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Molecular Oncology, Markers, Clinical Correlates |
Department of Otolaryngology/Head and Neck Surgery, University of California Irvine College of Medicine [T. Y. S., S. K.], and Chao Family Comprehensive Cancer Center, University of California Irvine Medical Center [T. Y. S., C. E. M., K-T. L., S. K.], Orange, California 92868; Departments of Otolaryngology/Head and Neck Surgery [N. N., G. H. Y.], of Immunology/Microbiology [W-Z. W.], of Medicine [J. E.], and of Pathology [W. S.], Wayne State University School of Medicine, Detroit, Michigan 48201; Biostatistics Shared Resource, Chao Family Comprehensive Cancer Center and the Epidemiology Division, Department of Medicine, University of California-Irvine, Irvine, California 92697 [C. E. M.]; and Karmanos Cancer Institute, Detroit, Michigan 48201 [W-Z. W., A. R., J. E., W. S.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design: Lymph node mononuclear cells were purified from cancer patients and stimulated with immobilized anti-CD3 in vitro for 8 days. Two populations were identified, nonresponders (NRs) with [3H]thymidinecounts per min (cpm) <3500 and responders (Rs) with cpm 
3500. NRs and Rs were prospectively followed for a minimum of 24 months, and clinical outcomes were compared. Postoperative complications, length of hospitalization, toxicities associated with chemotherapy or radiation therapy, survival, and disease-free status were measured.
Results: Twenty-six patients were followed, of which 19 Rs {[3H]X = 37,819 ± 24,979 cpm (mean proliferative count ± SD)} and 7 NRs ([3H]X = 1,375 ± 1,102 cpm) were identified. There were no phenotypic differences in lymph node T-cell subpopulations (CD3, CD4, CD8, CD28, CD45RO) between groups. There was a 71% (5/7) incidence of recurrent cancer in NRs compared with 16% (3/19) in Rs; the median disease-free interval was significantly less in NRs (P = 0.03). The risk ratio of Rs to develop a recurrent cancer was 0.237 (95% confidence interval, 0.057–0.994), much less than for NRs.
Conclusions: Patients with an unresponsive CD3 receptor as measured by in vitro response to anti-CD3 monoclonal antibodies had a significantly higher incidence of recurrent cancer. Analyses using Cox proportion hazards models demonstrated that CD3 response was the single greatest predictor of reduced disease-free interval. This is the first prospective study to confirm the importance of regional lymph node mononuclear cell CD3 receptor function in head and neck squamous cell carcinoma patients for tumor control.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CD3) MoAb, and a functionally suppressed subpopulation of patients was identified. In continuation of this earlier study (7) , we prospectively accrued more patients and followed this population of anti-CD3 NRs and Rs for a minimum of 2 years. In this study, we prospectively examine what the clinical implications of an unresponsive CD3 receptor has on the outcome by comparing NRs to a group of matched HNSCC patients who were CD3 responsive or Rs. We examined the disease-free interval, recurrence rate, and overall survival between groups. Medical complications commonly associated with poor immune function were studied prospectively as well. During treatment, groups were monitored and compared for postoperative wound infections, length of hospitalization, and complications related to chemotherapy and radiotherapy. To test whether poor CD3 responses could be reversed, stimulation with anti-CD3/anti-CD28 coated beads (8) was tested. To our knowledge, this is the first study to prospectively follow a group of patients with poor regional LNMC CD3 receptor function and examine the clinical outcome.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PBMC.
Peripheral blood was drawn from patients before surgery. Blood was suspended in an equal volume of PBS and PBMCs were isolated by centrifugation over a Ficoll-Hypaque (Pel-Freez, Brown Deer, WI) density gradient (10 min at 2400 rpm).
LNMC.
