Persistent lymphopenia after chemotherapy and/or radiotherapy predicted unfavorable outcome in breast cancer patients - a retrospective cohort study

doi:10.21203/rs.3.rs-1603322/v1

Background

Lymphopenia was often observed during follow-up for cancer patients, this study aimed to evaluate the prognostic value of prolonged lymphopenia for breast cancer patients.

Methods

This was a retrospective cohort analysis of 296 patients with clinical stage I-III operable breast cancer, who had received chemotherapy and/or radiotherapy. Absolute lymphocyte count lower than 1.1×10^3/ul was defined as lymphopenia. Lymphopenia around the 6th month after chemo-radiotherapy was defined as cohort L, while normal Absolute lymphocyte count was defined as cohort N. Predictors of lymphopenia were assessed using univariate and multivariate logistic regression analyses. Kaplan–Meier and Cox regression analyses were used to evaluate the relationship between the two cohorts.

Results

The baseline characteristics analysis: comparing to cohort N, cohort L had more Stage III(47.4% vs. 25.5%) and less Stage I cancer(12.3% vs. 20.1%)(p = 0.005), more patients experienced combined chemotherapy regimen(86.0% vs. 69.5%, p = 0.012), more patients experienced radiotherapy(78.9% vs. 64.9%, p = 0.041), and less patients experienced endocrine therapy(54.4% vs. 68.2%, p = 0.049). Multivariate logistic regression analysis revealed two negative independent factors associated with DFS: pTNM staging (stage II vs. I: HR = 5.346, 95% CI: 2.286–12.504, p < 0.001; stage III vs. II: HR = 3.089, 95% CI:1.739–5.487, p < 0.001), and ALC status 6 months after chemo- and/or radiotherapy (HR = 2.445, 95% CI: 1.577–3.788, p < 0.001). Cohort L had significantly shorter DFS (log rank: p < 0.001), as well as significantly shorter OS (log rank: p < 0.001).

Conclusion

Persistent lymphopenia after chemotherapy and/or radiotherapy was an independent predictor for breast cancer relapse and unfavorable survival.

breast cancer

persistent lymphopenia

immunity

relapse

Breast cancer is one of the most common and life-threatening malignancy globally[1, 2]. Although the early diagnosis and comprehensive treatment have greatly improved the outcome of majority breast cancer patients, cancer recurrence is still the biggest threat[3]. Patients with high recurrent risk were widely treated with chemotherapy and/or radiotherapy, which was necessary but would bring harm to the body, such as persistent lymphopenia. As lymphocytes played an important role in the immune system and cancer surveillance[4], we speculate that prolonged lymphopenia after chemotherapy and/or radiotherapy may be a negative factor for patients’ outcome.

Anti-cancer treatment related lymphopenia may last from the beginning to several months or even years after the end of treatment. A few studies reported that lymphopenia during chemotherapy and/or radiotherapy was associated with worse survival in patients with solid tumors, such as breast cancer, lung cancer, liver cancer and esophageal cancer[5–11], but we did not find any reports about the impact of the prolonged lymphopenia on the recurrence of breast cancer.

In this study, we aimed to find out whether prolonged lymphopenia could be an independent predictor for the breast cancer patients’ recurrence and survival.

Study population

This retrospective cohort study was reviewed and approved by the Institutional Review Board of Sir Run Run Shaw Hospital affiliated to Zhejiang University, School of Medicine. All protocols were in accordance with the Declaration of Helsinki.

From Jan-1st, 2012 to Dec-30th, 2016, 1995 breast cancer patients received radical surgery in the department of Surgical Oncology and were regularly followed up. Adjuvant chemotherapy was given to 1028 patients. After chemotherapy and/or radiotherapy, patients follow up every 3 months for two years, the blood test results of the first year were collected as well as the initiate result at the time of cancer diagnosis. Exclusion criteria were: (1) the primary cancer was not operable; (2) the primary cancer treatment and follow up were not performed in our hospital; (3) non-invasive cancer; (4) The patient received neither chemotherapy nor radiotherapy; (5) the patient recurred within one year after diagnosis; (6) complicated with other type of cancer; (7) the patient took immunosuppressive medications or corticosteroids.

