Wengang Chen

Lack of association of cytomegalovirus with human brain tumors

Cytomegalovirus (CMV) is thought to possess oncogenic properties and has been linked with a number of human malignancies. CMV infection was recently described in association with malignant gliomas. The intent of the present study was to further investigate the reported association between CMV and malignant gliomas. Tissue from 22 brain tumors of various histologic types and grades, four normal brains, six breast carcinomas, six colon carcinomas, six lung carcinomas, and six sarcomas were evaluated for the presence of CMV by polymerase chain reaction (PCR), in situ hybridization, and immunohistochemical methods. None of the brain tumors or normal brain tissue tested demonstrated evidence of CMV pp65 or early nuclear proteins by immunohistochemistry. In addition, no CMV RNA or DNA was detected in these cases by in situ hybridization and PCR. None of the carcinomas or sarcomas evaluated were positive for CMV by immunohistochemistry, in situ hybridization, or PCR. The findings of the present study suggest that CMV is not significantly associated with brain tumors in humans.

No Significant Association of Epstein-Barr Virus Infection with Invasive Breast Carcinoma

We studied 48 cases of invasive breast carcinoma for evidence of Epstein-Barr virus (EBV), which is associated with many human malignancies. In situ hybridization studies to detect the presence of EBV-encoded small nonpolyadenylated RNA (EBER)-1 were performed in paraffin sections. Immunohistochemical studies to detect EBV nuclear antigen (EBNA)-1, latent membrane protein (LMP)-1, and the transactivating immediate-early BZLF1 (ZEBRA) protein were also performed in paraffin sections. The presence of EBV genomic DNA was studied by polymerase chain reaction (PCR) amplification using sets of primers flanking the EBNA-4 and the EBV-LMP-1 genes in frozen tissues. Southern blot analysis using a probe flanking the EBV terminal repeat region was then attempted in cases that were PCR-positive. Five of 48 cases (10%) of breast carcinoma showed focal EBER-positive tumor cells. Twelve cases (25%) were positive for EBNA-1 by immunohistochemistry, all but one different from the EBER-positive cases. None of the cases were positive for LMP-1 or ZEBRA protein by immunohistochemistry. PCR studies for EBNA-4 and LMP-1 were each positive in five cases (including three cases in common). However, Southern blot studies successfully performed in all but one of the PCR-positive cases were completely negative. The identification of EBV by any methodology was not correlated with tumor size, grade, or lymph node status. This study demonstrated evidence of EBV infection in tissues involved by invasive breast carcinomas in a significant subset of cases. However, the lack of localization of EBV infection to a significant population of the tumor cells in any case, the negativity by Southern blot hybridization, and the lack of expression of multiple antigens in any case strongly argue against a significant role for EBV in the pathogenesis of breast carcinoma.

Deletion of Epstein-Barr virus latent membrane protein 1 gene in united states and Brazilian Hodgkin's disease and reactive lymphoid tissue: High frequency of a 30-bp deletion

A 30-basepair (bp) deletion in the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) gene has been reported in nasopharyngeal carcinoma and EBV-associated malignant lymphomas. Prior studies have found the deletion in about 10% to 28% of cases of Hodgkins disease (HD), particularly in cases with aggressive histology. We studied the prevalence of 30-bp LMP1 gene deletion in EBV-positive HD in the United States (US) (12 cases) and Brazil (26 cases) with comparison to reactive lymphoid tissues (21 cases) and HD without EBV-positive Reed-Sternberg cells (15 cases). We studied the status of the LMP1 gene by Southern blot hybridization of polymerase chain reaction (PCR) products obtained after amplification with primers spanning the site of the deletion. We also performed EBV typing, EBER1 in situ hybridization, and LMP1 protein immunohistochemistry. EBV was detected in 12/26 (46%) cases of HD from the US and 26/27 (96%) cases of Brazilian HD. The 30-bp LMP1 gene deletion was observed in 4/12 (33%) cases of EBV-positive HD from US, and 12/26 (46%) cases of Brazilian EBV-positive HD, including 3 cases of type B EBV, as compared with 12/21 (57%) reactive lymphoid tissues and 9/15 (60%) cases of EBV-negative HD. US and Brazilian HD showed a higher prevalence of the 30-bp LMP1 gene deletion, compared with studies of others. The unexpected finding of high incidence of 30-bp deletion in LMP1 gene in reactive lymphoid tissue and HD without EBV-positive Reed-Sternberg cells suggests that this deletion may not be relevant to HD pathogenesis in most cases.

