by Victoria A. Fisher, M.P.H.
Investigators in the Infections and Immunoepidemiology Branch (IIB) are leading a DCEG strategic initiative to define the link between immune and inflammatory responses and cancer. One of the major objectives has been to develop a carefully characterized panel of immune markers that could be made available to researchers in DCEG for use in their epidemiologic studies.
DCEG investigators have made significant progress toward this goal, having recently completed evaluation of a multiplex immune panel capable of measuring a large number of immune and inflammatory biomarkers simultaneously. The panel already has been used in several investigations, including studies of cancers of the lung, cervix, gallbladder, and ovary as well as non-Hodgkin lymphoma (NHL).
Normally, inflammation is a healthy part of the immune response in that it heals injury and fights infection. Inflammation is the body’s attempt at self-protection; whether you’ve cut a finger, broken a bone, or been exposed to an infectious disease, your body is innately programmed to respond.
Prolonged, chronic inflammation arises when this process fails to turn off, and instead continues to stimulate pro-inflammatory immune cells when they may no longer be needed. This excessive response can occur for a variety of reasons, including persistent infection, exposure to harmful substances in the environment, and even obesity.
Chronic inflammation is thought to increase the risk of cancer, both directly through DNA damage and indirectly through tissue remodeling and scarring. Studies show that inflammation and altered immunity play a critical role in several stages of the carcinogenic process, including tumor initiation, promotion, progression, metastasis, and response to therapy. Scientists believe that chronic inflammation and altered immunity are important etiologic factors for several cancers, including malignancies of the lung, esophagus, stomach, gallbladder, liver, cervix, pancreas, colon and rectum, prostate, and urinary bladder as well as hematopoietic malignancies.
Scientists seek to measure circulating levels of inflammatory markers to study the relationship of cancer to chronic inflammation. Until recently, most such epidemiologic investigations have been limited in scope, because scientists have been able to measure only a few markers at a time. Moreover, the markers evaluated in different studies have varied, making comparisons across studies difficult. As a result, scientists are still unclear on the exact biomarkers and pathways involved in carcinogenesis.
Today, emerging technology has opened the door for scientists to measure many immune-related markers simultaneously in a high-throughput, multiplex format using small volumes of specimens. As in any scientific field, investigators had to test and validate the methods of applying this technology.
Anil K. Chaturvedi, Ph.D. (IIB), Allan Hildesheim, Ph.D., Chief of IIB, Ruth M. Pfeiffer, Ph.D., Biostatistics Branch, Ligia A. Pinto, Ph.D., Head of the Human Papillomavirus (HPV) Immunology Laboratory at the Frederick National Laboratory for Cancer Research and an investigator in IIB, and colleagues led a study of 100 healthy individuals from the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial to evaluate the performance and reproducibility of various multiplex immune marker testing kits and specimen types. The results from this important methodologic study (Cancer Epidemiol Biomarkers Prev 2011) were used to define an optimal multiplex immune panel. The panel, which was validated and tested in Dr. Pinto’s laboratory, is being produced by EMD Millipore (Billerica, Massachusetts). DCEG investigators are using the panel to conduct studies evaluating the role of many circulating inflammatory markers in cancer etiology and exploring their utility in prognosis.
“Evaluation of existing multiplex platforms was only the first, but an important, step to provide investigators throughout DCEG with a well-characterized tool to comprehensively evaluate the association between immune and inflammatory markers and various cancers,” Dr. Hildesheim said. “With the new panel, DCEG is poised to make important contributions to our understanding of the role of inflammation in cancer through carefully designed studies nested within its well-established cohorts.”
Although smoking is the main risk factor for lung cancer, mounting evidence supports the role of chronic inflammation in the etiology of lung cancer.
In two nested case-control studies within PLCO, DCEG investigators showed that candidate markers of chronic pulmonary inflammation, including Chlamydia pneumoniae seropositivity (Cancer Epidemiol Biomarkers Prev 2010), C-reactive protein (CRP, an inflammation biomarker, J Clin Oncol 2010), interleukin 6 (IL-6) and IL-8 (J Natl Cancer Inst 2011), polymorphisms in innate immunity and inflammation pathways (Cancer 2012), and circulating markers of interstitial lung disease (Cancer Epidemiol Biomarkers Prev 2011), were associated with significantly increased risk of lung cancer.
To evaluate the role of inflammation in lung carcinogenesis more comprehensively, IIB investigators are conducting ongoing studies in PLCO, utilizing the multiplex immune panel.
