results indicate that intermediate-size DNA sequences can be amplified and analyzed when DNA is extracted from formalin-fixed bone marrow specimens.(ABSTRACT TRUNCATED AT 250 WORDS)

Inherited thrombophilia due to factor V Leiden mutation

Inherited thrombophilia due to activated protein C resistance is now recognized as one of the major genetic risk factors in the development of venous thromboembolic disease. Activated protein C resistance is secondary to a point mutation in the factor V gene, factor V Leiden. The high prevalence of this mutation in the general population, mainly in Caucasians of European descent, is a major contributing factor to the high incidence of venous thromboembolic disease in the United States, affecting one in 1000 individuals annually. Heterozygosity and homozygosity for factor V Leiden increase the risk for thrombosis 5- to 10-fold and 50- to 100-fold, respectively, compared with genotypically normal individuals. Factor V Leiden is more common than all other known genetic risk factors for thrombosis, and its presence results in a compounded risk in patients with simultaneous inherited abnormalities such as protein C, protein S, antithrombin III deficiencies, hyperhomocysteinemia, and/or acquired risk factors. Therefore, detection of activated protein C resistance and genotyping for factor V Leiden are important for establishing risk for thrombosis and ultimately for patient management.

Molecular Analysis in a Patient With Waldenström's Macroglobulinemia Reveals a Rare Case of Biclonality.

Background: The immunoglobulin and T-cell receptor gene rearrangement test is used to identify monoclonal populations in B- and T-cell malignancies and has become an important adjunct to morphologic analysis and immunophenotyping by flow cytometry. Waldenströms macroglobulinemia (WM) is typically a monoclonal proliferation of B cells with morphology of plasmacytoid lymphocytes and production of monoclonal IgM. Methods and Results: We report a case of WM with biclonal gammopathy (IgM kappa and IgM lambda) involving the blood and a diffuse lymphoplasmacytic infiltrate in the bone marrow in an 83-year-old man. Immunophenotyping of the blood and bone marrow aspirate revealed B cells expressing IgM lambda surface immunoglobulins and CD5+, CD19+, and CD20+ surface markers. Gene-rearrangement analysis with the Southern blot technique revealed multiple rearranged bands in each lane of restricted patient DNA after probing with both immunoglobulin heavy (J(H))- and light (J(kappa))-chain gene probes. Conclusions: Biclonal gammopathy in WM and biclonal B-cell proliferations as determined by gene-rearrangement studies are rare entities, and few evaluations of them are reported in the literature. To our knowledge, this case is the first one of biclonal WM to have been evaluated by serum protein immunofixation, immunohistologic staining, immunophenotyping by flow cytometry, and immunoglobulin gene-rearrangement analysis.

Blastic transformation in a case of hairy cell leukemia

Background: Hairy cell leukemia (HCL) is a slowly progressive lymphoproliferative disorder that tends to afflict middle-aged adults, especially men. Blastic transformation of this form of leukemia is extremely rare. To date, a single case has been reported. Methods and Results: A case of HCL, evolving with blastic transformation after a 9- year clinical course, is reported. Routine histology, cytochemistry, flow cytometry immunophenotyping, and Southern blot analysis for B- and T-cell gene rearrangements were used in the evaluation. Although morphology at the time of presentation was characteristic of HCL, the cells were initially tartrate-resistant acid phosphatase (TRAP) negative. During a clinical course over several years, the hairy cells became progressively TRAP positive. The morphology of the leukemic cells changed 9 years after initial diagnosis, with blastic transformation and retaining strong TRAP positivity. Immunophenotypic analysis showed evolution from a characteristic hairy cell leukemic phenotype to a phenotype indicative of marked immaturity. Genotypic analysis showed an evolving pattern of immunoglobulin gene rearrangements, paralleling the morphology and phenotypic evolution and ruling out a second B-cell malignancy. Conclusions: This case report of blastic transformation in a patient with HCL is only the second such case identified in the medical literature to date.

FLT3 mutations in myeloproliferative neoplasms: the Beaumont experience.

FLT3 is one of the most frequently mutated genes in acute myeloid leukemia. Previous studies have reported FLT3 mutation in as many as 9.2% of myeloproliferative neoplasms (MPNs) and myelodysplastic/myeloproliferative neoplasms (MDS/MPNs), as well as in chronic myelogenous leukemia, that are negative for the JAK2 V617F gene mutation; no FLT3 mutation has been found in JAK2-positive MPNs, suggesting that the mutations are mutually exclusive. The goal of our study is to evaluate the mutational status of the FLT3 gene in patients with an MPN or MDS/MPN, in correlation with the JAK2 mutational status. Patient specimens were retrospectively identified on the basis of MPN or MDS/MPN diagnosis and JAK2 analysis from February 2006 to December 2011. FLT3 mutation analysis was performed on DNA extracted from 152 patients using polymerase chain reaction amplification and analysis of amplicons by gel electrophoresis for internal tandem duplication mutations and by restriction endonuclease digestion fragment analysis for the D835 point mutation. FLT3 mutation analysis was performed on 90 cases of JAK2-negative MPN or MDS/MPN and 62 cases of JAK2-positive MPN. One FLT3 internal tandem duplication mutation was identified in the JAK2-negative group (1.1%), and none were identified in the JAK2-positive group, confirming the absence of FLT3 mutations in JAK2-positive specimens. The FLT3-positive MPN patient was diagnosed with MPN, unclassifiable, and was later found to have myeloid sarcoma; thus, FLT3 mutation was not seen in the usual types of MPN in our series. Our result of 1.1% FLT3 mutations in JAK2-negative MPN and MDS/MPN cases is lower than the 9.2% previously reported.