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Prognostic Analysis of Early Lymphocyte Recovery in Patients with Advanced Breast Cancer Receiving High-Dose Chemotherapy with an Autologous Hematopoietic Progenitor Cell Transplant

Yago Nieto, Elizabeth J. Shpall, Ian K. McNiece, Samia Nawaz, Julie Beaudet, Steve Rosinski, Julie Pellom, Victoria Slat-Vasquez, Peter A. McSweeney, Scott I. Bearman, James Murphy and Roy B. Jones
DOI: 10.1158/1078-0432.CCR-04-0117 Published August 2004

Abstract

Purpose: The purpose of this study was to evaluate the prognostic effect of early posttransplant lymphocyte recovery in patients with advanced breast cancer receiving high-dose chemotherapy with autologous hematopoietic progenitor cell transplantation.

Experimental Design: We analyzed the effect of the absolute lymphocyte count on day +15 posttransplant on freedom from relapse and overall survival in patients with high-risk primary breast cancer or metastatic breast cancer, enrolled between 1990 and 2001 in prospective high-dose chemotherapy trials, using a uniform regimen of cyclophosphamide, cisplatin, and 1,3-bis(2-chloroethyl)-1-nitrosourea.

Results: Four hundred and seventy-six patients (264 high-risk primary breast cancer and 212 metastatic breast cancer patients) were evaluated at median follow-up of 8 years (range, 1.5–11 years). The disease-free survival and overall survival rates in the high-risk primary breast cancer group were 67% and 70%, respectively. Patients with metastatic breast cancer patients had 21.8% disease-free survival and 31.5% overall survival rates. Day +15 absolute lymphocyte count correlated with freedom from relapse (P = 0.007) and overall survival (P = 0.04) in the metastatic breast cancer group, but not in the high-risk primary breast cancer group (P = 0.5 and 0.8, respectively). The prognostic effect of absolute lymphocyte count in metastatic breast cancer was restricted to those patients receiving unmanipulated peripheral blood progenitor cells (P = 0.04). In contrast, absolute lymphocyte count had no significant effect in those metastatic breast cancer patients receiving bone marrow or a CD34-selected product. In multivariate analyses, the prognostic effect of day +15 absolute lymphocyte count in metastatic breast cancer was independent of other predictors, such as disease status, pre-high-dose chemotherapy treatment, number of tumor sites, or HER2.

Conclusions: Early lymphocyte recovery is an independent outcome predictor in metastatic breast cancer patients receiving high-dose chemotherapy and an autologous peripheral blood progenitor cell transplant. These observations suggest that immune strategies targeting minimal posttransplant residual disease may prove worthwhile.

INTRODUCTION

Patients with advanced breast cancer have poor prognosis after standard treatment. High-risk primary breast cancer, commonly defined as ≥4 involved lymph nodes or inflammatory breast cancer, presents a relapse risk of >50%. In turn, metastatic breast cancer is considered incurable in the vast majority of cases. Whereas the use of high-dose chemotherapy with autologous hematopoietic progenitor cell transplant has been investigated in both settings for almost two decades, its relative efficacy compared with standard therapy is still the subject of intense controversy. Numerous randomized trials have addressed this question, most of which have only been analyzed in preliminary fashion (1) . The most recent Cochrane reviews of their pooled data show a significant advantage in favor of high-dose chemotherapy in early event-free survival in high-risk primary breast cancer (2) and metastatic breast cancer (3) , albeit without overall survival differences in either setting at the time of the analyses. Thus, longer follow-up of the randomized trials is needed to obtain a firm answer.

