Galus 2023. Vitamin D supplementation increases objective response rate and prolongs progression‐free time in patients with advanced melanoma undergoing anti–PD‐1 therapy.

Title: Vitamin D supplementation increases objective response rate and prolongs progressionfree time in patients with advanced melanoma undergoing antiPD1 therapy URL Source: blob://pdf/25b3a74a-d721-47d2-8144-63ac9b697e98 Markdown Content: Received: 5 December 2022 # - Revised: 28 January 2023 # - Accepted: 31 January 2023 DOI: 10.1002/cncr.34718 O R I G I N A L A R T I C L E # Vitamin D supplementation increases objective response rate and prolongs progression free time in patients with advanced melanoma undergoing antiPD 1 therapy # ukasz Galus MD 1 | Micha Michalak PhD 2 | Mateusz Lorenz MD 3 | # Renata Stoiska Swiniarek BSc 4 | Daria Tusie Maecka MD 1 | # Agnieszka Galus MSc 4 | Tomasz Kolenda PhD 5 | Ewa Leporowska PhD 6 | # Jacek Mackiewicz MD, PhD 1,7 > 1 Department of Medical and Experimental Oncology, Institute of Oncology, Poznan University of Medical Sciences, Pozna, Poland > 2 Department of Computer Science and Statistics, Poznan University of Medical Sciences, Pozna, Poland > 3 Department of Diagnostic Imaging, Heliodor Swiecicki Clinical Hospital, Poznan University of Medical Sciences, Pozna, Poland > 4 Department of Medical and Experimental Oncology, Heliodor Swiecicki Clinical Hospital, Poznan University of Medical Sciences, Pozna, Poland > 5 Laboratory of Cancer Genetics, Greater Poland Cancer Centre, Pozna, Poland > 6 Department of Laboratory Diagnostics, Greater Poland Cancer Centre, Pozna, Poland > 7 Department of Diagnostics and Cancer Immunology, Greater Poland Cancer Centre, Pozna, Poland Correspondence ukasz Galus, Klinika Onkologii Klinicznej i Dowiadczalnej, ul. Grunwaldzka 16/18, 60 780 Pozna, Poland. Email: [email protected] Funding information Poznan University of Medical Sciences, Poland Abstract Background: Vitamin D3 is a prohormone with pleiotropic effects, including modulating the functions of the immune system and may affect the effectiveness of antiPD 1 treatment in patients with cancer. According to the literature, the po-tential mechanism of vitamin D's influence on the effectiveness of therapy is most likely related to the amount and activity of tumor infiltrating lymphocytes. There are data showing the effect of vitamin D on cells regulating the activity of CD8 lymphocytes. Methods: A total of 200 patients with advanced melanoma were included in the study. All patients received antiPD 1 immunotherapy (nivolumab or pem-brolizumab) as first line treatment. Serum vitamin D levels were measured in pa-tients both before and every 12 weeks during treatment. Part of the group had vitamin D measured retrospectively from the preserved serum. The other part of the supplementation group was tested prospectively. Results: The response rate in the group with low vitamin D levels and not supple-mented was 36.2%, whereas in the group with normal baseline levels or a normal level obtained with supplementation was 56.0% ( p = .01). Moreover, progression free survival in these groups was 5.75 and 11.25 months, respectively ( p = .03). In terms of overall survival, there was also a difference in favor of the group with normal vitamin D levels (27 vs. 31.5 months, respectively; p = .39). Conclusions: In our opinion, maintaining the vitamin D level within the normal range during anti PD 1 immunotherapy in advanced melanoma patients should be a standard procedure allowing the improvement of treatment outcomes. K E Y W O R D S anti PD1, immunotherapy, increase in response, melanoma, vitamin D Cancer. 2023;129:2047 2055. wileyonlinelibrary.com/journal/cncr 2023 American Cancer Society. # - 2047 INTRODUCTION Calcitriol, which is the active, hormonally active form of vitamin D, through its specific intracellular vitamin D receptor, acts as a tran-scription factor for many genes. In the body, it is formed from externally supplied prohormones (i.e., ergocalciferol [vitamin D2] and cholecalciferol [vitamin D3]). The latter is also produced in the skin under the influence of ultraviolet radiation. In addition to the well known effects on calcium metabolism, in recent years further new mechanisms of vitamin D's influence on functions of the body have been described, including the nervous system and regulating the immune system and the phenomenon of apoptosis. The multitude of seemingly unrelated functions of vitamin D are referred to as pleio-tropic effects. 1 Insufficient vitamin D supply is a common phenomenon world-wide. According to data published by Holick et al. in 2013, vitamin D deficiency affects 31% to 36% of the general population of the United States (in which milk and some juices are fortified with vitamin D) and Australia, from approximately 60% of the population of northern Africa, New Zealand, and parts of Asian regions, to more than 90% of the general population of northern Europe, Middle Eastern regions, and parts of Asia. The most important factors responsible for vitamin D deficiency include sun avoidance, dark skin color, obesity, older age, pregnancy, and air pollution. In addition, the lower limit of the norm for these data was a vitamin D level of 20 ng/mL (currently, 30 ng/mL is considered the lower limit of the norm). In Poland, deficiency in varying degrees of severity has been estimated at 90% of the population. 15 The present study compared the effectiveness of antiPD 1therapy in patients with locally advanced, inoperable, or metastatic melanoma in relation to serum vitamin D levels. METHODS Patients included in the study Patients with locally advanced, inoperable, or metastatic melanoma undergoing immunotherapy with antiPD 1 antibodies (pem-brolizumab or nivolumab) as a first line treatment at our center were included in the study. Patients with central nervous system metas-tases who did not undergo radical local treatment (neurosurgery/ stereotactic radiotherapy) before or during immunotherapy were excluded. Patients who started immunotherapy before July 2018 had their vitamin D levels determined retrospectively (serum was pre-served and frozen before the first dose of medication and after 9 12 weeks [i.e., before the first assessment of treatment effective-ness]). Patients accepted for immunotherapy from July 2018 had their vitamin D levels determined before the start of treatment (prospectively), after 9 to 12 weeks (i.e., before the first assessment of treatment effectiveness), and as needed at subsequent scheduled laboratory tests (every 3 months). All patients included in the treatment as of July 2018 were on vitamin D supplementation. Patients with baseline normal levels received a prophylactic dose of 2000 IU, and patients with reduced levels received 4000 to6000 IU until normalization was observed, and then continued supplementa-tion at the prophylactic dose. A result of 30 ng/dL was considered a reduced level. Patients in the two previously mentioned groups were further divided according to baseline vitamin D levels and after 9 to 12 weeks of immunotherapy. One subgroup consisted of patients who obtained vitamin D levels within normal limits at baseline or as a result of supplementation, whereas the other subgroup consisted of patients with reduced baseline levels and no supplementation, or patients who did not obtain normal levels despite supplementation. Vitamin D assessment Patients enrolled in the study before July 2018 had serum protection before and during treatment. Blood samples (5 mL) were taken in test tubes without anticoagulants (S Monovette, Sarsted AG&Co., Num-brecht, Germany). They were then incubated 20 minutes after the collection and centrifuged for 15 minutes at 3000 rpm and cooled. Next, the upper serum layer was transferred into a new, clean tube without disturbing the blood clot, and frozen in 20C. Samples were stored at 80C until use. The samples were gently thawed on ice and the occurrence of hemolysis was assessed by a change in serum color; samples showing hemoglobin staining were rejected. Next, samples were pipetted and 200 L of serum were taken to assess vitamin. The 25(OH) vitamin D levels were measured using Elecsys Vitamin D total III kit Cobas (Roche Diagnostics GmbH, Mannheim, Germany) according to man-ufacturers protocol using Cobas e 801 analyzer (Roche Diagnostics GmbH) with the measurement range 3.00 to 120 ng/mL. Vitamin D test results were obtained against the manufacturer's standard curve and recalibrated against the two standards included in the kit. The results for all samples were automatically calculated based on analyte concentration of each sample by the analyzer and presented in ng/mL and nmol/L. For samples with the value below the limit of detection were set as <3.00 ng/mL ( <7.50 nmol/L). Samples above the limit of detection were >120 ng/mL. The influence of hemoglobin in a con-centration 0.373 mmol/L or 600 mg/dL in the sample had no impact on observed results as was indicated in the protocol. Vitamin D was prospectively measured in patients enrolled in the study from July 2018. Measurements were performed in the hospital laboratory with standard analyses required before and during treatment. Statistical analyses Both subgroups were subjected to an analysis of the oncological treatment effectiveness according to generally accepted standards (i.e., by computed tomography or magnetic resonance imaging). RECIST 1.1 criteria were used to assess the type of response ach-ieved. Both subgroups, group 1 (reduced vitamin D levels) and group > 2048 # - EFFECT OF VITAMIN D ON MELANOMA IMMUNOTHERAPY 10970142, 2023, 13, Downloaded from https://acsjournals.onlinelibrary.wiley.com/doi/10.1002/cncr.34718 by Test, Wiley Online Library on [23/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 2 (normal vitamin D levels/patients with effective supplementation), were then subjected to comparative analysis of objective response rate (ORR) using a 2 test. KaplanMeier curves were used to assess progression free survival (PFS) and overall survival (OS), which were compared using the log rank test between group 1 and group 2. Finally, univariate and multivariate regression analysis was also conducted to determine the prognostic significance of factors char-acterizing patients, including serum vitamin D levels. All statistical analysis was made using StatSoft Polska software. All results with p < .05 were considered statistically significant. RESULTS The study included 200 patients with inoperable, locally advanced, or metastatic melanoma. The 62 patients who started treatment before July 2018 had their serum preserved before the first dose of medication and addi-tionally after 9 to 12 weeks. A control determination was performed only in patients with normal levels to confirm the maintenance