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Does Unintentional Splenic Radiation Predict Outcomes After Pancreatic Cancer Radiation Therapy?
Accepted Manuscript Does unintentional splenic radiation predict severe lymphopenia following pancreatic cancer radiotherapy? Awalpreet S. Chadha, M.D., Guan Liu, M.D., Hsiang-Chun Chen, Ph.D., Prajnan Das, M.D., Bruce D. Minsky, M.D., Usama Mahmood, M.D., Marc E. Delclos, M.D., Yelin Suh, Ph.D., Gabriel O. Sawakuchi, Ph.D., Sam Beddar, Ph.D., Matthew H. Katz, M.D., Jason B. Fleming, M.D., Milind M. Javle, M.D., Gauri R. Varadhachary, M.D., Robert A. Wolff, M.D., Christopher H. Crane, M.D., Xuemei Wang, M.S., Howard Thames, Ph.D., Sunil Krishnan, M.D. PII:
S0360-3016(16)33422-8
DOI:
10.1016/j.ijrobp.2016.10.046
Reference:
ROB 23889
To appear in:
International Journal of Radiation Oncology • Biology • Physics
Received Date: 20 May 2016 Revised Date:
26 October 2016
Accepted Date: 31 October 2016
Please cite this article as: Chadha AS, Liu G, Chen H-C, Das P, Minsky BD, Mahmood U, Delclos ME, Suh Y, Sawakuchi GO, Beddar S, Katz MH, Fleming JB, Javle MM, Varadhachary GR, Wolff RA, Crane CH, Wang X, Thames H, Krishnan S, Does unintentional splenic radiation predict severe lymphopenia following pancreatic cancer radiotherapy?, International Journal of Radiation Oncology • Biology • Physics (2016), doi: 10.1016/j.ijrobp.2016.10.046. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title: Does unintentional splenic radiation predict severe lymphopenia following pancreatic cancer radiotherapy? Awalpreet S. Chadha, M.D.,1 Guan Liu, M.D.,1 Hsiang-Chun Chen, Ph.D.,2 Prajnan
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Das, M.D.,1 Bruce D. Minsky, M.D.1 Usama Mahmood, M.D.,1 Marc E. Delclos, M.D.,1 Yelin Suh, Ph.D.,3 Gabriel O. Sawakuchi, Ph.D.,3,4 Sam Beddar, Ph.D.,3 Matthew H. Katz, M.D.,5 Jason B. Fleming, M.D.,5 Milind M. Javle, M.D.,6 Gauri R. Varadhachary,
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Howard Thames, Ph.D.,1 Sunil Krishnan, M.D.1
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M.D.,6 Robert A. Wolff, M.D.,6 Christopher H. Crane, M.D.,1 Xuemei Wang, M.S.,2
Departments of Radiation Oncology1, Biostatistics,2 Radiation Physics,3 Surgical Oncology,5 and Gastrointestinal Medical Oncology,6 The University of Texas MD Anderson Cancer Center, Houston, Texas 77030
77030
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Acknowledgements:
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Graduate School of Biomedical Sciences,4 The University of Texas, Houston, Texas
This work was supported in part by Cancer Center Support (Core) Grant P30
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CA16672 to The University of Texas MD Anderson Cancer Center and the John E. and Dorothy J. Harris Endowed Professorship to SK. The authors also thank Ms. Christine Wogan (Program Manager, Divisional Publications, Radiation Oncology Department, MD Anderson Cancer Center) for carefully editing the manuscript. Corresponding Author: Sunil Krishnan M.D. Department of Radiation Oncology
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The University of Texas MD Anderson Cancer Center 1515 Holcombe Boulevard, Houston, TX 77030-4009
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Telephone: (713) 563-2377 Fax: (713) 745-2186 E-mail: [email protected]
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Running Title: Splenic radiation during pancreatic cancer radiotherapy
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Presented in part at the American Society of Clinical Oncology Gastrointestinal Cancers Symposium, San Francisco, 2016.
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Disclaimer: The authors report no conflict of interest.
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Summary
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We document an association between post-chemoradiation lymphopenia and survival of locally advanced pancreatic cancer patients. Furthermore, splenic dose-volume histogram parameters are associated with the observed lymphopenia, suggesting a correlation between unintentional splenic irradiation and lymphocyte depletion. This knowledge could be used to triage treatment plans that could cause lymphopenia or design treatment plans that spare the spleen. Presumably, preserving lymphocytes would also increase the possibility of mounting an anti-tumor immune response, especially when immunotherapy is planned in the future.
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Title: Does unintentional splenic radiation predict severe lymphopenia following pancreatic cancer radiotherapy?
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Abstract Purpose: To determine if severity of lymphopenia is dependent on radiation dose and fractional volume of spleen irradiated unintentionally during definitive
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chemoradiation (CRT) in patients with locally advanced pancreatic cancer (LAPC). Methods: 177 patients with LAPC received induction chemotherapy (mainly
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gemcitabine-based regimens) followed by CRT (median 50.4 Gy with concurrent capecitabine) from 1/2006 to 12/2012. Absolute lymphocyte count (ALC) was recorded at baseline, prior to CRT and 2-10 weeks after CRT. Splenic dose-volume histogram (DVH) parameters were reported as mean splenic dose (MSD) and percentage of splenic volume receiving at least 5 (V5), 10 (V10), 15 (V15) and 20 Gy
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(V20) dose. Overall survival (OS) was analyzed using the Cox model and development of post-CRT severe lymphopenia (ALC <0.5 K/UL) was assessed by multivariate logistic regression using baseline and treatment factors.
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Results: Median post-CRT ALC (0.68 K/UL, range 0.13-2.72) was significantly lower than both baseline (1.42 K/UL, range 0.34-3.97; p<0.0001) and pre-CRT ALC (1.32
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K/UL, range 0.36-4.82; p<0.0001). Post-CRT ALC <0.5 K/UL was associated with inferior OS on univariate (median: 11.1 vs. 15.3 months, p=0.01) and multivariate analysis (HR=1.66, p=0.01). MSD (9.8 vs. 6 Gy, p=0.03), median V10 (32.6 vs. 16%, p=0.04), V15 (23.2 vs. 9.5%, p=0.03) and V20 (15.4 vs. 4.6%, p=0.02) were significantly higher in patients with severe lymphopenia compared to those without. On multivariate analysis, post-induction lymphopenia (p<0.001, OR=5.25)
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and MSD (p=0.002, OR= 3.42) were independent predictors for development of severe post-CRT lymphopenia. Conclusion: Severe post-CRT lymphopenia is an independent predictor of poor OS
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in LAPC patients receiving CRT. Higher splenic doses increase the risk of developing severe post-CRT lymphopenia. When clinically indicated, assessment of splenic DVHs prior to acceptance of treatment plans may minimize the risk of severe post-
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CRT lymphopenia.
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Introduction
The immune system is generally regarded as a primary defense against cancer by recognizing and eliminating incipient tumors. Cancer immune surveillance, first proposed by Burnet and Thomas, may not always eradicate tumors but rather competes with tumor progression in a dynamic interplay that is referred to as
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immunoediting.(1-3) Transiting through or residing within tumors, lymphocytes play a key role in protecting the host and sculpting emerging tumors through their interactions with tumor associated antigens (TAAs).(4) Several clinical studies have
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highlighted the positive prognostic value of both CD8+ tumor-infiltrating lymphocytes and circulating lymphocytes in pancreatic, colorectal, head and neck,
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urothelial and lung cancer.(5-12) Unlike many malignancies that have abundant immune infiltrates, pancreatic cancer thrives in an immune-privileged milieu that has a conspicuous scarcity of effector T cells and a relative abundance of regulatory T cells, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages.(13; 14) This imbalance of pro- vs. anti-tumor T-cell populations may be mediated through surface expression of immunosuppressive molecules like programmed death-ligand 1 and Fas ligand and/or active secretion of GM-CSF by
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pancreatic cancer cells to promote recruitment and differentiation of precursor cells into MDSCs that counteract the activity of cytotoxic CD8+ T-cells.(15-19) Together with the exuberant desmoplastic stroma, the lack of effective immune
interventions and the emergence of treatment resistance.
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surveillance contributes to a hostile tumor microenvironment for therapeutic
Radiation therapy (RT) is known to have both immune-stimulating and immune-
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suppressive effects.(20-22) The latter has traditionally been linked to direct bone marrow suppression and/or depletion of circulating lymphocytes. Recent evidence
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suggests that patients with high-grade gliomas and pancreatic cancer have an increased incidence of and a poor prognosis from lymphopenia following chemoradiation therapy (CRT).(6; 21; 23) However, none of these studies have tried to explore possible mechanisms behind this phenomenon.
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We demonstrate that chemoradiation-induced lymphopenia portends a poor prognosis in patients with locally advanced pancreatic cancer (LAPC) treated with induction chemotherapy followed by CRT and that the severity of lymphopenia is dependent on the radiation dose and fractional volume of the spleen irradiated
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unintentionally during treatment.
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Patients and methods Patient identification
The xxx pancreatic cancer database was used to retrospectively identify consecutive
patients
with
biopsy-proven
LAPC
treated
with
induction
chemotherapy followed by CRT from 2006 to 2012. Patients having primarily resectable or borderline resectable disease, or distant metastasis at presentation were excluded.(24)
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Clinical evaluation and treatment characteristics Patients were evaluated by a multidisciplinary team consisting of a medical and a radiation oncologist and selected patients were later seen by surgical oncologists.
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Clinical and laboratory parameters were routinely assessed in all patients (Table 1 and Table 2). Absolute lymphocyte counts (ALC) were recorded prior to treatment (baseline), after completion of induction chemotherapy, and after CRT (2-10 weeks
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after completion of CRT).