It is clear that most metastatic breast cancer patients and a substantial fraction of high-risk primary breast cancer patients experience tumor relapse after high-dose chemotherapy. Potential ways to decrease recurrence include new high-dose chemotherapy combinations with greater antitumor effect or novel therapies (e.g., immune-mediated) targeting posttransplant residual disease. Whereas a wealth of data demonstrates a critical antitumor effect of the immune system after allogeneic transplantation for hematological malignancies, very little is known about its potential role after autologous hematopoietic progenitor cell transplant for solid tumors. Patients with advanced breast cancer who receive high-dose chemotherapy undergo a prolonged period of T-cell dysfunction and inversion of the CD4/CD8 ratio after unmanipulated (4) or CD34-selected autologous hematopoietic progenitor cell transplant (5 , 6) , whereas the number and function of B lymphocytes, natural killer, and dendritic cells remain less compromised. The correlation of these observations with patient outcome remains to be elucidated.

A prognostic value has been shown for early posttransplant absolute lymphocyte counts in patients with hematological malignancies receiving an allogeneic (7 , 8) or autologous transplant (9, 10, 11) . These observations suggest that early lymphocyte recovery might be a valid surrogate of effective posttransplant tumor immunity in those settings. We present here long-term prognostic analyses of absolute lymphocyte count recovery in advanced breast cancer patients enrolled in prospective trials of high-dose chemotherapy with autologous hematopoietic progenitor cell transplant.

PATIENTS AND METHODS

Patient Population.

We evaluated all breast cancer patients enrolled in prospective research trials of high-dose cyclophosphamide, cisplatin, and 1,3-bis(2-chloroethyl)-1-nitrosourea (STAMP-I regimen) at the University of Colorado Bone Marrow Transplant Program between 1990 and 2001. Our analysis included patients with metastatic breast cancer (n = 212; Table 1⇓ ) and high-risk primary breast cancer (n = 264; Table 2⇓ ). Trials for high-risk primary breast cancer included Phase II and III studies for patients with 4–9 positive axillary nodes (12) , ≥10 positive nodes (13) , or inflammatory breast cancer (14) . Metastatic breast cancer patients were enrolled in Phase II trials of first-line high-dose chemotherapy for measurable metastases (15) , bone-only disease (15) , and oligometastases (16) . These trials were approved by the University of Colorado Cancer Center Protocol Review Committee and the Institutional Review Board. All patients gave written informed consent before study entry. Absence of resistance to chemotherapy, defined as relapse or progression during pretransplant conventional chemotherapy, was required in these trials.

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Table 1

Demographics of metastatic breast cancer patients (N = 212)

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Table 2

Demographics of high-risk primary breast cancer patients (N = 264)

High-risk primary breast cancer patients received high-dose chemotherapy within 6 months of primary definitive surgery (mastectomy or lumpectomy with negative margins). Inflammatory breast cancer patients received preoperative chemotherapy, followed by surgery and high-dose chemotherapy. Non-inflammatory breast cancer patients received four to six cycles of Adriamycin-containing chemotherapy before high-dose chemotherapy. Metastatic breast cancer patients were not allowed more than one conventional chemotherapy regimen for metastatic disease before high-dose chemotherapy.

Protocols required adequate visceral organ function, as described previously (11, 12, 13, 14, 15) . Pretransplant staging tests were computed tomography scans of the head, chest, abdomen, and pelvis; bone scan; and bilateral bone marrow biopsies. Peripheral blood progenitor cells were mobilized with granulocyte colony-stimulating factor (G-CSF) at 10 μg/kg/day for 5 days and cryopreserved as described previously (17) . Seventy-nine metastatic breast cancer patients had their hematopoietic fractions CD34-selected, as part of a specific Phase II trial of this procedure (17) . Patients then received high-dose chemotherapy, with cyclophosphamide (5625 mg/m2), cisplatin (165 mg/m2), and 1,3-bis(2-chloroethyl)-1-nitrosourea (600 mg/m2). This was followed by stem cell infusion on days −1 to +1 (unselected graft) or on day −1 (CD34-selected graft) and administration of G-CSF at 5 μg/kg/day from day −1 until the third day with an absolute neutrophil count of >5000/mm3. No other immunologically active cytokines were used to mobilize peripheral blood progenitor cells or after infusion of the graft.

Determination of the CD34+ content of the grafts was performed by flow cytometry following the guidelines of the International Society for Hematology and Graft Engineering (18) .

