Analysis of the dynamic mutation in the SCA7 gene shows marked parental effects on CAG repeat transmission.

Journal ArticleThe gene for spinocerebellar ataxia 7 (SCA7) includes a transcribed, translated CAG tract that is expanded in SCA7 patients. We have determined expansions in 73 individuals from 17 SCA7 kindreds and compared them with repeat lengths of 180 unaffected individuals. Subjects with abnormal expansions comprise 59 clinically affected individuals and 14 at-risk currently unaffected individuals predicted to carry the mutation by haplotype analysis. For expanded alleles, CAG repeat length correlates with disease progression and severity and correlates inversely with age of onset. Increased repeat lengths are seen in generational transmission of the disease allele, consistent with the pattern of clinical anticipation seen in these kindreds. Repeat lengths in expanded alleles show somatic mosaicism in leukocyte DNA, suggesting that these alleles are unstable within individuals as well as between generations. Although dynamic repeat expansions from paternal transmissions are greater than those from maternal transmissions, maternal transmission of disease is more common, suggesting germline or embryonic effects of the repeat expansion

Sarcoma immunotherapy.

Much of our knowledge regarding cancer immunotherapy has been derived from sarcoma models. However, translation of preclinical findings to bedside success has been limited in this disease, though several intriguing clinical studies hint at the potential efficacy of this treatment modality. The rarity and heterogeneity of tumors of mesenchymal origin continues to be a challenge from a therapeutic standpoint. Nonetheless, sarcomas remain attractive targets for immunotherapy, as they can be characterized by specific epitopes, either from their mesenchymal origins or specific alterations in gene products. To date, standard vaccine trials have proven disappointing, likely due to mechanisms by which tumors equilibrate with and ultimately escape immune surveillance. More sophisticated approaches will likely require multimodal techniques, both by enhancing immunity, but also geared towards overcoming innate mechanisms of immunosuppression that favor tumorigenesis

Exploiting antitumor immunity to overcome relapse and improve remission duration

Cancer survivors often relapse due to evolving drug-resistant clones and repopulating tumor stem cells. Our preclinical study demonstrated that terminal cancer patient’s lymphocytes can be converted from tolerant bystanders in vivo into effective cytotoxic T-lymphocytes in vitro killing patient’s own tumor cells containing drug-resistant clones and tumor stem cells. We designed a clinical trial combining peginterferon α-2b with imatinib for treatment of stage III/IV gastrointestinal stromal tumor (GIST) with the rational that peginterferon α-2b serves as danger signals to promote antitumor immunity while imatinib’s effective tumor killing undermines tumor-induced tolerance and supply tumor-specific antigens in vivo without leukopenia, thus allowing for proper dendritic cell and cytotoxic T-lymphocyte differentiation toward Th1 response. Interim analysis of eight patients demonstrated significant induction of IFN-γ-producing-CD8+, -CD4+, -NK cell, and IFN-γ-producing-tumor-infiltrating-lymphocytes, signifying significant Th1 response and NK cell activation. After a median follow-up of 3.6 years, complete response (CR) + partial response (PR) = 100%, overall survival = 100%, one patient died of unrelated illness while in remission, six of seven evaluable patients are either in continuing PR/CR (5 patients) or have progression-free survival (PFS, 1 patient) exceeding the upper limit of the 95% confidence level of the genotype-specific-PFS of the phase III imatinib-monotherapy (CALGB150105/SWOGS0033), demonstrating highly promising clinical outcomes. The current trial is closed in preparation for a larger future trial. We conclude that combination of targeted therapy and immunotherapy is safe and induced significant Th1 response and NK cell activation and demonstrated highly promising clinical efficacy in GIST, thus warranting development in other tumor types