All patients received induction chemotherapy followed by CRT. RT was
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administered using either a four-field 3-dimensional conformal technique or an intensity-modulated radiation therapy (IMRT) technique based on physician preference. Follow-up visits for restaging were scheduled at roughly 6 weeks (after completion of CRT) and then every 3-4 months. Subsequent therapy was
Statistical analysis
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individualized based on treatment response.
Lymphopenia was defined based on the Common Terminology Criteria for Adverse
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Events, version 4.0.(25) Grade I and II lymphopenia (ALC between 0.5 and 1 K/UL), and grade III and IV lymphopenia (ALC < 0.5 K/UL) were categorized as mild and
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severe, respectively. Patient characteristics were tabulated by severe lymphopenia. Differences between continuous covariates were compared by the Wilcoxon ranksum test, and differences between categorical covariates were compared using Fisher's exact test or Chi-square tests as appropriate. To test for the prognostic significance of post-CRT lymphocyte nadirs, overall survival (OS) time was chosen as the outcome of interest. A landmark analysis of OS was performed to account for lead time bias between the start of induction chemotherapy and the assessment of
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post-CRT ALC. OS was calculated from a fixed landmark time point to the death date or last follow-up date, whichever occurred first. Patients who died or were lost to follow-up prior to the landmark time were excluded. Kaplan-Meier product limit
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method was used to estimate survival probabilities and differences between groups were compared using log-rank test.(26) Cox proportional hazards models were fit to assess the association between potential predictors and OS.(27) Predictors with
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a p-value ≤0.2 on univariate analysis were included in the multivariate model, and backward elimination was used to obtain the final multivariate Cox model (Table 3).
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For patients with available post-CRT ALC and RT plans, we contoured the spleen and obtained dose-volume histogram (DVH) parameters using Pinnacle 9.8. DVHs were reported in terms of mean splenic dose (MSD) and the percentage of splenic volume receiving at least 5 Gy (V5), 10 Gy (V10), 15 Gy (V15) and 20 Gy (V20) dose. We then compared MSD, and V5, V10, V15 and V20 (stratified at increments of
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10%) between patients with and without severe lymphopenia using Wilcoxon rank sum test or Fisher’s exact test, as appropriate. Finally, we analyzed the dose-bin data using the Lyman-Kutcher-Burman (LKB) model with post-CRT ALC as the
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dependent variable.(28; 29)
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ALCs at baseline, after induction chemotherapy and after CRT were compared using paired t-test. Predictors of post-CRT lymphocyte nadir were classified into (1) baseline and (2) treatment factors (Table 4). Univariate and multivariate logistic regression analysis (using factors found to be significant at the 0.2 level in the univariate analyses) were performed to assess the impact of these factors on development of severe lymphopenia. A two-tailed p-value of ≤0.05 was considered
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statistically significant. Statistical analyses were performed using SAS 9.3 (SAS Institute, Cary, NC) and R (The R Project for Statistical Computing, Vienna, Austria).
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Results Patient characteristics
177 patients with LAPC were treated with induction chemotherapy followed by
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definitive CRT. Median clinical follow-up time from start of CRT was 12 months (range, 1-87). Table 1 summarizes the patient characteristics. Median baseline ALC
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was 1.42 K/UL (range, 0.34-3.97). Median ALC after induction chemotherapy was 1.32 K/UL (range, 0.36-4.82) which was within normal range and not different from baseline (p=0.14). Median ALC further decreased to 0.68 K/UL (range, 0.13-2.72) after CRT which was significantly lower than both baseline (p<0.0001) and postinduction levels (p<0.0001). The incidence of severe lymphopenia was only 3% after
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induction chemotherapy but increased to 27% after CRT. Median time for recording ALC was 38 days (interquartile range, IQR, 32-44) after completion of CRT. Changes in neutrophil to lymphocyte ratio (NLR) mirrored the changes in ALC with increases
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noted following induction chemotherapy and after CRT (Figure 1).
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Treatment characteristics
Table 2 summarizes tumor and treatment characteristics. Median time for initiating treatment was 22 days (IQR, 12-34) from diagnosis. Induction chemotherapy was administered for a median duration of 3 months (IQR, 2–5) using predominantly gemcitabine-based (n=149, 84%) therapy. Median radiation dose was 50.4Gy (range, 25.2-70.4) in 28 fractions administered over 38 days (IQR, 37-39). Concurrent chemotherapy was primarily capecitabine-based (n=150, 85%).
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Lymphocyte counts and survival outcomes Post-CRT ALC was available for 162 patients. Characteristics of the 15 patients lacking ALC values were comparable to those of patients included in the analysis
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except for their being older in age and having higher NLR values after induction chemotherapy (Supplementary table 1). Median time for post-CRT ALC measurement was 5.5 months (range, 3.6-13) from start of induction
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chemotherapy. Therefore, a landmark analysis at 6 months from the start of induction chemotherapy was performed. Five patients who died within 6 months
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after the start of induction chemotherapy were excluded, and 157 patients (132 deaths) were included in the 6-month landmark analysis. Median OS from the landmark time point was 14 months (95% confidence interval, CI, 11.7-15.7 months). The 1-, 2- and 3-year OS rates were 57%, 26% and 14% respectively. Patients with post-CRT severe lymphopenia had an inferior OS (Figure 2) compared
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to those without severe lymphopenia (median: 11.1 vs. 15.3 months, p=0.01) and compared to patients with mild lymphopenia (median: 11.1 vs. 13.6 months, p=0.03). Other parameters found to be significant at the 0.2 level in the univariate
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analyses included age, KPS, baseline platelet count, baseline NLR, post-CRT NLR and radiation dose level. Because of the high correlation between ALC and NLR, only
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ALC was included in the final Cox model. On multivariate analysis, post-CRT ALC < 0.5 K/µL (HR=1.66, p=0.01) and baseline platelet count > 300 K/µL (HR=1.91, p=0.02) were the only independent predictors of inferior OS (Table 3). This is in accordance with data published previously by our group.(30) Same results were obtained with the forward selection method. Similar trends were seen when the predictors were divided into 4 quartiles, which confirms the appropriateness of using dichotomized predictors (Supplementary table 2).
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Predictors of lymphopenia DVH data were available for 157 patients. MSD ranged from 0.1 to 42 Gy with a mean of 8.8 Gy. Patients with severe lymphopenia had a higher MSD than those
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without severe lymphopenia (9.8 vs. 6 Gy, p=0.03). A univariate logistic analysis indicated that MSD was a strong predictor of severe lymphopenia (odds ratio, OR, 3.41; p=0.001). Furthermore, there was a dose-response relationship between MSD
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and severity of lymphopenia with the median MSD for patients with normal ALC, mild, and severe lymphopenia being 3.8 Gy, 6.8 Gy and 9.8 Gy, respectively. When
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analyzed as a continuous variable, MSD did not show a correlation with post-CRT ALC, which was possibly because of the dispersion within the data. Normalized MSD, however, showed a weaker but statistically significant negative correlation (r= -0.19, p=0.02) with post-CRT ALC. Similar trends were seen when MSD was
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divided into 4 quartiles and analyzed (Supplementary table 3). On further DVH analysis, we found that median V10 (32.6 vs. 16%, p=0.04), V15 (23.2 vs. 9.5%, p=0.03) and V20 (15.4 vs. 4.6%, p=0.02) were significantly higher in patients with severe lymphopenia compared to those without severe lymphopenia.
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Additionally, a greater proportion of patients with severe lymphopenia had V10
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(p=0.03), V15 (p=0.01) and V20 (p=0.04) >20% compared to those without severe lymphopenia. Similar results were obtained at 10% for V15 and V20 and 30% for V5, V10 and V15 but not at higher increments due to fewer patients in the cohort with high volumes and high doses. The most significant p values were obtained for the following stratifications: V5 at 30% (p=0.04), V10 at 30% (p=0.01), V15 at 20% (p=0.01) and V20 at 20% (p=0.03). The significance of thresholds at multiple points
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along the DVH curve also validate the clinical relevance of dose-dependent occurrence of severe lymphopenia. Finally, univariate analyses of baseline and other treatment factors showed that the
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development of post-CRT severe lymphopenia was also predicted by baseline lymphopenia (p=0.001) and post-induction lymphopenia (p<0.001). It was not affected by the type of induction chemotherapy regimen (p=0.87), radiation dose
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(biologically effective dose, BED, of ≤70 Gy vs. >70 Gy, p=0.56) or concurrent radiosensitizers used (p=0.44). Due to strong associations between MSD and V5,
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V10, V15 or V20 as well as between baseline and post-induction lymphopenia, only MSD and post-induction lymphopenia were included in the multivariate analysis. On the multivariate logistic regression model, post-induction lymphopenia (p<0.001, OR=5.25) and MSD dichotomized at the mean (p=0.002, OR= 3.42) were the most important predictors for development of post-CRT severe lymphopenia
scatter in the data.
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Discussion
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(Table 4). The LKB model, however, did not converge possibly due to amount of
Our data suggest that severe lymphopenia occurs relatively frequently following
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CRT in patients with LAPC and is an independent predictor of poor OS. We also observed a significant dose-response relationship between post-CRT ALC and MSD which offers a potential explanation for development of severe lymphopenia. In addition to MSD, post-induction lymphopenia was a significant predictor of severe post-CRT lymphopenia. The strong association between OS and post-CRT ALC as well as post-CRT ALC and MSD, however, do not imply an association between MSD and OS due to intransitivity of these relations. This is consistent with other DVH
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parameters that predict toxicity which, in turn, has an influence on survival but the DVH parameter per se does not directly impact survival.(31) Nevertheless, these DVH metrics have clinical utility in comparing treatment plans and choosing the
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most appropriate treatment plan.