LNs were harvested at the time of surgery and placed in balanced salt solution with 20% heat-inactivated FCS (Hyclone, Logan, UT), 1% penicillin/streptomycin, and 1% fung. LNs were minced, filtered through a nylon mesh, and washed twice in balanced salt solution with 5% FCS, 1% penicillin/streptomycin, and 1% fung. LNMCs were obtained by Ficoll-Hypaque density gradient centrifugation. Only pathologically confirmed negative LNs were used in this study.
MoAbs.
CD3 (OKT3) MoAb was purchased from Caltag Corp. (Burlingame, CA).
Lymphocyte Activation.
Lymphocytes were incubated in triplicate with uncoated plastic or CD3-coated plastic for 8 days. PBMCs (1.5 x 105) were cultured in 200 µl of culture media in 96-well flat-bottomed plates (Costar, Cambridge, MA). Culture media consisted of RPMI 1640 (Life Technologies, Paisley, United Kingdom) supplemented with 10% FCS (Hyclone, Logan, UT), 2 mM glutamine (Life Technologies), penicillin (100 units/ml; Life Technologies), streptomycin (100 µg/ml; Life Technologies), and fung (100 units/ml; Life Technologies). For phenotyping, 7.5 x 105 PBMCs or LNMCs were cultured in 2.0 ml of culture media in 24-well flat-bottomed plates for 6 days. All of the cells were cultured at 37°C and 5% CO2.
Plastic Stimulation.
The 96- and 24-well microtiter plates were precoated with CD3 at a concentration of 2 µg/ml in PBS for 4–18 h at 37°C and were washed three times with PBS. All CD3 stimulation was performed with MoAb immobilized on tissue culture plastic and performed in triplicate.
Bead Stimulation.
CD3/CD28 beads were generated as described previously (8)
. CD3/CD28 beads were mixed with lymphocytes at a 3:1 ratio for stimulation. CD3/CD28 bead stimulation was performed to test whether poor regional LNMC CD3 response could be reversed. LNMCs from NRs were stimulated for 6 days in vitro, and proliferative responses were measured.
[3H]Thymidine Incorporation by LNMCs or PBMCs.
On days 2,4, 6, and 8, the cultures were pulsed with 2 µCi of [3H]thymidine for 4 h and harvested onto glass fiber discs using a PHD cell harvester (Cambridge Technology, Cambridge, MA). The glass fiber discs were counted in vials containing 6 ml of scintillation-counting mixture in a Beckman scintillation counter.
Phenotypic Analysis.
On days 0, before incubation, cell suspensions were prepared from LNMC or PBMC cultures and stained with MoAbs to CD3, CD4, CD8, CD28, or CD45RO conjugated to phycoerythrin (PharMingen, San Diego, CA) as described previously (7
, 8)
. Surface marker expression was measured by flow cytometry using a Becton Dickinson FACScan.
Clinical Comparison.
NR and R groups were identified based on proliferative counts measuring [3H]thymidine incorporation within the first 6 days of stimulation. CD3 stimulation was performed in triplicate and the mean proliferative responses were calculated on days 2, 4, and 6. The highest mean value for day 2, 4, or 6 was used to determine whether individuals were NR or R. A LNL response to CD3 with [3H]thymidine incorporation 3500 cpm within the first 6 day of stimulation was defined as a "responder" (R); and a response <3500 cpm was defined as a "nonresponder" (NR), as published previously(7)
. Group demographics were compared that included age, sex, primary tumor site, stage of disease, and frequency of alcohol and tobacco exposure (Table 1)
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Present survival data as well as disease status were compared between groups.
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CD3 with [3H]thymidine incorporation were compared for Rs and NRs using the Wilcoxon rank-sum test. For the head and neck cancer patients studied, the Kaplan-Meier method was used to estimate the survival and disease-free interval functions for each group. The log-rank test was applied to test for equality of survival and disease-free interval function of Rs and NRs. Multivariable analyses were performed using Cox proportional hazards models with time-to-recurrence as the outcome variable, CD3 response as the main predictor, and potential confounders. The best fitting and most parsimonious models were selected using multivariable, step-wise, and forward- and backward-selection procedures. A significance level of P = 0.05 was used for all of the statistical tests. |
RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Demographics were compared between Rs and NRs. There was no significant difference in age, sex, stage of disease, primary tumor site, years of tobacco use, or alcohol consumption (Table 1)
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Response to Plastic-adherent CD3.