PhD

dissertationThis dissertation describes a hereditary neurological disorder, autosomal dominant cerebellar ataxia with retinal degeneration, from initial clinical findings through analysis and isolation of the responsible gene, to study of the affected protein. The work described herein follows a research arc beginning with a patient presenting with worsening coordination and progressive blindness. A detailed clinicopathologic description of the signs, symptoms and autopsy findings occurring in affected related individuals ensued, creating strict criteria for the disease and establishing it as a distinct genetic entity. Subsequent linkage analysis allowed me to localize the gene responsible for the disease to chromosome 3p14-21.1. I named the disease spinocerebellar ataxia type 7 and the gene SCA7. I undertook experiments to isolate SCA7, which was eventually cloned. As I had hypothesized, SCA7 harbored a dynamic mutation due to an unstable region of DNA within the gene. This region, a trinucleotide repeat, is relatively short and stable in the general population, but expanded and unstable in affected individuals. I observed a direct correlation between repeat length and disease severity, and an inverse correlation with age-of-onset. I found allele instability and rate of disease transmission depended upon the sex of the parent transmitting the allele. The dissertation concludes with specific observations of SCA7's protein, ataxin-7. The SCA7 trinucleotide repeat generates a tract of glutamine amino acids within ataxin-7; I noted ataxin-7 had a nuclear distribution, forming subnuclear aggregates with a morphology that depended, in part, on glutamine tract length. My data infers ataxin-7s role in apoptosis, or programmed cell death. I demonstrated that ataxin-7 is specifically cleaved by caspase-7, an executioner of apoptosis. Furthermore, I noted longer glutamine tracts within ataxin-7 correlated with increased proapoptotic effects in cells expressing the protein. I modified ataxin-7 to confer caspase-7 cleavage resistance to prepare for further investigation into the protein's apoptotic role. The findings in this dissertation offer insights into the function and pathology of SCA7, the gene responsible for spinocerebellar ataxia with retinal degeneration. It is hoped that the information described in this manuscript may eventually lead to therapeutic modalities for what is currently an inexorable, often fatal disease

Sarcoma Immunotherapy

Much of our knowledge regarding cancer immunotherapy has been derived from sarcoma models. However, translation of preclinical findings to bedside success has been limited in this disease, though several intriguing clinical studies hint at the potential efficacy of this treatment modality. The rarity and heterogeneity of tumors of mesenchymal origin continues to be a challenge from a therapeutic standpoint. Nonetheless, sarcomas remain attractive targets for immunotherapy, as they can be characterized by specific epitopes, either from their mesenchymal origins or specific alterations in gene products. To date, standard vaccine trials have proven disappointing, likely due to mechanisms by which tumors equilibrate with and ultimately escape immune surveillance. More sophisticated approaches will likely require multimodal techniques, both by enhancing immunity, but also geared towards overcoming innate mechanisms of immunosuppression that favor tumorigenesis

Sarcoma immunotherapy.

Much of our knowledge regarding cancer immunotherapy has been derived from sarcoma models. However, translation of preclinical findings to bedside success has been limited in this disease, though several intriguing clinical studies hint at the potential efficacy of this treatment modality. The rarity and heterogeneity of tumors of mesenchymal origin continues to be a challenge from a therapeutic standpoint. Nonetheless, sarcomas remain attractive targets for immunotherapy, as they can be characterized by specific epitopes, either from their mesenchymal origins or specific alterations in gene products. To date, standard vaccine trials have proven disappointing, likely due to mechanisms by which tumors equilibrate with and ultimately escape immune surveillance. More sophisticated approaches will likely require multimodal techniques, both by enhancing immunity, but also geared towards overcoming innate mechanisms of immunosuppression that favor tumorigenesis

KIT activation & up-regulation, concomitant parallel induction of ET3, KIT⁺Melan-A^–- progenitor cells, and melanocyte regeneration in proportion to sun-exposure.

<p>(<b><i>A</i></b>), IHC of KIT and ET3 on serial sections of human skin specimen obtained from a lower extremity-amputation. Sole represents active suppression of melanogenesis (<i>a</i> and <i>d</i>), dorsum of big toe represents intermediate sun-exposure (<i>b</i> and <i>e</i>), and lateral lower leg represents heavy sun-exposure (<i>c</i> and <i>f</i>). (<b><i>B</i></b>), IHC of KIT, Melan-A, and ET3 on serial sections of human skin punch biopsy specimens obtained from sun-protected axilla (<i>g</i>, <i>i</i>, <i>k</i>) and chronic heavy sun-exposed forearm (<i>h</i>, <i>j</i>, <i>l</i>) from the same individual. Lymphocytes serve as internal negative control for KIT, ET3 and Melan-A; mast cells serve as internal positive control for KIT. Together, these images demonstrate that human skin exhibits sun-exposure-dependent up-regulation of KIT (<i>a-c</i>) and concomitant parallel sun-exposure-induced increasing induction of ET3 (<i>d-f</i>). Chronic sun-exposure induces intense dendritic pattern of KIT expression as well as a large increase in the number of KIT-expressing-cells in the basal layer (<i>h</i>) consisting of KIT⁺Melan-A⁺ mature melanocytes (<i>j</i>) and KIT⁺Melan-A^–melanocyte progenitor cells as evidenced by the difference between (<i>h</i>) and (<i>j</i>).</p