Although our patients received induction chemotherapy followed by CRT, lymphopenia is predominantly a consequence of CRT because median ALC for the
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entire cohort after induction chemotherapy was normal and not significantly different from baseline. In fact, only 3% of patients developed severe lymphopenia
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following induction chemotherapy and this increased to 27% following CRT. Neither the type of induction chemotherapy nor that of concurrent chemotherapy was associated with severe post-chemoradiation lymphopenia. Though similar findings for pancreatic cancer have been previously described, the underlying mechanisms are largely unknown.(6; 7) The potential mechanisms for radiation-induced
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lymphopenia include depletion of progenitor cells in the bone marrow and/or circulating lymphocytes owing to large gross tumor volume (GTV) coverage.(21; 32) The anatomic location of the pancreas away from active marrow-producing bone
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and the lack of correlation between tumor size (a surrogate for GTV) and severe post-CRT lymphopenia largely precludes these mechanistic explanations. The
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anatomical proximity of the spleen to the pancreas, the relative abundance of lymphocytes in splenic white pulp and the exquisite radiosensitivity of lymphocytes to doses of radiation as low as 2 Gy support the hypothesis that splenic radiation dose is a predictor and potential mechanism for the development of severe lymphopenia.(33) In our dataset, the percentage change in ALC following CRT (compared to post-induction ALC) was directly related to the loge MSD (r=0.17, p=0.03), further implicating splenic irradiation as a mediator of lymphopenia. In
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addition, the lack of neutropenia associated with lymphopenia is distinctly different from that observed with either myelosuppression from chemotherapy or large irradiation volumes(33; 34) such as total body irradiation and craniospinal
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irradiation. The correlation between NLR and survival has been described in many cancers. While the precise mechanism remains unknown, it is believed that high neutrophil counts are (i) indicative of an immunosuppressive inflammatory
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response that suppresses cytotoxic T-cell and natural killer cell activity and (ii) associated with increased secretion of growth factors that promote tumor
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growth.(34)
The clinical significance of this finding may be more pronounced when lymphocyte activation for anti-tumor effects are the desired outcome of combination with immunotherapeutic agents. Recent data on the synergy between RT and immune check point inhibitors suggest that radiation can prime effector T-cells to recognize
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tumor-specific and/or radiation-induced tumor neoantigens.(21; 35; 36) Irradiated tumor cells express ‘eat me’ signals (calreticulin) and elevated levels of MHC class I molecules, and release ‘danger’ signals like high-mobility group protein B1 and ATP lead
to
dendritic
cell-mediated,
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which
cytotoxic
T-lymphocyte-induced
immunogenic death.(21; 37; 38) In addition, radiation conditions the tumor
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microenvironment by inducing expression of proinflammatory mediators (IFN-γ, CXCL16) which enhance T-cell trafficking and infiltration within tumors.(39; 40) This has led to profound interest in using radiation to convert tumors into an in-situ vaccines which constantly feed immune-adjuvant neoantigens to tumor-specific Tcells and fuels their recruitment to the tumor microenvironment.(41) The translation of this radiotherapy-immunotherapy alliance into a successful therapeutic model is especially relevant in pancreatic cancer where treatment
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resistance is extremely common and multiple drugs targeting various pathways have failed clinically. To this end, we believe that any attempt at combining immunotherapy and RT for pancreatic cancer will be considerably undermined by
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depletion of lymphocytes after CRT and as a corollary, preservation of ALCs through the use of spleen-sparing radiation techniques may be a reasonable consideration. In our practice, as is the custom at most centers in the United States, we have
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traditionally ignored the spleen while developing RT plans. If validated in other cohorts of LAPC patients receiving CRT, this finding would argue strongly for more
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routine assessment of splenic DVHs while triaging competing treatment plans and tailoring treatments to minimize gratuitous splenic irradiation, especially in the setting of preexisting lymphopenia. Furthermore, our data suggest that either an MSD threshold or a splenic dose-volume threshold must be crossed before severe lymphopenia develops, which is around an MSD of 9 Gy and a V15 of 20% in our
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dataset. From a clinical perspective, we believe that newer techniques like IMRT and stereotactic body RT could confer better spleen sparing while still allowing dose escalation to the GTV.(42; 43)
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These findings should be interpreted with caution given its retrospective nature.
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First, the results cannot be generalized to tumors located in other sites receiving CRT. The geographic proximity of the spleen to the pancreas and the frequent inclusion of portions of the spleen in radiation fields may, however, be similar to that of other upper abdominal tumors. Second, there is always a possibility of selection bias as we only included patients who had both post-CRT ALC and DVH data available. As noted in supplementary table 1, minor differences between the included and excluded groups of patients are an inevitable consequence of
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retrospective analyses that temper enthusiasm for broad generalization. This is especially notable because high BED was not an independent predictor of overall survival in this analysis, in discordance with our earlier report on high BED being a
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predictor of better outcome. We attribute this to the reduced numbers of patients with high BED in this analysis due to a shorter time window of patient accrual and patients dropping out from analysis due to the lack of lymphocyte counts (as noted
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above). Despite the use of appropriate statistical tools to analyze the validity of each predictive variable, this discordance is a reflection of inherent challenges
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associated with retrospective analyses where outcomes are studied by probing small subsets of patients and even a small decrease in the size of this subset impacts its predictive value. We consider both reports to be hypothesis-generating, needing independent validation before they influence individual clinical practice patterns and plan to embark on such validation efforts with other large single- and
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multi-institution datasets. Third, there was some variation in assessing post-CRT ALCs. To be consistent, we only included patients with ALC available 2-10 weeks after CRT and performed survival analysis from a landmark point to account for
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lead-time bias. Fourth, we did not record ALCs during subsequent follow-up visits and so we cannot conclude if this effect persists long-term or not. However, studies
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by other groups confirm that treatment-related lymphopenia persists up to 12 months after starting CRT.(6) Finally, the lack of convergence of the LKB model may reflect scatter within the data, and the likelihood that radiobiological models traditionally used for normal tissue complication probability predictions may not apply to lymphocyte depletion by splenic radiation, as observed in other scenarios.(44-46) The dependence of LKB model fitting on the specific endpoint analyzed and the underlying pathophysiology of the toxicity, and the need for
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collective institutional experiences suggest that validation of these findings in other datasets is warranted. In conclusion, severe lymphopenia occurs relatively frequently following
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chemoradiation and is an independent predictor of inferior OS. In addition, the risk increases as the MSD and the fractional volume of spleen receiving low radiation doses increases. The existence of a specific splenic dose-volume threshold needs
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further validation. The thresholds defined here could serve as guideposts for assessing RT plans for LAPC, and to customize individual plans to ensure adequate
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spleen sparing when clinically indicated.
Figure legends
Figure 1: Absolute lymphocyte count (ALC) and neutrophil-to-lymphocyte ration
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(NLR) at baseline, after induction chemotherapy and after chemoradiation. Figure 2: Kaplan-Meier estimates of overall survival based on landmark analysis in patients with locally advanced pancreatic cancer and grouped by post-
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chemoradiation therapy (CRT) absolute lymphocyte count (ALC).
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Supplementary figure 1: Axial isodose plots and dose volume histograms corresponding to a planned course of treatment for a representative patient with severe post-chemoradiation lymphopenia (a, b) and without severe postchemoradiation lymphopenia (c, d). REFERENCES 1. 2.
Teng MW, Swann JB, Koebel CM, et al. Immune-mediated dormancy: an equilibrium with cancer. J Leukoc Biol 2008; 84:988-93. Swann JB, Smyth MJ. Immune surveillance of tumors. J Clin Invest 2007; 117:1137-46.
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Tables Table 1: Patient characteristics
98 (55) 79 (45)
P-value
0.57
64 years 37 - 84 years
0.58
26 (59) 18 (41)
64 (54) 54 (46)
132 (75) 22 (12) 16 (9) 7 (4)
35 (80) 3 (7) 5 (11) 1 (2)
87 (74) 17 (14) 9 (8) 5 (4)
1.42 (0.34- 3.97) 1.32 (0.36- 4.82) 0.68 (0.13- 2.72)
1.12 (0.34- 2.24) 1.04 (0.36- 2.35) 0.37 (0.13- 0.49)
1.51 (0.52- 3.97) 1.51 (0.37- 4.82) 0.84 (0.5- 2.72)
<0.001 <0.001 <0.001
4.18 (1.32, 16.14) 3.18 (0.61, 32.43) 3.07 (0.96, 21.05)
4.1 (2.21, 10.34) 3.21 (0.95, 32.43) 2.76 (0.96, 21.05)
4.15 (1.32, 16.14) 3.02 (0.61, 23.71) 3.09 (1, 8.03)
0.86 0.21 0.62
0.51 (0.17, 1.42) 0.61 (0.03, 3.01) 0.49 (0.03, 2.18)
0.47 (0.23, 1.06) 0.53 (0.1, 1.33) 0.42 (0.09, 2.18)
0.52 (0.17, 1.32) 0.68 (0.03, 3.01) 0.53 (0.03, 1.85)
0.41 0.01 0.01
2.86 (0.94, 26.17) 2.37 (0.23, 20.26) 4.74 (0.81, 44.85)
3.87 (1.44, 16.5) 3.3 (0.97, 18.85) 7.55 (2.39, 43.85)
2.62 (0.94, 10.35) 1.99 (0.23, 20.26) 3.72 (0.81, 11.81)
0.001 <0.001 <0.001
219.5 95 - 588 138 (84) 26 (16)
212 95 - 588 37 (90) 4 (10)
226 130 - 580 92 (84) 17 (16)
0.04
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63 years 39 - 79 years
Postchemoradiation ALC ≥ 0.5 K/UL N=118
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64 years 37 - 88 years
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Age Median Range Gender Male Female Race White African- American Hispanic Asian ALC (K/UL) – median (range) Baseline Post-induction chemotherapy Post-chemoradiation Neutrophil (K/UL) – median (range) Baseline Post-induction chemotherapy Post-chemoradiation Monocyte (K/UL) – median (range) Baseline Post-induction chemotherapy Post-chemoradiation Neutrophil to ALC Ratio – median (range) Baseline Post-induction chemotherapy Post-chemoradiation Baseline Platelet Count (K/UL) Median Range ≤300 >300 KPS
Postchemoradiation ALC < 0.5 K/UL N=44
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All Patients N=177 N (%)
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Characteristic
0.51
0.44 0.78
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<80 22 (13) 4 (9) 14 (12) ≥80 154 (87) 40 (91) 104 (88) % Weight Loss 0.53 <10 92 (54) 21 (50) 64 (56) ≥10 79 (46) 21 (50) 51 (44) Baseline Hemoglobin (g/dL) 0.68 >12 115 (70) 30 (73) 76 (70) ≤12 49 (30) 11 (27) 33 (30) Abbreviations: ALC, absolute lymphocyte count; KPS, Karnofsky performance status; N, number of patients Where numbers do not add to column title value of N, insufficient data was available in remaining patients. Post-chemoradiation ALC fell within the prescribed range in 162 of 177 patients.