of normal levels. Determination of vitamin D levels in this group was performed retrospectively. Results are shown in Table 1. As of July 2018, 138 patients were included in the analysis. Their vitamin D levels were determined during qualification for immuno-therapy. All patients in the group subsequently underwent vitamin D supplementation. In five patients undergoing supplementation, normalization of vitamin D levels could not be obtained at the first follow up test performed after 9 to 12 weeks. These patients were advised to increase their vitamin D intake, which thus obtained the desired value in the next determination. The results of vitamin D levels for this group are also shown in Table 1. Then the patients in both groups mentioned were divided into subgroups, which were compared. The first subgroup consisted of 58 patients with reduced vitamin D levels (without supplementation or with ineffective supplementation). The second subgroup consisted of 141 patients with normal vitamin D levels (without supplementation or as a result of effective supply). One patient, whose baseline vitamin D level was normal and who did not receive supplementation and developed hypovitaminosis by the time of a follow up test, was not included in either subgroup and was not considered in further analyses. Both subgroups described were well balanced in terms of disease severity, age, and lactate dehydrogenase (LDH) levels, and differed statistically significantly only in the proportion of women to men. Characteristics of the patients are provided in Table 2. Each of the patients in both groups was treated until progression (or unaccept-able toxicity). The number of cycles was similar, but direct compari-son is difficult because of the higher rate of primary progression in the retrospective group. The study showed a higher percentage of objective responses as a result of therapy in the group with normal vitamin D levels (either originally or as a result of supplementation): 56.0% (79/141) compared with the group with reduced levels at 36.2% (21/58). The difference between the two groups was 19.8% and statistically sig-nificant ( p = .0111). Detailed data on response rates are shown in Table 3 and Figure 1. In addition, differences in PFS were observed ( p = .0378). The median PFS in group 1 was 5.75 months (95% CI, 3.011.50), whereas that of group 2 was 11.25 months (95% CI, 6.2520.25). During the 12 month observation period, 34% of patients in the first group were progression free, whereas 48.4% were progression free in group 2. At 24 months, the percentages were 22.7% and 33.7%, respectively. The PFS curves are shown in Figure 2. In terms of OS time, a difference was not observed. The OS was 27 and 31.5 months, in groups 1 and 2, respectively ( p = .39). By the time of the present analysis, disease progression had been observed in 86% of patients in the first group and 60% in the second group. In the first group, 66% of patients received subsequent lines of treatment (range, 17), whereas 77% of patients received subsequent lines of treatment in the second subgroup (range, 14). There were also differences in the T A B L E 1 Vitamin D levels in patients starting immunotherapy before July 2018 (analyzed retrospectively and assessed in the serum during qualification for antiPD 1 treatment) and vitamin D levels assessed in the serum of patients during qualification for antiPD 1treatment as of July 2018. Patients with preserved serum and retrospectively assessed vitamin D levels: 62 (without supplementation) Normal ( >30 ng/mL) 9 (14.5%) Reduced level ( <31 ng/mL) 53 (85.5%) Normal in follow up test after 3 months 8 (12.9%) Reduced level in follow up test after 3 months 1 (1.6%) not reassessed Patients with assessed vitamin D levels and included supplementation during qualification for immunotherapy: 138 Normal ( >30 ng/mL) 35 (25.4%) Reduced level ( 30 ng/mL) 103 (74.6%) Normal in follow up test after 3 months 35/35 (100%) Normal in follow up test after 3 months 98 (95.1%) Reduced level in follow up test after 3 months 5 (4.9%) > GALUS ET AL . # - 2049 10970142, 2023, 13, Downloaded from https://acsjournals.onlinelibrary.wiley.com/doi/10.1002/cncr.34718 by Test, Wiley Online Library on [23/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License percentage of patients treated with ipilimumab, BRAF and MEK in-hibitors, chemotherapy, or included in clinical trials. In the analyses conducted, there was no statistically significant difference, between both groups, in the incidence of adverse effects, either in all stages or in stages 3 and 4. Detailed results are presented in Table 2. Univariate and multivariate analyses were also conducted to determine the prognostic significance of factors characterizing pa-tients, including serum vitamin D levels. There was a statistically significant higher risk of progression for patients in stages M1c or M1d, with elevated LDH and with reduced vitamin D levels. A sta-tistically significant higher risk of death was also found for patients in stages M1c and M1d and with elevated LDH, and additionally for patients in ECOG performance stage 1. Detailed results are shown in Table 4. DISCUSSION The development of immuno oncology and the incorporation of anti PD 1 antibodies (i.e., nivolumab and pembrolizumab) into general clinical practice have significantly improved the prognosis of patients with advanced melanoma. The importance of these antibodies has been demonstrated in a number of clinical trials. These include T A B L E 3 Response rates following antiPD 1 treatment in patients with advanced melanoma showing normal and reduced vitamin levels. ORR Clinical advantage CR PR SD PD Group 1: reduced vitamin D levels 36.2% (21/58) 53.4% (31/58) 10.3% (6/58) 25.9% (15/58) 17.2% (10/58) 46.6% (27/58) Group 2: normal vitamin D levels/patients with effective supplementation 56.0% (79/141) 65.9% (93/141) 7.8% (11/141) 48.2% (68/141) 9.9% (14/141) 34.0% (48/141) Abbreviations: CR, complete response; ORR, objective response rate; PD, progression of disease; PR, partial response; SD, stabilization of disease. T A B L E 2 Characteristics of advanced melanoma patients showing normal and reduced vitamin D levels during antiPD 1 treatment. Group 1: reduced vitamin D levels, n = 5Group 2: normal vitamin D levels/ patients with effective supplementation, n = 14 Difference Stage III (LDH ULN), M1a0 10 (17.2%) 33 (23.4%) 6.2% ( p = .3341) III (LDH > ULN), M1a1 10 (17.2%) 15 (10.6%) 6.6% ( p = .2011) M1b0 9 (15.5%) 22 (15.6%) 0.1% ( p = .9859) M1b1 3 (5.2%) 6 (4.3%) 0.9% ( p = .7822) M1c0 8 (13.8%) 28 (19.9%) 6.1% ( p = .3100) M1c1 14 (24.1%) 25 (17.7%) 6.4% ( p = .3010) M1d0 3 (5.2%) 6 (4.3%) 0.9% ( p = .7822) M1d1 1 (1.7%) 6 (4.3%) 2.6% ( p = .3672) Other characteristics LDH > 1.5x ULN 12 (21%) 17 (12%) 9% ( p = .1025) Sex Women 59% Women 41% 18% ( p = .0206) Men 41% Men 59% Median age, years 67.5 (31 87) 68 (26 90) 0.5% ( p = .5647) BRAF mutation BRAF negative 46 (79%) BRAF negative 96 (68%) 11% ( p = .1194) BRAF positive 12 (21%) BRAF positive 45 (32%) ECOG 0 41 (70.7%) 0 100 (70.9%) 0.2% ( p = .9775) 1 17 (29.3%) 1 41 (29.1%) AE 1 4 55% 45% 10% ( p = .1989) AE 3 4 9% 4% 5% ( p = .1576) Abbreviations: AE, adverse events; ECOG, Eastern Cooperative Oncology Group; LDH, lactate dehydrogenase; ULN, upper limit of normal. 2050 - EFFECT OF VITAMIN D ON MELANOMA IMMUNOTHERAPY 10970142, 2023, 13, Downloaded from https://acsjournals.onlinelibrary.wiley.com/doi/10.1002/cncr.34718 by Test, Wiley Online Library on [23/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License CheckMate 066, CheckMate 067, Keynote 001, or Keynote 006. In these studies, for the arms receiving antiPD 1 as part of first line treatment, ORR ranged from 41.9% to 46%, whereas median PFS ranged from 5.1 to 16.9 months. 68 In a large retrospective analysis including 499 patients with advanced melanoma treated with anti PD 1 immunotherapy at leading Polish centers, ORR was 35% and median PFS was 7.9 months. 9 In comparison, in the present study in the group with normal vitamin D levels, these parameters were 56% and 11.25 months, respectively. The analysis shows that normal vitamin D levels in patients with advanced melanoma are related to a higher percentage of objective responses and a longer median PFS in patients undergoing antiPD 1 therapy compared with patients with levels below laboratory normal. The lack of statistical significance in terms of OS difference cannot conclusively prove the effect of vitamin D on the parameter in question during antiPD 1 immuno-therapy. However, after progression during the applied treatment, patients received subsequent lines of treatment. It is important to note the extent of reduced vitamin D levels in the patients included in the study, which was nearly 84% and 75% in both study groups. This deficiency is most likely related to the high F I G U R E 1 Response rates following antiPD 1 treatment in patients with advanced melanoma showing normal and reduced vitamin levels. F I G U R E 2 KaplanMeier curves showing progression free survival in patients with advanced melanoma depending on immunotherapy in relation to vitamin D levels. GALUS ET AL . - 2051 10970142, 2023, 13, Downloaded from https://acsjournals.onlinelibrary.wiley.com/doi/10.1002/cncr.34718 by Test, Wiley Online Library on [23/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License latitude where study patients reside and a lifestyle characterized by spending a lot of time indoors and avoiding exposure to sunlight. It would be advisable to popularize vitamin D supplementation. The subgroups studied differed statistically significantly in the proportion of men and women. Although some studies support a potential association with sex difference in relation to antiPD 1activity, there is no consensus to state that sex has a clear association with response to immunotherapy. There are times when freezing has no effect on vitamin D level. 25(OH)D is stable under common storage conditions: 4 hours at room temperature, 24 hours at 2 to 8C, 7 days at 20C, and 3 months at 80C. 10 Despite the significant improvement in the prognosis of meta-static melanoma patients, the vast majority experience disease pro-gression at some stage. Primary disease progression, in patients undergoing immunotherapy, occurs with a frequency of 33% to 38%. 68 It is possible to improve the prognosis of patients receiving antiPD 1 by introducing a biomarker that allows personalization of treatment or combine it with other drugs. 