Association between Chemotherapy-Response Assays and Subsets of Tumor-Infiltrating Lymphocytes in Gastric Cancer: A Pilot Study

Gastric cancer is the fifth leading cause of cancer deaths globally.1 The prognosis of gastric cancer is poor because even after curative resection, advanced cancer has a high risk of recurrence despite adjuvant chemotherapy.2, 3, 4, 5

To improve the response rate of chemotherapy, adenosine triphosphate-based chemotherapy response assays (ATP-CRAs) have been employed to individualize treatment.6, 7 It would be an ideal method for choosing the most effective patient-specific chemotherapy agent, provided that an in vitro assay could predict the in vivo chemo-responsiveness. The most attractive feature of this assay is that it can simultaneously test the sensitivity of multiple chemotherapy agents.

The distribution of tumor infiltrating lymphocytes (TILs) could also predict responses to neoadjuvant8, 9, 10, 11, 12 and adjuvant chemotherapies13, 14, 15 in solid cancers. TIL distribution is an independent prognostic marker for gastric cancer and other solid cancers.8, 10, 16, 17 Gastric medullary carcinomas that have extensive infiltration of lymphocytes often show excellent prognosis.18

Thus, improving predictions for chemo-responsiveness is a highly desirable possibility when combining ATP-CRA results and TIL distribution. The aim of the present study was to explore the possibility of selecting patient-specific sensitive chemotherapeutic agents based on TIL-related immune microenvironments.

At Severance Hospital, Yonsei University College of Medicine, from February 2007 to January 2011, 15 patients were enrolled for the study with histologically proven gastric cancewr ho had undergone gastric resection surgery. All data on patients' characteristics and pathological features of resected tumors were collected by a retrospective review of a prospectively maintained database. No patients were treated with neoadjuvant chemotherapies or had histories of another primary tumor. All patients agreed to the chemosensitivity test of their resected tumors and gave informed consent. This study was approved by the Yonsei Institutional Review Board (4-2011-0864).

ATP-CRAs were performed as previously described.6 Briefly, a 0.5-cm³ sample of cancer tissue was collected, stored in Hank's balanced salt solution (GIBCO BRL, Rockville, MD, USA) containing 100 IU/ml penicillin (Sigma, St. Louis, MO, USA), 100 µg/ml streptomycin (Sigma), 100 µg/ml gentamicin (GIBCO BRL), 2.5 µg/ml amphotericin B (GIBCO BRL), and 5% fetal bovine serum (GIBCO BRL), and immediately sent to the pathology laboratory. Tissues were washed, quantified, minced, and enzymatically dissociated. Cells were purified by density centrifugation to eliminate debris. After dilution of the separated tumor cells to 2×10⁴ cells/ml, cells were seeded in triplicate in 96-well microplates (Costar, Cambridge, MA, USA).

In the treated groups, 100 µl of chemotherapeutic agents were added to the seeded cells, and 100 µl of Iscove's modified Dulbecco's medium (GIBCO BRL) without chemotherapeutic agent s were added to the untreated control groups. Samples from each patient were individually treated with each of the chemotherapeutic agents. The test drug concentrations were determined based one apk plasma concentrations according to previous reports: etoposid(e3 .57 µg/ml), doxorubicin (1.5 µg/ml), epirubicin (1.2 µg/ml), mitomycin (0.2 µg/ml), 5-fluorouracil (5-FU, 10 µg/ml), oxaliplatin (2.9 µg/ml), irinotecan (4.7 µg/ml), docetaxel (3.7 µg/ml), paclitaxel (8.5 µg/ml), methotrexate (0.37 µg/ml), and cisplatin (2.5 µg/ml).19, 20, 21 Three different doses (0.2-, 1-, and 5-fold) of the test drug were used in triplicate. Microplates were cultured for 48 hours at 37℃ in 5% CO₂. ATP levels in the cell lysates were measured using flash type luminescence measurements (Roche, Mannheim, Germany). Cancer cell death rates were determined as the ratio of ATP luminescence reduction in the treated groups compared to that of the untreated control.