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Table 2: Tumor and treatment characteristics
149 (84) 28 (16)
37 (84) 7 (16)
P-value Postchemoradiation ALC ≥ 0.5 K/UL N=118 0.87 98 (83) 20 (17)
3 (2) 1 (1) 22 (13) 150 (85)
1 (2) 0 (0) 4 (9) 39 (89)
1 (1) 1 (1) 17 (14) 98 (84)
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Postchemoradiation ALC < 0.5 K/UL N=44
6.8 0.1 - 42
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59.5 29.7 - 100 34 (19) 143 (81)
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0.57
59.5 29.7 - 87.5 10 (23) 34 (77)
9.79 0.51 - 37.06
59.5 39 - 100 22 (19) 96 (81)
56 (49) 58 (51)
99 (63) 58 (37)
20 (46) 23 (54)
79 (69) 35 (31)
97 (62) 60 (38)
19 (44) 24 (56)
78 (68) 36 (32)
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105 (60) 35 (20) 14 (8) 8 (5) 12 (7)
0.56 0.03
0.03
13 (30) 30 (70)
115 (73) 42 (27)
0.66
5.98 0.12 - 41.97
69 (44) 88 (56)
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Induction Chemotherapy* Gemcitabine-based FOLFIRINOX Concurrent Chemotherapy# 5-fluorouracil Vorinostat Gemcitabine-based Capecitabine- based Radiation Dose (BED, Gy) Median Range BED > 70 BED ≤ 70 Mean Splenic Dose (Gy) Median Range V5 ≤ 30% > 30% V10 ≤ 30% > 30% V15 ≤ 20% > 20% V20 ≤ 20% > 20% Tumor Location Head Body Head/ Body Body/ Tail Tail Tumor size $
All Patients N=177 N (%)
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Characteristic
0.01
0.01
0.03 26 (60) 17 (40)
89 (78) 25 (22) 0.49
23 (52) 10 (23) 3 (7) 4 (9) 4 (9)
73 (63) 21 (18) 9 (8) 4 (3) 8 (7) 0.92
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3.5 cm 0.9- 8 cm
3.55 cm 1.7- 7.2 cm
3.55 cm 0.9- 8 cm 0.30
0 (0) 11 (69) 5 (31)
5 (14) 18 (51) 12 (34)
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5 (9) 31 (55) 20 (36)
0.47
163 (95) 5 (3) 1 (1) 2 (1)
43 (98) 0 (0) 0 (0) 1 (2)
110 (94) 5 (4) 1 (1) 1 (1)
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Median Range Grade Well differentiated Moderately differentiated Poorly differentiated Histology Adenocarcinoma Mucinous Signet ring Adenosquamous
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Abbreviations: ALC, absolute lymphocyte count; BED, Biologically Effective Dose; Gy, Gray; N, Vx, volume of spleen receiving x Gy of radiation; number of patients * Gemcitabine-based induction therapy consisted of gemcitabine alone or in combination with paclitaxel, cisplatin, oxaliplatin, erlotinib, bevacizumab or cetuximab; # Gemcitabine-based concurrent therapy consisted of gemcitabine alone or in combination with erlotinib; Capecitabine-based concurrent therapy consisted of capecitabine alone or in combination with bevacizumab, erlotinib or cetuximab $ Based on single largest dimension on abdominopelvic computed tomography scan Where numbers do not add to column title value of N, insufficient data was available in remaining patients. Post-chemoradiation ALC fell within the prescribed range in 162 of 177 patients.
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Table 3: Univariate and multivariate landmark analyses of prognostic factors for overall survival (OS), with OS time defined starting from 6 months after induction chemotherapy (n=157) p Hazard Ratio (95% CI) value
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Prognostic factor
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Univariate analysis for OS Baseline Factors Age (≥60 vs. <60 years) 0.13 1.33 (0.92-1.91) Gender (Female vs. male) 0.97 0.99 (0.70-1.40) Race (White vs. others) 0.95 0.99 (0.66-1.47) Tumor location (Head vs. others) 0.78 1.05 (0.74-1.50) Tumor grade (Moderately/well vs. poorly differentiated) 0.01 0.42 (0.22-0.83) Tumor size (maximum; >3.5 vs. ≤3.5 cm) 0.97 1.01 (0.67-1.50) % weight loss (<10 vs. ≥10 %) 0.70 1.07 (0.75-1.52) KPS < 80 vs. ≥80 0.12 1.49 (0.90-2.46) Baseline hemoglobin (>12 vs. ≤ 12 g/dL) 0.34 0.83 (0.56-1.22) Baseline platelet count (>300 vs. ≤300 K/UL) 0.04 1.71 (1.02-2.86) Baseline ALC (<1 vs. ≥1 K/UL) 0.02 1.73 (1.09-2.73) Baseline neutrophil to ALC ratio (≤3 vs.>3)$ 0.10 0.75 (0.53-1.06) Treatment Factors 0.24 1.39 (0.81-2.38) Induction chemotherapy (Gemcitabine vs. others) 0.25 1.27 (0.84-1.91) Post-induction ALC (<1 vs. ≥1 K/UL) Post-induction neutrophil to ALC ratio (≤2 vs.>2)$ 0.95 1.01 (0.71-1.43) Concurrent chemotherapy (Capecitabine vs. others) 0.26 0.76 (0.48-1.22) 0.09 0.68 (0.44-1.06) BED (>70 vs. ≤ 70 Gy) 0.01 1.66 (1.13-2.43) Post-chemoradiation ALC (< 0.5 vs. ≥0.5 K/UL) $ 0.06 0.72 (0.51-1.02) Post-chemoradiation neutrophil to ALC ratio (≤5 vs.>5) Mean splenic dose (>8.76 vs. ≤ 8.76 Gy) 0.36 0.84 (0.58- 1.21) Multivariate Analysis for OS (after backward elimination) Baseline platelet count (>300 vs. ≤300 K/UL) 0.02 1.91 (1.13- 3.23) Post-chemoradiation ALC (< 0.5 vs. ≥0.5 K/UL) 0.01 1.66 (1.11- 2.48) Abbreviations: ALC, absolute lymphocyte count; BED, Biologically Effective Dose; CI: 95% confidence interval; KPS, Karnofsky performance status * Grade was not included in the multivariate analysis because 68% of patients had missing data. $ Cut-offs were defined using the integers closest to the median
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Table 4: Predictors of development of severe lymphopenia (n=162)
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Odds Ratio (95% CI)
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p value
0.85 (0.41-1.73) 0.74 (0.23-2.39) 1.18 (0.53-2.64) 4.62 (1.84-11.61)
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0.65 0.62 0.68 0.001
0.87 0.44 <0.001 0.56 0.001 0.04 0.01 0.01 0.03
1.08 (0.42-2.76) 1.51 (0.53-4.33) 5.33 (2.37-11.99) 1.28 (0.55-2.98) 3.41 (1.65-7.06) 2.23 (1.06-4.70) 2.60 (1.26-5.33) 2.74 (1.33-5.62) 2.33 (1.09-4.96)
<0.001 0.002
5.25 (2.24-12.30) 3.42 (1.57- 7.44)
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Prognostic factor Univariate analysis of severe lymphopenia Baseline Factors Age (≥60 vs. <60 years) KPS (< 80 vs. ≥80) Baseline hemoglobin (>12 vs. ≤ 12 g/dL) Baseline ALC (<1 vs. ≥1 K/UL) Treatment Factors Induction chemotherapy (Gemcitabine vs. FOLFIRINOX) Concurrent chemotherapy (Capecitabine vs. others) Post-induction ALC (<1 vs. ≥1 K/UL) BED (>70 vs. ≤ 70 Gy) Mean splenic dose (>8.8 vs. ≤ 8.8 Gy) V5 (>30 vs. ≤ 30%) V10 (>30 vs. ≤ 30%) V15 (>20 vs. ≤ 20%) V20 (>20 vs. ≤ 20%) Multivariate analysis of severe lymphopenia Post-induction ALC (<1 vs. ≥1 K/UL) Mean splenic dose (>8.76 vs. ≤ 8.76 Gy)
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Abbreviations: ALC, absolute lymphocyte count; BED, Biologically Effective Dose; CI: 95% confidence interval; KPS, Karnofsky performance status; Vx, volume of spleen receiving x Gy of radiation
Figure 1
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Neutrophil/Lymphocyte Count Neutrophil to Absolute Lymphocyte Count Ratio
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Absolute Lymphocyte Countcount Absolute Lymphocyte Lymphocyte Count
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Normalized Volume
0.9
GTV
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Liver
0.7
Right Kidney
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Spleen Cord
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0.1 0
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Figure 2
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S0360-3016(16)33422-8
DOI:
10.1016/j.ijrobp.2016.10.046
Reference:
ROB 23889
To appear in:
International Journal of Radiation Oncology • Biology • Physics
Received Date: 20 May 2016 Revised Date:
26 October 2016
Accepted Date: 31 October 2016
Please cite this article as: Chadha AS, Liu G, Chen H-C, Das P, Minsky BD, Mahmood U, Delclos ME, Suh Y, Sawakuchi GO, Beddar S, Katz MH, Fleming JB, Javle MM, Varadhachary GR, Wolff RA, Crane CH, Wang X, Thames H, Krishnan S, Does unintentional splenic radiation predict severe lymphopenia following pancreatic cancer radiotherapy?, International Journal of Radiation Oncology • Biology • Physics (2016), doi: 10.1016/j.ijrobp.2016.10.046. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title: Does unintentional splenic radiation predict severe lymphopenia following pancreatic cancer radiotherapy? Awalpreet S. Chadha, M.D.,1 Guan Liu, M.D.,1 Hsiang-Chun Chen, Ph.D.,2 Prajnan
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Das, M.D.,1 Bruce D. Minsky, M.D.1 Usama Mahmood, M.D.,1 Marc E. Delclos, M.D.,1 Yelin Suh, Ph.D.,3 Gabriel O. Sawakuchi, Ph.D.,3,4 Sam Beddar, Ph.D.,3 Matthew H. Katz, M.D.,5 Jason B. Fleming, M.D.,5 Milind M. Javle, M.D.,6 Gauri R. Varadhachary,
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Howard Thames, Ph.D.,1 Sunil Krishnan, M.D.1
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M.D.,6 Robert A. Wolff, M.D.,6 Christopher H. Crane, M.D.,1 Xuemei Wang, M.S.,2
Departments of Radiation Oncology1, Biostatistics,2 Radiation Physics,3 Surgical Oncology,5 and Gastrointestinal Medical Oncology,6 The University of Texas MD Anderson Cancer Center, Houston, Texas 77030
77030
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Acknowledgements:
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Graduate School of Biomedical Sciences,4 The University of Texas, Houston, Texas