1117 A potential determi-nant of the efficacy of antiPD 1 antibodies also appears to be the serum vitamin D levels of patients. Currently, there are many data in the literature confirming its anticancer effect and its influence on immune function. The role of vitamin D as a molecule with potential anticancer effects has been and continues to be the subject of many studies. To our knowledge, the first significant reports on the anticancer effects of vitamin D appeared in 1940, in which a study was presented comparing the incidence of cancer among those exposed to ultravi-olet radiation (farmers and soldiers) compared with those with lower exposure. The former group had a significant increase in skin cancers, T A B L E 4 Single factor and multifactor Cox proportional hazards analysis for PFS and OS. Parameter PFS OS Univariate analysis Multivariate analysis Univariate analysis Multivariate analysis HR 95% CI p HR 95% CI p HR 95% CI p HR 95% CI p Age, years 60 1.00 1.00 vs. >60 1.00 0.991.01 .97 1.00 0.991.02 .57 Stage III, M1a, M1b 1.00 1.00 1.00 vs. M1c, M1d 1.70 1.212.41 .02 1.65 1.16 2.33 .01 1.96 1.263.02 .00 1.81 1.16 2.81 .01 Presence of BRAF mutations none 1.00 1.00 vs. current 1.40 0.962.04 .08 1.04 0.651.67 .87 CNS metastasis none 1.00 1.00 vs. current 1.35 0.682.63 .39 1.03 0.502.14 .93 Sex W 1.00 1.00 vs. M 1.05 0.751.48 .77 1.26 0.811.95 .31 LDH ULN 1.00 1.00 1.00 1.00 vs. > ULN 1.82 1.292.56 .00 1.71 1.21 2.41 .00 2.37 1.543.63 .00 1.89 1.21 2.96 .00 ECOG 0 1.00 1.00 vs. 1 1.49 1.042.15 .03 2.60 1.69 4.00 .00 2.09 1.33 3.29 .00 Vitamin D reduced level 1.00 1.00 vs. normal 0.69 0.480.99 .04 0.65 0.49 0.94 .02 0.81 0.511.30 .39 Abbreviations: CNS, central nervous system; ECOG, Eastern Cooperative Oncology Group; HR, hazard ratio; LDH, lactate dehydrogenase; OS, overall survival; PFS, progression free survival; ULN, upper limit of normal. 2052 - EFFECT OF VITAMIN D ON MELANOMA IMMUNOTHERAPY 10970142, 2023, 13, Downloaded from https://acsjournals.onlinelibrary.wiley.com/doi/10.1002/cncr.34718 by Test, Wiley Online Library on [23/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License but at the same time a lower rate of other cancers. 18 Since then, the effect of vitamin D has been subjected to various in vitro tests. In 1981, Stucci and colleagues described the inhibitory effect of vitamin D on melanoma cell proliferation in vitro, and using it, they differ-entiated mouse acute leukemia cells into macrophages. 1921 Its anticancer effects were also reported by Bochen and colleagues, in which inhibiting the development of hamster buccal mucosal tumors previously induced by 7,12 dimethylbenzanthracene, and then sup-plementing the animals with vitamin D. 22,23 The potential anticancer effect of cholecalciferol has also been proven in clinical trials. In a randomized, placebo controlled, blinded study, daily vitamin D supplementation of 1100 IU/d along with calcium 1500 mg/d by postmenopausal women was proven to sta-tistically significantly reduce the risk of cancer. 19,24,25 There has also been a correlation between higher vitamin D levels and lower mor-tality from breast cancer and colorectal cancer, and a large meta analysis showed a protective effect of vitamin D in preventing the development of lung cancer. 19,26 The effects of vitamin D on the immune system are complex and involve multiple components. Vitamin D receptors are found on almost all cell types of this system: B lymphocytes, T lymphocytes, antigen presenting cells, neutrophils, macrophages, and dendritic cells. Its effects on individual cells are related to, among others, activation or inhibition of proliferation/function via the MAPK pathway. Most significantly, in the context of antitumor response, an increase in the amount of CD8 as well as the number of CD4 T cells in the tumor, and increased expression of their activation marker CD69 under the influence of vitamin D was shown. Independent of this, it has also been observed that during activation of T cell re-ceptors, expression of vitamin D receptors occurs. Regardless of the direct effect of vitamin D on CD8 lymphocytes, its effect on CD8 lymphocyte regulatory cells such as dendritic cells, CD4 lymphocytes, and macrophages has also been observed. 27 A suspensory effect of vitamin D on the secretion of interleukin (IL) 12, IL 23 and IL 6 has also been shown, with the former being associated with lymphocyte differentiation toward Th1, and the latter two being Th17 stimula-tors. Therefore, ultimately, vitamin D is associated with a reduction in Th1 and Th17 and, as a result, Th2 dominance, which is related to a reduction in inflammation generated by Th1 and Th17. Proin-flammatory cytokines such as IL 12 are necessary locally for activa-tion of CD8 T cells in cytotoxic T lymphocytes. However, generalized inflammation adversely affects the antitumor response. There are reports indicating levels of IL 6, for example, as a negatively pre-dictive biomarker during antiPD 1/PDL 1 therapy. The promotion of Th2 lymphocytes (at the expense of Th1 and Th 17) cannot be indicated as a factor that unequivocally develops a humoral response because it simultaneously inhibits the proliferation and differentia-tion of B lymphocytes, as well as their secretion of immunoglobu-lins. 19,28,29 Vitamin D also has an inhibitory effect on the production of downstream inflammatory cytokines such as IL 1, tumor necrosis factor , and IL 8. Vitamin D exerts an inhibitory effect on Th17 lymphocytes, which are mainly responsible for production of inflammatory cytokines and neutrophil migration. The expansion of lymphocytes in question is also associated with the frequency of adverse events (AEs) during therapy with antiPD 1 and antiCTLA 4 antibodies. Vitamin D may therefore have a reducing effect on the frequency of immune com-plications of such treatment. 19,3033 The literature has described a relationship between vitamin D supplementation and reduced risk of intestinal inflammation during immunotherapy with immune check-point inhibitors. 34 No statistically significant differences in the inci-dence of AEs were observed in the patients analyzed in this study. However, there was a trend indicating a lower incidence of AEs with normal vitamin D levels (both in the analysis for severity stages 14 and 34 according to Common Terminology Criteria for AEs). In a large study of 231 patients with head and neck carcinoma, Bochen and colleagues showed a correlation between normal vitamin D levels and longer patient survival. However, in the described study, higher vitamin D levels were simultaneously found in patients who were human papilloma viruspositive (HPV +) compared with pa-tients who were HPV, and the mere presence of HPV DNA is associated with a better prognosis. In the subgroup of patients with HPV + alone, there was no difference in survival between patients with reduced vs. normal vitamin D levels. 22 In the same study, the authors also showed significantly greater infiltration of the tumor and its lining by cells such as T lymphocytes (CD3 +), helper T lymphocytes (CD4 +), cytotoxic lymphocytes (CD8 +), natural killer (NK) cells (CD56 +), and macrophages and M1 macrophages among patients with higher vitamin D levels, with less infiltration of the lining by M2 macrophages (CD163 +). Increased tumor infiltration by CD8 + T cells and NK cells and infiltration of the tumor lining by CD3 + and CD4 + lymphocytes appeared to be pre-dictive of longer survival, whereas conversely, greater infiltration of M2 macrophages (CD163 +) was related to worse prognosis. In the same study, a positive correlation between vitamin D levels and NK cell activity was shown based on in vitro analyses of NK cell activity collected from patients. 22 Based on studies of tumor tissues from 35 patients with squa-mous cell carcinoma of the head and neck undergoing vitamin D supplementation and 18 nonsupplemented control patients, vitamin D was shown to increase the levels of proinflammatory cytokines such as IL 6, IL 10, and interferon , importantly, locally, inside the tumor. An analogous study conducted in 11 and 12 patients with squamous cell carcinoma of the head and neck, respectively, demonstrated that vitamin D supplementation was related to reduction in the number of immunosuppressive CD34 + cells and immature dendritic cells in tumor tissue. 22,35,36 Vitamin D has also been shown to stimulate PDL 1 expression on immune cells in inflammatory bowel disease and human epithelial cells. Taking into account the correlation of the intensity of PDL 1expression with the response to therapy with immune checkpoint inhibitors, it can be presumed that vitamin D is positively correlated with the response to treatment with antiPD 1/antiPDL 1 anti-bodies. 19,3739 Furthermore, according to the analysis of Custao et al., vitamin D levels determined at specific time points in patients with nonsmall > GALUS ET AL . # - 2053 10970142, 2023, 13, Downloaded from https://acsjournals.onlinelibrary.wiley.com/doi/10.1002/cncr.34718 by Test, Wiley Online Library on [23/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License cell lung cancer treated with nivolumab correlate with the levels of administered antibodies. This is due to its effect on the expression of genes related to metabolism or elimination of various medications. Importantly, according to previous reports, the levels of administered antibodies are statistically higher in patients who have achieved a response to treatment compared with those who are progressing. 40,41 Moreover, in addition to its effects on the immune system, vitamin D is known to have anticancer effects through anti-proliferative activity, induction of apoptosis, inhibition of invasion, and metastasis formation, as well as antiangiogenic effects. 42 CONCLUSIONS The analysis showed a statistically significant, positive effect of normal serum vitamin D levels on increasing ORR and PFS as a result of antiPD 1 immunotherapy in patients with locally advanced inoperable or metastatic melanoma. An undeniable disadvantage of this study is the comparative analysis of a population evaluated retrospectively with a population in which vitamin D supplementa-tion was prospectively intervened. However, the nature of inter-vention and the medical wide indications for vitamin D supply, in case of vitamin D deficiency, make it impossible for ethical reasons to construct a study comparing patients fully prospectively, with pla-cebo control and randomization. Both American and European endocrine guidelines, including those in Poland, recommend vitamin D supplementation in case of deficiency. 