A rapid, one step assay for simultaneous detection of FLT3/ITD and NPM1 mutations in AML with normal cytogenetics

Baglin, T., Barrowcliffe, T.W., Cohen, A. & Greaves, M. (2006) Guidelines on the use and monitoring of heparin. British Journal of Haematology, 133, 19–34. Buller, H.R., Davidson, B.L., Decousus, H., Gallus, A., Gent, M., Piovella, F., Prins, M.H., Raskob, G., van den Berg-Segers, A.E., Cariou, R., Leeuwenkamp, O. & Lensing, A.W. (2003) Subcutaneous fondaparinux versus intravenous unfractionated heparin in the initial treatment of pulmonary embolism. New England Journal of Medicine, 349, 1695–1702. Fiessinger, J.N., Lopez-Fernandez, M., Gatterer, E., Granqvist, S., Kher, A., Olsson, C.G. & Soderberg, K. (1996) Once-daily subcutaneous dalteparin, a low molecular weight heparin, for the initial treatment of acute deep vein thrombosis. Thrombosis and Haemostasis, 76, 195–199. Kovacs, M.J., Anderson, D., Morrow, B., Gray, L., Touchie, D. & Wells, P.S. (2000) Outpatient treatment of pulmonary embolism with dalteparin. Thrombosis and Haemostasis, 83, 209–211. Lindmarker, P., Holmstrom, M., Granqvist, S., Johnsson, H. & Lockner, D. (1994) Comparison of once-daily subcutaneous Fragmin with continuous intravenous unfractionated heparin in the treatment of deep vein thrombosis. Thrombosis and Haemostasis, 72, 186–190. Meyer, G., Brenot, F., Pacouret, G., Simonneau, G., Gillet Juvin, K., Charbonnier, B. & Sors, H. (1995) Subcutaneous low-molecularweight heparin fragmin versus intravenous unfractionated heparin in the treatment of acute non massive pulmonary embolism: an open randomized pilot study. Thrombosis and Haemostasis, 74, 1432–1435. Simonneau, G., Sors, H., Charbonnier, B., Page, Y., Laaban, J.P., Azarian, R., Laurent, M., Hirsch, J.L., Ferrari, E., Bosson, J.L., Mottier, D. & Beau, B. (1997) A comparison of low-molecularweight heparin with unfractionated heparin for acute pulmonary embolism. The THESEE Study Group. Tinzaparine ou Heparine Standard: Evaluations dans l’Embolie Pulmonaire. New England Journal of Medicine, 337, 663–669. The Columbus Investigators (1997) Low-molecular-weight heparin in the treatment of patients with venous thromboembolism. New England Journal of Medicine, 337, 657–662. Wells, P.S., Anderson, D.R., Rodger, M.A., Forgie, M.A., Florack, P., Touchie, D., Morrow, B., Gray, L., O’Rourke, K., Wells, G., Kovacs, J. & Kovacs, M.J. (2005) A randomized trial comparing 2 lowmolecular-weight heparins for the outpatient treatment of deep vein thrombosis and pulmonary embolism. Archives of Internal Medicine, 165, 733–738.

Implant-Associated Primary Anaplastic Large-Cell Lymphoma With Simultaneous Involvement of Bilateral Breast Capsules

Introduction Primary anaplastic large cell lymphoma (ALCL) associated with breast implants is a rare and usually indolent T-cell lymphoproliferative disorder first described by Keech and Creech in 1997. Currently, > 100 occurrences have been reported worldwide, with approximately 60 cases described in the literature. The incidence of this disease is estimated at 0.1 to 0.3 per 100,000 women with breast implants per year. Most patients have unilateral breast involvement by ALCL, with only rare cases having bilateral involvement, and 1 reported patient in whom subsequent bilateral axillary lymph node metastases developed. In 2011, the United States Food and Drug Administration recognized the possible association of ALCL with breast prostheses.