This work was supported in part by Cancer Center Support (Core) Grant P30
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CA16672 to The University of Texas MD Anderson Cancer Center and the John E. and Dorothy J. Harris Endowed Professorship to SK. The authors also thank Ms. Christine Wogan (Program Manager, Divisional Publications, Radiation Oncology Department, MD Anderson Cancer Center) for carefully editing the manuscript. Corresponding Author: Sunil Krishnan M.D. Department of Radiation Oncology
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The University of Texas MD Anderson Cancer Center 1515 Holcombe Boulevard, Houston, TX 77030-4009
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Telephone: (713) 563-2377 Fax: (713) 745-2186 E-mail: [email protected]
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Running Title: Splenic radiation during pancreatic cancer radiotherapy
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Presented in part at the American Society of Clinical Oncology Gastrointestinal Cancers Symposium, San Francisco, 2016.
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Disclaimer: The authors report no conflict of interest.
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Summary
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We document an association between post-chemoradiation lymphopenia and survival of locally advanced pancreatic cancer patients. Furthermore, splenic dose-volume histogram parameters are associated with the observed lymphopenia, suggesting a correlation between unintentional splenic irradiation and lymphocyte depletion. This knowledge could be used to triage treatment plans that could cause lymphopenia or design treatment plans that spare the spleen. Presumably, preserving lymphocytes would also increase the possibility of mounting an anti-tumor immune response, especially when immunotherapy is planned in the future.
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Title: Does unintentional splenic radiation predict severe lymphopenia following pancreatic cancer radiotherapy?
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Abstract Purpose: To determine if severity of lymphopenia is dependent on radiation dose and fractional volume of spleen irradiated unintentionally during definitive
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chemoradiation (CRT) in patients with locally advanced pancreatic cancer (LAPC). Methods: 177 patients with LAPC received induction chemotherapy (mainly
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gemcitabine-based regimens) followed by CRT (median 50.4 Gy with concurrent capecitabine) from 1/2006 to 12/2012. Absolute lymphocyte count (ALC) was recorded at baseline, prior to CRT and 2-10 weeks after CRT. Splenic dose-volume histogram (DVH) parameters were reported as mean splenic dose (MSD) and percentage of splenic volume receiving at least 5 (V5), 10 (V10), 15 (V15) and 20 Gy
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(V20) dose. Overall survival (OS) was analyzed using the Cox model and development of post-CRT severe lymphopenia (ALC <0.5 K/UL) was assessed by multivariate logistic regression using baseline and treatment factors.
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Results: Median post-CRT ALC (0.68 K/UL, range 0.13-2.72) was significantly lower than both baseline (1.42 K/UL, range 0.34-3.97; p<0.0001) and pre-CRT ALC (1.32
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K/UL, range 0.36-4.82; p<0.0001). Post-CRT ALC <0.5 K/UL was associated with inferior OS on univariate (median: 11.1 vs. 15.3 months, p=0.01) and multivariate analysis (HR=1.66, p=0.01). MSD (9.8 vs. 6 Gy, p=0.03), median V10 (32.6 vs. 16%, p=0.04), V15 (23.2 vs. 9.5%, p=0.03) and V20 (15.4 vs. 4.6%, p=0.02) were significantly higher in patients with severe lymphopenia compared to those without. On multivariate analysis, post-induction lymphopenia (p<0.001, OR=5.25)
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and MSD (p=0.002, OR= 3.42) were independent predictors for development of severe post-CRT lymphopenia. Conclusion: Severe post-CRT lymphopenia is an independent predictor of poor OS
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in LAPC patients receiving CRT. Higher splenic doses increase the risk of developing severe post-CRT lymphopenia. When clinically indicated, assessment of splenic DVHs prior to acceptance of treatment plans may minimize the risk of severe post-
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CRT lymphopenia.
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Introduction
The immune system is generally regarded as a primary defense against cancer by recognizing and eliminating incipient tumors. Cancer immune surveillance, first proposed by Burnet and Thomas, may not always eradicate tumors but rather competes with tumor progression in a dynamic interplay that is referred to as
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immunoediting.(1-3) Transiting through or residing within tumors, lymphocytes play a key role in protecting the host and sculpting emerging tumors through their interactions with tumor associated antigens (TAAs).(4) Several clinical studies have
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highlighted the positive prognostic value of both CD8+ tumor-infiltrating lymphocytes and circulating lymphocytes in pancreatic, colorectal, head and neck,
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urothelial and lung cancer.(5-12) Unlike many malignancies that have abundant immune infiltrates, pancreatic cancer thrives in an immune-privileged milieu that has a conspicuous scarcity of effector T cells and a relative abundance of regulatory T cells, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages.(13; 14) This imbalance of pro- vs. anti-tumor T-cell populations may be mediated through surface expression of immunosuppressive molecules like programmed death-ligand 1 and Fas ligand and/or active secretion of GM-CSF by
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pancreatic cancer cells to promote recruitment and differentiation of precursor cells into MDSCs that counteract the activity of cytotoxic CD8+ T-cells.(15-19) Together with the exuberant desmoplastic stroma, the lack of effective immune
interventions and the emergence of treatment resistance.
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surveillance contributes to a hostile tumor microenvironment for therapeutic
Radiation therapy (RT) is known to have both immune-stimulating and immune-
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suppressive effects.(20-22) The latter has traditionally been linked to direct bone marrow suppression and/or depletion of circulating lymphocytes. Recent evidence
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suggests that patients with high-grade gliomas and pancreatic cancer have an increased incidence of and a poor prognosis from lymphopenia following chemoradiation therapy (CRT).(6; 21; 23) However, none of these studies have tried to explore possible mechanisms behind this phenomenon.
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We demonstrate that chemoradiation-induced lymphopenia portends a poor prognosis in patients with locally advanced pancreatic cancer (LAPC) treated with induction chemotherapy followed by CRT and that the severity of lymphopenia is dependent on the radiation dose and fractional volume of the spleen irradiated
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unintentionally during treatment.
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Patients and methods Patient identification
The xxx pancreatic cancer database was used to retrospectively identify consecutive
patients
with
biopsy-proven
LAPC
treated
with
induction
chemotherapy followed by CRT from 2006 to 2012. Patients having primarily resectable or borderline resectable disease, or distant metastasis at presentation were excluded.(24)
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Clinical evaluation and treatment characteristics Patients were evaluated by a multidisciplinary team consisting of a medical and a radiation oncologist and selected patients were later seen by surgical oncologists.
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Clinical and laboratory parameters were routinely assessed in all patients (Table 1 and Table 2). Absolute lymphocyte counts (ALC) were recorded prior to treatment (baseline), after completion of induction chemotherapy, and after CRT (2-10 weeks
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after completion of CRT).
All patients received induction chemotherapy followed by CRT. RT was
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administered using either a four-field 3-dimensional conformal technique or an intensity-modulated radiation therapy (IMRT) technique based on physician preference. Follow-up visits for restaging were scheduled at roughly 6 weeks (after completion of CRT) and then every 3-4 months. Subsequent therapy was
Statistical analysis
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individualized based on treatment response.