43,44 The authors opinion is that assessment of vitamin D levels and appropriate vitamin D sup-plementation should be considered in every patient qualified for treatment with antiPD 1 immunotherapy. AUTHOR CONTRIBUTIONS ukasz Galus: Conceptualization, methodology, investigation, data curation, writing original draft, visualization, and project adminis-tration. Micha Michalak: Formal analysis and software. Mateusz Lorenz: Investigation. Renata Stoiska Swiniarek: Investigation. Daria Tusie Maecka: Investigation. Agnieszka Galus: Investigation. Tomasz Kolenda: Investigation. Ewa Leporowska: Investigation. Jacek Mackiewicz: Writing review and editing, funding acquisition, re-sources, and supervision. ACKNOWLEDGMENTS Poznan University of Medical Sciences, Poland. CONFLICT OF INTEREST STATEMENT ukasz Galus has received remuneration from BMS, Roche, MSD, Novartis, and Pierre Fabre. Daria Tusie Maecka has received remuneration from Merck, BMS, Novartis, and Pierre Fabre. Jacek Mackiewicz reports remuneration from BMS, GlaxoSmithKline, Roche, MSD, Novartis, and Pierre Fabre. The other authors made no disclosures. ORCID ukasz Galus https://orcid.org/0000-0002-9515-1834 REFERENCES 1. Rusiska A, Pudowski P, Walczak M, et al. Zasady suplementacji i leczenia witamin D nowelizacja 2018 r. Postpy Neonatologii .2018;24(1). 2. Sewerynek E, Cielak K, Janik M, Gowin E, Stuss M. Evaluation of vitamin D concentration in a population of young, healthy women the effects of vitamin D supplementation. Endokrynol Pol . 2017; 68(5):533 540. 3. upiska A, Chlebna Sok D. Czynniki wpywajce na stenie witaminy D w surowicy dzieci dzkich w wieku wczesnoszkolnym z nadmiarem masy ciaa. Post N Med . 2016;29(10):709 715. 4. Chlebna Sok D, Michaus I, Rusiska A, et al. Ocena stenia witaminy D w surowicy u dzieci hospitalizowanych z powodu obja-ww klinicznych sugerujcych zaburzenia w ukadzie kostnym. Endokrynol Pediatr . 2016;15(4):23 32. doi:10.18544/ep 01.15.04. 1653 5. Pudowski P, Ducki C, Konstantynowicz J, Jaworski M. Vitamin D status in Poland. Pol Arch Med Wewn . 2016;126(7 8):530 539. 6. Ascierto PA, Long GV, Robert C, et al. Survival outcomes in patients with previously untreated BRAF wild type advanced melanoma treated with nivolumab therapy: three year follow up of a ran-domized phase 3 trial. JAMA Oncol . 2019;5(2):187 194. doi:10.1001/ jamaoncol.2018.4514 7. Hamid O, Robert C, Daud A, et al. Five year survival outcomes for patients with advanced melanoma treated with pembrolizumab in KEYNOTE 001. Ann Oncol . 2019;30(4):582 588. doi:10.1093/ annonc/mdz011 8. Robert C, Ribas A, Schachter J, et al. Pembrolizumab versus ipili-mumab in advanced melanoma (KEYNOTE 006): post hoc 5 year results from an open label, multicentre, randomised, controlled, phase 3 study. Lancet Oncol . 2019;20(9):1239 1251. doi:10.1016/ s1470 2045(19)30388 29. Cybulska Stopa B, Pacholczak Madej R, Kamiska Winciorek G, et al. First line treatment of advanced/metastatic melanoma with anti PD 1 antibodies: multicenter experience in Poland. Immuno-therapy . 2021;13(4):297 307. doi:10.2217/imt 2020 0217 10. Colak A, Toprak B, Dogan N, Ustuner F. Effect of sample type, centrifugation and storage conditions on vitamin D concentration. Biochem Med . 2013;23:321 325. doi:10.11613/bm.2013.039, 325. 11. Sha D, Jin Z, Budzcies J, Kluck K, Stenzinger A, Sinicrope FA. Tumor mutational burden (TMB) as a predictive biomarker in solid tumors. Cancer Discov . 2020;10(12):1808 1825. doi:10.1158/2159 8290.cd 20 0522 12. Eisenstein A, Gonzalez EC, Raghunathan R, et al. Emerging bio-markers in cutaneous melanoma. Mol Diagn Ther , 2018;22(2): 203 218, doi:10.1007/s40291 018 0318 z13. Bai R, Lv Z, Xu D, Cui J. Predictive biomarkers for cancer immuno-therapy with immune checkpoint inhibitors. Biomark Res . 2020;8(1): 34. doi:10.1186/s40364 020 00209 014. Tumeh PC, Harview CL, Yearley JH, et al. PD 1 blockade induces responses by inhibiting adaptive immune resistance. Nature .2014;515(7528):568 571, doi:10.1038/nature13954 15. Wolchok JD, Chiarion Sileni V, Rene Gonzalez R, et al. Overall sur-vival with combined nivolumab and ipilimumab in advanced mela-noma. N Engl J Med . 2017;377(14):1345 1356, doi:10.1056/ nejmoa1709684 16. Liu D, Schilling B, Liu D, et al. Integrative molecular and clinical modeling of clinical outcomes to PD1 blockade in patients with metastatic melanoma. Nat Med . 2019;25(12):1916 1927. doi:10. 1038/s41591 019 0654 5 2054 - EFFECT OF VITAMIN D ON MELANOMA IMMUNOTHERAPY 10970142, 2023, 13, Downloaded from https://acsjournals.onlinelibrary.wiley.com/doi/10.1002/cncr.34718 by Test, Wiley Online Library on [23/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 17. Johnson DB, Frampton GM, Rioth MJ, et al. Targeted next genera-tion sequencing identifies markers of response to PD 1 blockade. Cancer Immunol Res . 2016;4(11):959 967. doi:10.1158/2326 6066. cir 16 0143 18. Apperly FL. The relation of solar radiation to cancer mortality in North America. Cancer Res . 1941;1:191 195. 19. Stucci LS, D'Oronzo S, Tucci M, et al. Vitamin D in melanoma: con-troversies and potential role in combination with immune check point inhibitors. Cancer Treat Rev . 2018;69:21 28. doi:10.1016/j. ctrv.2018.05.016 20. Colston K, Colston MJ, Feldman D. 1,25 dihydroxyvitamin D3 and malignant melanoma: the presence of receptors and inhibition of cell growth in culture. Endocrinology . 1981;108(3):1083 1086. doi:10. 1210/endo 108 31083 21. Abe E, Miyaura C, Sakagami H, et al. Differentiation of mouse myeloid leukemia cells induced by 1 alpha, 25 dihydroxyvitamin D3. Proc Natl Acad Sci USA . 1981;78(8):4990 4994. doi:10.1073/pnas.78.8.4990 22. Bochen F, Balensiefer B, Krner S, et al. Vitamin D deficiency in head and neck cancer patients prevalence, prognostic value and impact on immune function. OncoImmunology . 2018;7(9):e1476817. doi:10. 1080/2162402x.2018.1476817 23. Meier JD, Enepekides DJ, Poirier B, Bradley CA, Albala JS, Farwell DG. Treatment with 1 alpha,25 dihydroxyvitamin D3 (vitamin D3) to inhibit carcinogenesis in the hamster buccal pouch model. Arch Otolaryngol Head Neck Surg . 2007;133(11):1149 1152. doi:10.1001/ archotol.133.11.1149 24. Lappe JM, Travers Gustafson MD, Davies KM, Recker RR, Heany RP. Vitamin D and calcium supplementation reduces cancer risk; result of randomisation trial. Am J Clin Nutr . 2008;87(3):794. 25. Chlebowski RT, Johnson KC, Kooperberg C, et al. Calcium plus vitamin D supplementation and the risk of breast cancer. J Natl Cancer Inst . 2008;100(22):1581 1591. doi:10.1093/jnci/djn360 26. Zhang L, Wang S, Che X, Li X. Vitamin D and lung cancer risk: a comprehensive review and meta analysis. Cell Physiol Biochem .2015;36(1):299 305. doi:10.1159/000374072 27. Sarkar S, Hewison M, Studzinski GP, Li YCH, Kalia V. Role of vitamin D in cytotoxic T lymphocyte immunity to pathogens and cancer. Crit Rev Clin Lab Sci . 2016;53(2):132 145. doi:10.3109/10408363.2015. 1094443 28. Daniel C, Sartory NA, Zahn N, Radeke HH, Stein JM. Immune modulatory treatment of trinitrobenzene sulfonic acid colitis with calcitriol is associated with a change of a T helper (Th) 1/Th17 to a Th2 and regulatory T cell profile. J Pharmacol Exp Therapeut .2008;324(1):23 33. doi:10.1124/jpet.107.127209 29. Bui L, Zhu Z, Hawkins S, Cortez Resendiz A, Bellon A. Vitamin D regulation of the immune system and its implications for COVID 19: a mini review. SAGE Open Med . 2021;9:205031212110140. doi:10. 1177/20503121211014073 30. Sotirchos ES, Bhargava P, Eckstein C, et al. Safety and immuno-logic effects of high vs low dose cholecalciferol in multiple scle-rosis. Neurology . 2016;86(4):382 390. doi:10.1212/wnl.000000000 0002316 31. Joshi S, Pantalena LC, Liu XK, et al. 1,25dihydroxyvitamin D(3) ameliorates Th17 autoimmunity via transcriptional modulation of interleukin 17A. Mol Cell Biol . 2011;31(17):3653 3669. doi:10.1128/ mcb.05020 11 32. Tarhini AA, Zahoor H, Lin Y, et al. Baseline circulating IL 17 predicts toxicity while TGF 1 and IL 10 are prognostic of relapse in ipili-mumab neoadjuvant therapy of melanoma. J Immunother Cancer .2015;3(1):39. doi:10.1186/s40425 015 0081 133. Stucci S, Palmirotta R, Passarelli A, et al. Immune related adverse events during anticancer immunotherapy: pathogenesis and man-agement. Oncol Lett . 2017;14(5):5671 5680. 34. Grover S, Dougan M, Tyan K, et al. Vitamin D intake is associated with decreased risk of immune checkpoint inhibitor induced colitis. Cancer . 2020;126(16):3758 3767. doi:10.1002/cncr.32966 35. Walker DD, Reeves TD, de Costa AM, Schuyler C, Young MR. Immunological modulation by 1 ,25 dihydroxyvitamin D3 in pa-tients with squamous cell carcinoma of the head and neck. Cytokine .2012;58(3):448 454. doi:10.1016/j.cyto.2012.03.002 36. Kulbersh JS, Day TA, Gillespie MB, Young MR. 1 alpha,25 Dihy-droxyvitamin D(3) to skew intratumoral levels of immune inhibitory CD34( +) progenitor cells into dendritic cells. Otolaryngol Head Neck Surg . 2009;140(2):235 240. doi:10.1016/j.otohns.2008.11.011 37. Larkin J, Chiarion Sileni V, Gonzalez R, et al. Combined nivolumab and ipilimumab or monotherapy in untreated melanoma. N Engl J Med . 2015;373(1):23 34. doi:10.1056/nejmoa1504030 38. Dimitrov V, Bouttier M, Boukhaled G, et al. Hormonal vitamin D up regulates tissue specific PD L1 and PD L2 surface glycoprotein expression in humans but not mice. J Biol Chem . 2017;292(50): 20657 20668. doi:10.1074/jbc.m117.793885 39. Bendix M, Greisen S, Dige A, et al. Vitamin D increases programmed death receptor 1 expression in Crohn's disease. Oncotarget .2017;8(15):24177 24186. doi:10.18632/oncotarget.15489 40. Cusato J, Genova C, Tomasello C, et al. Influence of vitamin D in advanced non small cell lung cancer patients treated with nivolu-mab. Cancers . 2019;11:125. doi:10.3390/cancers11010125 41. Koolen SLW, Basak EA, Hurkmans et al. Correlation between nivo-lumab exposure and treatment outcome in NSCLC. In Proceedings of the 2018 ASCO Annual Meeting, Chicago, IL, USA , 15 June 2018. 42. Broyna A, Hoffman R, Slominski A. Relevance of vitamin D in melanoma development, progression and therapy. Anticancer Res .2020;40(1):473 489. doi:10.21873/anticanres.13976 43. Holick MF, Binkley NC, Gordon CM, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab . 2011;96(7):1911 1930. doi:10.1210/jc.2011 0385 44. Pudowski P, Karczmarewicz E, Bayer M, et al. Practical guidelines for the supplementation of vitamin D and the treatment of deficits in Central Europe recommended vitamin D intakes in the general population and groups at risk of vitamin D deficiency. Endokrynol Pol .2013;64(4):319 327. doi:10.5603/ep.2013.0012 How to cite this article: Galus , Michalak M, Lorenz M, et al. Vitamin D supplementation increases objective response rate and prolongs progression free time in patients with advanced melanoma undergoing antiPD 1 therapy. Cancer .2023;129(13):2047 2055. doi:10.1002/cncr.34718 GALUS ET AL . - 2055 10970142, 2023, 13, Downloaded from https://acsjournals.onlinelibrary.wiley.com/doi/10.1002/cncr.34718 by Test, Wiley Online Library on [23/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Title: Vitamin D supplementation increases objective response rate and prolongs progressionfree time in patients with advanced melanoma undergoing antiPD1 therapy