Immunohistochemical staining and quantification of TIL subsets was performed as previously described.16 Paraffin-embedded gastric cancer tissue sections were serially sectioned at 4-µm, deparaffinized in xylene, and rehydrated in decreasing concentrations of ethanol. Antigen retrieval was performed in citrate buffer in a microwave. Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxide in methan ol for 5 minutes. Sections were incubated for 60 minutes at room temperature (20℃ to 25℃) with primary monoclonal antibodies: CD3 (1:100; Lab Vision Corporation, Fremont, CA, USA), CD4 (1:100; Novocastra, Newcastle Upon Tyne, UK), CD8 (1:100; Novocastra), Foxp3, (forkhead/winged helix transcription factor 3, 1:100, ab20034; Abcam, Cambridge, UK), and Granzyme B (1:100; Lab Vision Corporation), which were used to identify the following T lymphocyte subsets: total T lymphocytes, helper T lymphocytes, cytotoxic T lymphocytes, regulatory T cells, and activated cytotoxic T lymphocytes, respectively. Incubation in horseradish peroxidase-conjugated secondary antibody was subsequently performed, followed by development with diaminobenzidine and counterstaining with hematoxylin. Five high-power fields (×400) from each slide were selected for manual counting using an Olympus CX31 microscope (Olympus America, Center Valley, PA, USA). The absolute number of lymphocytes per high-power field was determined for each antibody (CD3, CD4, CD8, Foxp3, and Granzyme B). The median count number was used to divide the patients into low- and high-density groups.

Pearson's correlation tests were performed for cancer cell death rates and TIL subsets. Absolute numbers of cells positive for each stain were dichotomized using cut-off values derived by the median. Survival curves were constructed using the Kaplan-Meier method, and the log-rank test was used to evaluate the significance. A statistical significance level was defined as a P-value of 0.05 or less. All statistical analyses were performed with SAS 9.2 software (SAS Institute, Cary, NC, USA).

Accumulating evidence suggests that the immune microenvironment alters chemo-responsiveness.22, 23 Previous studies on chemo-responsiveness used various chemotherapeutic agents for different types of cancer. Thus, translation of the results to gastric cancer treatment is challenging. For example, high posttreatment levels of CD3 or CD8 TIL subsets in the tumor micro-environment correlates with more robust responses to paclitaxel neoadjuvant chemotherapy in breast cancer.24 Additionally, the density of CD8 TILs affects the chemo-responsiveness to 5-FU in stage III colon cancer.25 Higher densities of CD3, CD8, and Granzyme B, but not of Foxp3 TILs at invasive margins are related to improved chemo-responsiveness to irinotecan- and platinum-based chemotherapies in metastatic colorectal cancer.26 Furthermore, the level of regulatory T cells prior to chemotherapy is a predictive marker for early breast cancer.27 However, the relationship of the immune microenvironment and chemoresponsiveness in gastric cancer has never been studied. To explore whether cancer cell response to chemotherapeutic agents has any relationship with immune microenvironments, we investigated the association of cancer cell death rates in ATP-CRAs and the distribution of TIL subsets within the tumor tissue microenvironment. However, no significant associations were identified.

It is unknown whether TILs cause or enhance susceptibility to chemotherapeutic agents or are simply chemosensitivity markers. The current study suggests that TILs are more representative of susceptibility to chemotherapy rather than a marker of chemosensitivity. Our ATP-CRA and TIL analyses were separately performed using cultured cancer cells and immunohistochemical staining of tumor tissues. Thus, it may not completely and accurately represent the in situ interactive effects of chemotherapy on the tumor/host immune system. A subset of TILs in tumor microenvironments can modulate the susceptibility of chemotherapy against cancer cells.9, 28, 29 Conversely, chemotherapy can enhance the efficacy of host immune functions by reducing tumor burdens and enhancing tumor cell susceptibility.30 It is also known that tumor-associated antigens are released when chemotherapeutic agents destroy tumor cells.9

Limitations of this study include the small number of the patients, emphasizing the need for further validation of these observations in additional data sets; only a small portion of the patients were treated according to ATP-CRA results; and the biological mechanism underlying these observations was not studied. Despite these limitations, we tried to assess the association of chemosensitivity test results and TIL subsets in gastric cancer. No significant association between the two suggests that current ATP-CRA has limitations for predicting cancer cell destruction, which could be affected by the immune system.

In conclusion, cancer cell death rates in response to specific chemotherapeutic agents had a poor association with the distribution of TIL subsets.