Lymphopenia was defined based on the Common Terminology Criteria for Adverse
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Events, version 4.0.(25) Grade I and II lymphopenia (ALC between 0.5 and 1 K/UL), and grade III and IV lymphopenia (ALC < 0.5 K/UL) were categorized as mild and
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severe, respectively. Patient characteristics were tabulated by severe lymphopenia. Differences between continuous covariates were compared by the Wilcoxon ranksum test, and differences between categorical covariates were compared using Fisher's exact test or Chi-square tests as appropriate. To test for the prognostic significance of post-CRT lymphocyte nadirs, overall survival (OS) time was chosen as the outcome of interest. A landmark analysis of OS was performed to account for lead time bias between the start of induction chemotherapy and the assessment of
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post-CRT ALC. OS was calculated from a fixed landmark time point to the death date or last follow-up date, whichever occurred first. Patients who died or were lost to follow-up prior to the landmark time were excluded. Kaplan-Meier product limit
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method was used to estimate survival probabilities and differences between groups were compared using log-rank test.(26) Cox proportional hazards models were fit to assess the association between potential predictors and OS.(27) Predictors with
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a p-value ≤0.2 on univariate analysis were included in the multivariate model, and backward elimination was used to obtain the final multivariate Cox model (Table 3).
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For patients with available post-CRT ALC and RT plans, we contoured the spleen and obtained dose-volume histogram (DVH) parameters using Pinnacle 9.8. DVHs were reported in terms of mean splenic dose (MSD) and the percentage of splenic volume receiving at least 5 Gy (V5), 10 Gy (V10), 15 Gy (V15) and 20 Gy (V20) dose. We then compared MSD, and V5, V10, V15 and V20 (stratified at increments of
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10%) between patients with and without severe lymphopenia using Wilcoxon rank sum test or Fisher’s exact test, as appropriate. Finally, we analyzed the dose-bin data using the Lyman-Kutcher-Burman (LKB) model with post-CRT ALC as the
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dependent variable.(28; 29)
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ALCs at baseline, after induction chemotherapy and after CRT were compared using paired t-test. Predictors of post-CRT lymphocyte nadir were classified into (1) baseline and (2) treatment factors (Table 4). Univariate and multivariate logistic regression analysis (using factors found to be significant at the 0.2 level in the univariate analyses) were performed to assess the impact of these factors on development of severe lymphopenia. A two-tailed p-value of ≤0.05 was considered
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statistically significant. Statistical analyses were performed using SAS 9.3 (SAS Institute, Cary, NC) and R (The R Project for Statistical Computing, Vienna, Austria).
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Results Patient characteristics
177 patients with LAPC were treated with induction chemotherapy followed by
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definitive CRT. Median clinical follow-up time from start of CRT was 12 months (range, 1-87). Table 1 summarizes the patient characteristics. Median baseline ALC
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was 1.42 K/UL (range, 0.34-3.97). Median ALC after induction chemotherapy was 1.32 K/UL (range, 0.36-4.82) which was within normal range and not different from baseline (p=0.14). Median ALC further decreased to 0.68 K/UL (range, 0.13-2.72) after CRT which was significantly lower than both baseline (p<0.0001) and postinduction levels (p<0.0001). The incidence of severe lymphopenia was only 3% after
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induction chemotherapy but increased to 27% after CRT. Median time for recording ALC was 38 days (interquartile range, IQR, 32-44) after completion of CRT. Changes in neutrophil to lymphocyte ratio (NLR) mirrored the changes in ALC with increases
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noted following induction chemotherapy and after CRT (Figure 1).
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Treatment characteristics
Table 2 summarizes tumor and treatment characteristics. Median time for initiating treatment was 22 days (IQR, 12-34) from diagnosis. Induction chemotherapy was administered for a median duration of 3 months (IQR, 2–5) using predominantly gemcitabine-based (n=149, 84%) therapy. Median radiation dose was 50.4Gy (range, 25.2-70.4) in 28 fractions administered over 38 days (IQR, 37-39). Concurrent chemotherapy was primarily capecitabine-based (n=150, 85%).
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Lymphocyte counts and survival outcomes Post-CRT ALC was available for 162 patients. Characteristics of the 15 patients lacking ALC values were comparable to those of patients included in the analysis
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except for their being older in age and having higher NLR values after induction chemotherapy (Supplementary table 1). Median time for post-CRT ALC measurement was 5.5 months (range, 3.6-13) from start of induction
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chemotherapy. Therefore, a landmark analysis at 6 months from the start of induction chemotherapy was performed. Five patients who died within 6 months
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after the start of induction chemotherapy were excluded, and 157 patients (132 deaths) were included in the 6-month landmark analysis. Median OS from the landmark time point was 14 months (95% confidence interval, CI, 11.7-15.7 months). The 1-, 2- and 3-year OS rates were 57%, 26% and 14% respectively. Patients with post-CRT severe lymphopenia had an inferior OS (Figure 2) compared
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to those without severe lymphopenia (median: 11.1 vs. 15.3 months, p=0.01) and compared to patients with mild lymphopenia (median: 11.1 vs. 13.6 months, p=0.03). Other parameters found to be significant at the 0.2 level in the univariate
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analyses included age, KPS, baseline platelet count, baseline NLR, post-CRT NLR and radiation dose level. Because of the high correlation between ALC and NLR, only
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ALC was included in the final Cox model. On multivariate analysis, post-CRT ALC < 0.5 K/µL (HR=1.66, p=0.01) and baseline platelet count > 300 K/µL (HR=1.91, p=0.02) were the only independent predictors of inferior OS (Table 3). This is in accordance with data published previously by our group.(30) Same results were obtained with the forward selection method. Similar trends were seen when the predictors were divided into 4 quartiles, which confirms the appropriateness of using dichotomized predictors (Supplementary table 2).
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Predictors of lymphopenia DVH data were available for 157 patients. MSD ranged from 0.1 to 42 Gy with a mean of 8.8 Gy. Patients with severe lymphopenia had a higher MSD than those
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without severe lymphopenia (9.8 vs. 6 Gy, p=0.03). A univariate logistic analysis indicated that MSD was a strong predictor of severe lymphopenia (odds ratio, OR, 3.41; p=0.001). Furthermore, there was a dose-response relationship between MSD
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and severity of lymphopenia with the median MSD for patients with normal ALC, mild, and severe lymphopenia being 3.8 Gy, 6.8 Gy and 9.8 Gy, respectively. When
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analyzed as a continuous variable, MSD did not show a correlation with post-CRT ALC, which was possibly because of the dispersion within the data. Normalized MSD, however, showed a weaker but statistically significant negative correlation (r= -0.19, p=0.02) with post-CRT ALC. Similar trends were seen when MSD was
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divided into 4 quartiles and analyzed (Supplementary table 3). On further DVH analysis, we found that median V10 (32.6 vs. 16%, p=0.04), V15 (23.2 vs. 9.5%, p=0.03) and V20 (15.4 vs. 4.6%, p=0.02) were significantly higher in patients with severe lymphopenia compared to those without severe lymphopenia.
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Additionally, a greater proportion of patients with severe lymphopenia had V10
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(p=0.03), V15 (p=0.01) and V20 (p=0.04) >20% compared to those without severe lymphopenia. Similar results were obtained at 10% for V15 and V20 and 30% for V5, V10 and V15 but not at higher increments due to fewer patients in the cohort with high volumes and high doses. The most significant p values were obtained for the following stratifications: V5 at 30% (p=0.04), V10 at 30% (p=0.01), V15 at 20% (p=0.01) and V20 at 20% (p=0.03). The significance of thresholds at multiple points
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along the DVH curve also validate the clinical relevance of dose-dependent occurrence of severe lymphopenia. Finally, univariate analyses of baseline and other treatment factors showed that the
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development of post-CRT severe lymphopenia was also predicted by baseline lymphopenia (p=0.001) and post-induction lymphopenia (p<0.001). It was not affected by the type of induction chemotherapy regimen (p=0.87), radiation dose
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(biologically effective dose, BED, of ≤70 Gy vs. >70 Gy, p=0.56) or concurrent radiosensitizers used (p=0.44). Due to strong associations between MSD and V5,
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V10, V15 or V20 as well as between baseline and post-induction lymphopenia, only MSD and post-induction lymphopenia were included in the multivariate analysis. On the multivariate logistic regression model, post-induction lymphopenia (p<0.001, OR=5.25) and MSD dichotomized at the mean (p=0.002, OR= 3.42) were the most important predictors for development of post-CRT severe lymphopenia
scatter in the data.
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Discussion
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(Table 4). The LKB model, however, did not converge possibly due to amount of
Our data suggest that severe lymphopenia occurs relatively frequently following
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CRT in patients with LAPC and is an independent predictor of poor OS. We also observed a significant dose-response relationship between post-CRT ALC and MSD which offers a potential explanation for development of severe lymphopenia. In addition to MSD, post-induction lymphopenia was a significant predictor of severe post-CRT lymphopenia. The strong association between OS and post-CRT ALC as well as post-CRT ALC and MSD, however, do not imply an association between MSD and OS due to intransitivity of these relations. This is consistent with other DVH
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parameters that predict toxicity which, in turn, has an influence on survival but the DVH parameter per se does not directly impact survival.(31) Nevertheless, these DVH metrics have clinical utility in comparing treatment plans and choosing the
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most appropriate treatment plan.