Anti-CD3 MoAb was immobilized on tissue culture plastic and was used to stimulate LNMCs and PBMCs in vitro from patients with HNSCC. Stimulation of CD3 was chosen because altered signaling via the TcR has been reported in other cancers. The optimal CD3-stimulating conditions had been previously identified in our lab by measuring proliferative responses of normal control PBMCs (7)
. LNMCs from 26 patients with advanced-stage cancer were stimulated with CD3-coated plastic over an 8-day period. Nineteen patients responded to CD3 with [3H]thymidine incorporation 3500 cpm within the first 6 days of stimulation (Fig. 1)
. Seven patients who did not respond to CD3 and had 3[H]thymidine counts <3500 cpm were defined as NRs (Fig. 1)
. The difference between groups in LNMC responses was highly significant (Wilcoxon rank-sum test, P = 0.0001). Comparison of the highest PBMC response to CD3 stimulation within the first 6 days of stimulation between R and NR was not significantly different (Wilcoxon rank-sum test, P = 0.066; Fig. 2
).
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CD3 stimulation. If there were differences in the percentage of CD3 positive cells between groups, then testing anti-CD3 responses would be an invalid method of differentiating patients. There was no difference in phenotype between R and NR, measuring CD3, CD4, CD8, CD28, and CD45RO positive cells (Table 2)
. Attempts at phenotyping the T-cell population present after 6 days of in vitro CD3 stimulation did not allow for adequate expansion of T cells for analysis.
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CD3/CD28 Stimulation.CD3/CD28 beads. All NRs had enhanced proliferative responses to bead stimulation, above CD3 responses (Wilcoxon rank-sum test, P = 0.017; Table 2
; Fig. 3
). Four of seven had responses with [3H]thymidine counts >3500 cpm, whereas three of seven had improved responses but not >3500 cpm (Fig. 3)
. All of the Rs tested had enhanced proliferative responses as well (Table 2
; Fig. 3
), although not statistically higher (P = 0.25) than CD3 stimulation alone.
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; Fisher’s exact test, P = 0.66). There was no difference in the length of hospitalization (Table 3
; Wilcoxon rank-sum test, P = 0.58).
With regard to chemotherapy and radiation therapy, there was a higher participation in chemotherapy for NRs (86%) compared with Rs (26%; Table 3
; Fisher’s exact test, P = 0.02). There was no difference between groups in the frequency of radiation therapy (Table 3)
. Fifty-eight % of Rs and 86% of NRs received postoperative radiation therapy (Fisher’s exact test, P = 0.36). Complications associated with chemotherapy and/or radiation therapy were graded on the NCI toxicity grading scale (Table 3)
, but small patient numbers did not allow for statistical analysis.
Clinical Outcome.
There was a higher incidence of recurrent cancer in NRs compared with Rs within the first 24 months after treatment (Table 4)
. The disease-free interval between groups was significantly different (P = 0.03; Fig. 3
). The disease-free survival between groups was not significantly different because a number of R patients died of causes other than their cancer (P = 0.36; Table 4
). Five-year survival data will not be available for several more years. Multivariable analyses were performed on the data set from the 26 patients. A Cox proportional hazards model was used with time-to-recurrence as the outcome variable and CD3 response as the main predictor adjusted for potential confounding variables. The risk ratio for CD3 Rs for recurrence was 0.237 (95% CI, 0.057–0.994). Patients who responded to anti-CD3 MoAb stimulation were much less likely to develop a recurrent cancer than were NRs. Although stage of disease was predictive of recurrent cancer (log-rank test, P = 0.04), it did not add information to the multivariant model, including CD3 response.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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expression in the T lymphocytes from a murine colon carcinoma model. Nakagomi et al. (12)
found decreased expression of CD3 chain in TIL and natural killer cells in 14 patients with colorectal carcinoma, and Finke et al. (2)
found similar deficits in 10 cases of renal cell carcinoma. Cardi et al. (13)
question whether these findings were valid and attributed these alterations to laboratory method. Others have continued to show alteration in CD3 and CD3
expression using different techniques and have retrospectively identified lack of expression as important to clinical outcome (14
, 15)
. Unfortunately, there are no prospective studies examining what the clinical significance of an altered CD3 receptor has on outcome. This is the first study to prospectively address this question.
