Tissue Microarrays & Colorectal Cancer: survival, treatment and diagnosis
Maximillian A. Rogers
Dartmouth Medical School MCB Graduate Program


This report is published as a partial requirement for the graduate class in Oncogenomics, Genetics 144 taught by Dr. Charles Brenner, Dartmouth Medical School, Winter Term 2006.
The course is based on material covered in Oncogenomics: Molecular Approaches to Cancer.

PLEASE NOTE: Online subscription to the New England Journal of Medicine may be required to view some material on this page.


COLON CANCER

The colon is the first 6 feet of the large intestine, also referred to as the large bowel. It is part of the digestive system, which removes and processes nutrients from food sources along with removing waste material from the body.



Colon Cancer: Typically colon cancer starts off as a non-cancerous polyp that changes into cancer, which may eventually grow through the layers of the colon and spread to surrounding tissues, lymph nodes and organs. While very curable in the early stages, colon cancer, as noted by the National Cancer Institute, is the number two cause of cancer related deaths in the US.

RISK FACTORS

There are several risk factors for the development of colon cancer, including:

-Age, with higher risk in individuals 50 years old or older.
-Family history of colon or rectum cancer.
-Individual history of colon, rectum, ovary, endometrium or breast cancer.
-Individual history of ulcers within the lining of the large intestine.
-Various hereditary disease like Lynch Syndrome and familial adenomatous polyposis.

DIAGNOSIS/PROGNOSIS

Diagnosis can be most easily be made by various tests, which exam the rectum and its tissue, along with blood.

The chance of recovery, prognosis, depends on:
-The cancer's stage. Meaning whether the cancer has spread beyond the inner lining of the colon.
-If the cancer has either blocked or generated a whole in the colon.
-The level of carcinoembryonic antigen in the blood prior to treatment, which may be increased in the presence of cancer.
-If the cancer is a reoccurrence.
-The overall health of the patient.

STAGING

Following diagnosis several tests can be done to see if the cancer has spread within the colon or to other areas of the body, such as: CAT scan, lymph node biopsy, complete blood count, carcinoembryonic antigen assay, MRI, chest x-ray and surgery.

These tests are used in the staging process. The cure rates using standard therapies are high within patients whose cancer has yet to spread outside of the colon, but drastically fall as the cancer spreads. Colon cancer is typically divided into 5 stages , which include:



Stage O (Carcinoma in Situ): Here the cancer is found only in the innermost lining of the colon.

Stage 1 or Dukes’ A: The cancer at this stage has spread beyond the innermost lining of the colon wall into the middle layers.

Stage 2 or Dukes’ B: The cancer has spread beyond the middle layers or to nearby tissues or organs.

Stage 3 or Dukes’ C: The cancer has spread to as many as three or four lymph nodes and/or to nearby tissues or organs.

Stage 4 or Dukes’ D: The cancer has spread to nearby lymph nodes and various parts of the body like the liver or lungs.



Effector Memory T Cells, Early Metastasis and Survival in Colorectal Cancer
Franck Pages, M.D., Ph.D., Anne Berger, M.D., Ph.D., Matthieu Camus, M.Sc., Fatima Sanchez-Cabo, Ph.D., Anne Costes, B.S., Robert Molidor, Ph.D., Bernhard Mlecnik, M.Sc., Amos Kirilovsky, M.Sc., Malin Nilsson, B.S., Diane Damotte, M.D., Ph.D., Tchao Meatchi, M.D., Patrick Bruneval, M.D., Ph.D., Paul-Henri Cugnenc, M.D., Ph.D., Zlatko Trajanoski, Ph.D., Wolf-Herman Fridman, M.D., Ph.D., and Jerome Galon, Ph.D.

New England Journal of Medicine, Vol 353, 2654-2666, December 2005





Background
Despite several modern medical advances and gained knowledge in the area of colon cancer there still remains large fluctuations of survival times with patients diagnosed with the same stage and who also undergo the same treatment regimen. Why this is the case is still unknown, but a recent paper in the New England Journal of Medicine by Pages et al. (2005) attempts to provide some insight to the question.

Recent developments have indicated inflammation as a factor in tumor progression, once thought mainly to be governed by genetic changes, epigenetic and environmental factors (Steeg 2003 and Coussens 2002). In addition, studies in mice have shown that an increased production of inflammatory mediators are found during colorectal cancer progression (Greten 2004, Pikarsky 2004 and Clevers 2004). However, the role that tumor-infiltrating immune cells play in the early metastatic invasion of colorectal cancer still remains unknown.

Therefore, it was the goal of the Pages et al. (2005) study to see whether the early steps of the metastatic processes of colorectal cancers are associated with inflammatory or immunosuppressive mediators, or both, and whether the absence of the early metastatic process is associated with an adaptive immune response.

For the purpose of the study the authors termed the early metastatic invasion "VELIPI". VELIPI involves the presence of vascular emboli, lymphatic invasion, and perineural invasion, alone or in combination.

Vascular emboli: Cancer cells that travel through the blood stream, lodges in a blood vessel and blocks it.



Lymphatic invasion: Cancer cells invading the lymph nodes.



Perineural invasion: Cancer cells invading in or around a nerve or group of nerves.



Methods

Pages et al. (2005) studied the pathological signs of early metastatic invasion, VELIPI, in 959 specimens of resected colorectal cancer. Observation time for the patient records was between diagnosis and their last contact. The local immune response within the tumor was examined by flow cytometry using 39 tumors, low density-array real-time polymerase-chain-reaction assay using 75 tumors, and tissue microarrays for 415 tumors.