Although our patients received induction chemotherapy followed by CRT, lymphopenia is predominantly a consequence of CRT because median ALC for the
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entire cohort after induction chemotherapy was normal and not significantly different from baseline. In fact, only 3% of patients developed severe lymphopenia
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following induction chemotherapy and this increased to 27% following CRT. Neither the type of induction chemotherapy nor that of concurrent chemotherapy was associated with severe post-chemoradiation lymphopenia. Though similar findings for pancreatic cancer have been previously described, the underlying mechanisms are largely unknown.(6; 7) The potential mechanisms for radiation-induced
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lymphopenia include depletion of progenitor cells in the bone marrow and/or circulating lymphocytes owing to large gross tumor volume (GTV) coverage.(21; 32) The anatomic location of the pancreas away from active marrow-producing bone
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and the lack of correlation between tumor size (a surrogate for GTV) and severe post-CRT lymphopenia largely precludes these mechanistic explanations. The
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anatomical proximity of the spleen to the pancreas, the relative abundance of lymphocytes in splenic white pulp and the exquisite radiosensitivity of lymphocytes to doses of radiation as low as 2 Gy support the hypothesis that splenic radiation dose is a predictor and potential mechanism for the development of severe lymphopenia.(33) In our dataset, the percentage change in ALC following CRT (compared to post-induction ALC) was directly related to the loge MSD (r=0.17, p=0.03), further implicating splenic irradiation as a mediator of lymphopenia. In
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addition, the lack of neutropenia associated with lymphopenia is distinctly different from that observed with either myelosuppression from chemotherapy or large irradiation volumes(33; 34) such as total body irradiation and craniospinal
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irradiation. The correlation between NLR and survival has been described in many cancers. While the precise mechanism remains unknown, it is believed that high neutrophil counts are (i) indicative of an immunosuppressive inflammatory
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response that suppresses cytotoxic T-cell and natural killer cell activity and (ii) associated with increased secretion of growth factors that promote tumor
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growth.(34)
The clinical significance of this finding may be more pronounced when lymphocyte activation for anti-tumor effects are the desired outcome of combination with immunotherapeutic agents. Recent data on the synergy between RT and immune check point inhibitors suggest that radiation can prime effector T-cells to recognize
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tumor-specific and/or radiation-induced tumor neoantigens.(21; 35; 36) Irradiated tumor cells express ‘eat me’ signals (calreticulin) and elevated levels of MHC class I molecules, and release ‘danger’ signals like high-mobility group protein B1 and ATP lead
to
dendritic
cell-mediated,
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which
cytotoxic
T-lymphocyte-induced
immunogenic death.(21; 37; 38) In addition, radiation conditions the tumor
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microenvironment by inducing expression of proinflammatory mediators (IFN-γ, CXCL16) which enhance T-cell trafficking and infiltration within tumors.(39; 40) This has led to profound interest in using radiation to convert tumors into an in-situ vaccines which constantly feed immune-adjuvant neoantigens to tumor-specific Tcells and fuels their recruitment to the tumor microenvironment.(41) The translation of this radiotherapy-immunotherapy alliance into a successful therapeutic model is especially relevant in pancreatic cancer where treatment
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resistance is extremely common and multiple drugs targeting various pathways have failed clinically. To this end, we believe that any attempt at combining immunotherapy and RT for pancreatic cancer will be considerably undermined by
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depletion of lymphocytes after CRT and as a corollary, preservation of ALCs through the use of spleen-sparing radiation techniques may be a reasonable consideration. In our practice, as is the custom at most centers in the United States, we have
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traditionally ignored the spleen while developing RT plans. If validated in other cohorts of LAPC patients receiving CRT, this finding would argue strongly for more
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routine assessment of splenic DVHs while triaging competing treatment plans and tailoring treatments to minimize gratuitous splenic irradiation, especially in the setting of preexisting lymphopenia. Furthermore, our data suggest that either an MSD threshold or a splenic dose-volume threshold must be crossed before severe lymphopenia develops, which is around an MSD of 9 Gy and a V15 of 20% in our
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dataset. From a clinical perspective, we believe that newer techniques like IMRT and stereotactic body RT could confer better spleen sparing while still allowing dose escalation to the GTV.(42; 43)
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These findings should be interpreted with caution given its retrospective nature.
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First, the results cannot be generalized to tumors located in other sites receiving CRT. The geographic proximity of the spleen to the pancreas and the frequent inclusion of portions of the spleen in radiation fields may, however, be similar to that of other upper abdominal tumors. Second, there is always a possibility of selection bias as we only included patients who had both post-CRT ALC and DVH data available. As noted in supplementary table 1, minor differences between the included and excluded groups of patients are an inevitable consequence of
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retrospective analyses that temper enthusiasm for broad generalization. This is especially notable because high BED was not an independent predictor of overall survival in this analysis, in discordance with our earlier report on high BED being a
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predictor of better outcome. We attribute this to the reduced numbers of patients with high BED in this analysis due to a shorter time window of patient accrual and patients dropping out from analysis due to the lack of lymphocyte counts (as noted
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above). Despite the use of appropriate statistical tools to analyze the validity of each predictive variable, this discordance is a reflection of inherent challenges
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associated with retrospective analyses where outcomes are studied by probing small subsets of patients and even a small decrease in the size of this subset impacts its predictive value. We consider both reports to be hypothesis-generating, needing independent validation before they influence individual clinical practice patterns and plan to embark on such validation efforts with other large single- and
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multi-institution datasets. Third, there was some variation in assessing post-CRT ALCs. To be consistent, we only included patients with ALC available 2-10 weeks after CRT and performed survival analysis from a landmark point to account for
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lead-time bias. Fourth, we did not record ALCs during subsequent follow-up visits and so we cannot conclude if this effect persists long-term or not. However, studies
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by other groups confirm that treatment-related lymphopenia persists up to 12 months after starting CRT.(6) Finally, the lack of convergence of the LKB model may reflect scatter within the data, and the likelihood that radiobiological models traditionally used for normal tissue complication probability predictions may not apply to lymphocyte depletion by splenic radiation, as observed in other scenarios.(44-46) The dependence of LKB model fitting on the specific endpoint analyzed and the underlying pathophysiology of the toxicity, and the need for
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collective institutional experiences suggest that validation of these findings in other datasets is warranted. In conclusion, severe lymphopenia occurs relatively frequently following
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chemoradiation and is an independent predictor of inferior OS. In addition, the risk increases as the MSD and the fractional volume of spleen receiving low radiation doses increases. The existence of a specific splenic dose-volume threshold needs
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further validation. The thresholds defined here could serve as guideposts for assessing RT plans for LAPC, and to customize individual plans to ensure adequate
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spleen sparing when clinically indicated.
Figure legends
Figure 1: Absolute lymphocyte count (ALC) and neutrophil-to-lymphocyte ration
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(NLR) at baseline, after induction chemotherapy and after chemoradiation. Figure 2: Kaplan-Meier estimates of overall survival based on landmark analysis in patients with locally advanced pancreatic cancer and grouped by post-
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chemoradiation therapy (CRT) absolute lymphocyte count (ALC).
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Supplementary figure 1: Axial isodose plots and dose volume histograms corresponding to a planned course of treatment for a representative patient with severe post-chemoradiation lymphopenia (a, b) and without severe postchemoradiation lymphopenia (c, d). REFERENCES 1. 2.
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Tables Table 1: Patient characteristics
98 (55) 79 (45)
P-value
0.57
64 years 37 - 84 years
0.58
26 (59) 18 (41)
64 (54) 54 (46)
132 (75) 22 (12) 16 (9) 7 (4)
35 (80) 3 (7) 5 (11) 1 (2)
87 (74) 17 (14) 9 (8) 5 (4)
1.42 (0.34- 3.97) 1.32 (0.36- 4.82) 0.68 (0.13- 2.72)
1.12 (0.34- 2.24) 1.04 (0.36- 2.35) 0.37 (0.13- 0.49)
1.51 (0.52- 3.97) 1.51 (0.37- 4.82) 0.84 (0.5- 2.72)
<0.001 <0.001 <0.001
4.18 (1.32, 16.14) 3.18 (0.61, 32.43) 3.07 (0.96, 21.05)
4.1 (2.21, 10.34) 3.21 (0.95, 32.43) 2.76 (0.96, 21.05)
4.15 (1.32, 16.14) 3.02 (0.61, 23.71) 3.09 (1, 8.03)
0.86 0.21 0.62
0.51 (0.17, 1.42) 0.61 (0.03, 3.01) 0.49 (0.03, 2.18)
0.47 (0.23, 1.06) 0.53 (0.1, 1.33) 0.42 (0.09, 2.18)
0.52 (0.17, 1.32) 0.68 (0.03, 3.01) 0.53 (0.03, 1.85)
0.41 0.01 0.01
2.86 (0.94, 26.17) 2.37 (0.23, 20.26) 4.74 (0.81, 44.85)
3.87 (1.44, 16.5) 3.3 (0.97, 18.85) 7.55 (2.39, 43.85)
2.62 (0.94, 10.35) 1.99 (0.23, 20.26) 3.72 (0.81, 11.81)
0.001 <0.001 <0.001
219.5 95 - 588 138 (84) 26 (16)
212 95 - 588 37 (90) 4 (10)
226 130 - 580 92 (84) 17 (16)
0.04
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63 years 39 - 79 years
Postchemoradiation ALC ≥ 0.5 K/UL N=118
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64 years 37 - 88 years
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Age Median Range Gender Male Female Race White African- American Hispanic Asian ALC (K/UL) – median (range) Baseline Post-induction chemotherapy Post-chemoradiation Neutrophil (K/UL) – median (range) Baseline Post-induction chemotherapy Post-chemoradiation Monocyte (K/UL) – median (range) Baseline Post-induction chemotherapy Post-chemoradiation Neutrophil to ALC Ratio – median (range) Baseline Post-induction chemotherapy Post-chemoradiation Baseline Platelet Count (K/UL) Median Range ≤300 >300 KPS
Postchemoradiation ALC < 0.5 K/UL N=44
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All Patients N=177 N (%)
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Characteristic
0.51
0.44 0.78
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SC
RI PT
<80 22 (13) 4 (9) 14 (12) ≥80 154 (87) 40 (91) 104 (88) % Weight Loss 0.53 <10 92 (54) 21 (50) 64 (56) ≥10 79 (46) 21 (50) 51 (44) Baseline Hemoglobin (g/dL) 0.68 >12 115 (70) 30 (73) 76 (70) ≤12 49 (30) 11 (27) 33 (30) Abbreviations: ALC, absolute lymphocyte count; KPS, Karnofsky performance status; N, number of patients Where numbers do not add to column title value of N, insufficient data was available in remaining patients. Post-chemoradiation ALC fell within the prescribed range in 162 of 177 patients.