URL Source: blob://pdf/25b3a74a-d721-47d2-8144-63ac9b697e98

Markdown Content:
Received: 5 December 2022

# - Revised: 28 January 2023

# - Accepted: 31 January 2023 DOI: 10.1002/cncr.34718

O R I G I N A L A R T I C L E

# Vitamin D supplementation increases objective response rate and prolongs progression free time in patients with advanced melanoma undergoing antiPD 1 therapy

# ukasz Galus MD 1 | Micha Michalak PhD 2 | Mateusz Lorenz MD 3 |

# Renata Stoiska Swiniarek BSc 4 | Daria Tusie Maecka MD 1 |

# Agnieszka Galus MSc 4 | Tomasz Kolenda PhD 5 | Ewa Leporowska PhD 6 |

# Jacek Mackiewicz MD, PhD 1,7

> 1

Department of Medical and Experimental Oncology, Institute of Oncology, Poznan University of Medical Sciences, Pozna, Poland

> 2

Department of Computer Science and Statistics, Poznan University of Medical Sciences, Pozna, Poland

> 3

Department of Diagnostic Imaging, Heliodor Swiecicki Clinical Hospital, Poznan University of Medical Sciences, Pozna, Poland

> 4

Department of Medical and Experimental Oncology, Heliodor Swiecicki Clinical Hospital, Poznan University of Medical Sciences, Pozna, Poland

> 5

Laboratory of Cancer Genetics, Greater Poland Cancer Centre, Pozna, Poland

> 6

Department of Laboratory Diagnostics, Greater Poland Cancer Centre, Pozna, Poland

> 7

Department of Diagnostics and Cancer Immunology, Greater Poland Cancer Centre, Pozna, Poland

Correspondence

ukasz Galus, Klinika Onkologii Klinicznej i Dowiadczalnej, ul. Grunwaldzka 16/18, 60

780 Pozna, Poland. Email: lukasz_galus@wp.pl

Funding information

Poznan University of Medical Sciences, Poland

Abstract

Background: Vitamin D3 is a prohormone with pleiotropic effects, including modulating the functions of the immune system and may affect the effectiveness of antiPD 1 treatment in patients with cancer. According to the literature, the po-tential mechanism of vitamin D's influence on the effectiveness of therapy is most likely related to the amount and activity of tumor infiltrating lymphocytes. There are data showing the effect of vitamin D on cells regulating the activity of CD8 lymphocytes.

Methods: A total of 200 patients with advanced melanoma were included in the study. All patients received antiPD 1 immunotherapy (nivolumab or pem-brolizumab) as first line treatment. Serum vitamin D levels were measured in pa-tients both before and every 12 weeks during treatment. Part of the group had vitamin D measured retrospectively from the preserved serum. The other part of the supplementation group was tested prospectively.

Results: The response rate in the group with low vitamin D levels and not supple-mented was 36.2%, whereas in the group with normal baseline levels or a normal level obtained with supplementation was 56.0% ( p = .01). Moreover, progression

free survival in these groups was 5.75 and 11.25 months, respectively ( p = .03). In terms of overall survival, there was also a difference in favor of the group with normal vitamin D levels (27 vs. 31.5 months, respectively; p = .39).

Conclusions: In our opinion, maintaining the vitamin D level within the normal range during anti PD 1 immunotherapy in advanced melanoma patients should be a standard procedure allowing the improvement of treatment outcomes.

K E Y W O R D S

anti PD1, immunotherapy, increase in response, melanoma, vitamin D

Cancer. 2023;129:2047 2055. wileyonlinelibrary.com/journal/cncr  2023 American Cancer Society.

# - 2047 INTRODUCTION

Calcitriol, which is the active, hormonally active form of vitamin D, through its specific intracellular vitamin D receptor, acts as a tran-scription factor for many genes. In the body, it is formed from externally supplied prohormones (i.e., ergocalciferol [vitamin D2] and cholecalciferol [vitamin D3]). The latter is also produced in the skin under the influence of ultraviolet radiation. In addition to the well

known effects on calcium metabolism, in recent years further new mechanisms of vitamin D's influence on functions of the body have been described, including the nervous system and regulating the immune system and the phenomenon of apoptosis. The multitude of seemingly unrelated functions of vitamin D are referred to as pleio-tropic effects. 1

Insufficient vitamin D supply is a common phenomenon world-wide. According to data published by Holick et al. in 2013, vitamin D deficiency affects 31% to 36% of the general population of the United States (in which milk and some juices are fortified with vitamin D) and Australia, from approximately 60% of the population of northern Africa, New Zealand, and parts of Asian regions, to more than 90% of the general population of northern Europe, Middle Eastern regions, and parts of Asia. The most important factors responsible for vitamin D deficiency include sun avoidance, dark skin color, obesity, older age, pregnancy, and air pollution. In addition, the lower limit of the norm for these data was a vitamin D level of 20 ng/mL (currently, 30 ng/mL is considered the lower limit of the norm). In Poland, deficiency in varying degrees of severity has been estimated at 90% of the population. 15

The present study compared the effectiveness of antiPD 1therapy in patients with locally advanced, inoperable, or metastatic melanoma in relation to serum vitamin D levels.

METHODS Patients included in the study

Patients with locally advanced, inoperable, or metastatic melanoma undergoing immunotherapy with antiPD 1 antibodies (pem-brolizumab or nivolumab) as a first line treatment at our center were included in the study. Patients with central nervous system metas-tases who did not undergo radical local treatment (neurosurgery/ stereotactic radiotherapy) before or during immunotherapy were excluded. Patients who started immunotherapy before July 2018 had their vitamin D levels determined retrospectively (serum was pre-served and frozen before the first dose of medication and after 9 12 weeks [i.e., before the first assessment of treatment effective-ness]). Patients accepted for immunotherapy from July 2018 had their vitamin D levels determined before the start of treatment (prospectively), after 9 to 12 weeks (i.e., before the first assessment of treatment effectiveness), and as needed at subsequent scheduled laboratory tests (every 3 months). All patients included in the treatment as of July 2018 were on vitamin D supplementation. Patients with baseline normal levels received a prophylactic dose of 2000 IU, and patients with reduced levels received 4000 to6000 IU until normalization was observed, and then continued supplementa-tion at the prophylactic dose. A result of 30 ng/dL was considered a reduced level. Patients in the two previously mentioned groups were further divided according to baseline vitamin D levels and after 9 to 12 weeks of immunotherapy. One subgroup consisted of patients who obtained vitamin D levels within normal limits at baseline or as a result of supplementation, whereas the other subgroup consisted of patients with reduced baseline levels and no supplementation, or patients who did not obtain normal levels despite supplementation.

Vitamin D assessment

Patients enrolled in the study before July 2018 had serum protection before and during treatment. Blood samples (5 mL) were taken in test tubes without anticoagulants (S Monovette, Sarsted AG&Co., Num-brecht, Germany). They were then incubated 20 minutes after the collection and centrifuged for 15 minutes at 3000 rpm and cooled. Next, the upper serum layer was transferred into a new, clean tube without disturbing the blood clot, and frozen in 20C. Samples were stored at 80C until use. The samples were gently thawed on ice and the occurrence of hemolysis was assessed by a change in serum color; samples showing hemoglobin staining were rejected. Next, samples were pipetted and 200 L of serum were taken to assess vitamin. The 25(OH) vitamin D levels were measured using Elecsys Vitamin D total III kit Cobas (Roche Diagnostics GmbH, Mannheim, Germany) according to man-ufacturers protocol using Cobas e 801 analyzer (Roche Diagnostics GmbH) with the measurement range 3.00 to 120 ng/mL. Vitamin D test results were obtained against the manufacturer's standard curve and recalibrated against the two standards included in the kit. The results for all samples were automatically calculated based on analyte concentration of each sample by the analyzer and presented in ng/mL and nmol/L. For samples with the value below the limit of detection were set as <3.00 ng/mL ( <7.50 nmol/L). Samples above the limit of detection were >120 ng/mL. The influence of hemoglobin in a con-centration 0.373 mmol/L or 600 mg/dL in the sample had no impact on observed results as was indicated in the protocol. Vitamin D was prospectively measured in patients enrolled in the study from July 2018. Measurements were performed in the hospital laboratory with standard analyses required before and during treatment.