Posttransplant treatment included radiotherapy to locoregional sites (high-risk primary breast cancer) or to amenable metastases (metastatic breast cancer), on platelet recovery. Hormone therapy was prescribed for 5 years to patients with estrogen receptor/progesterone receptor-positive tumors. Bisphophonates were administered to most patients with widespread bone disease. Trastuzumab was not available for clinical use during these trials.

Variables Analyzed.

We previously identified the following variables as independent predictors of outcome in high-risk primary breast cancer: (a) pathological tumor size; (b) estrogen receptor/progesterone receptor; (c) nodal ratio (number of involved axillary nodes/number of dissected nodes); and (d) HER2 (19 , 20) . We now studied the value of whole blood absolute lymphocyte count on posttransplant day +15 and the infused number of CD34+ cells in this population.

In the metastatic breast cancer group, we analyzed in the present study the potential prognostic value of the following variables: (a) day +15 absolute lymphocyte count; (b) age; (c) primary breast tumor size; (d) original inflammatory breast cancer; (e) primary axillary nodal ratio; (f) primary number of positive axillary nodes; (g) HER2; (h) estrogen receptor; (i) progesterone receptor; (j) histological tumor grade; (k) previous exposure to doxorubicin or to any adjuvant chemotherapy; (l) prior adjuvant radiotherapy; (m) metastases at diagnosis or at relapse; (n) disease-free interval; (o) number of metastatic sites; (p) bone marrow involvement; (q) specific metastatic locations (liver versus other visceral versus bone/bone marrow versus soft tissue); (r) infusion of a CD34-selected graft or an unselected graft; (s) number of CD34+ cells infused; (t) delivery of post-high-dose chemotherapy radiotherapy; (u) stem cell source (peripheral blood versus bone marrow); and (v) disease status at high-dose chemotherapy [complete remission (achieved with either chemotherapy or surgery)/single bone lesion versus multiple bone lesions versus partial remission with or without concurrent bone lesions/stable disease].

Immunohistochemical HER2 analyses used monoclonal antibody CB-11 (Ventana, Tucson, AZ) as described previously (20 , 21) . Paraffin-embedded tumor blocks were obtained from the referring hospitals. All immunostained slides were reviewed by the same pathologist (S. N.), who was blinded to patient outcome.

Statistical Methods.

Correlations between categorical and continuous variables were assessed using the χ2 or Fisher’s exact test and Student’s t test, respectively. Disease-free survival was defined as the time from study entry to documented relapse/progression or to death from any cause. Overall survival was defined as the time from study entry to death from any cause. Freedom from relapse analyses, used in the prognostic studies but not in the descriptive overall outcome analyses, excluded those patients who died from early treatment-related complications. In the freedom from relapse curves, patients who died from secondary malignancy with no evidence of breast cancer at the time of their deaths or at latest follow-up were censored as free of breast cancer at the time of death.

All survival times were analyzed using the Kaplan-Meier method (22) . The log-rank test was used to study the correlation of potential prognostic variables with survival times (23) . Unless mentioned otherwise, the median values of the continuous variables were chosen as cutoffs. Associations between continuous variables were assessed using the Spearman correlation test.

Multivariable proportional hazard Cox regression models for disease-free survival or overall survival included patient- or tumor-related variables with significance at the P < 0.05 level in univariate analyses (24) . The significance of the overall model was evaluated with the likelihood ratio test. Individual coefficients were tested using the Wald test. The proportionality assumption for all variables was assessed with Kaplan-Meier curves. All P values presented are two-tailed.

RESULTS

Overall Patient Outcome.

A total of 476 patients (264 high-risk primary breast cancer and 212 metastatic breast cancer patients) were analyzed at median follow-up of 8 years (range, 1.5–11 years). The disease-free survival and overall survival rates of the high-risk primary breast cancer group were 67% (95% confidence interval, 61–73%) and 70% (95% confidence interval, 64–76%), respectively (Fig. 1A)⇓ . Median disease-free survival and overall survival times have not been reached. In the metastatic breast cancer group, the disease-free survival and overall survival rates were 21.8% (95% confidence interval, 19–25%) and 31.5% (95% confidence interval, 25–38%), respectively (Fig. 1B)⇓ . The median disease-free survival and overall survival times in the metastatic group were 1.3 and 3.2 years, respectively.

