Biomarkers and Correlative Endpoints for Immunotherapy Trials: What Can We Learn in Lung Cancer from Other Tumor Types?

Immunotherapies for lung cancer are reaching phase III clinical trial, but the ultimate success likely will depend on developing biomarkers to guide development and choosing patient populations most likely to benefit. Because the immune response to cancer involves multiple cell types and cytokines, some spatially and temporally separated, it is likely that multiple biomarkers will be required to fully characterize efficacy of the vaccine and predict eventual benefit. Peripheral blood markers of response, such as the ELISPOT assay and cytokine flow cytometry analyses of peripheral blood mononuclear cells following immunotherapy, remain the standard approach, but it is increasingly important to obtain tissue to study the immune response at the site of the tumor. Earlier clinical endpoints such as response rate and progression-free survival do not correlate with overall survival demonstrated for some immunotherapies, suggesting the need to develop other intermediary clinical endpoints. Insofar as all these biomarkers and surrogate endpoints are relevant in multiple malignancies, it may be possible to extrapolate findings to immunotherapy of lung cancer.

Comparison of Intracellular Cytokine Flow Cytometry and an Enzyme Immunoassay for Evaluation of Cellular Immune Response to Active Tuberculosis

ABSTRACT A prospective cross-sectional blinded study of 28 patients (21 male and 7 female patients; mean age, 44 years) with suspected active tuberculosis (TB) attending a TB and chest clinic is described. Blood was taken for immune cell enumeration, a whole-blood enzyme-linked immunosorbent assay (ELISA) for the detection of gamma interferon (IFN-γ) by the QuantiFERON-TB Gold (QFT-G) assay, and intracellular cytokine flow cytometry (ICC) analysis; and sputum was simultaneously taken for bacteriological culture for Mycobacterium tuberculosis. Twelve healthy subjects were included as controls. The performance characteristics of the QFT-G and ICC assays for the detection of active TB were compared. Among the patients with active TB, we found (i) normal to slightly elevated peripheral CD4+ and CD8+ T-cell counts but a significant reduction in the number of NK cells; (ii) CD4+ T cells were the major cell type producing IFN-γ, a type 1 cytokine; (iii) small percentages of CD8+ T cells were also primed for IFN-γ production; (iv) the production of interleukin-4 (IL-4), a type 2 cytokine, was not prominent; and (v) the sensitivity and the specificity of the QFT-G assay were 88.2% and 18%, respectively, and those of the ICC assay were 94.1% and 36.4%, respectively. The specificities of the blood tests were likely underestimated due to cross-reaction to a non-M. tuberculosis mycobacterial infection and the lack of a confirmatory test that could be used to diagnose latent M. tuberculosis infection. Flow cytometry accurately locates the pool of immunological effector cells responsible for cytokine production during active TB. The ICC assay is an additional useful tool for the diagnosis of active TB.

Phase II trial of vaccination with WT1 peptide, GM-CSF, and KLH in patients with acute myeloid leukemia and myelodysplasia: Final immunological, molecular, and clinical results

3008 Purpose: A phase II trial of vaccination with the HLA-A2-restricted WT1.126–134 peptide was performed in patients (pts) with AML and MDS and overexpression of WT1 to determine toxicity, immunogenicity, and molecular and clinical activity. Methods: Patients received intra/subcut. vaccinations of 0.2 mg WT1.126–134 peptide (day 3), 62.5 mcg GM-CSF (days 1–4) as DC-stimulant and 1 mg keyhole limpet hemocyanin (day 3) as T helper protein. Vaccination was biweekly x 4 followed by 4-weekly in the first 13 patients and continuously biweekly in the subsequent 13. Early disease progression until vaccine # 6 was allowed, if not requiring chemotherapy. WT1-specific T cell responses were measured by tetramer and cytokine flow cytometry. WT1 levels were assessed by qRT-PCR. Clinical response assessment followed IWG-MDS criteria, capturing stable disease and hematologic improvement. Statistical analysis was performed by frequency tables and exploratory comparisons using Fishers test. Results: Of 29 pts enrolled 25 were evaluable, 23 with AML and 2 with RAEB. 15 AML pts had >5% marrow blasts and 8 had high-risk CR with detectable WT1 mRNA. A median of 11 (range 3 - 25) vaccinations was administered, 3 pts are ongoing. No relevant toxicity occurred. There were no obvious differences in outcome parameters between the 2 vaccination schedules. The percentage of patients with WT1 tetramer response increased from 28% prior to vaccination to 80% at week 10 (p=0.003), while the WT1 peptide specific cytokine response increased from 20% to 57% (p=0.012) of patients. WT1 mRNA-levels increased in 22% of patients, were stable in 26%, and decreased in 52% (2 to>50-fold). One CR (514 days) and 13 SD (99 to 339 days) were observed, 5 SD with >50% blast reduction and 3 with hematologic improvement. The CR and 3 SD occurred after initial PD. The median time to treatment failure (TTF) was 143 days. There was a significant association between decrease in WT1 mRNA levels and TTF (p=0.026). Conclusions: This study proves immunological, molecular and clinical efficacy of WT1 peptide vaccination in AML. Further investigations are indicated in WT1 expressing carcinomas and in combination with agents targeting CTLA4 or PD1. No significant financial relationships to disclose.