Statistical analyses

Both subgroups were subjected to an analysis of the oncological treatment effectiveness according to generally accepted standards (i.e., by computed tomography or magnetic resonance imaging). RECIST 1.1 criteria were used to assess the type of response ach-ieved. Both subgroups, group 1 (reduced vitamin D levels) and group

> 2048

# - EFFECT OF VITAMIN D ON MELANOMA IMMUNOTHERAPY 10970142, 2023, 13, Downloaded from https://acsjournals.onlinelibrary.wiley.com/doi/10.1002/cncr.34718 by Test, Wiley Online Library on [23/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 2 (normal vitamin D levels/patients with effective supplementation), were then subjected to comparative analysis of objective response rate (ORR) using a 2 test. KaplanMeier curves were used to assess progression free survival (PFS) and overall survival (OS), which were compared using the log rank test between group 1 and group 2. Finally, univariate and multivariate regression analysis was also conducted to determine the prognostic significance of factors char-acterizing patients, including serum vitamin D levels. All statistical analysis was made using StatSoft Polska software. All results with

p < .05 were considered statistically significant.

RESULTS

The study included 200 patients with inoperable, locally advanced, or metastatic melanoma. The 62 patients who started treatment before July 2018 had their serum preserved before the first dose of medication and addi-tionally after 9 to 12 weeks. A control determination was performed only in patients with normal levels to confirm the maintenance of normal levels. Determination of vitamin D levels in this group was performed retrospectively. Results are shown in Table 1. As of July 2018, 138 patients were included in the analysis. Their vitamin D levels were determined during qualification for immuno-therapy. All patients in the group subsequently underwent vitamin D supplementation. In five patients undergoing supplementation, normalization of vitamin D levels could not be obtained at the first follow up test performed after 9 to 12 weeks. These patients were advised to increase their vitamin D intake, which thus obtained the desired value in the next determination. The results of vitamin D levels for this group are also shown in Table 1. Then the patients in both groups mentioned were divided into subgroups, which were compared. The first subgroup consisted of 58 patients with reduced vitamin D levels (without supplementation or with ineffective supplementation). The second subgroup consisted of 141 patients with normal vitamin D levels (without supplementation or as a result of effective supply). One patient, whose baseline vitamin D level was normal and who did not receive supplementation and developed hypovitaminosis by the time of a follow up test, was not included in either subgroup and was not considered in further analyses. Both subgroups described were well balanced in terms of disease severity, age, and lactate dehydrogenase (LDH) levels, and differed statistically significantly only in the proportion of women to men. Characteristics of the patients are provided in Table 2. Each of the patients in both groups was treated until progression (or unaccept-able toxicity). The number of cycles was similar, but direct compari-son is difficult because of the higher rate of primary progression in the retrospective group. The study showed a higher percentage of objective responses as a result of therapy in the group with normal vitamin D levels (either originally or as a result of supplementation): 56.0% (79/141) compared with the group with reduced levels at 36.2% (21/58). The difference between the two groups was 19.8% and statistically sig-nificant ( p = .0111). Detailed data on response rates are shown in Table 3 and Figure 1. In addition, differences in PFS were observed ( p = .0378). The median PFS in group 1 was 5.75 months (95% CI, 3.011.50), whereas that of group 2 was 11.25 months (95% CI, 6.2520.25). During the 12 month observation period, 34% of patients in the first group were progression free, whereas 48.4% were progression free in group 2. At 24 months, the percentages were 22.7% and 33.7%, respectively. The PFS curves are shown in Figure 2. In terms of OS time, a difference was not observed. The OS was 27 and 31.5 months, in groups 1 and 2, respectively ( p = .39). By the time of the present analysis, disease progression had been observed in 86% of patients in the first group and 60% in the second group. In the first group, 66% of patients received subsequent lines of treatment (range, 17), whereas 77% of patients received subsequent lines of treatment in the second subgroup (range, 14). There were also differences in the

T A B L E 1 Vitamin D levels in patients starting immunotherapy before July 2018 (analyzed retrospectively and assessed in the serum during qualification for antiPD 1 treatment) and vitamin D levels assessed in the serum of patients during qualification for antiPD 1treatment as of July 2018.

Patients with preserved serum and retrospectively assessed vitamin D levels: 62 (without supplementation) Normal ( >30 ng/mL) 9 (14.5%) Reduced level ( <31 ng/mL) 53 (85.5%) Normal in follow up test after 3 months 8 (12.9%) Reduced level in follow up test after 3 months 1 (1.6%) not reassessed Patients with assessed vitamin D levels and included supplementation during qualification for immunotherapy: 138 Normal ( >30 ng/mL) 35 (25.4%) Reduced level ( 30 ng/mL) 103 (74.6%) Normal in follow up test after 3 months 35/35 (100%) Normal in follow up test after 3 months 98 (95.1%) Reduced level in follow up test after 3 months 5 (4.9%)

> GALUS ET AL .

# - 2049 10970142, 2023, 13, Downloaded from https://acsjournals.onlinelibrary.wiley.com/doi/10.1002/cncr.34718 by Test, Wiley Online Library on [23/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License percentage of patients treated with ipilimumab, BRAF and MEK in-hibitors, chemotherapy, or included in clinical trials. In the analyses conducted, there was no statistically significant difference, between both groups, in the incidence of adverse effects, either in all stages or in stages 3 and 4. Detailed results are presented in Table 2. Univariate and multivariate analyses were also conducted to determine the prognostic significance of factors characterizing pa-tients, including serum vitamin D levels. There was a statistically significant higher risk of progression for patients in stages M1c or M1d, with elevated LDH and with reduced vitamin D levels. A sta-tistically significant higher risk of death was also found for patients in stages M1c and M1d and with elevated LDH, and additionally for patients in ECOG performance stage 1. Detailed results are shown in Table 4.

DISCUSSION

The development of immuno oncology and the incorporation of anti PD 1 antibodies (i.e., nivolumab and pembrolizumab) into general clinical practice have significantly improved the prognosis of patients with advanced melanoma. The importance of these antibodies has been demonstrated in a number of clinical trials. These include

T A B L E 3 Response rates following antiPD 1 treatment in patients with advanced melanoma showing normal and reduced vitamin levels.

ORR Clinical advantage CR PR SD PD

Group 1: reduced vitamin D levels 36.2% (21/58) 53.4% (31/58) 10.3% (6/58) 25.9% (15/58) 17.2% (10/58) 46.6% (27/58) Group 2: normal vitamin D levels/patients with effective supplementation 56.0% (79/141) 65.9% (93/141) 7.8% (11/141) 48.2% (68/141) 9.9% (14/141) 34.0% (48/141) Abbreviations: CR, complete response; ORR, objective response rate; PD, progression of disease; PR, partial response; SD, stabilization of disease.

T A B L E 2 Characteristics of advanced melanoma patients showing normal and reduced vitamin D levels during antiPD 1 treatment.

Group 1: reduced vitamin D levels,

n = 5Group 2: normal vitamin D levels/ patients with effective supplementation, n = 14 Difference

Stage III (LDH  ULN), M1a0 10 (17.2%) 33 (23.4%) 6.2% ( p = .3341) III (LDH > ULN), M1a1 10 (17.2%) 15 (10.6%) 6.6% ( p = .2011) M1b0 9 (15.5%) 22 (15.6%) 0.1% ( p = .9859) M1b1 3 (5.2%) 6 (4.3%) 0.9% ( p = .7822) M1c0 8 (13.8%) 28 (19.9%) 6.1% ( p = .3100) M1c1 14 (24.1%) 25 (17.7%) 6.4% ( p = .3010) M1d0 3 (5.2%) 6 (4.3%) 0.9% ( p = .7822) M1d1 1 (1.7%) 6 (4.3%) 2.6% ( p = .3672) Other characteristics LDH > 1.5x ULN 12 (21%) 17 (12%) 9% ( p = .1025) Sex Women 59% Women 41% 18% ( p = .0206) Men 41% Men 59% Median age, years 67.5 (31 87) 68 (26 90) 0.5% ( p = .5647) BRAF mutation BRAF negative 46 (79%) BRAF negative 96 (68%) 11% ( p = .1194) BRAF positive 12 (21%) BRAF positive 45 (32%) ECOG 0 41 (70.7%) 0 100 (70.9%) 0.2% ( p = .9775) 1 17 (29.3%) 1 41 (29.1%) AE 1 4 55% 45% 10% ( p = .1989) AE 3 4 9% 4% 5% ( p = .1576) Abbreviations: AE, adverse events; ECOG, Eastern Cooperative Oncology Group; LDH, lactate dehydrogenase; ULN, upper limit of normal.

2050 - EFFECT OF VITAMIN D ON MELANOMA IMMUNOTHERAPY 10970142, 2023, 13, Downloaded from https://acsjournals.onlinelibrary.wiley.com/doi/10.1002/cncr.34718 by Test, Wiley Online Library on [23/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License CheckMate 066, CheckMate 067, Keynote 001, or Keynote 006. In these studies, for the arms receiving antiPD 1 as part of first line treatment, ORR ranged from 41.9% to 46%, whereas median PFS ranged from 5.1 to 16.9 months. 68 In a large retrospective analysis including 499 patients with advanced melanoma treated with anti PD 1 immunotherapy at leading Polish centers, ORR was 35% and median PFS was 7.9 months. 9 In comparison, in the present study in the group with normal vitamin D levels, these parameters were 56% and 11.25 months, respectively. The analysis shows that normal vitamin D levels in patients with advanced melanoma are related to a higher percentage of objective responses and a longer median PFS in patients undergoing antiPD 1 therapy compared with patients with levels below laboratory normal. The lack of statistical significance in terms of OS difference cannot conclusively prove the effect of vitamin D on the parameter in question during antiPD 1 immuno-therapy. However, after progression during the applied treatment, patients received subsequent lines of treatment. It is important to note the extent of reduced vitamin D levels in the patients included in the study, which was nearly 84% and 75% in both study groups. This deficiency is most likely related to the high

F I G U R E 1 Response rates following antiPD 1 treatment in patients with advanced melanoma showing normal and reduced vitamin levels.

