Uma N. M. Rao

High-Dose Interferon Alfa-2b Significantly Prolongs Relapse-Free and Overall Survival Compared With the GM2-KLH/QS-21 Vaccine in Patients With Resected Stage IIB-III Melanoma: Results of Intergroup Trial E1694/S9512/C509801

PURPOSE Vaccine alternatives to high-dose interferon alfa-2b therapy (HDI), the current standard adjuvant therapy for high-risk melanoma, are of interest because of toxicity associated with HDI. The GM2 ganglioside is a well-defined melanoma antigen, and anti-GM2 antibodies have been associated with improved prognosis. We conducted a prospective, randomized, intergroup trial to evaluate the efficacy of HDI for 1 year versus vaccination with GM2 conjugated to keyhole limpet hemocyanin and administered with QS-21 (GMK) for 96 weeks (weekly x 4 then every 12 weeks x 8). PATIENTS AND METHODS Eligible patients had resected stage IIB/III melanoma. Patients were stratified by sex and number of positive nodes. Primary end points were relapse-free survival (RFS) and overall survival (OS). RESULTS Eight hundred eighty patients were randomized (440 per treatment group); 774 patients were eligible for efficacy analysis. The trial was closed after interim analysis indicated inferiority of GMK compared with HDI. For eligible patients, HDI provided a statistically significant RFS benefit (hazard ratio [HR] = 1.47, P = .0015) and OS benefit (HR = 1.52, P = .009) for GMK versus HDI. Similar benefit was observed in the intent-to-treat analysis (RFS HR = 1.49; OS HR = 1.38). HDI was associated with a treatment benefit in all subsets of patients with zero to > or = four positive nodes, but the greatest benefit was observed in the node-negative subset (RFS HR = 2.07; OS HR = 2.71 [eligible population]). Antibody responses to GM2 (ie, titers > or = 1:80) at days 29, 85, 365, and 720 were associated with a trend toward improved RFS and OS (P2 = .068 at day 29). CONCLUSION This trial demonstrated a significant treatment benefit of HDI versus GMK in terms of RFS and OS in melanoma patients at high risk of recurrence.

A Pooled Analysis of Eastern Cooperative Oncology Group and Intergroup Trials of Adjuvant High-Dose Interferon for Melanoma

Purpose: Nearly 2000 patients with stage IIB and III melanoma have participated in four multicenter, randomized trials, conducted by the Eastern Cooperative Oncology Group and the Intergroup, investigating adjuvant high-dose IFN-α 2b therapy. The objectives of this study were to update the analyses of each individual trial and to analyze prognostic factors and treatment effects based on pooled data. Experimental Design: Survival and disease status were updated to April 2001. Analysis of prognostic factors using optimized statistical models was based on data from patients in E1684, E1690, E1694, and E2696. Analysis of treatment effects versus observation (Obs) was based on data from 713 patients randomized to high-dose IFN-α 2b (HDI) or Obs in Trials E1684 and E1690. Results: Updated analysis of E1684, E1690, and E1694 confirmed their original conclusions, now at median follow-up intervals of 2.1–12.6 years. Based on two-sided univariate log-rank analysis of pooled data from E1684 and E1690 (median follow-up, 7.2 years), relapse-free survival (RFS)—but not overall survival (OS)—was significantly prolonged (two-sided log-rank P value = 0.006) for patients treated with HDI versus Obs. Among all patients, prognostic factors that significantly negatively impacted RFS and OS included ulceration, recurrent disease at entry, enrollment in E1684, and age > 49 years. Multivariate statistical models adjusting for these factors confirmed the statistically significant RFS benefit of HDI versus Obs but did not demonstrate a significant OS benefit in the pooled populations. Conclusions: In patients with high-risk resected melanoma, HDI is effective adjuvant therapy with strong evidence for improved RFS and evidence for moderate improvement in OS based on two prospective randomized studies but not the pooled analysis. Analyses of predictors of relapse and response are now needed to improve the therapeutic value of this modality.