In continuation and expansion of a previously initiated study (7)
, we prospectively followed a group of advanced-stage HNSCC patients in whom altered CD3 responses had been identified. In vitro LNL and PBL responses to CD3 MoAb stimulation were measured by [3H]thymidine cpm incorporation. Using this simple screening test, we identified two populations of patients, Rs and NRs (Figs. 1
and 2
). The presence of CD3 on the surface of LNLs and PBLs was checked and confirmed by flow cytometry, and no differences in T-cell subpopulations existed between the groups (Table 2)
.
Patient demographics were compared, and no differences between groups were identified (Table 1)
. Groups were then followed clinically for a minimum of 24 months to identify whether there were higher complication rates during surgery, radiation therapy, or chemotherapy. No differences in postoperative complications, length of hospitalization, or wound infections were identified (Table 3)
. Small sample size did not allow for statistical comparison of chemotherapy or radiation therapy complications between groups (Table 3)
. Disease-free interval was compared between groups and 71% of anti-CD3 NRs had recurrent or metastatic cancer compared with 16% of Rs (P = 0.03; Table 4
). There was no difference in survival between groups (Fig. 4)
, but because all patients have been followed for only 2 years and not 5 years, no definitive comment regarding survival can be made at this time. In addition, a number of responsive patients died from causes other than their cancer, making survival data difficult to analyze and interpret (Table 4)
. Frequently, advanced-stage HNSCC patients have complex health problems in addition to their cancer, usually reflecting their referral to a tertiary healthcare institute.
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and CD3 chain from paraffin-embedded oral squamous cell carcinoma tumors. They found that a lack of CD3 chain expression in the TILs, combined with tumor stage or nodal status, defined a group of patients with extremely poor prognosis. We found that T lymphocytes from noncancerous regional LNs could predict the disease-free interval after treatment. By measuring CD3 responses using a simple proliferation assay, which cross-links the TcR with anti-CD3 MoAbs, a subpopulation of cancer patients at high risk for recurrent or metastatic cancer was identified. The response of T lymphocytes from regional lymphatics was an even greater predictor of the disease-free interval than was tumor staging. This study prospectively confirms the retrospective findings of other investigators who have associated poor T-cell function with poor prognosis in HNSCC (15
, 16)
. Our study is unique in demonstrating that poor TcR function is present in the regional noncancerous lymphatics of a subpopulation of HNSCC patients. Others have identified this problem in TILs (15)
.
In an attempt to determine whether poor CD3 response was caused by lack of costimulation (CD28 signal), we stimulated NR LNL with CD3/CD28 beads (8)
. Interestingly all of the LNLs responded to this costimulation and had enhanced proliferative responses. Of the seven NRs tested, four had proliferative responses well above the 3500 cpm cutoff for nonresponsiveness, whereas three did not (Fig. 3)
. It is possible that poor CD3 responses may be caused by lack of costimulation in a subgroup of HNSCC patients, but this does not fully explain the deficiency. Future studies are needed to identify the specific causes.
