Lynn Abbott

Prognostic Significance of K-ras Codon 12 Mutations in Patients With Resected Stage I and II Non–Small-Cell Lung Cancer

PURPOSE The aim of this study was to investigate the prognostic importance of codon 12 K-ras mutations in patients with early-stage non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS We identified 260 patients with surgically resected stage I (n = 193) and stage II (n = 67) NSCLC with at least a 5-year follow-up. We performed polymerase chain reaction analysis of DNA obtained from paraffin-embedded NSCLC tissue, using mutation-specific probes for codon 12 K-ras. RESULTS K-ras mutations were detected in 35 of 213 assessable specimens (16.4%). K-ras mutations were detected in 27 of 93 adenocarcinomas (29.0%), one of 61 squamous cell carcinomas (1.6%), five of 39 large-cell carcinomas (12.8%), and two of 20 adenosquamous carcinomas (10%) (P = .001). G to T transversions accounted for 71% of the mutations. There was no statistically significant difference in overall survival for all patients with K-ras mutations (median survival, 39 months) compared with patients without K-ras mutations (median survival, 53 months; P = .33). There was no statistically significant difference in overall or disease-free survival for subgroups with stage I disease, adenocarcinoma, or non-squamous cell carcinoma or for specific amino acid substitutions. The median survival time for stage II patients with K-ras mutations was 13 months, compared with 38 months for patients without K-ras mutations (P = .03). CONCLUSION Codon 12 K-ras mutations were more common in adenocarcinomas than in squamous cell carcinomas. For the subgroup with stage II NSCLC, there was a statistically significant adverse effect on survival for the presence of K-ras mutations. However, when the entire group was considered, the presence of K-ras mutations was not of prognostic significance in this cohort of patients with resected early-stage NSCLC.

The complete genomic sequence of an in vivo low replicating BLV strain

DNA was extracted from lamb lymphocytes that were infected in vivo with a BLV strain after inoculation with the peripheral blood mononuclear cells from a persistently sero-indeterminate, low viral load, BLV-infected Holstein cow (No. 41) from Argentina. The DNA was PCR amplified with a series of overlapping primers encompassing the entire BLV proviral DNA. The amplified BLV ARG 41 DNA was cloned, sequenced, and compared phylogenetically to other BLV sequences including an in vivo high replicating strain (BLV ARG 38) from the same herd in Argentina. Characterization of BLV ARG 41s deduced proteins and its relationship to other members of the PTLV/BLV genus of retroviruses are discussed.

Expression of Tropomyosin 1 Gene Isoforms in Human Breast Cancer Cell Lines

Nine malignant breast epithelial cell lines and 3 normal breast cell lines were examined for stress fiber formation and expression of TPM1 isoform-specific RNAs and proteins. Stress fiber formation was strong (++++) in the normal cell lines and varied among the malignant cell lines (negative to +++). Although TPM1γ and TPM1δ were the dominant transcripts of TPM1, there was no clear evidence for TPM1δ protein expression. Four novel human TPM1 gene RNA isoforms were discovered (λ, μ, ν, and ξ), which were not identified in adult and fetal human cardiac tissues. TPM1λ was the most frequent isoform expressed in the malignant breast cell lines, and it was absent in normal breast epithelial cell lines. By western blotting, we were unable to distinguish between TPM1γ, λ, and ν protein expression, which were the only TPM1 gene protein isoforms potentially expressed. Some malignant cell lines demonstrated increased or decreased expression of these isoforms relative to the normal breast cell lines. Stress fiber formation did not correlate with TPM1γ RNA expression but significantly and inversely correlated with TPM1δ and TPM1λ expression, respectively. The exact differences in expression of these novel isoforms and their functional properties in breast epithelial cells will require further study.

CD8+ cutaneous T-cell lymphoma successfully treated with bexarotene: a case report and review of the literature.