Neoadjuvant Treatment of Regional Stage IIIB Melanoma With High-Dose Interferon Alfa-2b Induces Objective Tumor Regression in Association With Modulation of Tumor Infiltrating Host Cellular Immune Responses

PURPOSE Adjuvant high-dose interferon-alfa-2b (HDI) improves disease-free and overall survival in patients with high-risk melanoma. However, its mechanism of action is largely unknown. Therefore, HDI was investigated in the neoadjuvant setting to assess clinical and pathologic responses after 4 weeks of HDI and to perform immunohistochemical evaluation of immune cell subsets and melanoma-associated antigens. PATIENTS AND METHODS Patients with palpable regional lymph node metastases from melanoma (American Joint Committee on Cancer stage IIIB-C) underwent surgical biopsy at study entry and then received standard intravenous HDI (20 million units/m2, 5 days per week) for 4 weeks followed by complete lymphadenectomy and standard maintenance subcutaneous HDI (10 million units/m2 3 times per week) for 48 weeks. Biopsy samples were obtained before and after intravenous HDI and subjected to immunohistochemical analysis as well as routine pathologic study. RESULTS Twenty patients were enrolled, and biopsy samples were informative for 17. Eleven patients (55%) demonstrated objective clinical response, and 3 patients (15%) had complete pathologic response. At a median follow-up of 18.5 months (range, 7 months to 50 months) 10 patients had no evidence of recurrent disease. Clinical responders had significantly greater increases in endotumoral CD11c+ and CD3+ cells and significantly greater decreases in endotumoral CD83+ cells compared with nonresponders. No changes in the expression of melanoma-associated lineage antigens, tumor cell proliferation, angiogenesis, or apoptosis were evident. CONCLUSION Neoadjuvant HDI is highly effective for the treatment of palpable stage IIIB-C melanoma, and the findings of this study implicate an indirect immunomodulatory mechanism rather than a direct antitumor mechanism.

Immune Monitoring of the Circulation and the Tumor Microenvironment in Patients with Regionally Advanced Melanoma Receiving Neoadjuvant Ipilimumab

We evaluated neoadjuvant ipilimumab in patients with surgically operable regionally advanced melanoma in order to define markers of activity in the blood and tumor as assessed at baseline (before ipilimumab) and early on-treatment. Patients were treated with ipilimumab (10 mg/kg intravenously every 3 weeks ×2 doses) bracketing surgery. Tumor and blood biospecimens were obtained at baseline and at surgery. Flow cytometry and immunohistochemistry for select biomarkers were performed. Thirty five patients were enrolled; IIIB (3; N2b), IIIC (32; N2c, N3), IV (2). Worst toxicities included Grade 3 diarrhea/colitis (5; 14%), hepatitis (2; 6%), rash (1; 3%), elevated lipase (3; 9%). Median follow up was 18 months: among 33 evaluable patients, median progression free survival (PFS) was 11 months, 95% CI (6.2–19.2). There was a significant decrease in circulating myeloid derived suppressor cells (MDSC). Greater decrease in circulating monocyte gate MDSC Lin1−/HLA-DR−/CD33+/CD11b+ was associated with improved PFS (p = 0.03). There was a significant increase in circulating regulatory T cells (Treg; CD4+CD25hi+Foxp3+) that, unexpectedly, was associated with improved PFS (HR = 0.57; p = 0.034). Baseline evidence of fully activated type I CD4+ and CD8+ antigen-specific T cell immunity against cancer-testis (NY-ESO-1) and melanocytic lineage (MART-1, gp100) antigens was detected and was significantly potentiated after ipilimumab. In tumor, there was a significant increase in CD8+ T cells after ipilimumab (p = 0.02). Ipilimumab induced increased tumor infiltration by fully activated (CD69+) CD3+/CD4+ and CD3+/CD8+ T cells with evidence of induction/potentiation of memory T cells (CD45RO+). The change in Treg observed within the tumor showed an inverse relationship with clinical benefit and greater decrease in tumor MDSC subset Lin1−/HLA-DR−/CD33+/CD11b+ was associated with improved PFS at one year. Neoadjuvant evaluation revealed a significant immunomodulating role for ipilimumab on Treg, MDSC and effector T cells in the circulation and tumor microenvironment that warrants further pursuit in the quest for optimizing melanoma immunotherapy.

Modulation of Signal Transducers and Activators of Transcription 1 and 3 Signaling in Melanoma by High-Dose IFNα2b