At last, totally 296 patients were included, and according to the second round follow up lymphocyte result, they were defined as Lymphopenia group(cohort L) and Normal group(cohort N)(Fig1.).

Pathologic and blood test criteria

The following criteria were used to identify the patients’ molecular characteristics: (1) the ER/PR expressing <10% was defined as negative, and both ER and PR negative was defined as hormone negative, either ER or PR positive was defined as hormone positive. (2) HER2/neu receptor was identified by ASCO CAP guidelines 2013.

Absolute lymphocyte count below 1.1×10^3/ul was defined as lymphopenia, equal or above 1.1×10^3/ul was defined as normal.

Statistical analysis

Statistical analysis was performed using SPSS 19 for Windows® (IBM Corporation, Armonk, NY, USA). Patient baseline characteristics were summarized using descriptive statistics. Associations between two categorical characteristics were examined by Pearson’s Chi-square test. Wilcoxon rank sum test was used to compare quantitative variables including age, absolute lymphocyte count(ALC) between two groups. For other quantitative variables, two-sample T-test was performed to derive the p-value. OS was defined as the time interval from date of diagnosis to date of death or latest follow-up visit, whichever came first. DFS was defined as the time interval from the date of diagnosis to the date of relapse. The Cox proportional hazard model was used to estimate Hazard ratio (HR) with 95% confidence interval (CI). The time-varying Cox model was applied to estimate the value of the variable on survival endpoints with HR (95% CI) and Wald test P-value reported. The multivariate logistic regression model was further fitted by incorporating other covariates including pathological stage and chemotherapy strength to estimate the adjusted HR of the DFS. DFS and OS were analyzed by the Kaplan–Meier and compared with the log-rank test. All statistical tests were two-sided and a p-value of less than 0.05 was considered statistically significant.

1. Baseline characteristics of the two cohorts

A total of 296 invasive breast cancer patients were included, among them 57 patients who were detected lymphopenia around the 6^th month after chem- and/or radiotherapy entered into cohort L, and 239 patients with normal ALC the same follow up time entered into cohort N. Table1. showed that the age, menopausal status, molecular subtype, type of surgery, histological type and Herceptin therapy rate of the two cohorts had no significant difference. Comparing to cohort N, cohort L had more Stage III(47.4% vs. 25.5%) and less Stage I cancer(12.3% vs. 20.1%)(p=0.005), more patients experienced combined chemotherapy regimen(86.0% vs. 69.5%, p=0.012), more patients experienced radiotherapy(78.9% vs. 64.9%, p=0.041), and less patients experienced endocrine therapy(54.4% vs. 68.2%, p=0.049).

Table1. Baseline characteristics of the two cohorts
Variable	Cohort L n=57(%)	Cohort N n=239(%)	p value
Age(year)	49.7±10.9	51.2±9.5	0.281
Menopausal status			0.593
Premenopausal	33(57.9%)	129(54%)
Postmenopausal	24(42.1%)	110(46%)
Pathologic TNM staging			0.005
I	7(12.3%)	48(20.1%)
II	23(40.4%)	130(54.4%)
III	27(47.4%)	61(5.5%)
Molecular subtype			0.635
HR pos+, HER2 neg-	25(43.9%)	122(51.0%)
HR pos+, HER2 pos+	9(15.8%)	42(17.6%)
HR neg-, HER2 pos+	10(17.5%)	34(14.2%)
Tipple neg-	13(22.8%)	41(17.2%)
Type of surgery			0.337
Breast conserving	10(17.5%)	56(23.4%)
Mastectomy	47(82.5%)	183(76.6%)
Histological type			0.565
invasive ductal carcinoma	49(86.0%)	212(88.7%)
other invasive type	8(14.0%)	27(11.3%)
Chemotherapy regimens			0.012
Anthracycline or Paclitaxel	8(14.0%)	73 (30.5%)
Anthracycline and Paclitaxel	49(86.0%)	166 (69.5%)
Radiotherapy	45 (78.9%)	155 (64.9%)	0.041
Herceptin therapy	8(14.0%)	21(8.8%)	0.231
Endocrine therapy	31(54.4%)	163(68.2%)	0.049
Abbreviations: +ve: positive, -ve: negative.