CD8+ cutaneous T‐cell lymphoma (CTCL) is a relatively rare subset of the non‐Hodgkins lymphomas. Bexarotene has been FDA‐approved for the treatment of CTCL, but previous studies have been conducted on CD4+ CTL and there have been no reports about its use in CD8+ CTCL. Herein, we report on a patient whose CD8+ CTCL completely responded to treatment with bexarotene. Am. J. Hematol., 2008.

Absence of Human Herpes Virus 8 DNA Sequences in Large Granular Lymphocyte (LGL) Leukemia

The etiology of large granular lymphocyte (LGL) leukemia is uncertain. Recently, a Kaposis sarcoma-associated herpes virus, denoted as human herpes virus 8 (HHV-8), has been identified. Some data suggest that HHV-8 and Epstein-Barr virus (EBV) may interact to induce malignant transformation. Infection with EBV has been implicated in the pathogenesis of some cases of LGL leukemia. Therefore, we performed PCR analyses for HHV-8 detection in samples from nineteen patients with LGL leukemia; three of these samples contained the EBV genome. We could not detect HHV-8 sequences in any of these patients. Therefore, HHV-8 infection is not involved in the pathogenesis of T-LGL leukemia.

Short Communication: No Evidence of HTLV-3 and HTLV-4 Infection in New York State Subjects at Risk for Retroviral Infection

The primate T-cell lymphoma viruses (PTLV) are divided into six distinct species. The biology and epidemiology of PTLV-1 and PTLV-2 are very well understood. However, that of PTLV-3, 4, 5, and 6 are not. Recently, in Cameroon, three and one humans were shown to be infected with HTLV-3 and HTLV-4, respectively. We undertook a study to ascertain whether any of these two retroviruses were present in the peripheral blood mononuclear cell DNA of New York State subjects deemed at risk for PTLV infection. Samples were analyzed by PTLV-3 and PTLV-4 specific PCR assays from the following human and simian subject types: African-American medical clinic patients; HTLV EIA+, WB indeterminate blood donors; intravenous drug users; patients with leukemia, lymphoma, myelopathy, polymyositis, or AIDS; and African chimpanzees. None of the 1200 subjects was positive for HTLV-3 or 4. The data indicate that, at the time of sample collection, no evidence exists for the dissemination of HTLV-3 or 4 to New York State. Continued epidemiological studies are warranted to explore the worldwide prevalence rates and dissemination patterns of HTLV-3 and 4 infections, and their possible disease associations.

Increased seroreactivity to HERV-K10 peptides in patients with HTLV myelopathy.

BackgroundPreviously, we had shown that persons infected with human T-cell lymphoma leukemia virus 1 or 2 (HTLV-1 or 2) had an increased prevalence of antibodies to a peptide in the Pol protein of the retrovirus HERV-K10, homologous to a peptide in HTLV gp21 envelope protein. The prevalence rate was higher in those with myelopathy vs. non-myelopathy. We have now extended our observations to a cohort restricted to North America in whom the diagnosis of HTLV myelopathy was rigorously confirmed to also test for reactivity to another HERV-K10 peptide homologous to the HTLV p24 Gag protein.MethodsSera from 100 volunteer blood donors (VBD), 53 patients with large granular lymphocytic leukemia (LGLL), 74 subjects with HTLV-1 or 2 infection (58 non-myelopathy and 16 myelopathy) and 83 patients with multiple sclerosis (MS) were evaluated in ELISA assays using the above peptides.ResultsThe HTLV myelopathy patients had a statistically significant increased prevalence of antibodies to both HERV-K10 peptides (87.5%) vs. the VBD (0%), LGLL patients (0%), MS patients (4.8%), and the HTLV positive non-myelopathy subjects (5.2%).ConclusionThe data suggest that immuno-cross-reactivity to HERV-K10 peptides and/or transactivation of HERV-K10 expression by the HTLV Tax protein may be involved in the pathogenesis of HTLV-associated myelopathy/tropical spastic paraparesis and spastic ataxia.

