Antibodies to HSP70 and HSP90 in serum in non-small cell lung cancer patients

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Abstract

Heat shock proteins (HSPs) are components of a physiologic stress response that are also over-expressed in various cancers including non-small cell lung cancer (NSCLC). During NSCLC serum-antibody screening of a NSCLC cDNA T7 phage library for immunogenic proteins we isolated HSP70 and HSP90 proteins. Isolation of these proteins suggested that corresponding antibodies could be elevated in NSCLC patient sera, a novel finding that could pilot their use as markers of NSCLC. We showed histochemically that patient sera were more reactive with each phage-expressed protein than normal sera. Antibody affinity for each phage-expressed protein was confirmed by limiting the dilution of individual sera assayed by Ab enzyme-linked immunosorbent assay (ELISA). Sera from 49 NSCLC patients assayed by Ab ELISA and normalized to 40 controls showed that HSP70 antibodies were significantly greater in patient sera than in normals (P=0.0002), while HSP90 antibodies were not significantly different (P=0.11). Analysis of the results with logistic regression and receiver operating characteristics (ROC) curves showed that HSP70 antibodies were modest markers of NSCLC (sensitivity 0.74 and specificity 0.73; area under the curve or AUC=0.731), while HSP90 antibodies appeared to be poor in both criteria with an AUC of 0.602. Further evaluation of HSP70 antibodies as potential markers of disease may be rational.

Introduction

Heat shock proteins (HSPs) comprise of a group of highly conserved and stress-related proteins with regulatory, homeostatic and chaperoning activities [1], [2], [3]. HSP70 and HSP90 are over-expressed by a number of solid tumors, including non-small cell lung cancer (NSCLC) suggesting a role in tumor cell growth and survival [4], [5], [6]. Recently, HSP70 over-expression, has been identified as a marker of poor prognosis in adenocarcinomas of the lung [7]. Notably, HSPs are immunogenic [8], [9], [10] and antibodies to HSP70 and HSP90, have been identified in sera of individuals with a variety of cancers [11], [12], [13], [14], [15], [16], a number of nonmalignant conditions [17], [18], [19], [20], [21], [22], [23], and in a percentage of normals [20], [24], [25], [26]. In breast cancer and osteosarcoma, elevated levels of antibodies to HSP90 have been shown to have independent prognostic significance [11], [12]. In osteosarcoma, HSP70 antibodies have also been shown to correlate with metastastic spread [13]. Antibodies to neither the protein have been reported in NSCLC.

During library screening of a T7 phage-expressed NSCLC cDNA library for immunogenic tumor proteins using preferential affinity of antibodies in NSCLC patient versus normal sera, we isolated phages expressing HSP70 and HSP90 proteins. The selection of these proteins using this approach indicated the presence of HSP70 and HSP90 antibodies in our NSCLC patient sera. Since HSP70 and HSP90 antibodies are potentially novel markers in NSCLC with putative diagnostic and prognostic importance, we further investigated these findings, including an assessment of sensitivity and specificity of antibody levels in NSCLC compared to normal controls.

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Human subjects

Following informed consent, peripheral blood was obtained from individuals with histologically confirmed NSCLC. The majority of patients were stage 3a/b (stages 1–2b: n=11; stage 3a/b: n=25; stage 4: n=13). NSCLC was biopsy-proven in all subjects and comprised of a variety of cell types (squamous cell: n=17; adenocarcinoma: n=14; large cell carcinoma: n=1), although specific cell types were not discernable from available biopsies from a number of subjects (undifferentiated or poorly

Isolating HSP70 and HSP90 from a T7 phage library

Following a series of sequential selection and enrichment steps, we isolated phages that had high immunochemical reactivity with patient sera. After PCR amplification and sequencing, by comparison to known cDNA sequences in GeneBank, we identified HSP70 and HSP90 from the T7 NSCLC cDNA phage library. Relative specificity for these two proteins using this approach was confirmed by the comparison of immunochemical reactivity of normal and NSCLC patient sera (Fig. 1).

Quantitative ELISA

To quantify the antibodies to

Discussion

We interrogated a NSCLC cDNA T7 phage-expressed library with antibodies in NSCLC patient sera to identify aberrantly expressed tumor proteins in lung cancer, and extracted phage-expressed HSP70 and HSP90 proteins. Since the selection technique uses the preferential affinity of antibodies found in NSCLC patient sera but not in normal sera, the isolation of these proteins suggested corresponding antibodies could be specifically elevated in NSCLC. Notably, HSP70 and HSP90 are abundantly expressed

Acknowledgements

These studies were supported by the Veterans Administration Career Development Award project #596-416905007-0001 and from a generous donation from the McDowell Foundation—Papa Johns/Valvano fund for cancer research. We would like to thank Basil Hirschowitz for his help in writing this manuscript.

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