Jeff Longmate

Speed congenics: a classic technique in the fast lane (relatively speaking)

Marker-assisted selection protocol (MASP)-based strategies produce congenic strains with the target gene contained on clearly defined donor-derived genomic intervals in less than half the member of generations required by the classic protocol. Thus, the quality and speed of congenic strain construction are enhanced by this methodology. Here, Edward Wakeland and colleagues compare various MASP-based strategies and discuss their advantages with reference to immunological traits.

Trastuzumab Plus Docetaxel in HER-2/neu-Positive Prostate Carcinoma Final Results from the California Cancer Consortium Screening and Phase II Trial

Overexpression of the HER‐2/neu oncoprotein has been reported to occur in ≤ 60% of patients with prostate carcinoma and to correlate with shortened survival. Trastuzumab is a humanized monoclonal antibody to the HER‐2 receptor and has activity against HER‐2–positive breast carcinoma, more so when combined with a taxane. The authors screened for HER‐2 overexpression in patients developing hormone‐refractory prostate carcinoma (HRPC) and conducted a Phase II trial of trastuzumab plus docetaxel in HER‐2–positive patients.

Relative dominance of HLA-B*07 restricted CD8+ T-Lymphocyte immune responses to human cytomegalovirus pp65 in persons sharing HLA-A*02 and HLA-B*07 alleles

CD8(+) T-cell responses to three human cytomegalovirus (CMV) pp65 epitopes were studied in panels of healthy seropositive HLA-A*02/HLA-B*07 individuals, and HLA-A*02 donors mismatched for HLA-B*07. The majority of the latter had significant responses to a HLA-A*02-restricted epitope within the CMV pp65 antigen. By contrast, the strongest responses to CMV in the first group were to HLA-B*07-restricted epitopes. Similar immunodominance of HLA-B*07 over HLA-A*02 was found in two immunocompromised HIV-infected HLA-A*02/HLA-B*07 patients, and in the reconstituting immune system of three stem cell transplant recipients. In vitro stimulation of peripheral blood mononuclear cells (PBMC) from two immunocompetent HLA-A*02/HLA-B*07 individuals indicated that cytotoxic T lymphocyte (CTL) precursors specific for both HLA-A*02 and HLA-B*07 restricted epitopes were present and could be expanded by stimulation with the cognate peptides. However, if stimulation was performed by antigen presenting cells infected with recombinant vaccinia expressing full-length native pp65, only HLA-B*07 epitope-specific cells were seen. In one patient the HLA-B*07 dominance was partially broken by using recombinant vaccinia expressing ubiquitinated pp65, suggesting that enhanced protein processing can reveal weaker immune responses. Our results indicate that CMV-specific cellular immune responses restricted by HLA-B*07 dominate those restricted by HLA-A*02 in both immunocompetent and immunocompromised individuals. This may have significant consequences for the design of epitope-specific vaccines.

MULTIPLEX INHERITANCE OF COMPONENT PHENOTYPES IN A MURINE MODEL OF LUPUS

Abstract. We analyzed the linkage of GN and a wide spectrum of serological phenotypes associated with systemic lupus erythematosus in a (NZM2410 × C57BL/6)F2 cross. Some phenotypes, such as glomerulonephritis (GN) and anti-chromatin IgG antibody production, were more penetrant in females, but others, such as anti-dsDNA antibody production, did not show a gender bias. These results suggest that gender bias affects only a subset of SLE-component phenotypes, and that NZM2410 can be used to dissect the genetic basis of this phenomenon. Genome scanning linked six chromosomal intervals with the expression of one or more component phenotypes. These loci included two Sle loci previously identified in an (NZM2410 × B6)F1× NZM2410 backcross, loci identified by others in the NZB/W model. Our analysis also suggested two new intervals on chromosomes (Chrs.) 10 and 11. Detailed analysis of the segregation of different phenotypes within these intervals suggests that they encompass more than one susceptibility locus. This clustering has been a common finding in several murine polygenic traits. Each of NZM2410 susceptibility loci can be aligned with a specific genetic pathways contributing to SLE pathogenesis on the basis of the spectrum of component phenotypes expressed.