F I G U R E 2 KaplanMeier curves showing progression free survival in patients with advanced melanoma depending on immunotherapy in relation to vitamin D levels.

GALUS ET AL . - 2051 10970142, 2023, 13, Downloaded from https://acsjournals.onlinelibrary.wiley.com/doi/10.1002/cncr.34718 by Test, Wiley Online Library on [23/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License latitude where study patients reside and a lifestyle characterized by spending a lot of time indoors and avoiding exposure to sunlight. It would be advisable to popularize vitamin D supplementation. The subgroups studied differed statistically significantly in the proportion of men and women. Although some studies support a potential association with sex difference in relation to antiPD 1activity, there is no consensus to state that sex has a clear association with response to immunotherapy. There are times when freezing has no effect on vitamin D level. 25(OH)D is stable under common storage conditions: 4 hours at room temperature, 24 hours at 2 to 8C, 7 days at 20C, and 3 months at 80C. 10

Despite the significant improvement in the prognosis of meta-static melanoma patients, the vast majority experience disease pro-gression at some stage. Primary disease progression, in patients undergoing immunotherapy, occurs with a frequency of 33% to 38%. 68 It is possible to improve the prognosis of patients receiving antiPD 1 by introducing a biomarker that allows personalization of treatment or combine it with other drugs. 1117 A potential determi-nant of the efficacy of antiPD 1 antibodies also appears to be the serum vitamin D levels of patients. Currently, there are many data in the literature confirming its anticancer effect and its influence on immune function. The role of vitamin D as a molecule with potential anticancer effects has been and continues to be the subject of many studies. To our knowledge, the first significant reports on the anticancer effects of vitamin D appeared in 1940, in which a study was presented comparing the incidence of cancer among those exposed to ultravi-olet radiation (farmers and soldiers) compared with those with lower exposure. The former group had a significant increase in skin cancers,

T A B L E 4 Single factor and multifactor Cox proportional hazards analysis for PFS and OS.

Parameter PFS OS Univariate analysis Multivariate analysis Univariate analysis Multivariate analysis HR 95% CI p HR 95% CI p HR 95% CI p HR 95% CI p

Age, years 60 1.00 1.00 vs.

>60 1.00 0.991.01 .97    1.00 0.991.02 .57

Stage III, M1a, M1b 1.00 1.00 1.00 vs. M1c, M1d 1.70 1.212.41 .02 1.65 1.16 2.33 .01 1.96 1.263.02 .00 1.81 1.16 2.81 .01 Presence of BRAF mutations none 1.00 1.00 vs. current 1.40 0.962.04 .08    1.04 0.651.67 .87

CNS metastasis none 1.00 1.00 vs. current 1.35 0.682.63 .39    1.03 0.502.14 .93

Sex W 1.00 1.00 vs. M 1.05 0.751.48 .77    1.26 0.811.95 .31

LDH  ULN 1.00 1.00 1.00 1.00 vs.

> ULN 1.82 1.292.56 .00 1.71 1.21 2.41 .00 2.37 1.543.63 .00 1.89 1.21 2.96 .00 ECOG 0 1.00 1.00 vs. 1 1.49 1.042.15 .03    2.60 1.69 4.00 .00 2.09 1.33 3.29 .00 Vitamin D reduced level 1.00 1.00 vs. normal 0.69 0.480.99 .04 0.65 0.49 0.94 .02 0.81 0.511.30 .39

Abbreviations: CNS, central nervous system; ECOG, Eastern Cooperative Oncology Group; HR, hazard ratio; LDH, lactate dehydrogenase; OS, overall survival; PFS, progression free survival; ULN, upper limit of normal.

2052 - EFFECT OF VITAMIN D ON MELANOMA IMMUNOTHERAPY 10970142, 2023, 13, Downloaded from https://acsjournals.onlinelibrary.wiley.com/doi/10.1002/cncr.34718 by Test, Wiley Online Library on [23/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License but at the same time a lower rate of other cancers. 18 Since then, the effect of vitamin D has been subjected to various in vitro tests. In 1981, Stucci and colleagues described the inhibitory effect of vitamin D on melanoma cell proliferation in vitro, and using it, they differ-entiated mouse acute leukemia cells into macrophages. 1921 Its anticancer effects were also reported by Bochen and colleagues, in which inhibiting the development of hamster buccal mucosal tumors previously induced by 7,12 dimethylbenzanthracene, and then sup-plementing the animals with vitamin D. 22,23

The potential anticancer effect of cholecalciferol has also been proven in clinical trials. In a randomized, placebo controlled, blinded study, daily vitamin D supplementation of 1100 IU/d along with calcium 1500 mg/d by postmenopausal women was proven to sta-tistically significantly reduce the risk of cancer. 19,24,25 There has also been a correlation between higher vitamin D levels and lower mor-tality from breast cancer and colorectal cancer, and a large meta

analysis showed a protective effect of vitamin D in preventing the development of lung cancer. 19,26

The effects of vitamin D on the immune system are complex and involve multiple components. Vitamin D receptors are found on almost all cell types of this system: B lymphocytes, T lymphocytes, antigen presenting cells, neutrophils, macrophages, and dendritic cells. Its effects on individual cells are related to, among others, activation or inhibition of proliferation/function via the MAPK pathway. Most significantly, in the context of antitumor response, an increase in the amount of CD8 as well as the number of CD4 T cells in the tumor, and increased expression of their activation marker CD69 under the influence of vitamin D was shown. Independent of this, it has also been observed that during activation of T cell re-ceptors, expression of vitamin D receptors occurs. Regardless of the direct effect of vitamin D on CD8 lymphocytes, its effect on CD8 lymphocyte regulatory cells such as dendritic cells, CD4 lymphocytes, and macrophages has also been observed. 27 A suspensory effect of vitamin D on the secretion of interleukin (IL) 12, IL 23 and IL 6 has also been shown, with the former being associated with lymphocyte differentiation toward Th1, and the latter two being Th17 stimula-tors. Therefore, ultimately, vitamin D is associated with a reduction in Th1 and Th17 and, as a result, Th2 dominance, which is related to a reduction in inflammation generated by Th1 and Th17. Proin-flammatory cytokines such as IL 12 are necessary locally for activa-tion of CD8 T cells in cytotoxic T lymphocytes. However, generalized inflammation adversely affects the antitumor response. There are reports indicating levels of IL 6, for example, as a negatively pre-dictive biomarker during antiPD 1/PDL 1 therapy. The promotion of Th2 lymphocytes (at the expense of Th1 and Th 17) cannot be indicated as a factor that unequivocally develops a humoral response because it simultaneously inhibits the proliferation and differentia-tion of B lymphocytes, as well as their secretion of immunoglobu-lins. 19,28,29 Vitamin D also has an inhibitory effect on the production of downstream inflammatory cytokines such as IL 1, tumor necrosis factor  , and IL 8. Vitamin D exerts an inhibitory effect on Th17 lymphocytes, which are mainly responsible for production of inflammatory cytokines and neutrophil migration. The expansion of lymphocytes in question is also associated with the frequency of adverse events (AEs) during therapy with antiPD 1 and antiCTLA 4 antibodies. Vitamin D may therefore have a reducing effect on the frequency of immune com-plications of such treatment. 19,3033 The literature has described a relationship between vitamin D supplementation and reduced risk of intestinal inflammation during immunotherapy with immune check-point inhibitors. 34 No statistically significant differences in the inci-dence of AEs were observed in the patients analyzed in this study. However, there was a trend indicating a lower incidence of AEs with normal vitamin D levels (both in the analysis for severity stages 14 and 34 according to Common Terminology Criteria for AEs). In a large study of 231 patients with head and neck carcinoma, Bochen and colleagues showed a correlation between normal vitamin D levels and longer patient survival. However, in the described study, higher vitamin D levels were simultaneously found in patients who were human papilloma viruspositive (HPV +) compared with pa-tients who were HPV, and the mere presence of HPV DNA is associated with a better prognosis. In the subgroup of patients with HPV + alone, there was no difference in survival between patients with reduced vs. normal vitamin D levels. 22

In the same study, the authors also showed significantly greater infiltration of the tumor and its lining by cells such as T lymphocytes (CD3 +), helper T lymphocytes (CD4 +), cytotoxic lymphocytes (CD8

+), natural killer (NK) cells (CD56 +), and macrophages and M1 macrophages among patients with higher vitamin D levels, with less infiltration of the lining by M2 macrophages (CD163 +). Increased tumor infiltration by CD8 + T cells and NK cells and infiltration of the tumor lining by CD3 + and CD4 + lymphocytes appeared to be pre-dictive of longer survival, whereas conversely, greater infiltration of M2 macrophages (CD163 +) was related to worse prognosis. In the same study, a positive correlation between vitamin D levels and NK cell activity was shown based on in vitro analyses of NK cell activity collected from patients. 22

Based on studies of tumor tissues from 35 patients with squa-mous cell carcinoma of the head and neck undergoing vitamin D supplementation and 18 nonsupplemented control patients, vitamin D was shown to increase the levels of proinflammatory cytokines such as IL 6, IL 10, and interferon  , importantly, locally, inside the tumor. An analogous study conducted in 11 and 12 patients with squamous cell carcinoma of the head and neck, respectively, demonstrated that vitamin D supplementation was related to reduction in the number of immunosuppressive CD34 + cells and immature dendritic cells in tumor tissue. 22,35,36

Vitamin D has also been shown to stimulate PDL 1 expression on immune cells in inflammatory bowel disease and human epithelial cells. Taking into account the correlation of the intensity of PDL 1expression with the response to therapy with immune checkpoint inhibitors, it can be presumed that vitamin D is positively correlated with the response to treatment with antiPD 1/antiPDL 1 anti-bodies. 19,3739

Furthermore, according to the analysis of Custao et al., vitamin D levels determined at specific time points in patients with nonsmall

> GALUS ET AL .