Heimdal et al. have examined the response of PBL T cells and monocytes to Concanavalin A in HNSCC patients and found that responses were an independent predictor of outcome (17
, 18)
. Concanavalin A is a polyclonal mitogen that broadly stimulates T cells. We similarly tested PBL T-cell function by measuring responses to anti-CD3 MoAb alone. We found no significantly suppressed responses to anti-CD3 in PBLs but found significant suppression in the regional lymphatic T-cell population (Figs. 1
and 2
). These poor T-cell responses were an independent predictor of outcome.
HNSCC is a unique cancer, which "fights back" and uses a number of local immune suppressive mechanisms to shut down the host immune response. Fas-L, prostaglandins, P15-E, tumor growth factor ß, and several other immune escape mechanisms have been suggested (19, 20, 21, 22, 23)
. The regional immunosuppression can enhance recurrent cancer (Table 4)
. Which factor is responsible for poor CD3 dysfunction in our patient population has not been clearly identified. It is conceivable that a lack of costimulation (CD28 signal) may be a contributing factor for poor TcR function in some cases but may not explain the entire cause. T-cell function of regional LNMCs seems to be a very important factor in local cancer control. There is a need for future development of immune enhancing strategies that specifically target the regional lymphatics of HNSCC patients. Enhancement of regional T-lymphocyte function may reduce recurrent cancer and improve local control of this disease and survival.
Our conclusions are: (a) HNSCC patients with poor CD3 receptor function had a significantly higher incidence of recurrent cancer; (b) CD3 response was the single greatest predictor of reduced disease-free interval after multivariable analysis and Cox proportional hazard model analyses were performed. The risk ratio of Rs to develop a recurrent cancer was 0.237 (95% CI, 0.057–0.994), which was much less than that of NRs; (c) this study is the first to prospectively confirm the importance of cancer-free regional LNMC CD3-receptor function and its effect on clinical outcome in HNSCC patients; (d) anti-CD3/anti-CD28 costimulation was able to overcome poor anti-CD3 responses in a subgroup of HNSCC patients; and (e) future immune-enhancing strategies that target the regional lymphatics of HNSCC patients should be developed.
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FOOTNOTES |
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1 Supported by NIH Grants T32DC00026 and ACS IN-162 (to T. Y. S.). 
2 To whom requests for reprints should be addressed, at Department of Otolaryngology/Head and Neck Surgery, University of California Irvine Medical Center, 101 The City Drive South Building 25, Orange, CA 92868. E-mail: TShibuya{at}uci.edu
3 The abbreviations used are: HNSCC, head and neck squamous cell carcinoma; MoAb, monoclonal antibody; LN, lymph node; LNMC, LN mononuclear cell; TIL, tumor-infiltrating lymphocyte; LNL, LN lymphocyte; PBL, peripheral blood lymphocyte; TcR, T-cell receptor; R, responder; NR, nonresponder; PB, peripheral blood; PBMC, PB mononuclear cell; fung, fungizone; NCI, National Cancer Institute; cpm, counts per min.
4 National Cancer Institute Cancer Therapy Evaluation Program, Common Toxicity Criteria Manual, Version 2.0. June 1, 1999.
Received 6/27/01; revised 11/ 6/01; accepted 11/12/01.
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REFERENCES |
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chain and p56lck in T-cells infiltrating human renal cell carcinoma. Cancer Res., 53: 5613-5616, 1993. chains in tumor-infiltrating T-cells and NK cells of patients with colorectal carcinoma. Cancer Res., 53: 5610-5612, 1993.-chain expression on tumor-infiltrating lymphocytes from renal cell carcinoma. Cancer Res., 57: 3517-3519, 1997. chain in T cells at the tumor site correlates with poor survival in patients with oral carcinoma. Cancer Res., 58: 5344-5347, 1998. chain expression in peripheral blood lymphocytes of patients with head and neck cancer. Clin. Cancer Res., 5: 329-334, 1999. in head and neck cancer. Cancer Immunol. Immunother., 38: 178-184, 1994.[Medline]
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