Fig. 1.
Fig. 1.

Disease-free survival (DFS) and overall survival (OS) in patients with high-risk primary breast cancer (n = 264; A) and metastatic breast cancer (n = 212; B).

Twenty-five patients (5.3%; 14 high-risk primary breast cancer patients and 11 metastatic breast cancer patients) died from early direct high-dose chemotherapy-related organ complications. They were considered events in the overall outcome analyses (Fig. 1, A and B)⇓ but were excluded from all prognostic analyses. In addition, three high-risk primary breast cancer patients and two metastatic breast cancer patients developed secondary acute myelogenous leukemia more than 5 years after high-dose chemotherapy. All of them died from their leukemia or from complications related to salvage treatment. No breast cancer was identified in their postmortem exams. They were considered events in the descriptive outcome analyses (Fig. 1, A and B)⇓ but were censored as free of breast cancer at the time of death for the purpose of prognostic analyses (Figs. 2⇓ 3⇓ 4⇓ 5⇓ 6)⇓ .

Fig. 2.
Fig. 2.

Outcome of high-risk primary breast cancer patients with high absolute lymphocyte count (ALC; >930/mm3) and low absolute lymphocyte count (≤930/mm3) on day +15. A, freedom from relapse (P = 0.54). B, overall survival (P = 0.8).

Fig. 3.
Fig. 3.

Freedom from relapse (P = 0.007; A) and overall survival (P = 0.03; B) in metastatic breast cancer patients with low (≤500/mm3) and high (>500/mm3) absolute lymphocyte count (ALC) on day +15.

Fig. 4.
Fig. 4.

Effect of day +15 absolute lymphocyte count (ALC) on freedom from relapse of metastatic breast cancer patients per stem cell source. A, peripheral blood progenitor cells (P = 0.04). B, bone marrow (P = 0.86).

Fig. 5.
Fig. 5.

Differential prognostic effect of the absolute lymphocyte count (ALC) in metastatic breast cancer patients receiving unselected (A; P = 0.04) or CD34-selected grafts (B; P = 0.8).

Fig. 6.
Fig. 6.

Freedom from relapse (P = 0.0001; A) and overall survival (P = 0.007; B) in metastatic breast cancer patients with high (>1000/mm3), intermediate (300–1000/mm3), and low (<300/mm3) day +15 absolute lymphocyte count (ALC).

Evaluation of Day +15 Absolute Lymphocyte Count.

The absolute lymphocyte count on day +15, +30, +45, and +60 was known for 434, 52, 24, and 10 patients, respectively. Therefore, we focused on day +15 as the time point of early lymphocyte recovery.

Day +15 absolute lymphocyte count was available in 98% of high-risk primary breast cancer patients and 88% of metastatic breast cancer patients. Day +15 absolute lymphocyte count was significantly higher in high-risk primary breast cancer patients (median, 930/mm3; range, 18–8,220/mm3) than in those with metastatic breast cancer (median, 500/mm3; range, 10–11,500/mm3; P = 0.01).

The correlation of day +15 absolute lymphocyte count and CD34+ graft content was statistically significant, albeit very weak, in the following groups: (a) all patients; (b) metastatic breast cancer patients; (c) all patients receiving peripheral blood progenitor cells; and (d) all patients receiving bone marrow (Table 3)⇓ . In the high-risk primary breast cancer subset, the correlation between absolute lymphocyte count and CD34+ cell dose did not reach significance. No significant correlation between the absolute lymphocyte count and the total nucleated cell count in the graft was observed in any group.