2. Univariate and multivariate Cox regression survival analyses

As regard to DFS, based on univariate analysis, the significant prognostic factors were pTNM staging, chemotherapy regimens strength and ALC status 6 months after chemo- and/or radiotherapy. Based on the multivariate Cox regression analysis, there were two negative independent factors associated with DFS: pTNM staging (stage II vs. I: HR=5.346, 95% CI: 2.286-12.504, p<0.001; stage III vs. II: HR=3.089, 95% CI:1.739-5.487, p<0.001), and ALC status 6 months after chemo- and/or radiotherapy (HR=2.445, 95% CI: 1.577-3.788, p<0.001). (Table 2.)

Table 2. Univariate and Multivariate analysis of prognostic factors for DFS
characteristics	Univariate		Multivariate
characteristics	HR(95%CI)	p value	HR(95%CI)	p value
Pathologic TNM staging
II VS. I	6.329(2.985-13.514)	<0.001	5.346(2.286-12.504)	<0.001
III VS. II	3.165(2.049-4.878)	<0.001	3.089(1.739-5.487)	<0.001
Hormone Receptor
+ve VS. -ve	0.730(0.476-1.120)	0.149	-
HER2 status
+ve VS -ve	1.298(0.476-1.120)	0.217	-
Surgery type
Mestectomy vs. Breast conserving	1.181(0.706-1.976)	0.526	-
Radiotherapy
yes VS. no	1.985(1.216-3.241)	0.006	1.144(0.661-1.980)	0.631
Chemotherapy regimens
Combined vs. single	2.081(1.215-3.565)	0.008	1.488(0.725-3.049)	0.279
ALC status 6^th month
lymphopenia vs. Normal	2.933(1.905-4.505)	<0.001	2.445(1.577-3.788)	<0.001

3. Survival analysis

The median follow up time was 157.8 months. Cohort L had significantly shorter DFS (log rank: p< 0.001) (Fig 2.), as well as significantly shorter OS (log rank: p< 0.001)(Fig 3.).

The CBC test is routinely used from primary evaluation to follow-up surveillance. In this retrospective cohort study, we proved the hypothesis that persistent lymphopenia was an independent predictor of the breast cancer relapse and unfavorable survival.

The immune system played a key role to restrict the cancer from growing and escaping. It had been widely accepted that cancer development went through three processes while fighting against immune system: elimination, equilibrium and escape[4]. Chemotherapy and radiotherapy was provided to kill the subclinical cancer lesion or micrometastasis, while at the same time they inhibited hematopoietic function[12] and immunity[13–15].

The harmful effect of chemotherapy and radiotherapy on immunity had been demonstrated[16], and the effect could last long. Lorna etc. studied 40 children with acute lymphoblastic leukaemia and revealed that both plasma cell and immunoglobulin production was reduced apparently up to 5 years after the end of treatment[17]. Three different studies reported the same finding that chemotherapy for malignancy had harm on children’s humoral immunity within 6 months after the completion of treatment[18–20]. Fukuchi etc. reported that for elderly patients with unresectable gastric cancer, relatively higher regulatory T cell (Treg) ratio within 7 weeks after chemotherapy was a favorable factor affecting OS, which provided evidence that better immunity brought better survival[21]. Radiation therapy was also reported to bring harm to the immune cells at least 2 weeks after cessation of therapy[10, 22]. Surgery induced lymphopenia could also be negative predictor for esophageal cancer prognosis .

Our data demonstrated that persistent lymphopenia, as an indicator of impaired immune system, might participate in the breast cancer recurrence and lead to worse survival. Although cancer staging and molecular subtype played great role in deciding patients’ outcome, and the post treatment lymphopenia was influenced by the strength of chemotherapy and radiotherapy, the cohort study and multivariate analysis showed that prolonged lymphopenia independently predict more and earlier relapse.

ALC was mainly composed by T and B lymphocytes, which were the major immune cells to perform the antitumor function, lymphopenia reflect the decrease of antitumor ability. Some researchers had analyzed the lymphocytes subgroup change after chemotherapy, from two weeks following chemotherapy to as long as one year[24–26]. For example, Head JF et al. revealed that after 2–4 weeks of chemotherapy, T-lymphocyte and B-lymphocyte functional competence was impaired in 83/142 (58%) and 34/142 (24%) of these patients, respectively[27]. B and CD4 + T cells remained significantly depleted even 9 months post-chemotherapy[24].