Low frequency of BK virus in prostatic adenocarcinomas

BK virus (BKV) exhibits many oncogenic properties and has been associated with a variety of tumors in humans. BKV has not been well studied in the context of prostate neoplasia; however, an association of BKV with prostatic adenocarcinoma has been suggested based on the detection of viral DNA sequences and expression of viral proteins in clinical samples. To further investigate the reported association of BKV with prostatic adenocarcinoma and the potential role of the virus in prostate tumorigenesis, 30 cases of adenocarcinoma of the prostate were analyzed for evidence of BKV infection by in situ hybridization and immunohistochemistry. In situ hybridization analysis detected BKV DNA in 2 of 30 (7%) prostatic adenocarcinomas, with positive signals focally identified in less than 1% of the neoplastic cells in both cases. However, none of the tumors evaluated demonstrated evidence of BKV large tumor antigen expression by immunohistochemistry. Among prostatic adenocarcinomas that showed no evidence of BKV infection, BKV DNA was focally observed in the adjacent non‐neoplastic prostate tissue in four cases by in situ hybridization in the absence of BKV large tumor antigen immunoreactivity. The findings of the present study indicate rare cases of prostatic adenocarcinoma may be associated with BKV infection. However, lack of localization of BKV to a large population of the neoplastic cells and absence of BKV large tumor antigen expression suggest that the virus does not play a role in the pathogenesis of prostate cancer.

Coexisting Thymic and Gastric Lymphomas of Mucosa-Associated Lymphoid Tissues in a Patient With Sjogren Syndrome

Lymphomas of mucosa-associated lymphoid tissues (MALTomas) arising from the thymus are extremely rare. In this case report, we describe a 36-year-old woman with an 11-year history of Sjögren syndrome who was found to have a thymic MALToma coexisting with a gastric MALToma. Both tumors shared similar histologic features, showing clusters of centrocytic-like B cells, lymphoepithelial lesions, and prominent plasmacytic differentiation. They also showed the following identical immunohistochemical features: CD20(+), IgA/lambda(+), CD5(-), and CD43(-). Molecular studies using polymerase chain reaction methods revealed monoclonal gene rearrangement of the immunoglobulin heavy chain in the gastric MALToma, but not in the thymic MALToma. The possible pathogenesis of this unusual case is discussed.

C/EBPA gene mutation and C/EBPA promoter hypermethylation in acute myeloid leukemia with normal cytogenetics†‡

In the current study, we investigated C/EBPA gene mutations and promoter hypermethylation in a series of 53 patients with CN‐AML. In addition, we also analyzed two other frequent mutations (FLT3/ITD and NPM1) in these patients and correlated them with C/EBPA gene alterations. 13/53 patients were FLT3/ITD+/NPM1‐, 11/53 patients were FLT3/ITD+/NPM1+, 9/53 patients were FLT3/ITD‐/NPM1+, and 20/53 patients were FLT3/ITD‐/NPM1‐. Four of 53 cases displayed C/EBPA mutations, whereas 49 cases had only C/EBPA wild‐type alleles. Of the four positive cases, three patients had N‐terminal mutations only, whereas one patient had mutations in both the N‐ and C‐terminal region. Two of the four positive cases also harbored both FLT3/ITD and NPM1 mutation simultaneously, whereas the other two patients had neither FLT3/ITD nor NPM1 mutations. Furthermore, 7/53 cases displayed C/EBPA promoter hypermethylation. Interestingly, they were all in CN‐AML cases without FLT3/ITD or NPM1 mutations. None of the seven patients with C/EBPA promoter hypermethylation showed C/EBPA mutation. In conclusion, C/EBPA mutation and promoter hypermethylation can be detected at a relatively low frequency in de novo CN‐AML patients, suggesting they may contribute to leukemogenesis. C/EBPA mutation appears to be seen in “high‐risk” AML (FLT3/ITD+/NPM1+; FLT3/ITD+/NPM1‐ or FLT3/ITD‐/NPM1‐), while C/EBPA hypermethylation appears to be more common in AML with FLT3/ITD‐ /NPM1‐ and is not associated with C/EBPA mutation. Am. J. Hematol. 2010.

The use of archival bone marrow specimens in detecting B-cell non-Hodgkin's lymphomas using polymerase chain reaction methods.