General information on the methods used in this study:
Tissue Microarray
Flow Cytometry
Real Time
Kaplan-Meier

Results

Early Metastatic Invasion and Clinical Outcome
The data from 959 patients with colorectal cancer was investigated to determine the prognostic significance of the presence of VELIPI, which delineates early metastatic invasion (Table 1) . Of note the presence or absence of VELIPI as well as the tumor stage significantly influenced disease-free and overall survival (P less than .001 for all comparisons). In addition, the presence of more than one sign of early metastatic invasion conferred a worse prognosis than the presence of a single sign.

Kaplan-Meier curves were used to asses the influence of the signs of early metastatic invasion, VELIPI, on overall and disease-free survival (Figure 1) . The Kaplan-Meier analysis suggested longer overall survival and disease free survival among patients with VELIPI-negative tumors than among patients with VELIPI positive tumors.

Immune-cell Infiltration, Inflammation, Early Metastatic Invasion, and Prognosis
Using the theories that increased inflammation might be connected to tumor progression along with increased immunosuppressive mediators being connected to spontaneous tumors the study next looked to see if this might be the case with their study samples. Extracting total RNA from 100 randomly chosen samples, the authors generated 75 samples of sufficient quality to observe the expression of inflammatory genes, immunosuppressive genes and genes related to the adaptive immune response (Figure 2). In regards to the expression of inflammatory genes the authors saw no significant diffence between VELIPI positive or negative tumors. As was the case when they examined the expression of immunosuppressive genes. However, when the examined the expression levels of genes related to the adaptive immune response the noted several significant differences. Of note, tumors without signs of early metastatic invasion had increased levels of mRNA for the products of type 1 helper effector T cells, CD8, T-BET, IRF-1, Interferon-gamma, granulizing and granzyme B. Part of which suggest an increase in Th1 response in VELIPI negative tumors, but not an increase in the Th2 response.

Phenotypes of Tumor-Infiltrating Immune Cells
To analyze subpopulations of immune cells from 39 freshly resected colon cancers Pages et al. (2005) used large-scale flow cytometry (Figure 3). They measured 410 combinations of surface markers, from which they saw CD3+ T cells (Th1 subset) as the most prevalent tumor infiltrating immune cells. They then used clustering to show that markers of T cell migration, activation and differentiation were increased in tumors without signs of early metastatic invasion. Looking at the differentiation process they saw that VELIPI - tumors had increased numbers of CD8 + T cells from early memory (CD45RO+, CCR7-, CD28+ and CD27+) to effector memory (CD45RO+, CCR7-, CD28- and CD27-).

Effector Memory T Cells and Survival
In order to get at whether the increased expression of memory T cells played a role in a good clinical outcome they performed immunohistochemical analysis of tissue microarrays prepared from 415 colorectal cancers (Figure 4).To count CD45RO+ cells the used automatic-image software and did a validation study with optical counts. A comparison of the optical and automatic cell counts showed a close correlation. (R2=0.914, P<0.001). They then used Kaplan-Meier curves to correlate the CD45RO+ cell counts to the patient data. As a result they noticed that the presence of high levels of infiltrating memory CD45RO+ cells correlated with the absence of signs of early metastatic invasion, a less advanced pathological stage, along with an increased survival.

Conclusions

One of the key interpretations of this study is that the tumor microenvironment and the host’s immune response are of paramount importance to tumor progression. The study's finding of large numbers of memory T-cells, CD45RO+, within a colorectal tumor as being correlated with increased survival for patients, leads to numerous interpretations of the role of tumor-infiltrating immune cells. Primarily that immune cells appear to control the growth of some colorectal tumors by preventing early metastasis. Therefore, an immune response could be occurring in some colorectal tumors with beneficial results, a theory contrary to earlier views that immune reactions in colorectal cancer were mostly ineffective and perhaps even harmful to the individual.

Therefore, new attempts to use immunotherapy to treat colorectal cancer, or new clinical trials of immunotherapy in patients with early disease or seemingly disease-free after surgery seems reasonable if not a needed next step.

Class Discussion

During the class presentation, on which this web report was based, there was some discussion as to how to correctly interpret the results of the Pages et al. (2005) study. Essentially what some students felt the study needed to do was take a more in-depth look at the patient genotypes. They theorized that those cancers associated VELIPI could be less immunogenic than those that lacked any sign of early metastasis. In addition to the use of the VELIPI work, they wanted a greater comparison to be made between tumors that had large numbers of immune cells and those that did not. They essentially argue that because of this the immune response can not be said to protect individuals from colon cancer.

However, I feel this argument ignores the fact that whether or not the tumors of the various patients were immunogenic, the presence of memory T cells did correlate with a statistically significant positive survival in patients. Do memory T cells directly or indirectly positively effect patient survival, and is it the patient's immune system or the tumor environment that is more important in regards to early metastasis? I can not say for sure, nor did this paper attempt to generate the definitive answers. What it did attempt to do was generate the potential for research in an area that may have been previously overlooked.

References

Clevers H. At the crossroads of inflammation and cancer. Cell 2004;118:671-674.

Coussens LM, Werb Z. Inflammation and cancer. Nature 2002;420:860-867.

Greten FR, Eckmann L, Greten TF, et al. IKKbeta links inflammation and tumorigenesis in a mouse model of colitis-associated cancer. Cell 2004;118:285-296.

Pages F, Berger A, Camus M, et al. Effector memory T cells, early metastasis, and survival in colorectal cancer. NEJM 2005; 353:2654-2666.

Pikarsky E, Porat RM, Stein I, et al. NF-kappaB functions as a tumour promoter in inflammation-associated cancer. Nature 2004;431:461-466.

Pollard JW. Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer 2004;4:71-78.

Steeg PS. Metastasis suppressors alter the signal transduction of cancer cells. Nat Rev Cancer 2003;3:55-63.