[9–12]. Multiple retrospective and small prospective studies have shown radiation related lymphopenia to be associated with detrimental survival endpoints [13 – 17]. Currently, there are no standardized dose constraints that are available to limit the radiation dose to the resident and circulating lymphocyte populations. This is much more pertinent in the current immunotherapy era wherein ongoing clinical trials are trying to optimally time and sequence radiation and immunotherapy combinations for potentially synergistic and or additive effects [18].

We performed a systematic review of studies in published literature to create the LymphoTEC guideline of dose constraint for reduction of radiation related lymphopenia (attached in appendix). The LymphoTEC guideline (published in radiotherapy and oncology 10.1016/j.radonc.2022.10.019) provides an initial framework for dosimetric constraints to reduce radiation related lymphopenia. The studies included in the generation of the guideline is summarized in appendix 1.0. The authors of the guideline conclude that ‘The dose constraints described herein may be considered for prospective validation and future use in clinical trials to limit risk of radiation-related lymphopenia.’ The prospective validation is much more pertinent in the current immunotherapy era wherein ongoing clinical trials are trying to optimally time and sequence radiation and immunotherapy combinations for potentially synergistic and or additive effects [19]. We therefore attempted a prospective validation study wherein dose constraints proposed by LymphoTEC was applied to a homogenous population of prostate cancer receiving pelvic radiotherapy (MPMMCC Study ID: 11000598; Approval date: 28/10/2022). A total of 42 patients were recruited in the single arm cohort study between November 2022 and November 2023. All patients included (n = 42) in study were high risk prostate cancer as per the NCCN criteria. All patients received pelvic and prostate radiotherapy with simultaneous integrated boost intensity modulated radiation therapy (SIB-IMRT) to a dose of 68 Gray in 25 fractions to the prostate and 50 Gray in 25 fractions to the pelvis. The study assumption was that if 2 of 3 constraints (LymphoTEC) are met i.e. V10 pelvic bone < 80%, V40 pelvic bone < 600 cc and Mean Body dose < 15 Gy, acute grade 3 lymphopenia with RT will decrease from 50% to 25%.  In the study, (MPMMCC Study ID: 11000598) all patients completed planned course of radiotherapy without interruptions. 38 patients were included in final analysis. 4 patients were excluded from final analysis as they received short course steroids for obstructing voiding symptoms or antibiotics for suspected urinary tract infection during course of pelvic radiotherapy (n = 2 each).

The study dosimetric constraints i.e. atleast 2 of 3 parameters was met in all patients. Median Mean body dose was 14.3 Gy (Range: 12.79 Gy – 16.49 Gy). Median V10 pelvic bone was 81 % (Range: 63.4% - 95 %) and Median V40 was 241 cc (Range: 139 cc – 452 cc). Pelvic bone V40 < 600 cc was met in all patients (n = 38). Pelvic bone V10 < 80% and mean body dose < 15 Gy was met in 23 (60.5%) and 29 (76.3%) patients respectively. All 3 constraints were met in 21 (55.2%) patients.

The median baseline lymphocyte count was 1930 / mm³ (Range: 950 – 3440 / mm3). Grade 3 or higher lymphopenia (i.e. < 500 / mm3) occurred in 22 (57.9%) patients while grade 2 lymphopenia (< 800 / mm3) occurred in 37 (97.4%) patients. On chi square testing, baseline lymphocyte count being < median (1930/ mm3) was significantly associated with grade 3 or higher lymphopenia (p = 0.02). All constraints being met, dose rate (> 1000 MU vs 600 MU) and V10 Pelvic bone < 80% was not statistically associated with lymphopenia rates.

The results of the study showed that baseline lymphocyte count seems to be the most important predictor of lymphopenia with RT. Dosimetric constraints alone could not reliably prevent radiation induced lymphopenia.

The possible reasons for failure of hypothesis of LymphoTEC GU may be – first at any given time, 70% of lymphocytes reside in lymph nodes while only 5% reside in blood. Nodal irradiation is therefore expected to cause lymphopenia even if radiation dose to rest of blood pool and marrow are restricted. Second, lymphocyte homeostasis also depends on interleukins like levels of IL7, IL 15 and also TGF beta 1 in blood (mechanisms explained below) [20, 22]. At onset of lymphopenia due to other causes (not RT), compensatory increase in IL7 and IL15 has been shown in various studies which in turn leads to increased lymphocyte count. In patients receiving radiotherapy or chemo-radiotherapy (CTRT) developing lymphopenia, studies have resulted in conflicting results.  In a study by Ellsworth et al. [23]. patients with high-grade glioma treated with concurrent chemo-radiotherapy showed lymphocyte depletion, but no increase in IL-7 levels. Contrary to this, a study by Byun et al on hepatocellular carcinoma patients receiving radiotherapy, compensatory rise of IL7 at onset of severe lymphopenia was documented. [24]. Interestingly, murine studies have also shown that lympho-depletion due to radiotherapy alone is not associated with a compensatory increase in the homeostatic T cell cytokines Interleukin-7 and Interleukin-15. Murine studies have also suggested that use of IL15 super-agonist could possible alleviate RIL and survival [21]. The above human studies had the confounding of chemotherapy (Ellsworth et al) and absence of utilization of any dose constraints to mitigate lymphopenia. In the absence of clear data, it is imperative to perform a prospective study using LymphoTEC dose constraints and correlate with cytokine levels.