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Table 3

Spearman analyses of the correlations between day +15 absolute lymphocyte count and TNC/CD34+ cells infused

In the metastatic breast cancer group, day +15 absolute lymphocyte count was higher among those unexposed to adjuvant chemotherapy than those previously exposed to adjuvant chemotherapy, whose median absolute lymphocyte counts were 690 and 500/mm3, respectively (P = 0.04). No associations were observed between day +15 absolute lymphocyte count and age (P = 0.8), prior exposure to adjuvant anthracyclines (P = 0.15), pre-high-dose chemotherapy radiotherapy (P = 0.1), number of chemotherapy cycles for metastatic disease (P = 0.6), or length of disease-free interval (P = 0.27).

Prognostic Analyses of Day +15 Absolute Lymphocyte Count.

We did not observe significant associations of absolute lymphocyte count with freedom from relapse (P = 0.54; Fig. 2A⇓ ) or overall survival (P = 0.81; Fig. 2B⇓ ) in the high-risk primary breast cancer group. No prognostic absolute lymphocyte count thresholds were detected after testing higher (up to 2000/mm3) or lower (down to 100/mm3) cutoffs (P values not shown). No association of absolute lymphocyte count with outcome was observed within the high-risk primary breast cancer subsets receiving peripheral blood progenitor cells (n = 232) or bone marrow (n = 21).

In contrast, the median absolute lymphocyte count in metastatic breast cancer patients (500 lymphocytes/mm3) established two groups with significantly different freedom from relapse (Fig. 3A)⇓ and overall survival (Fig. 3B)⇓ . Patients with high absolute lymphocyte count (median 1230 lymphocytes/mm3) presented superior freedom from relapse rates (33% versus 20.5%) and median freedom from relapse times (2.3 years versus 1 year; P = 0.007), as well as improved overall survival rates (42% versus 29%) and median overall survival times (4 versus 2.5 years; P = 0.03), compared with the low absolute lymphocyte count group (median, 185 lymphocytes/mm3).

The correlation of absolute lymphocyte count with freedom from relapse was observed in those metastatic breast cancer patients receiving a peripheral blood progenitor cell graft (n = 137), whose median freedom from relapse times were 1.1 (low absolute lymphocyte count) and 1.75 years (high absolute lymphocyte count; P = 0.04; Fig. 4A⇓ ). In contrast, we did not observe a prognostic effect of absolute lymphocyte count in metastatic breast cancer patients receiving bone marrow (n = 43; Fig. 4B⇓ ). In this subgroup, median freedom from relapse times were 0.6 (low absolute lymphocyte count) and 0.7 year (high absolute lymphocyte count; P = 0.86).

Similarly, the prognostic impact of absolute lymphocyte count was restricted to those metastatic breast cancer patients who received unselected cells of any source (Fig. 5A)⇓ , with median freedom from relapse times of 1.25 (low absolute lymphocyte count) and 2.9 years (high absolute lymphocyte count; P = 0.04). However, absolute lymphocyte count had no effect on metastatic breast cancer patients receiving a CD34-selected graft, with median freedom from relapse of 1.1 years (low absolute lymphocyte count) and 1 year (high absolute lymphocyte count; Fig. 5B⇓ ; P = 0.8).

Additionally, absolute lymphocyte count was analyzed as a semicontinuous variable, with three metastatic breast cancer groups: (a) high (>1000/mm3; n = 59); (b) intermediate (300–1000/mm3; n = 62); and (c) low absolute lymphocyte count (<300/mm3; n = 67). There were significant differences in freedom from relapse (P = 0.0001; Fig. 6A⇓ ) and overall survival (P = 0.007; Fig. 6B⇓ ) between these groups.

Prognostic Analysis of the Graft CD34+ Cell Content.

The median numbers of CD34+ cells infused did not differ significantly between high-risk primary breast cancer (median, 4.2 × 106/kg; range, 0.5–32.6 × 106/kg) and metastatic breast cancer patients (median, 3.87 × 106/kg; range, 0.47–35.4 × 106/kg; P = 0.2). Within both populations, the numbers of cells infused were higher in patients receiving peripheral blood progenitor cells than in patients receiving bone marrow (Table 4)⇓ .