Autologous Hematopoietic Progenitor Cell Transplant had been reported to be worthwhile for metastatic breast cancer patients to achieve early lymphocyte recovery, which was an independent outcome predictor[28]. Would this strategy be meaningful to help early stage breast cancer patients, or would any medication to rise lymphocyte level and enhance the immunity be helpful for patients’ survival? More researches need to be done.

Persistent lymphopenia after chemotherapy and/or radiotherapy was an independent predictor for more recurrence and worse survival. Any strategy to raise the serum lymphocyte level may potentially contribute to improve patients’ outcome.

Ethical Approval and Consent to participate

This study was reviewed and approved by the Institutional Review Board of Sir Run Run Shaw Hospital affiliated to Zhejiang University, School of Medicine. All protocols were in accordance with the Declaration of Helsinki. All patients gave their written informed consent prior to study inclusion.

Consent for publication

Not applicable.

Availability of data and materials

The datasets generated during and/or analysed during the present study are not publicly available, owing to confidentiality reasons, but anonymised versions may be available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by National Natural Science Foundation of China [81672729], by Zhejiang Natural Science Funding [LY19H160059], by Zhejiang Provincial Natural Science Funding [LQ19H160034], by Youth Talent Project of Zhejiang Provincial Population and Family Planning Commission [2018254316] and by the Zhejiang Provincial Medical and Healthy Science and Technology Projects [2018RC053].

Authors' contributions

Minjun Dong and Linbo Wang designed the study, Minjun Dong wrote the manuscript text. Wenying Zhuo, LiangLuo, Zhongyu Wu, Haitao Yu, Lixi Luo, Yi Lu, Danzhi Chen collected the patients' blood examing data and followed up each patients. Jianguo Shen, Wenjun Chen, Wenhe Zhao, Wang Linbo were the attendings providing data and communicated with the patients while follow up.

Acknowledgements

We thank Breast Cancer Now and The Institute of Cancer Research for support of the Generations Study, as well as the study participants; the study staff; and the doctors, nurses and other health care providers and health information sources who have contributed to the study.