Purpose: The Janus-activated kinase/signal transducers and activators of transcription (STAT) pathway of IFN signaling is important to immunoregulation and tumor progression. STAT1 plays a prominent role in the effector immune response, whereas STAT3 is implicated in tumor progression and down-regulation of the response to type I IFNs. The goal of this study was to understand the effects of high-dose IFNα2b (HDI) in relation to the balance of pSTAT1 and pSTAT3. Experimental Design: We evaluated STAT1 and STAT3 jointly as mediators of IFNα effects in the setting of a prospective neoadjuvant trial of HDI, in which tissue samples were obtained before and after 20 doses of HDI therapy. Double immunohistochemistry for pSTAT1 and pSTAT3 was done on paired fixed (9 patients) or frozen (12 patients) biopsies. Results: HDI was found to up-regulate pSTAT1, whereas it down-regulates pSTAT3 and total STAT3 levels in both tumor cells and lymphocytes. Higher pSTAT1/pSTAT3 ratios in tumor cells pretreatment were associated with longer overall survival (P = 0.032). The pSTAT1/pSTAT3 ratios were augmented by HDI both in melanoma cells (P = 0.005) and in lymphocytes (P = 0.022). Of the immunologic mediators and markers tested, TAP2 was augmented by HDI (but not TAP1 and MHC class I/II). Conclusion: IFNα2b significantly modulates the balance of STAT1/STAT3 in tumor cells and host lymphocytes, leading to up-regulation of TAP2 and augmented host antitumor response. The pSTAT1/pSTAT3 ratio in tumor cells at baseline may serve as a useful predictor of clinical outcome in cutaneous melanoma; the modulation of this ratio may serve as a predictor of therapeutic effect.

Microrna profiling analysis of differences between the melanoma of young adults and older adults

BackgroundThis study represents the first attempt to perform a profiling analysis of the intergenerational differences in the microRNAs (miRNAs) of primary cutaneous melanocytic neoplasms in young adult and older age groups. The data emphasize the importance of these master regulators in the transcriptional machinery of melanocytic neoplasms and suggest that differential levels of expressions of these miRs may contribute to differences in phenotypic and pathologic presentation of melanocytic neoplasms at different ages.MethodsAn exploratory miRNA analysis of 666 miRs by low density microRNA arrays was conducted on formalin fixed and paraffin embedded tissues (FFPE) from 10 older adults and 10 young adults including conventional melanoma and melanocytic neoplasms of uncertain biological significance. Age-matched benign melanocytic nevi were used as controls.ResultsPrimary melanoma in patients greater than 60 years old was characterized by the increased expression of miRs regulating TLR-MyD88-NF-kappaB pathway (hsa-miR-199a), RAS/RAB22A pathway (hsa-miR-204); growth differentiation and migration (hsa-miR337), epithelial mesenchymal transition (EMT) (let-7b, hsa-miR-10b/10b*), invasion and metastasis (hsa-miR-10b/10b*), hsa-miR-30a/e*, hsa-miR-29c*; cellular matrix components (hsa-miR-29c*); invasion-cytokinesis (hsa-miR-99b*) compared to melanoma of younger patients. MiR-211 was dramatically downregulated compared to nevi controls, decreased with increasing age and was among the miRs linked to metastatic processes. Melanoma in young adult patients had increased expression of hsa-miR-449a and decreased expression of hsa-miR-146b, hsa-miR-214*. MiR-30a* in clinical stages I-II adult and pediatric melanoma could predict classification of melanoma tissue in the two extremes of age groups. Although the number of cases is small, positive lymph node status in the two age groups was characterized by the statistically significant expression of hsa-miR-30a* and hsa-miR-204 (F-test, p-value < 0.001).ConclusionsOur findings, although preliminary, support the notion that the differential biology of melanoma at the extremes of age is driven, in part, by deregulation of microRNA expression and by fine tuning of miRs that are already known to regulate cell cycle, inflammation, Epithelial-Mesenchymal Transition (EMT)/stroma and more specifically genes known to be altered in melanoma. Our analysis reveals that miR expression differences create unique patterns of frequently affected biological processes that clearly distinguish old age from young age melanomas. This is a novel characterization of the miRnomes of melanocytic neoplasms at two extremes of age and identifies potential diagnostic and clinico-pathologic biomarkers that may serve as novel miR-based targeted modalities in melanoma diagnosis and treatment.

Cytogenetic and histologic correlation of peripheral nerve sheath tumors of soft tissue.

Cytogenetic analysis was performed on 11 peripheral nerve sheath tumors of soft tissue from 10 patients. They include 6 benign and 5 malignant schwannomas. Five cases which include two benign, one cellular and two malignant schwannomas had a known association with a nerve, but only one patient with malignant schwannoma has clinically documented neurofibromatosis type I. All the patients had a normal diploid constitutional karyotype. Two cases of cellular schwannoma were analyzed by routine cytogenetic analysis and fluorescence in situ hybridization (FISH). One tumor was karyotyped as 45, XX,-13,-22 +mar; and the other case had a 45,X,-Y,t(1;17) (p12;q11.2) karyotype. In the latter, the breakpoint in 17q occurred below the centromere and is at or in the region of the Neurofibromatosis Type 1 (NF1) gene. Four benign tumors had a normal diploid karyotype. One hypodiploid malignant schwannoma with myxoid features demonstrated monosomy of chromosomes 17 and 22 by FISH analysis. The rest of the malignant schwannomas showed a wide range of numerical and structural aberrations, with frequent loss of 22q and gains of chromosomes 2 and 7. Loss of a sex chromosome was observed in cellular as well as malignant schwannomas. Regional karyotypic evolution was noted in one malignant schwannoma. Cytogenetic analysis may prove to be useful in identifying tumors, such as cellular schwannomas, which, because of their histologic features may be inadvertently categorized as malignant. Simultaneous involvement of NF1 and NF2 genes, which are located on chromosomes 17q and 22q, respectively, should be investigated at a molecular level in both benign and malignant tumors of peripheral nerves.

