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Molecular Oncology, Markers, Clinical Correlates |
1 University of Pittsburgh Cancer Institute and Departments of 2 Pathology and 3 Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design: Venous blood was obtained from 146 patients with SCCHN and 54 normal volunteers. Absolute numbers of CD3+, CD4+, and CD8+ T lymphocytes were determined using fluorobeads in a flow cytometry-based technique. Percentages of T lymphocyte subsets were also evaluated by flow cytometry. The patients were grouped at the time of blood draw [active versus no evidence of disease (NED), type of therapy administered, and the length of follow-up].
Results: Patients with SCCHN had significantly lower absolute numbers of CD3+ CD4+, and CD8+ T cells than normal controls. However, no differences in the percentages of T-cell subsets between patients and normal controls were observed. Patients with active disease had significantly lower CD3+ and CD4+ T-cell counts than those with NED. Patients who had NED after surgery and radiotherapy had the lowest T-cell counts among the NED cohort. Patients who had NED for >2 years did not recover their T-cell counts, and the T-cell imbalance was evident many years after curative surgery. The tumor-node-metastasis (TNM) stage or site of the disease was not related to the absolute T-cell count. Patients with recurrent disease at the time of blood draw tended to have the lowest CD4+ T-cell counts.
Conclusions: Patients with SCCHN have altered lymphocyte homeostasis, which persists for months or years after curative therapies.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Patients with cancer who do not receive conventional therapies are not generally considered to be lymphopenic. Nevertheless, abnormalities in T-cell counts have been observed in patients with breast cancer, ovarian cancer, myeloma, head and neck cancer, or liver cancer (8, 9, 10, 11) . More importantly, some reports show associations of T-cell subset abnormalities with poor clinical outcomes (11, 12, 13) . However, there is no consensus on the extent of changes in different T-lymphocyte subsets in the course of cancer progression or its relationship with response to tumor-specific therapy or patient survival.
Absolute numbers of lymphocyte subsets in the peripheral circulation have been traditionally measured by dual-platform technologies, which couple percentages of positive cell subsets determined by flow cytometry with the absolute lymphocyte count obtained by automated hematology analyzers. Until recently, this was a standard, universally used technology, and it has been suggested that it is responsible for substantial differences in absolute lymphocyte counts reported by different laboratories (14 , 15) . The more recent development of single-platform technologies, which are performed entirely on the flow cytometer, has significantly improved the assay precision and accuracy and allowed for greater uniformity of results between laboratories (14, 15, 16) .
To evaluate a possible impact of spontaneous apoptosis of circulating T lymphocytes (2, 3, 4, 5) on the peripheral T-cell pool, we investigated both the percentages and absolute numbers of CD3+, CD4+, and CD8+ T lymphocyte subsets in a large cohort of patients with SCCHN, using a single-platform flow cytometry-based method. We found that the presence of tumor as well as its recurrence had a significant impact on the absolute number of T-cell subsets. Both the type of therapy and length of the posttherapy period were considered, and the results suggested that regardless of these factors, lymphopenia was a persistent feature of the disease.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The characteristics of all of the patients included in this study are shown in Table 1
. The cohort of 146 patients included 109 men and 37 women with a median age of 62 years (range, 24–86 years), and the group of 54 volunteers comprised 25 males and 29 females with a median age of 54 years (range, 22–88 years). Of 146 patients, 48 had active disease, and 98 had no evidence of disease (NED) after surgery alone (n = 56) or after surgery and radiotherapy (n = 42; Table 1
).
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Absolute Lymphocyte Count Determination.
A standard single-platform technique, tetraONE System (Beckman Coulter, Miami, FL), based on four-color flow cytometry in the presence of counting beads was used. The identification of lymphocytes by expression of bright CD45 and low side scatter signals was followed by the identification of T-cell subsets based on the expression of CD3, CD4, and CD8, as described previously (16)
. Briefly, 100 µl of heparinized anticoagulated blood were incubated with 10 µl of tetraCHROME reagent containing anti-CD45-FITC-, anti-CD4-RD1-, anti-CD8-ECD-, and anti-CD3-PE-Cy5 (Beckman Coulter)-labeled antibodies. Specimens were then lysed with the ImmunoPrep Reagent System at the Coulter TQ-Prep Workstation. Leukocyte morphology and cell surface integrity were maintained by a gentle, no-wash erythrocyte lysing method. Immediately before analysis, 100 µl of Flow-count Fluorospheres (Beckman Coulter) were added to the stained cells, and the beads were counted along with cells. The sample acquisition and analysis were performed on the EPICS CL flow cytometer with a fully automated software-reagent combination. The number of cells (or cell subsets) per microliter was obtained by calculating the number of cells counted x concentration of beads/number of beads counted. In each experiment, blood obtained from patients was evaluated together with blood from at least one NC. The one-platform method was validated in our laboratory by comparisons with the previously established two-platform method.