Expression of various sarcomeric tropomyosin isoforms in equine striated muscles

In order to better understand the training and athletic activity of horses, we must have complete understanding of the isoform diversity of various myofibrillar protein genes like tropomyosin. Tropomyosin (TPM), a coiled-coil dimeric protein, is a component of thin filament in striated muscles. In mammals, four TPM genes (TPM1, TPM2, TPM3, and TPM4) generate a multitude of TPM isoforms via alternate splicing and/or using different promoters. Unfortunately, our knowledge of TPM isoform diversity in the horse is very limited. Hence, we undertook a comprehensive exploratory study of various TPM isoforms from horse heart and skeletal muscle. We have cloned and sequenced two sarcomeric isoforms of the TPM1 gene called TPM1α and TPM1κ, one sarcomeric isoform of the TPM2 and one of the TPM3 gene, TPM2α and TPM3α respectively. By qRT-PCR using both relative expression and copy number, we have shown that TPM1α expression compared to TPM1κ is very high in heart. On the other hand, the expression of TPM1α is higher in skeletal muscle compared to heart. Further, the expression of TPM2α and TPM3α are higher in skeletal muscle compared to heart. Using western blot analyses with CH1 monoclonal antibody we have shown the high expression levels of sarcomeric TPM proteins in cardiac and skeletal muscle. Due to the paucity of isoform specific antibodies we cannot specifically detect the expression of TPM1κ in horse striated muscle. To the best of our knowledge this is the very first report on the characterization of sarcmeric TPMs in horse striated muscle.

Cloning, Sequencing, and the Expression of the Elusive Sarcomeric TPM4α Isoform in Humans

In mammals, tropomyosin is encoded by four known TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, the sarcomeric isoform(s) of each of the TPM genes, except for the TPM4, have been known for a long time. Recently, on the basis of computational analyses of the human genome sequence, the predicted sequence of TPM4α has been posted in GenBank. We designed primer-pairs for RT-PCR and showed the expression of the transcripts of TPM4α and a novel isoform TPM4δ in human heart and skeletal muscle. qRT-PCR shows that the relative expression of TPM4α and TPM4δ is higher in human cardiac muscle. Western blot analyses using CH1 monoclonal antibodies show the absence of the expression of TPM4δ protein (~28 kDa) in human heart muscle. 2D western blot analyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass ~32 kD and above in adult heart. By Mass spectrometry, we determined the amino acid sequences of the extracted proteins from these spots. Spot “G” reveals the putative expression of TPM4α along with TPM1α protein in human adult heart.

Expression of Tropomyosin 2 Gene Isoforms in Human Cardiac Tissue

Previous studies have shown that although the transcript levels of TPM1α and TPM1k are expressed in human hearts in comparable levels, the level of TPM1α protein is ~90%. The proteins of TPM1κ and TPM2α are about 5% of the total sarcomeric TM. The TPM2 gene is known to generate three alternatively spliced isoforms, which are designated as TPM2α, TPM2β, and TPM2γ. The expression level of TPM2β and TPM2γ in human hearts is unknown. Using a series of primers pairs and probes for RNA PCR, we found that both TPM2α and β but not γ were expressed in fetal and adult heart tissue, with about the same amounts of each isoform in fetal hearts and more β than α in adult hearts. Four new isoforms of TPM2 RNA were identified (TPM2δ - η). Most of these were present in very small amounts in both the fetal and adult hearts with the exception of TPM2ξ, which was present at about 40% of the level of TPM2α in adult heart tissue. Western blot analyses using a series of anti-tropomyosin antibodies indicate that TPM2 protein is present in both fetal and adult hearts at about the same levels as TPM1κ and much less than TPM1α. We are unsure about the expression of TPM2δ, TPM2ζ, and TPM2η proteins in fetal and adult human hearts. The exact function of these new TPM2 isoforms in heart and their role(s) in cardiac disease remain to be elucidated.