We examined the prognostic significance of lymphocytic reaction to tumor in a population of stage I-IV colorectal cancer patients who were concurrently assessed for other clinical and molecular predictors of patient outcome. We observed a significant relation between lymphocytic reaction and patient survival, independent of patient characteristics and other related molecular variables including the number of lymph nodes, p53, KRAS
, microsatellite instability (MSI), the CpG island methylator phenotype (CIMP) and LINE-1 hypomethylation. Although each of the 4 components of lymphocytic reactions [Crohn's-like reaction, peritumoral reaction, intratumoral periglandular reaction and tumor infiltrating lymphocytes (TIL)] appeared to predict longer survival of patients, the association with survival was most robust when overall lymphocytic score was used. Our results may support the role of the host immune reaction to tumor as an independent prognostic factor among colorectal cancer patients. The 4 components of lymphocytic reactions can be assessed upon routine histopathologic examination of resected colorectal cancer, and an evaluation of these features can be implemented in clinical practice. Our study also supports the use of immune cells as potential cancer treatment. The stimulation of immune response has a number of theoretical advantages over other forms of cancer therapy (1
). Immune cells can direct to antigen-expressing tumor cells wherever they are located in the body, and can proliferate until all tumor cells are eradicated. Immunologic memory can be generated for surveillance against any tumor recurrence (1
). Finally, targeting host immune cells may avoid the emergence of resistance mutations that are commonly observed during targeted treatment against molecules within cancer cells.
Examining prognostic and predictive factors is important in cancer research (39
). Lymphocytic reaction to colorectal cancer has been associated with longer survival in colorectal cancer (2
). However, the mechanism underlying the survival advantage associated with lymphocytic reaction to tumor remains uncertain. Lymphocytic reaction may be an indicator of host immune response to tumor cells, leading to improved survival (7
). In addition, immune response may cause enlargement of lymph nodes, which may contribute to an increase in the recovered lymph node count and, thereby, more accurate staging of colorectal cancer. In fact, lymphocytic reaction to colorectal cancer have been associated with an increased lymph node count (13
), and the lymph node count has consistently been associated with improved survival of colorectal cancer patients (13
). Alternatively, lymphocytic reaction to tumor may reflect specific tumoral molecular alterations associated with indolent tumor behavior. Tumoral lymphocytic reaction has been associated with MSI-high (19
), which, in turn, is associated with longer patient survival (25
). Recent studies have further shown that MSI-high in colorectal cancer is associated with the CpG island methylator phenotype (CIMP), BRAF
), and high LINE-1 methylation level (35
), and all of these factors (MSI, CIMP, BRAF
mutation and LINE-1 methylation) have been independently related with survival of colon cancer patients (26
). Therefore, In fact, numerous pathologic and molecular features (the lymph node count, MSI, CIMP, BRAF
mutation, and LINE-1 methylation) could account for the beneficial effect of lymphocytic reaction to tumor. However, none of the previous studies of lymphocytic reaction and patient survival has comprehensively examined the aforementioned molecular features in colorectal cancer beyond MSI. In our analysis, the benefit associated with higher tumoral lymphocytic reaction remained significant after adjusting for these various pathologic and molecular features.
One unresolved question is whether subtyping of infiltrating lymphocytes provides any additional information beyond histopathologic evaluation of lymphocytic reaction patterns. Previous studies have shown that the presence, degree or localization of infiltrates by a specific subtype of lymphocytes (e.g., CD57+, CD8+, CD45RO+ or FOXP3+ lymphocytes) is associated with patient outcome in colorectal cancer (7
). In addition, high tumoral expression of chemokine CXCL16 has been associated with TIL and good prognosis (50
). However, none of these studies (7
) has comprehensively evaluated the distinct histopathologic patterns of lymphocytic infiltrates described in the current study. Moreover, none of the previous studies has examined potential confounding effect of the number of lymph nodes examined, and molecular features of colorectal cancer beyond MSI (i.e., CIMP, BRAF
mutation and LINE-1 methylation). Thus, confounding effect by these variables (the lymph node count and tumoral molecular features) cannot be excluded in the previous studies (7
). Additional studies are necessary to clarify whether lymphocyte subtyping adds any additional or independent prognostic information beyond histopathologic evaluation of lymphocytic reaction patterns.
We calculated the lymphocytic reaction score using the four components, i.e., Crohn's-like reaction, peritumoral reaction, intratumoral periglandular reaction and TIL. TIL appeared to be less significantly associated with patient survival, than the other three components. Although using an overall score of the three components yielded similar results (data not shown), the significance and the magnitude of the effect were attenuated. Thus, in this study, we provide the data based on the four components. Nonetheless, utilization of the three components without TIL may represent a potentially reasonable alternative approach.
We confirmed the positive relation between lymphocytic reaction and the lymph node count, which has been previously reported (13
). The recovered lymph node count has consistently been associated with longer survival of colorectal cancer patients (13
). Our data provide evidence supporting that the recovered node count is influenced by host immune reaction to tumor. Nonetheless, the best effort should be made to recover and examine as many lymph nodes as possible for accurate staging. Additional studies are necessary to assess whether the beneficial prognostic effect of the lymph node count is independent of lymphocytic reaction to tumor.
There are limitations in this study. For example, data on cancer treatment were limited. Nonetheless, it is unlikely that chemotherapy use substantially differed according to lymphocytic reactions to tumor, since such data were not typically used for treatment decision making. In addition, beyond cause of mortality, data on cancer recurrences were not available in these cohorts. Nonetheless, given the median survival for metastatic colon cancer was approximately 10-12 months during much of the time period of this study, colorectal cancer-specific survival should be a reasonable surrogate for cancer-specific outcomes.
There are advantages in utilizing the database of the two independent prospective cohort studies, the Nurses' Health Study and Health Professionals Follow-up Study to examine prognostic significance of lymphocytic reaction and its interactions with tumoral and host factors. Anthropometric measurements, family history, other clinical information, pathologic and tumor staging data, and tumoral molecular features were prospectively collected, blinded to patient outcome. Cohort participants who developed cancer were treated at hospitals throughout the U.S., and thus more representative of colorectal cancers in the general U.S. population than patients in a single to several hospitals. There were no demographic difference between cases with tumor tissue analyzed and those without tumor tissue analyzed (27
). Lymphocytic reaction to colorectal cancer was examined by the single study pathologist, and a subset of cases were re-examined by a second pathologist for the agreement study. Finally, our rich tumor database enabled us to simultaneously assess pathologic and tumoral molecular features and control for confounding by a number of tumoral molecular alterations. None of the previous studies on lymphocytic reactions and patient outcome has examined as many molecular variables as we did in this study.
In summary, our large cohort study suggests that lymphocytic reaction to tumor is associated with longer survival of colorectal cancer patients, independent of other clinical, pathologic and tumoral molecular characteristics. Our data suggest a possible role of host immune response as an independent prognostic factor in colorectal cancer patients. Future studies are needed to confirm these results as well as to elucidate exact mechanisms by which lymphocytic reaction to tumor affects clinical outcome in colorectal cancer.