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Table 2: Tumor and treatment characteristics
149 (84) 28 (16)
37 (84) 7 (16)
P-value Postchemoradiation ALC ≥ 0.5 K/UL N=118 0.87 98 (83) 20 (17)
3 (2) 1 (1) 22 (13) 150 (85)
1 (2) 0 (0) 4 (9) 39 (89)
1 (1) 1 (1) 17 (14) 98 (84)
RI PT
Postchemoradiation ALC < 0.5 K/UL N=44
6.8 0.1 - 42
M AN U
59.5 29.7 - 100 34 (19) 143 (81)
SC
0.57
59.5 29.7 - 87.5 10 (23) 34 (77)
9.79 0.51 - 37.06
59.5 39 - 100 22 (19) 96 (81)
56 (49) 58 (51)
99 (63) 58 (37)
20 (46) 23 (54)
79 (69) 35 (31)
97 (62) 60 (38)
19 (44) 24 (56)
78 (68) 36 (32)
AC C
105 (60) 35 (20) 14 (8) 8 (5) 12 (7)
0.56 0.03
0.03
13 (30) 30 (70)
115 (73) 42 (27)
0.66
5.98 0.12 - 41.97
69 (44) 88 (56)
EP
Induction Chemotherapy* Gemcitabine-based FOLFIRINOX Concurrent Chemotherapy# 5-fluorouracil Vorinostat Gemcitabine-based Capecitabine- based Radiation Dose (BED, Gy) Median Range BED > 70 BED ≤ 70 Mean Splenic Dose (Gy) Median Range V5 ≤ 30% > 30% V10 ≤ 30% > 30% V15 ≤ 20% > 20% V20 ≤ 20% > 20% Tumor Location Head Body Head/ Body Body/ Tail Tail Tumor size $
All Patients N=177 N (%)
TE D
Characteristic
0.01
0.01
0.03 26 (60) 17 (40)
89 (78) 25 (22) 0.49
23 (52) 10 (23) 3 (7) 4 (9) 4 (9)
73 (63) 21 (18) 9 (8) 4 (3) 8 (7) 0.92
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3.5 cm 0.9- 8 cm
3.55 cm 1.7- 7.2 cm
3.55 cm 0.9- 8 cm 0.30
0 (0) 11 (69) 5 (31)
5 (14) 18 (51) 12 (34)
RI PT
5 (9) 31 (55) 20 (36)
0.47
163 (95) 5 (3) 1 (1) 2 (1)
43 (98) 0 (0) 0 (0) 1 (2)
110 (94) 5 (4) 1 (1) 1 (1)
SC
Median Range Grade Well differentiated Moderately differentiated Poorly differentiated Histology Adenocarcinoma Mucinous Signet ring Adenosquamous
AC C
EP
TE D
M AN U
Abbreviations: ALC, absolute lymphocyte count; BED, Biologically Effective Dose; Gy, Gray; N, Vx, volume of spleen receiving x Gy of radiation; number of patients * Gemcitabine-based induction therapy consisted of gemcitabine alone or in combination with paclitaxel, cisplatin, oxaliplatin, erlotinib, bevacizumab or cetuximab; # Gemcitabine-based concurrent therapy consisted of gemcitabine alone or in combination with erlotinib; Capecitabine-based concurrent therapy consisted of capecitabine alone or in combination with bevacizumab, erlotinib or cetuximab $ Based on single largest dimension on abdominopelvic computed tomography scan Where numbers do not add to column title value of N, insufficient data was available in remaining patients. Post-chemoradiation ALC fell within the prescribed range in 162 of 177 patients.
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Table 3: Univariate and multivariate landmark analyses of prognostic factors for overall survival (OS), with OS time defined starting from 6 months after induction chemotherapy (n=157) p Hazard Ratio (95% CI) value
RI PT
Prognostic factor
AC C
EP
TE D
M AN U
SC
Univariate analysis for OS Baseline Factors Age (≥60 vs. <60 years) 0.13 1.33 (0.92-1.91) Gender (Female vs. male) 0.97 0.99 (0.70-1.40) Race (White vs. others) 0.95 0.99 (0.66-1.47) Tumor location (Head vs. others) 0.78 1.05 (0.74-1.50) Tumor grade (Moderately/well vs. poorly differentiated) 0.01 0.42 (0.22-0.83) Tumor size (maximum; >3.5 vs. ≤3.5 cm) 0.97 1.01 (0.67-1.50) % weight loss (<10 vs. ≥10 %) 0.70 1.07 (0.75-1.52) KPS < 80 vs. ≥80 0.12 1.49 (0.90-2.46) Baseline hemoglobin (>12 vs. ≤ 12 g/dL) 0.34 0.83 (0.56-1.22) Baseline platelet count (>300 vs. ≤300 K/UL) 0.04 1.71 (1.02-2.86) Baseline ALC (<1 vs. ≥1 K/UL) 0.02 1.73 (1.09-2.73) Baseline neutrophil to ALC ratio (≤3 vs.>3)$ 0.10 0.75 (0.53-1.06) Treatment Factors 0.24 1.39 (0.81-2.38) Induction chemotherapy (Gemcitabine vs. others) 0.25 1.27 (0.84-1.91) Post-induction ALC (<1 vs. ≥1 K/UL) Post-induction neutrophil to ALC ratio (≤2 vs.>2)$ 0.95 1.01 (0.71-1.43) Concurrent chemotherapy (Capecitabine vs. others) 0.26 0.76 (0.48-1.22) 0.09 0.68 (0.44-1.06) BED (>70 vs. ≤ 70 Gy) 0.01 1.66 (1.13-2.43) Post-chemoradiation ALC (< 0.5 vs. ≥0.5 K/UL) $ 0.06 0.72 (0.51-1.02) Post-chemoradiation neutrophil to ALC ratio (≤5 vs.>5) Mean splenic dose (>8.76 vs. ≤ 8.76 Gy) 0.36 0.84 (0.58- 1.21) Multivariate Analysis for OS (after backward elimination) Baseline platelet count (>300 vs. ≤300 K/UL) 0.02 1.91 (1.13- 3.23) Post-chemoradiation ALC (< 0.5 vs. ≥0.5 K/UL) 0.01 1.66 (1.11- 2.48) Abbreviations: ALC, absolute lymphocyte count; BED, Biologically Effective Dose; CI: 95% confidence interval; KPS, Karnofsky performance status * Grade was not included in the multivariate analysis because 68% of patients had missing data. $ Cut-offs were defined using the integers closest to the median
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Table 4: Predictors of development of severe lymphopenia (n=162)
TE D
Odds Ratio (95% CI)
RI PT
p value
0.85 (0.41-1.73) 0.74 (0.23-2.39) 1.18 (0.53-2.64) 4.62 (1.84-11.61)
SC
0.65 0.62 0.68 0.001
0.87 0.44 <0.001 0.56 0.001 0.04 0.01 0.01 0.03
1.08 (0.42-2.76) 1.51 (0.53-4.33) 5.33 (2.37-11.99) 1.28 (0.55-2.98) 3.41 (1.65-7.06) 2.23 (1.06-4.70) 2.60 (1.26-5.33) 2.74 (1.33-5.62) 2.33 (1.09-4.96)
<0.001 0.002
5.25 (2.24-12.30) 3.42 (1.57- 7.44)
M AN U
Prognostic factor Univariate analysis of severe lymphopenia Baseline Factors Age (≥60 vs. <60 years) KPS (< 80 vs. ≥80) Baseline hemoglobin (>12 vs. ≤ 12 g/dL) Baseline ALC (<1 vs. ≥1 K/UL) Treatment Factors Induction chemotherapy (Gemcitabine vs. FOLFIRINOX) Concurrent chemotherapy (Capecitabine vs. others) Post-induction ALC (<1 vs. ≥1 K/UL) BED (>70 vs. ≤ 70 Gy) Mean splenic dose (>8.8 vs. ≤ 8.8 Gy) V5 (>30 vs. ≤ 30%) V10 (>30 vs. ≤ 30%) V15 (>20 vs. ≤ 20%) V20 (>20 vs. ≤ 20%) Multivariate analysis of severe lymphopenia Post-induction ALC (<1 vs. ≥1 K/UL) Mean splenic dose (>8.76 vs. ≤ 8.76 Gy)
AC C
EP
Abbreviations: ALC, absolute lymphocyte count; BED, Biologically Effective Dose; CI: 95% confidence interval; KPS, Karnofsky performance status; Vx, volume of spleen receiving x Gy of radiation
Figure 1
AC C
1
TE D
0 Baseline
EP
3
2
M AN U
4 40
32
Post-induction 24
16
8
Neutrophil/Lymphocyte Count Neutrophil to Absolute Lymphocyte Count Ratio
SC
5
RI PT
Absolute Lymphocyte Countcount Absolute Lymphocyte Lymphocyte Count
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0
Post-chemoradiation
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1
Normalized Volume
0.9
GTV
0.8
Liver
0.7
Right Kidney
0.6
Left Kidney
0.5
Spleen Cord
0.4 0.3 0.2
0.1 0
10
20
30 Dose (Gy)
40
50
M AN U
SC
0
RI PT
(b)
(a)
1
(d)
AC C
Figure 2
EP
TE D
Normalized Volume
(c)
0.9
GTV
0.8
Liver
0.7
Right Kidney
0.6
Left Kidney
0.5
Spleen Cord
0.4 0.3 0.2
0.1 0 0
10
20
30 Dose (Gy)
40
50