Impact of donor CMV status on viral infection and reconstitution of multifunction CMV-specific T cells in CMV-positive transplant recipients

Reconstitution of cytomegalovirus (CMV)-specific CD8(+) T cells is essential to the control of CMV infection in CMV-positive recipients (R(+)) after allogeneic hematopoietic stem cell transplantation (HCT). Six-color flow cytometry was used to assess the functional profile of CMV-specific CD8(+) T cells in 62 of 178 R(+) HCT recipients followed virologically for CMV reactivation. R(+) recipients receiving grafts from CMV-negative donors (D(-); D(-)/R(+)) reconstituted fewer multifunctional CD8(+) T cells expressing tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1beta (MIP-1beta), and CD107 in addition to interferon-gamma (IFN-gamma), compared with D(+)/R(+) recipients. Unlike monofunctional CD8(+) T cells secreting IFN-gamma, which were abundantly generated during CMV reactivation in D(-)/R(+) recipients, the relative lack of multifunctional CD8(+) T cells persisted until at least 1 year post-HCT. D(-)/R(+) recipients were more likely to require recurrent and prolonged use of antivirals. These findings were robust to statistical adjustment for pretransplant factors, as well as for posttransplant factors including graft-versus-host disease (GVHD) and its treatment by steroids. These analyses suggest that D(+)/R(+) transplants, on average, generate higher levels of multifunctional CMV-specific T cells and require less antiviral therapy compared with D(-)/R(+) HCT recipients. These results highlight the benefit of D(+) donors in improving outcomes of R(+) HCT recipients by reducing the duration and recurrent need of antiviral treatment, aided by increased levels of multifunctional CMV-specific T cells.

Longitudinal Assessment of Cytomegalovirus (CMV)—Specific Immune Responses in Liver Transplant Recipients at High Risk for Late CMV Disease

Cytomegalovirus (CMV)-seronegative recipients (R(-)) of a liver transplant from CMV-positive donors (D(+)) are at high risk for developing late CMV disease after discontinuation of antiviral prophylaxis. Levels of viremia and CMV-specific interferon (IFN)- gamma -producing CD4(+) and IFN- gamma -producing CD8(+) T cell responses were prospectively measured from discontinuation of antiviral prophylaxis until 1 year after transplantation in 17 consecutive D(+)/R(-) patients. CMV loads of >1000 copies/mL were strongly associated with CMV disease in the 6 symptomatic patients. Despite immunosuppression, broadly diverse T cells specific for CMV lysate or peptide libraries spanning pp65 and immediate early (IE) 1 immunodominant CMV antigens developed in all patients. A vigorous CD8(+) T cell response to pp65 and IE1 antigens characterized the D(+)/R(-) cohort. Unexpectedly, none of these responses were predictive of CMV disease or viremia. No significant lymphopenia or functional impairment of CMV-specific T cells was detected in the symptomatic patients, whose morbidity was resolved after antiviral treatment while measurable CMV immunity was maintained during the 1-year observation period.

The AKT inhibitor perifosine in biochemically recurrent prostate cancer: a phase II California/Pittsburgh cancer consortium trial.

BACKGROUND Perifosine is an oral alkylphospholipid that inhibits cancer cell growth through decreased Akt phosphorylation. We conducted a phase II trial of perifosine in patients with biochemically recurrent, hormone-sensitive prostate cancer. PATIENTS AND METHODS Eligible patients had histologically confirmed prostate cancer, previous prostatectomy and/or radiation therapy, and rising prostate-specific antigen (PSA) without radiographic evidence of metastasis. Previous androgen deprivation therapy < 9 months in duration (completed >or= 1 year before registration) was allowed. The primary endpoint was PSA response, defined as a decrease by >or= 50% from the pretreatment value. Treatment was composed of a loading dose of perifosine 900 mg orally on day 1, then 100 mg daily starting 24 hours later. RESULTS Of 25 patients, 24 were evaluable for response. After a median follow-up of 8 months, 5 patients (20%) had a reduction in serum PSA levels, but none met criteria for PSA response. Three patients immediately progressed with no response to therapy. Median progression-free survival was 6.64 months (range, 4.53-12.81 months). No change in the PSA doubling time (7 months) was observed before and after treatment initiation. Dose-limiting toxicities (all grade 3) included hyponatremia, arthritis, hyperuricemia, and photophobia. CONCLUSION Although well tolerated, perifosine did not meet prespecified PSA criteria for response as a single agent in biochemically recurrent prostate cancer. However, 20% of patients had evidence of PSA reduction, suggesting modest single-agent clinical activity. The role of perifosine in combination with androgen deprivation or chemotherapy is currently under investigation.