Dr. Chaturvedi, Meredith Shiels, Ph.D., M.H.S. (IIB), and collaborators recently measured serum levels of 77 inflammation markers and found several that were significantly associated with lung cancer risk, including acute-phase proteins (CRP and serum amyloid A [SAA]); pro- and anti-inflammatory cytokines (sTNFRII and IL-1RA); lymphoid differentiation cytokines (IL-7); growth factors (TGF-A); and chemokines (ENA78/CCXL5, MIG/CXCL9, BCA-1/CXCL13, TARC/CCL17, and MDC/CCL22) (JNCI 2013).
“These markers remained associated with lung cancer risk after careful adjustment for cigarette smoking, and associations for some of the markers preceded lung cancer diagnosis by several years,” Dr. Shiels said. “Our study adds to the body of evidence supporting a role of inflammation in lung cancer etiology by implicating several classes of inflammation markers in lung carcinogenesis.”
In addition, investigators used the Environment And Genetics in Lung cancer Etiology (EAGLE) study, a population-based study of 2,000 lung cancers, to evaluate the ability of inflammatory markers to predict survival. Clara Bodelon, Ph.D., M.S., Hormonal and Reproductive Epidemiology Branch (HREB), Maria Teresa Landi, M.D., Ph.D., Genetic Epidemiology Branch, and colleagues recently published evidence that deregulation of inflammatory responses may play a role in the survival of patients with early-stage lung cancer (Ann Oncol 2013).
Severe immune dysregulation, as seen, for example, in immunosuppression and in patients with AIDS, is an established risk factor for NHL. However, the role of more subtle immunologic disturbances in NHL is unclear.
In 2011, Mark Purdue, Ph.D., and Nathaniel Rothman, M.D., M.P.H., M.H.S., both of the Occupational and Environmental Epidemiology Branch, along with other collaborators, conducted a nested case-control study within PLCO to investigate whether circulating levels of 11 cytokines and other immune markers were associated with future risk of NHL (Cancer Res 2011). They found elevated circulating levels of the soluble immune activation markers sCD27 and sCD30 to be strongly associated with increased future risk of NHL. There was limited evidence of association with the pro-inflammatory markers soluble tumor necrosis factor receptor-1 (sTNFR1) and sTNFR2.
To more broadly investigate the effects of different immunologic and other mechanisms on NHL risk, Drs. Purdue, Rothman, and colleagues conducted a new nested case-control study within PLCO in 2013 (Blood 2013). They compared serum levels of 67 immune and inflammation markers between NHL cases diagnosed at least five years after blood collection and controls. Their findings suggest that several circulating immune markers, including circulating B-cell attracting chemokine-1 (BCA-1), sTNFR2, and soluble vascular endothelial growth factor receptor-2 (sVEGFR2), are associated with risk of NHL well in advance of diagnosis.
Using data from PLCO, Britton Trabert, Ph.D., and Nicolas Wentzensen, M.D., Ph.D., M.S. (both of HREB), along with other collaborators, are investigating whether circulating levels of immune markers are associated with risk of ovarian cancer. Preliminary results suggest a positive association between IL-8, CRP, and TNF-alpha and increased risk of ovarian cancer.
Jill Koshiol, Ph.D. (IIB), and colleagues evaluated the performance of a multiplex panel of immune-related markers in cervical secretions within the Study to Understand Cervical Cancer Early Endpoints and Determinants (SUCCEED) (Int J Cancer 2013). They found that over 70 percent of the immune markers were measured reliably. As a secondary objective, they explored potential associations of immune markers with HPV infection and cervical intraepithelial neoplasia (CIN) status. Evidence also suggested that higher levels of chemoattractant markers were associated with CIN 2/3.
Dr. Koshiol, Felipe Castro, Ph.D. (IIB), and other investigators also are evaluating the role of immune markers in the development of gallbladder cancer within a case-control study in Shanghai, China. Using multiplex technology, these researchers were able to identify a number of inflammatory markers in serum and bile that were associated with gallbladder cancer. These associations were strong in magnitude, reinforcing the belief that inflammation plays a critical role in this disease process.
As an important next step, DCEG investigators will seek to replicate the top associations from these initial studies. Other ongoing work includes:
Investigators are also considering:
In addition, Drs. Chaturvedi, Hildesheim, and Shiels are working to identify knowledge gaps to help focus future efforts in this area as part of a working group with Danielle Mercatante Carrick, Ph.D., M.H.S., Muin J. Khoury, M.D., Ph.D., and Mukesh Verma, Ph.D., in NCI’s Division of Cancer Control and Population Sciences.
“It is our hope,” Dr. Chaturvedi said, “that this series of studies using a comprehensive immune marker panel will lead to the identification of the most promising markers that can be the target of future analytical, laboratory, and clinical efforts to better understand the role of inflammation in cancer and their potential for clinical application.”