The detection of B-cell non-Hodgkins lymphoma (B-NHL) involving the bone marrow (BM) can be enhanced by assessing immunoglobulin heavy chain (IgH/JH) gene rearrangement using PCR. While the fresh BM aspirate has been the most commonly used specimen, the utility of archival BM tissues has not been extensively evaluated. We studied the BM from 13 patients with nodal B-NHL (7 low-grade and 6 intermediate grade), which were categorized into three groups based on the histologic finding of lymphoma (H) and the presence of a monoclonal IgH/JH band by PCR using fresh BM aspirates (M): (1) H(+)/M(+), 4 cases; (2) H(+)/M(-), 4 cases; and (3) H(equivocal)/M(-), 5 cases. Archival tissues available for study included paraffin-embedded trephine biopsy (TB)/aspirate clots (AC) and air-dried aspirate smears (AS). All TB (13/13) and a subset of AC (5/13) were B5-fixed, and all these tissues failed to yield analyzable DNA. In contrast, sufficient DNA was consistently obtained in AC that were formalin-fixed (8/13). Of these 8 cases, 2/3 of group 1, 3/3 of group 2, and 0/2 of group 3 had a monoclonal IgH band. Using DNA extracted from microdissected lymphoid aggregates morphologically evident in the AC sections, additional positive cases were identified: 1/3 of group 1 and 2/2 of group 3. In those 5 cases that did not have formalin-fixed TB/AC, sufficient DNA was extracted from AS in all cases; one additional positive case was identified in group 1. Overall, 4/4 (100%) of group 1, 3/4 (75%) of group 2, and 2/5 (40%) of group 3 showed molecular evidence of lymphoma. To conclude, archival BM specimens are a useful source of DNA for molecular detection of B-NHL involvement, and formalin appears to be a better fixative than B5. The use of these samples may improve the overall detection sensitivity.

Absence of JAK-2V617F point mutations in multiple myeloma.

Multiple myeloma (MM) is a terminal differentiated B-cell chronic lymphoproliferative disorder, characterized by the latent accumulation of plasma cells with a low proliferative index and an extended lifespan in bone marrow or extramedullary tissues. Studies on neoplastic cells isolated directly from MM patients or on MM-derived cell lines demonstrated that interleukin (IL)-6 is a major growth and survival factor for the initiation of signaling in MM cells in an autocrine or paracrine manner. However, some MM cells independent of on extracellular signaling such as IL-6 have been reported, suggesting other mechanisms may be involved in signal transduction pathways as well as pathogenesis in MM cells (Huang et al., Blood. 2000; 96: 3241, abstract). Accumulating evidence showed that IL-6 induces intracellular signaling through members of the signal transducers and activators of transcription (STAT) family of proteins by activating the Janus tyrosine kinase (JAK) family of protein tyrosine kinases, which subsequently phosphorylate and activate cytoplasmic STAT proteins. Activated STAT proteins dimerize and translocate to the nucleus, where they bind to specific DNA response elements and induce expression of STAT-regulated genes. Previous studies demonstrated that one of the STAT family proteins, STAT3, is being constitutively activated in the majority of MM patients, suggesting constitutively activated JAK-2 kinase activities upon extracellular IL-6 stimulation. However, other mechanisms may also be responsible for constitutive JAK2 activation in MM cells, in particular in IL-6-independent MM cells (Huang et al. Blood. 2000; 96: 3241, abstract). Recently, an aquired somatic mutation in exon 12 of JAK2 gene has been described in higher frequency of centain myeloproliferative diseases: Philadelphia chromosome negative as polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis. More recently this mutation has also been identified in lower frequency in acute myeloid leukemia, myelodysplastic syndrome and in atypical chronic myeloid leukemia (BCR-ABL negative). This point mutation results in a substitution of valine for phenylalanine at position 617 (V617F) in the JH2 domain of the gene, and leads to the constitutive tyrosine phosphorylation and cytokine hypersensitivity. Neoplastic cells carrying this mutation can be heterozygous or hemizygous, if they have loss of heterozygosity of 9p chromosome by mitotic recombination, where JAK2 is located. The point mutation yields constitutive activation of JAK2 kinase activity in the presence or absence of extracellular signaling and may result in the pathogenesis of those disorders. Furthermore, previous study showed that V617F JAK2 is a subtle mutation that induces a low gain of function in JAK2. There is increasing evidence that its activity requires the presence of cytokine receptors to induce signaling. In this study, we determine whether JAK-2 (Val617Phe) point mutation was present in MM patients by using an allele-specific polymerase chain reaction followed by separation and detection with capillary electrophoresis. The test has a detection sensitivity of