Siegel, R.L., K.D. Miller and A. Jemal, Cancer statistics, 2020. CA Cancer J Clin, 2020. 70(1): p. 7–30.
Chen, W., et al., Cancer statistics in China, 2015. CA: A Cancer Journal for Clinicians, 2016. 66(2): p. 115–132.
DeSantis, C.E., et al., Breast cancer statistics, 2019. CA: A Cancer Journal for Clinicians, 2019. 69(6): p. 438–451.
Gavin P Dunn, A.T.B.H. and L. Abeler-Doner, Cancer immunoediting: from immuno- surveillance to tumor escape. Nature immunology, 2002(Nov;3(11)): p. 991–8.
Araki, K., et al., Predictive impact of absolute lymphocyte counts for progression-free survival in human epidermal growth factor receptor 2-positive advanced breast cancer treated with pertuzumab and trastuzumab plus eribulin or nab-paclitaxel. BMC Cancer, 2018. 18(1): p. 982.
Kroese, T.E., et al., Severe lymphopenia acquired during chemoradiotherapy for esophageal cancer: Incidence and external validation of a prediction model. Radiother Oncol, 2021. 163: p. 192–198.
Sun, G.Y., et al., Radiation-Induced Lymphopenia Predicts Poorer Prognosis in Patients With Breast Cancer: A Post Hoc Analysis of a Randomized Controlled Trial of Postmastectomy Hypofractionated Radiation Therapy. Int J Radiat Oncol Biol Phys, 2020. 108(1): p. 277–285.
Upadhyay, R., et al., Risk and impact of radiation related lymphopenia in lung cancer: A systematic review and meta-analysis. Radiotherapy and Oncology, 2021. 157: p. 225–233.
Vicente, C.M., et al., Predictive value of peripheral blood lymphocyte count in breast cancer patients treated with primary chemotherapy. Breast, 2012. 21(4): p. 468–74.
Zhuang, Y., et al., Association Between Circulating Lymphocyte Populations and Outcome After Stereotactic Body Radiation Therapy in Patients With Hepatocellular Carcinoma. Front Oncol, 2019. 9: p. 896.
Wang, J., et al., Lymphopenia in Esophageal Cancer: What Have We Learned? Frontiers in Oncology, 2021. 11.
Lucas, D., et al., Chemotherapy-induced bone marrow nerve injury impairs hematopoietic regeneration. Nat Med, 2013. 19(6): p. 695–703.
Rollinghoff, M., et al., Cyclophosphamide-sensitive T lymphocytes suppress the in vivo generation of antigen-specific cytotoxic T lymphocytes. J Exp Med, 1977. 145(2): p. 455–9.
Cupps, T.R., L.C. Edgar and A.S. Fauci, Suppression of human B lymphocyte function by cyclophosphamide. J Immunol, 1982. 128(6): p. 2453–7.
Winkelstein, A., et al., Mechanisms of immunosuppression: effects of cyclophosphamide on lymphocytes. J Lab Clin Med, 1972. 80(4): p. 506–13.
Harris, J., et al., The effect of immunosuppressive chemotherapy on immune function in patients with malignant disease. Cancer, 1976. 37(2 Suppl): p. 1058–69.
Layward, L., R.J. Levinsky and M. Butler, Long-term abnormalities in T and B lymphocyte function in children following treatment for acute lymphoblastic leukaemia. Br J Haematol, 1981. 49(2): p. 251–8.
Keskin, Y.Z. and M. Buyukavci, Assessment of Humoral Immunity to Hepatitis B, Measles, Rubella, and Mumps in Children After Chemotherapy. J Pediatr Hematol Oncol, 2018. 40(2): p. e99-e102.
Zignol, M., et al., Assessment of humoral immunity to poliomyelitis, tetanus, hepatitis B, measles, rubella, and mumps in children after chemotherapy. Cancer, 2004. 101(3): p. 635–41.
Zuhal Keskin Yildirim, A.M.B., Assessment of Humoral Immunity to Hepatitis B, Measles, Rubella, and Mumps in Children After Chemotherapy. J Pediatr Hematol Oncol, 2018(Volume 00): p. 000–000.
Fukuchi, M., et al., [Evaluation of immunity in elderly patients with unresectable gastric cancer receiving S-1/Lentinan combination chemotherapy]. Gan To Kagaku Ryoho, 2014. 41(10): p. 1264–6.
Mehrdad Talebian Yazdi, M.S.S.N. and P.S.H.M. Taube, Standard radiotherapy but not chemotherapy impairs systemic immunity in non-small cell lung cancer. OncoImmunology, 2016. NO. 12(26 October 2016, VOL. 5): p. e1255393 (9 pages).
Hall, B.M., et al., Lack of Lymphocyte Recovery After Esophagectomy Predicts Overall and Recurrence-Free Survival. Journal of Surgical Research, 2020. 246: p. 379–383.
Verma, R., et al., Lymphocyte depletion and repopulation after chemotherapy for primary breast cancer. Breast Cancer Res, 2016. 18(1): p. 10.
Hakim, F.T., et al., Constraints on CD4 recovery postchemotherapy in adults: thymic insufficiency and apoptotic decline of expanded peripheral CD4 cells. Blood, 1997. 90(9): p. 3789–98.
Mackay, I.R., et al., Effect on immunologic and other indices of adjuvant cytotoxic chemotherapy including melphalan in breast cancer. Cancer, 1984. 53(12): p. 2619–27.
Head, J.F., R.L. Elliott and J.L. McCoy, Evaluation of lymphocyte immunity in breast cancer patients. Breast Cancer Res Treat, 1993. 26(1): p. 77–88.
Nieto, Y., et al., Prognostic analysis of early lymphocyte recovery in patients with advanced breast cancer receiving high-dose chemotherapy with an autologous hematopoietic progenitor cell transplant. Clin Cancer Res, 2004. 10(15): p. 5076–86.

No competing interests reported.

Persistent lymphopenia after chemotherapy and/or radiotherapy predicted unfavorable outcome in breast cancer patients - a retrospective cohort study

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Abstract

Background

Methods

Results

Conclusion

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Introduction

Methods

Results

Discussion

Conclusion

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Status:

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