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Table 4

CD34+ cell and TNC graft content in MBC and HRPBC patients

No associations were observed between the CD34+ content and freedom from relapse or overall survival in high-risk primary breast cancer (P = 0.68 and 0.6, respectively) or metastatic breast cancer patients (P = 0.2 and 0.25, respectively), using their median counts as cutoffs. Evaluation of higher or lower cutoffs also failed to show a prognostic effect (data not shown). Likewise, CD34+ counts did not correlate with outcome within any subset (those receiving peripheral blood progenitor cells or bone marrow, unselected or CD34-selected grafts; data not shown).

Other Univariate Analyses in Patients with Metastatic Breast Cancer.

In addition to day +15 absolute lymphocyte count (Table 5)⇓ , the following were significantly associated with freedom from relapse and overall survival: primary axillary nodal ratio; primary inflammatory breast cancer; HER2; estrogen receptor; progesterone receptor; prior adjuvant chemotherapy; prior adjuvant anthracyclines; metastases at diagnosis; disease status at transplant; number of metastatic sites; specific organ involved; stem cell source; and posttransplant radiotherapy. The beneficial effect of radiotherapy was observed separately in patients with one and more than one metastatic site. Tumor grade correlated with overall survival, but not with freedom from relapse.

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Table 5

Univariate analyses in metastatic breast cancer patients

No correlation with outcome was observed for age, primary breast tumor size, primary absolute number of positive axillary nodes, tumor grade, disease-free interval length, bone marrow involvement, and CD34 selection of the graft.

Multivariate Analyses in Metastatic Breast Cancer Patients.

Day +15 absolute lymphocyte count showed an independent predictive value for both freedom from relapse (P = 0.02) and overall survival (P = 0.04; Table 6⇓ ). Other independent outcome predictors were disease status, primary nodal ratio, HER2, primary inflammatory breast cancer, and number of metastatic sites.

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Table 6

Multivariate analyses in metastatic breast cancer (N = 201)

DISCUSSION

At median follow-up of 8 years, the disease-free survival and overall survival rates in our high-dose chemotherapy trials were 67% and 70%, respectively, for high-risk primary breast cancer patients and 22% and 31%, respectively, for metastatic breast cancer patients. These encouraging long-term results indicate the need for mature analyses of ongoing randomized studies. Other authors reached a similar conclusion after long-term analysis of their prospective Phase II trials of high-dose chemotherapy (25 , 26) .

Early lymphocyte recovery, defined as the absolute lymphocyte count on day +15, emerged as an important prognostic factor in patients with metastatic breast cancer, independent of other known predictors. The choice of day +15 for this analysis was driven by the availability of data for most patients, as compared with later time points. Other authors have also chosen day +15 for study of early lymphocyte recovery after autologous hematopoietic progenitor cell transplant for metastatic breast cancer and other tumors (9, 10, 11 , 27) . Our findings are consistent with those reported by Porrata et al. (27) in their analysis of 29 metastatic breast cancer patients, most of whom received unselected peripheral blood progenitor cells, after a median follow-up of 2 years. In their study, the median day +15 absolute lymphocyte count of 500/mm3 (the same as in our metastatic breast cancer population) was an independent predictor of freedom from relapse and overall survival.

In contrast to our observations in metastatic breast cancer patients, no significant effect from the absolute lymphocyte count was noticed in the nonmetastastic high-risk primary breast cancer group. These results would appear to contradict the well-established fact that in vitro cellular immunity is depressed in advanced stages of breast cancer and other solid tumors, compared with early-stage tumors (28, 29, 30, 31) . In keeping with this concept, our high-risk primary breast cancer patients had a substantially higher absolute lymphocyte count on day +15 than the ones with metastatic breast cancer. It is possible that the overall preservation of cellular immune function in the high-risk primary breast cancer group could have prevented the emergence of prognostic differences among those patients, in contrast to the greater heterogeneity of the metastatic breast cancer population. Alternatively, it is conceivable that the immune system may play a more active antitumor role in metastatic breast cancer than in high-risk primary breast cancer, despite a more marked immune suppression. This explanation would appear to be consistent with the “danger model” of tumor immunity proposed by Matzinger and colleagues, whereby anticancer immune reactions are not triggered by the expression of “non-self” antigens by tumor cells, as proposed by Burnet (32) , but rather by the immune cell recognition of tissue stress or destruction signals (33 , 34) , which presumably would be more intense in advanced stages than in earlier stages of the disease.