Statistical Analysis.
Differences in percentages and counts of lymphocyte subsets between patients and NCs were age-adjusted by linear regression models after suitable data transformations. If age adjustment was unnecessary, differences were tested with the t test or the Wilcoxon test. The associations among lymphocyte subsets and clinical (disease status and site of disease), pathological (stage), and behavioral (smoking) characteristics were tested with the t test or Wilcoxon test for two-group differences or the Kruskal-Wallis test for three or more groups. Tests of trend with ordinally scaled end points such as T and N stages were conducted with Jonckheere-Terpstra test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. However, the absolute counts of CD3+ and CD4+ but not CD8+ T cells decreased significantly with age in patients and NCs. As shown in Fig. 2
, the absolute counts for CD3+ cells decreased for patients and NCs with an estimated slope of –7.3 (P = 0.02) between the ages of 24 and 88 years. CD4+ cells decreased with a slope of –5.5 (P = 0.01). CD8+ T cells were unrelated to age in either group (P = 0.66).
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; Table 2
). Age-dependent comparisons of absolute lymphocyte counts in patients versus NCs were adjusted for age. The patients had 382 fewer CD3+ cells, 297 fewer CD4+ cells, and 85 fewer CD8+ cells per mm3 than NCs. These significant differences (P < 0.0001 for CD3+ and CD4+ cells; P < 0.03 for CD8+ cells) were detectable at any age (Fig. 2)
. Overall, the patients had significantly lower absolute T-cell counts but not percentages than NCs, and the absolute numbers of CD8+ T cells were the least depressed in patients with SCCHN relative to NCs.
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). However, the same CD4/CD8 ratio does not discriminate between patients with a high versus low absolute count of the two lymphocyte subsets. In our series, for example, one patient with a CD4/CD8 ratio of 2.44 had an absolute count of 800 CD4+ cells and 328 CD8+ cells, whereas another patient with the same CD4/CD8 ratio of 2.44 had 155 CD4+ cells and 64 CD8+ cells. For this reason, we undertook a more detailed analysis of the relationship between the numbers of CD4+ and CD8+ T lymphocytes in individual patients, as shown in Fig. 3
. Relative to NCs, whose mean CD4/CD8 ratio is 2.4 (Fig. 3A)
, patients with active disease, whose mean CD4/CD8 ratio is 2.3, have decreased numbers of CD4+ and CD8+ T cells. As a result, there is a dramatic shift of the individual CD4/CD8 ratios to the bottom left quadrant in Fig. 3B
. Not surprisingly, the patients with recurrent disease have the lowest absolute counts of CD4+ and CD8+ T cells (Fig. 3B)
. Among the patients with active disease, a small subgroup (8 of 48 patients) with high numbers of CD4+ T cells and relatively normal counts of CD8+ T cells (top right quadrant in Fig. 3B
) is evident.
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. Here, it can be seen that patients previously treated with radiation therapy (RT) in addition to surgery had the lowest CD4+ counts. Other patients with NED had somewhat depressed CD4+ counts but normal CD8+ counts when compared with NCs (Fig. 3A)
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Effects of Postoperative Radiation or Chemotherapy on Absolute Lymphocyte Counts.
Among 98 patients with NED at the time of blood draws, 42 had received postoperative RT. The postoperative RT consisted of 66–70 Gy divided into 1.8–2.0-Gy fractions over 35 individual daily fractions. This RT was applied within 6 weeks of surgery. No patient received hyperfractionated RT. The time duration between last RT and a blood draw for this study was variable, ranging from 1 to 302 months (mean ± SD, 47 ± 67 months). The patients who had RT also had significantly lower absolute numbers of CD4+ T cells (P < 0.0001) as well as CD3+ T cells (P = 0.0043) compared with 56 patients with NED without prior RT (Fig. 4)
. In contrast, the count of CD8+ T lymphocytes remained unchanged after RT, an indication that CD8+ T cells were not sensitive to RT. Nevertheless, it should be noted that 37 of 56 patients with NED treated with surgery alone also had comparably low CD4+ and CD8+ T-cell numbers, and, thus, RT was only one of the factors contributing to T-cell cytopenia in the patients.