AIDS-Related EBV-Associated Smooth Muscle Tumors: A Review of 64 Published Cases

The number of reported cases of smooth muscle tumor (SMT) arising in patients with AIDS has been increasing since the mid-1990s. The aim of this study is to characterize the epidemiology, clinical manifestations, pathologic features, prognosis and, management of Epstein-Barr virus-related SMT (EBV-SMT) in patients with AIDS. An English language literature search identified 53 articles including 64 reported cases of EBV-SMT. The majority of these reports involved patients who were young, severely immunosuppressed, and had multifocal tumors. The central nervous system was the most common site to be involved. Histologically, tumors had smooth muscle features and were immunoreactive for muscle markers and all but two tumors demonstrated the presence of EBV by either immunohistochemistry, in situ hybridization, and/or PCR. While mitoses and/or necrosis were used to separate leiomyoma from leiomyosarcoma, these features did not correlate with clinical outcome. Treatment included primarily resection, and less often radiotherapy, chemotherapy and highly active antiretroviral therapy (HAART). Overall, EBV-SMTs appear to have variable aggressiveness and clinical outcome and may exhibit a more favorable prognosis compared to conventional leiomyosarcoma. Tumor-related death from EBV-SMT occurred in only 4 of 51 patients.

Gene expression analysis of human soft tissue leiomyosarcomas

Leiomyosarcoma of the somatic soft tissue is a rare malignant mesenchymal neoplasm that metastasizes to other organs in a subset of cases. Much remains to be learned about the mechanisms underlying the development of aggressive behavior of this tumor. It has been difficult to predict the clinical behavior of leiomyosarcomas using the morphology-based grading system, even though tumor size and histological grade have correlated with biologic behavior in some studies. In this study we analyzed the gene expression patterns of 35 samples of mesenchymal origin, including 11 cases of leiomyosarcomas of different histological grades arising in soft tissue and the retroperitoneum, using the Affymetrix U133a chips, which contain more than 22,000 genes and expression sequence tags (ESTs). We identified a set of genes whose expression was commonly altered in all leiomyosarcoma samples. In addition, we identified specific gene expression patterns in several subsets of the tumor. We used these alterations of gene expression to subclassify the leiomyosarcomas into 3 groups. Interestingly, the grouping of these samples correlated well with tumor differentiation and clinical aggressiveness. The analysis identified 92 genes that distinguish low-grade, well-differentiated leiomyosarcomas from less well-differentiated, high-grade, and metastatic leiomyosarcoma. Thesse alterations of gene expression appear to be correlated with the clinical behavior and histological grade of the tumor. The striking differences in terms of gene expression pattern among leiomyosarcomas of different differentiation status and clinical aggressiveness imply that several genetic abnormalities are responsible for the genesis and progression of this tumor.

Hepatocyte growth factor and c-MET in benign and malignant peripheral nerve sheath tumors.

Hepatocyte growth factor (HGF), secreted by mesenchymal cells, has pleiotropic biological activities on several cell types. HGF and its receptor, the c-met proto-oncogene product (c-MET) have been implicated in the genesis and progression of several carcinomas and sarcomas. It has been suggested that MET/HGF autocrine signaling may contribute to tumorigenesis in sarcomas. HGF has been recently found to be a mitogen for rat Schwann cells and to be present in neurofibromas in NF1 patients. In this investigation, we assessed the immunoreactive patterns of HGF and MET in benign and malignant peripheral nerve sheath tumors (PNST) using archival formalin-fixed tissue. The standard avidin-biotin-peroxidase method was used. All benign tumors were negative with HGF. Eight cases of MPNST were positive with both HGF and MET. In some malignant PNST, positivity with both ligand and the receptor may be indicative of an autocrine mediated signal transduction and may implicate HGF/MET in tumor progression. Immunoreactivity with MET was strikingly greater in MPNST in contrast to benign PNST; this finding may prove to be helpful in distinguishing some histologically low-grade MPNST from cellular and atypical benign PNST.