There is a growing wealth of data suggesting that the immune system plays a role in breast cancer control (35) . Breast cancer cells can overexpress Fas ligand, inducing dysfunction and apoptosis of Fas-expressing activated T lymphocytes and natural killer cells and thus evading immune response (36) . Müschen et al. (37) observed that the expression of Fas ligand on breast cancer cells correlated with more advanced stages (metastatic versus nonmetastatic) and higher grading of the tumors in a series of 40 cases. Additionally, they observed that tumor-infiltrating lymphocytes in close proximity to breast cancer cells expressing Fas ligand were predominantly apoptotic. Further, Bewick et al. (38) observed an adverse effect of high pretransplant serum levels of Fas in 94 metastatic breast cancer patients who received high-dose chemotherapy.

Our observations suggest that day +15 absolute lymphocyte count may be a valid surrogate of those immune subsets that might be involved in immunity. Recovery of CD3+, CD4+, and CD8+ cells has been recently associated with outcome in patients with ovarian cancer receiving high-dose chemotherapy and autologous hematopoietic progenitor cell transplant (39) . It has been shown that post-autologous hematopoietic progenitor cell transplant levels of CD8+ T lymphocytes return to pretransplant levels more rapidly than those of CD4+ T cells (40 , 41) . Because naïve CD4+ cell immunity and responses to neoantigen stimuli remain depressed for longer periods of time (39 , 40) , it is conceivable that an effective antitumor immune response after transplant might depend on memory cells. To test this hypothesis and to further define the levels and function of memory and naïve T-cell populations, in a separate study we prospectively measured pre- and posttransplant levels of T-cell subsets in autologous hematopoietic progenitor cell transplant recipients. The results of that study (42) showed an association between pretransplant levels of CD4 cells (more specifically, the memory CD4RA-CD62- phenotype) and outcome in metastatic breast cancer patients, independently of other known predictors, such as HER2, number of sites, or chemosensitivity of the tumor. Additionally, other immune cells, such as natural killer lymphocytes, might have an important effect on tumor control after autologous hematopoietic progenitor cell transplant. A rapid posttransplant recovery of natural killer cells in breast cancer patients, as early as 1 month after high-dose chemotherapy, has been observed (4 , 43) . An antitumor effect of natural killer cells extracted from breast cancer patients has been demonstrated in vitro with or without augmentation of their cytotoxicity with cytokines such as interleukin 2 or G-CSF (44, 45, 46) . Thus, it can also be hypothesized that natural killer cells may play a major immunological surveillance role early after transplant. It would be of interest to evaluate the in vitro function of memory T cells in patients with nonmetastatic and metastatic disease after transplant, as well as the prognostic impact of pre- and posttransplant levels of natural killer and other immune populations, such as dendritic cells.

The effect of absolute lymphocyte count on outcome in metastatic breast cancer patients appears restricted to those patients receiving peripheral blood progenitor cells, as opposed to those receiving bone marrow, and to those who received an unselected cell product, in contrast to those receiving a CD34-selected graft. The T-cell content of the graft depends on its source and on whether it is manipulated ex vivo. Peripheral blood progenitor cell grafts contain approximately 1 log more T cells than bone marrow products with a similar number of CD34+ cells (47) . Likewise, the content of T cells in unselected hematopoietic-cell fractions is 2–4 logs higher than that in CD34-selected products (48) . Because formation of memory T cells requires release of T-cell progenitors from the bone marrow into the bloodstream and migration through the thymus and lymph nodes, the content of memory T cells will presumably be much greater in peripheral blood progenitor cells than in bone marrow grafts. Thus, it is conceivable that effective posttransplant immunity requires infusion of large numbers of T cells in the graft and, more specifically, of memory T cells.