Studies have also shown that hypo-fractionated radiotherapy and SBRT have led to less incidence of RIL [25, 26]. However, as ultra-hypofractionation is not technically feasible due to risk of toxicity in subsites like head and neck, CNS and abdomen, it is necessary that evaluation of role of cytokines in RIL with conventional or moderate hypofractionation (Dose per fraction < 3 Gy) be made as these fractionations are expected to be used more commonly in combination with immunotherapy in future.

Due to the above factors, we attempt to perform a study of cytokine levels and correlation with lymphopenia while utilizing the proposed LymphoTEC guidelines. If cytokines IL7, IL15 and TGFBeta1 independently predict acute lymphopenia and there is absence of compensatory rise in cytokine levels after RT, it paves way for future drug trials with cytokine super agonist before or during RT, particularly when planned with immunotherapy. As dose constraints alone failed to mitigate lymphopenia in LymphoTEC GU study (MPMMCC Study ID: 11000598), it is possible that multifactorial intervention i.e. cytokine super-agonist along with dose constraints may help alleviate RIL.

IL 7 and lymphocyte homeostasis:

T-cell homeostasis in the peripheral lymphoid compartment is rigorously regulated through turnover, survival, and death. IL-7 promotes T-cell survival by upregulating the expression level of the Bcl-2 family of molecules, especially Mcl-1 (Anti-apoptotic) and Bcl-2, which can extensively inhibit the mitochondrial apoptotic pathway [27]. In addition to dependence on a dynamic balance of pro-apoptotic and anti-apoptotic signals, IL-7 also affects glucose metabolism which is also critical for T-cell survival. Research has also shown that expression of IL-7 and CCL19 significantly improves T-cell infiltration and survival of CAR-T cells in mouse tumors, enhancing the anti-tumor activity against solid tumors [28]. The data cited indicates that IL 7 levels appear critical to maintain T cell levels. However, if downstream pathways like BcL 2 molecules are impaired, levels of IL 7 may not correlate with lymphopenia. In other words, if downstream effector molecules/ pathways are deranged with radiotherapy, a compensatory rise in IL 7 may not correlate with increased lymphocyte levels. Such a finding should stimulate further lab research in that direction.

IL 15 and lymphocyte homeostasis:

IL-15 promotes lymphocyte cell survival during lymphopenia through JAK/STAT and PI3K/AKT pathway-mediated regulation of both anti- and pro-apoptotic factors of the Bcl-2 protein family. In addition to regulation by Bcl2 family proteins, IL-15 could regulate homeostatic cell survival by modulating several ER stress response proteins through an unknown mechanism, thus inhibiting survival defects induced by intense ER stress and PKR-like ER kinase (PERK) expression [29, 30]. The data cited indicates that IL 15 levels appear critical to maintain T cell levels. However, if downstream pathways like BcL 2 molecules or PKR-like ER kinase (PERK) expression are impaired, levels of IL 15 may not correlate with lymphopenia. In other words, if downstream effector molecules/ pathways are deranged with radiotherapy, a compensatory rise in IL 15 may not correlate with increased lymphocyte levels. Such a finding should stimulate further lab research in that direction.

TGF beta and lymphocyte homeostasis:

TGF-β1 primarily helps in maintaining immune homeostasis and prevention of autoimmunity. Naive T cells released from thymus must interact with self-MHC to survive in the periphery. Self-activation is normally inhibited because [Ca²⁺]_i levels are not sufficient for synergy with costimulatory pathways. TGF-β1-deficient T cells have spontaneously high [Ca²⁺]_i levels, which alters the threshold level for activation upon TCR/MHC interaction. Upon self-Ag recognition they become activated, cause an autoimmune-type inflammatory response [31, 32]. TGF Beta therefor normally prevents auto-immunity. But in cancer, recognition of Tumor associated antigens (TAAs) is essential. Elevated TGF beta levels may lead to T cells not getting activated even if no lymphopenia is present. This in turn may prevent recognition of Tumor associated antigens (TAAs) and therefore poorer cancer control. The present study will look to document TGF beta levels at start of RT and look to correlate with initial lymphocyte counts. To decipher complete role of TGF beta further lab studies must be planned in future