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Effects of Surgery on Absolute Lymphocyte Counts.
The cohort of 56 patients with NED who underwent curative surgery alone was divided into those treated >2 years before the blood draw and those studied within 2 years of surgery. As seen in Fig. 5
, the absolute numbers of the T-cell subsets were significantly decreased in both groups relative to NCs (see Fig. 3A
; P < 0.0001). The data suggest that effects of the tumor on the homeostasis of lymphocytes are observed long after the tumor is removed and in the absence of any other lymphoablative therapy.
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Lymphocyte Counts and Disease.
Among 42 patients with active disease at the time of blood draw before surgery, 30 were diagnosed as having primary tumors, and 12 were diagnosed as having recurrent or second primary tumors (Table 1)
. CD4+ and CD8+ T-cell counts were significantly depressed in both these groups relative to NCs (Fig. 3B)
. Although a trend toward lower CD4+ T-cell counts was noted for patients with recurrence or second primary tumors, this difference did not reach statistical significance (data not shown). In addition, there were six patients with recurrent disease after previous surgery, and these subjects had the lowest T-cell counts (data not shown). Similarly, among the patients who, at the time of blood draw, had NED after curative therapy (n = 98), those treated for recurrence of a second primary tumor (n = 19) had, on average, a 25% lower number of CD4+ T cells than the patients treated for primary disease (n = 79), as shown in Fig. 6
(P < 0.06).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The most interesting finding of this study, however, was the observation that the absolute count of CD8+ T cells, although significantly decreased in the patients versus NCs (and especially so in patients with active disease), appeared to recover and normalize in most patients with NED, whereas the CD4+ T-cell count did not. In view of the data reported by us previously, which had demonstrated preferential apoptosis of CD8+ T cells in the circulation of SCCHN patients with clinical characteristics similar to those of the patient cohort studied here (2 , 3) , we had expected to find significantly decreased numbers of circulating CD8+ T lymphocytes rather than CD4+ T lymphocytes in these patients. A possible explanation for this unexpected finding could be that the homeostatic mechanisms compensate for the selective apoptosis by a rapid expansion of CD8+ T cells in the periphery of patients with SCCHN. It is now known that lymphocyte numbers are not regulated through control of their production from stem cells but rather by survival and homeostatic proliferation of mature lymphocytes (17 , 18) . Thus, if the number of lymphocytes is low, proliferation ensues and generates more cells. If the number of lymphocytes is high, excess lymphocytes die (17 , 18) . This is exactly what we have observed in the circulation of patients with SCCHN, where expansion of CD8+ effector subsets (specifically, the CD8+CD45RO–CD27– or CD8+CD28– subsets) was accompanied by their rapid demise by apoptosis (19 , 20) . More recently, we have reported that the majority of CD3+CD8+tetramer+ T cells detectable in the circulation of patients with head and neck cancer also bound annexin V (21) . Thus, the homeostasis of CD8+ T cells in the peripheral circulation of patients with head and neck cancer appears to be maintained by their rapid turnover, resulting in a relatively stable mean peripheral CD8+ cell count for the patient population as a whole. A situation associated with profound depression of the peripheral CD8+ T-cell count occurred in patients with stage IV disease, who failed to normalize CD8+ T-cell count after surgery. This observation suggests that in advanced disease, patients might fail to effectively compensate for the loss of effector cells.
Not surprisingly, the absolute counts of both CD8+ and CD4+ T cells were found to be highly variable in the patient cohort we studied. For CD8+ T cells, the range varied from >1500/mm3 to <100/mm3. For CD4+ T cells, the range was >2000/mm3 to <100/mm3. Patients with documented recurrence of disease or second primary tumor at the time of blood draw had the lowest T-cell counts of all studied. The observed trend was for CD8+ T cell counts to increase and for CD4+ T cell counts to decrease after tumor removal by surgery. However, one of the most interesting findings of this study was that SCCHN patients who had NED for 2 years or >2 years after curative surgery alone (no radiotherapy or chemotherapy) still showed an imbalance in the T-cell subsets. This imbalance was highly significant for CD4+ T cells, whereas CD8+ T cells tended to normalize. This observation suggests that the disease process has a profound and long-lasting impact on T lymphocyte homeostasis in patients with SCCHN. Others have suggested previously that lymphocyte counts have prognostic value in SCCHN (22) .