The immune cell content of the graft may be related to pretransplant immunity. In our study, the correlation between the early posttransplant absolute lymphocyte count and the number of infused total nucleated cell count or CD34+ cells was very weak or nonsignificant in high-risk primary breast cancer and metastatic breast cancer patients receiving either bone marrow or peripheral blood progenitor cells. In addition, the day +15 absolute lymphocyte count was significantly higher in the high-risk primary breast cancer group than in the metastatic breast cancer group, both of which received similar CD34+ or total nucleated cell count numbers. These observations suggest that the graft T-cell content depends more on pretransplant immunity than on the number of infused stem cells. It is possible that the increased tumor burden or more intense “danger signals” of tissue destruction in metastatic breast cancer compared with high-risk primary breast cancer may generate a greater breast-cancer-activated memory T-cell population before transplant, resulting in a larger or more effective presence of these cells in the graft and at the time of early lymphocyte recovery.

Finally, we did not identify any correlation between the CD34+ cell count and outcome in any of the following subgroups: (a) high-risk primary breast cancer or metastatic breast cancer; (b) metastatic breast cancer patients receiving peripheral blood progenitor cells or bone marrow; and (c) metastatic breast cancer patients receiving an unselected or a CD34-selected graft. Our observations contrast with previous reports (49 , 50) . This discrepancy may be due to differences in the flow cytometric techniques. Because the CD34+ cell fraction is a small percentage of the mononuclear cells, variations in immunolabeling resulting from the specific anti-CD34 antibody or fluorochrome used or in the gating strategy can lead to great differences in the CD34+ count. Contradictory data also exist with regard to the prognostic impact of CD34+ content after autologous hematopoietic progenitor cell transplant for hematological malignancies (51, 52, 53, 54) . Because variability in flow cytometry among centers is a well-recognized circumstance, adoption of the International Society for Hematology and Graft Engineering guidelines (18) has been recommended (55) .

In conclusion, we have identified an independent prognostic effect of the early lymphocyte recovery in metastatic breast cancer patients receiving high-dose chemotherapy with STAMP-I. Taken together, our observations and those of other authors suggest the possibility of an important role for the immune system in tumor control after high-dose chemotherapy for metastatic breast cancer and support the investigation of pre- and posttransplant immune-based strategies targeting minimal residual disease.

Acknowledgments

We acknowledge the outstanding patient care provided by the nurses and house staff of the Bone Marrow Transplant Unit. We thank Dr. Anthony Elias for comments on the manuscript. We are indebted to the referring physicians who entrusted us with their patients and, above all, to all of our patients, who agreed to participate in our studies.

Footnotes

  • Grant support: Supported by National Institutes of Health Grant 1 R21 CA095762-01 (Y. Nieto).

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • Requests for reprints: Yago Nieto, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, B-190, Denver, CO 80262. Phone: (303) 372-9000; Fax: (303) 372-9003; E-mail: yago.nieto{at}uchsc.edu

  • Received January 20, 2004.
  • Revision received April 13, 2004.
  • Accepted April 28, 2004.

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Clinical Cancer Research: 10 (15)
August 2004
Volume 10, Issue 15

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Prognostic Analysis of Early Lymphocyte Recovery in Patients with Advanced Breast Cancer Receiving High-Dose Chemotherapy with an Autologous Hematopoietic Progenitor Cell Transplant
Yago Nieto, Elizabeth J. Shpall, Ian K. McNiece, Samia Nawaz, Julie Beaudet, Steve Rosinski, Julie Pellom, Victoria Slat-Vasquez, Peter A. McSweeney, Scott I. Bearman, James Murphy and Roy B. Jones
Clin Cancer Res August 1 2004 (10) (15) 5076-5086; DOI: 10.1158/1078-0432.CCR-04-0117
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