Evidence for X-Chromosomal Schizophrenia Associated with microRNA Alterations

Background Schizophrenia is a severe disabling brain disease affecting about 1% of the population. Individual microRNAs (miRNAs) affect moderate downregulation of gene expression. In addition, components required for miRNA processing and/or function have also been implicated in X-linked mental retardation, neurological and neoplastic diseases, pointing to the wide ranging involvement of miRNAs in disease. Methods and Findings To explore the role of miRNAs in schizophrenia, 59 microRNA genes on the X-chromosome were amplified and sequenced in males with (193) and without (191) schizophrenia spectrum disorders to test the hypothesis that ultra-rare mutations in microRNA collectively contribute to the risk of schizophrenia. Here we provide the first association of microRNA gene dysfunction with schizophrenia. Eight ultra-rare variants in the precursor or mature miRNA were identified in eight distinct miRNA genes in 4% of analyzed males with schizophrenia. One ultra-rare variant was identified in a control sample (with a history of depression) (8/193 versus 1/191, p = 0.02 by one-sided Fishers exact test, odds ratio = 8.2). These variants were not found in an additional 7,197 control X-chromosomes. Conclusions Functional analyses of ectopically expressed copies of the variant miRNA precursors demonstrate loss of function, gain of function or altered expression levels. While confirmation is required, this study suggests that microRNA mutations can contribute to schizophrenia.

The Cyclin-Dependent Kinase Inhibitor UCN-01 Plus Cisplatin in Advanced Solid Tumors: A California Cancer Consortium Phase I Pharmacokinetic and Molecular Correlative Trial

Background: UCN-01 (7-hydroxy-staurosporine) is a novel antineoplastic agent targeting cyclin-dependent kinases, which shows potent in vitro and in vivo activity against a broad range of tumor types. Our group has previously shown that UCN-01 potentiates the apoptotic response of agents such as cisplatin in vitro by preventing sequence-specific abrogation of G2 arrest caused by DNA-damaging chemotherapies. Patients and Methods: This National Cancer Institute–sponsored phase I trial was designed to determine the safety, maximum tolerated dose, and pharmacokinetics of escalating doses of cisplatin in combination with UCN-01 in patients with advanced malignant solid tumors, as well as to do molecular correlative studies on tumor specimens. Cisplatin was infused over 1 hour before UCN-01 (45 mg/m2/d) given as a 72-hour continuous infusion. Escalation of cisplatin was planned through five dose levels at 20, 30, 45, 60, and 75 mg/m2. Results: Ten patients were accrued. Accrual was halted at dose level 2 (cisplatin, 30 mg/m2) due to dose-limiting toxicities consisting of grade 5 sepsis with respiratory failure associated with grade 3 creatinine (one patient) and grade 3 atrial fibrillation (one patient). Plasma and salivary pharmacokinetics of UCN-01 were unaffected by cisplatin. Pretreatment and posttreatment tumor biopsies showed that UCN-01 was active against a key molecular target, the checkpoint kinase Chk1. Conclusions: This phase I trial failed to achieve targeted therapeutic dose levels of cisplatin when combined with prolonged infusion UCN-01. However, because preclinical data indicate that UCN-01 potentiates response to platinum, further studies with alternative dose schedules of the combination, or with other platinum analogues, are warranted.

A phase II trial of the Src-kinase inhibitor AZD0530 in patients with advanced castration-resistant prostate cancer: A California Cancer Consortium study

Prostate cancer cells undergo neuroendocrine differentiation during androgen deprivation and secrete neuropeptides, hence activating androgen receptor-regulated genes. Src-family protein kinases are involved in neuropeptide-induced prostate cancer growth and migration. A phase II trial of AZD0530, an oral Src-family kinase inhibitor, in patients with advanced castration resistant prostate cancer was conducted. The primary endpoint was prostate cancer-specific antigen (PSA) response rate, defined as a 30% or greater decrease. A two-stage Simon design was used. Eligibility criteria included documentation of castration resistance (including antiandrogen withdrawal), adequate end-organ function, and performance status, and not more than one prior taxane-based chemotherapy regimen. AZD0530 was given at 175 mg orally once daily continuously. Rapid accrual led to 28 patients registering in the first stage. Median age was 67 years. Sixteen patients had performance status (PS) 0, eight patients had PS 1, and four patients had PS 2. Nine patients (32%) had prior docetaxel-based chemotherapy. Five patients had transient PSA reductions not meeting PSA response criteria. Median progression-free survival time was 8 weeks. Treatment was generally well tolerated. AZD0530, a potent oral Src kinase inhibitor, is feasible and tolerable in this pretreated patient population but possessed little clinical efficacy as monotherapy. Strong preclinical evidence warrants further investigation of AZD0530 in earlier-stage prostate cancer or as combination therapy.