Another factor that had a significant impact on T-cell counts was the history of previous RT. Only CD4+ cells, but not CD8+ T cells, appeared to be affected by previous RT (Fig. 4)
. Whereas this has been reported previously (e.g., Ref. 23
), the finding of decreased counts of CD4+ T cells long after the administration of RT was somewhat surprising. Apparently, CD4+ T cells are not only more sensitive to RT than CD8+ lymphocytes, but the restoration of the peripheral CD4+ T cell pool after RT is very slow. As indicated above, many patients with NED and no previous RT also had reduced CD4+ T-cell counts long after curative surgery. Thus, RT was not the only factor modulating the number of circulating CD4+ cells in a cohort of patients with SCCHN previously treated for their disease.
It has been reported recently that lymphocyte counts are predictive of survival in cancer (11) . Whereas the current study was not designed to address survival as end point, we considered the possibility that disturbed lymphocyte homeostasis might be especially prominent in patients with poor prognosis, i.e., stage IV disease and nodal metastases. However, we could not establish a statistically significant relationship between disease stage or its severity and lymphocyte counts based on retrospective data analysis. It should be noted that no predictive value for absolute lymphocyte counts or CD4/CD8 ratios for larynx preservation, response to therapy, or survival of patients with SCCHN was seen in another study (24) .
The observed imbalance in T lymphocyte counts in a heterogeneous cohort of patients with SCCHN is likely to be mediated by several distinct mechanisms. In patients with NED, it could be due to a decreased thymic output of recent thymic emigrants, as reported by us previously (25) , leading to alterations in the size of naïve, memory, and effector pools. In patients with active primary or recurrent disease, it could be mediated by increased cell death (apoptosis) of CD8+ T lymphocytes in the peripheral circulation (2 , 3) . We are currently investigating each of these possibilities. Clearly, the snapshot in time represented by each patient’s cell count, as reported here, does not reflect lymphocyte kinetics within the circulating lymphocyte pool. Dynamic techniques, taking advantage of novel methods of cellular labeling and serial measurements (6 , 7) , are necessary to confirm that the T-cell turnover is altered in patients with cancer relative to NCs. The use of CD4/CD8 ratios without considering changes in absolute pool sizes can be misleading because changes in either the denominator or numerator might lead to the same ratio, as we have shown here.
Our results for patients with SCCHN are complementary to the findings of others who also report lymphocyte imbalance in cancer (10, 11, 12, 13) . Combined with the presence of functional abnormalities in T cells of patients with SCCHN, as reported by us previously (3 , 5 , 19 , 20 , 25) , the overall impression is that of decreased immune competence in these patients. Lowered T-cell numbers in the circulation could predispose the patients to infections, disease recurrence, or a second malignancy. It is, therefore, advisable to pay attention to T-cell counts during posttreatment visits, even when the patients have NED long after curative therapy. Our data emphasize that decreased T-cell counts reflect effects of the disease process on T-cell homeostasis and not just therapy-mediated alterations and that the imbalance is long-lasting. In view of the possibility that such persistent changes in homeostasis of T-cell subsets might adversely influence antitumor responses and promote recurrence, therapies designed to increase lymphocyte counts could be considered even in patients with NED.
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FOOTNOTES |
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Requests for reprints: Theresa L. Whiteside, University of Pittsburgh Cancer Institute, Research Pavilion at the Hillman Cancer Center, Suite 1.27, 5117 Centre Avenue, Pittsburgh, PA 15213, Phone: (412) 624-0096; Fax: (412) 624-0264; E-mail: whitesidetl{at}msx.upmc.edu
Received 1/ 9/04; revised 2/18/04; accepted 2/23/04.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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chain expression and apoptosis in CD3+ peripheral T lymphocytes in patients with melanoma. Clin Cancer Res, 7: 947-57, 2001.
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