# - 2053 10970142, 2023, 13, Downloaded from https://acsjournals.onlinelibrary.wiley.com/doi/10.1002/cncr.34718 by Test, Wiley Online Library on [23/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License cell lung cancer treated with nivolumab correlate with the levels of administered antibodies. This is due to its effect on the expression of genes related to metabolism or elimination of various medications. Importantly, according to previous reports, the levels of administered antibodies are statistically higher in patients who have achieved a response to treatment compared with those who are progressing. 40,41

Moreover, in addition to its effects on the immune system, vitamin D is known to have anticancer effects through anti-proliferative activity, induction of apoptosis, inhibition of invasion, and metastasis formation, as well as antiangiogenic effects. 42

CONCLUSIONS

The analysis showed a statistically significant, positive effect of normal serum vitamin D levels on increasing ORR and PFS as a result of antiPD 1 immunotherapy in patients with locally advanced inoperable or metastatic melanoma. An undeniable disadvantage of this study is the comparative analysis of a population evaluated retrospectively with a population in which vitamin D supplementa-tion was prospectively intervened. However, the nature of inter-vention and the medical wide indications for vitamin D supply, in case of vitamin D deficiency, make it impossible for ethical reasons to construct a study comparing patients fully prospectively, with pla-cebo control and randomization. Both American and European endocrine guidelines, including those in Poland, recommend vitamin D supplementation in case of deficiency. 43,44 The authors opinion is that assessment of vitamin D levels and appropriate vitamin D sup-plementation should be considered in every patient qualified for treatment with antiPD 1 immunotherapy.

AUTHOR CONTRIBUTIONS ukasz Galus: Conceptualization, methodology, investigation, data curation, writing  original draft, visualization, and project adminis-tration. Micha Michalak: Formal analysis and software. Mateusz Lorenz: Investigation. Renata Stoiska Swiniarek: Investigation.

Daria Tusie Maecka: Investigation. Agnieszka Galus: Investigation.

Tomasz Kolenda: Investigation. Ewa Leporowska: Investigation. Jacek Mackiewicz: Writing  review and editing, funding acquisition, re-sources, and supervision.

ACKNOWLEDGMENTS

Poznan University of Medical Sciences, Poland.

CONFLICT OF INTEREST STATEMENT

ukasz Galus has received remuneration from BMS, Roche, MSD, Novartis, and Pierre Fabre. Daria Tusie Maecka has received remuneration from Merck, BMS, Novartis, and Pierre Fabre. Jacek Mackiewicz reports remuneration from BMS, GlaxoSmithKline, Roche, MSD, Novartis, and Pierre Fabre. The other authors made no disclosures.

ORCID

ukasz Galus https://orcid.org/0000-0002-9515-1834

REFERENCES

1. Rusiska A, Pudowski P, Walczak M, et al. Zasady suplementacji i leczenia witamin D  nowelizacja 2018 r. Postpy Neonatologii .2018;24(1). 2. Sewerynek E, Cielak K, Janik M, Gowin E, Stuss M. Evaluation of vitamin D concentration in a population of young, healthy women  the effects of vitamin D supplementation. Endokrynol Pol . 2017; 68(5):533 540. 3. upiska A, Chlebna Sok D. Czynniki wpywajce na stenie witaminy D w surowicy dzieci dzkich w wieku wczesnoszkolnym z nadmiarem masy ciaa. Post N Med . 2016;29(10):709 715. 4. Chlebna Sok D, Michaus I, Rusiska A, et al. Ocena stenia witaminy D w surowicy u dzieci hospitalizowanych z powodu obja-ww klinicznych sugerujcych zaburzenia w ukadzie kostnym.

Endokrynol Pediatr . 2016;15(4):23 32. doi:10.18544/ep 01.15.04. 1653 5. Pudowski P, Ducki C, Konstantynowicz J, Jaworski M. Vitamin D status in Poland. Pol Arch Med Wewn . 2016;126(7 8):530 539. 6. Ascierto PA, Long GV, Robert C, et al. Survival outcomes in patients with previously untreated BRAF wild type advanced melanoma treated with nivolumab therapy: three year follow up of a ran-domized phase 3 trial. JAMA Oncol . 2019;5(2):187 194. doi:10.1001/ jamaoncol.2018.4514 7. Hamid O, Robert C, Daud A, et al. Five year survival outcomes for patients with advanced melanoma treated with pembrolizumab in KEYNOTE 001. Ann Oncol . 2019;30(4):582 588. doi:10.1093/ annonc/mdz011 8. Robert C, Ribas A, Schachter J, et al. Pembrolizumab versus ipili-mumab in advanced melanoma (KEYNOTE 006): post hoc 5 year results from an open label, multicentre, randomised, controlled, phase 3 study. Lancet Oncol . 2019;20(9):1239 1251. doi:10.1016/ s1470 2045(19)30388 29. Cybulska Stopa B, Pacholczak Madej R, Kamiska Winciorek G, et al. First line treatment of advanced/metastatic melanoma with anti PD 1 antibodies: multicenter experience in Poland. Immuno-therapy . 2021;13(4):297 307. doi:10.2217/imt 2020 0217 10. Colak A, Toprak B, Dogan N, Ustuner F. Effect of sample type, centrifugation and storage conditions on vitamin D concentration.

Biochem Med . 2013;23:321 325. doi:10.11613/bm.2013.039, 325. 11. Sha D, Jin Z, Budzcies J, Kluck K, Stenzinger A, Sinicrope FA. Tumor mutational burden (TMB) as a predictive biomarker in solid tumors.

Cancer Discov . 2020;10(12):1808 1825. doi:10.1158/2159 8290.cd

20 0522 12. Eisenstein A, Gonzalez EC, Raghunathan R, et al. Emerging bio-markers in cutaneous melanoma. Mol Diagn Ther , 2018;22(2): 203 218, doi:10.1007/s40291 018 0318 z13. Bai R, Lv Z, Xu D, Cui J. Predictive biomarkers for cancer immuno-therapy with immune checkpoint inhibitors. Biomark Res . 2020;8(1): 34. doi:10.1186/s40364 020 00209 014. Tumeh PC, Harview CL, Yearley JH, et al. PD 1 blockade induces responses by inhibiting adaptive immune resistance. Nature .2014;515(7528):568 571, doi:10.1038/nature13954 15. Wolchok JD, Chiarion Sileni V, Rene Gonzalez R, et al. Overall sur-vival with combined nivolumab and ipilimumab in advanced mela-noma. N Engl J Med . 2017;377(14):1345 1356, doi:10.1056/ nejmoa1709684 16. Liu D, Schilling B, Liu D, et al. Integrative molecular and clinical modeling of clinical outcomes to PD1 blockade in patients with metastatic melanoma. Nat Med . 2019;25(12):1916 1927. doi:10. 1038/s41591 019 0654 5

2054 - EFFECT OF VITAMIN D ON MELANOMA IMMUNOTHERAPY 10970142, 2023, 13, Downloaded from https://acsjournals.onlinelibrary.wiley.com/doi/10.1002/cncr.34718 by Test, Wiley Online Library on [23/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 17. Johnson DB, Frampton GM, Rioth MJ, et al. Targeted next genera-tion sequencing identifies markers of response to PD 1 blockade.

Cancer Immunol Res . 2016;4(11):959 967. doi:10.1158/2326 6066. cir 16 0143 18. Apperly FL. The relation of solar radiation to cancer mortality in North America. Cancer Res . 1941;1:191 195. 19. Stucci LS, D'Oronzo S, Tucci M, et al. Vitamin D in melanoma: con-troversies and potential role in combination with immune check

point inhibitors. Cancer Treat Rev . 2018;69:21 28. doi:10.1016/j. ctrv.2018.05.016 20. Colston K, Colston MJ, Feldman D. 1,25 dihydroxyvitamin D3 and malignant melanoma: the presence of receptors and inhibition of cell growth in culture. Endocrinology . 1981;108(3):1083 1086. doi:10. 1210/endo 108 31083 21. Abe E, Miyaura C, Sakagami H, et al. Differentiation of mouse myeloid leukemia cells induced by 1 alpha, 25  dihydroxyvitamin D3. Proc Natl Acad Sci USA . 1981;78(8):4990 4994. doi:10.1073/pnas.78.8.4990 22. Bochen F, Balensiefer B, Krner S, et al. Vitamin D deficiency in head and neck cancer patients  prevalence, prognostic value and impact on immune function. OncoImmunology . 2018;7(9):e1476817. doi:10. 1080/2162402x.2018.1476817 23. Meier JD, Enepekides DJ, Poirier B, Bradley CA, Albala JS, Farwell DG. Treatment with 1 alpha,25 dihydroxyvitamin D3 (vitamin D3) to inhibit carcinogenesis in the hamster buccal pouch model. Arch Otolaryngol Head Neck Surg . 2007;133(11):1149 1152. doi:10.1001/ archotol.133.11.1149 24. Lappe JM, Travers Gustafson MD, Davies KM, Recker RR, Heany RP. Vitamin D and calcium supplementation reduces cancer risk; result of randomisation trial. Am J Clin Nutr . 2008;87(3):794. 25. Chlebowski RT, Johnson KC, Kooperberg C, et al. Calcium plus vitamin D supplementation and the risk of breast cancer. J Natl Cancer Inst . 2008;100(22):1581 1591. doi:10.1093/jnci/djn360 26. Zhang L, Wang S, Che X, Li X. Vitamin D and lung cancer risk: a comprehensive review and meta analysis. Cell Physiol Biochem .2015;36(1):299 305. doi:10.1159/000374072 27. Sarkar S, Hewison M, Studzinski GP, Li YCH, Kalia V. Role of vitamin D in cytotoxic T lymphocyte immunity to pathogens and cancer. Crit Rev Clin Lab Sci . 2016;53(2):132 145. doi:10.3109/10408363.2015. 1094443 28. Daniel C, Sartory NA, Zahn N, Radeke HH, Stein JM. Immune modulatory treatment of trinitrobenzene sulfonic acid colitis with calcitriol is associated with a change of a T helper (Th) 1/Th17 to a Th2 and regulatory T cell profile. J Pharmacol Exp Therapeut .2008;324(1):23 33. doi:10.1124/jpet.107.127209 29. Bui L, Zhu Z, Hawkins S, Cortez Resendiz A, Bellon A. Vitamin D regulation of the immune system and its implications for COVID 19: a mini review. SAGE Open Med . 2021;9:205031212110140. doi:10. 1177/20503121211014073 30. Sotirchos ES, Bhargava P, Eckstein C, et al. Safety and immuno-logic effects of high  vs low dose cholecalciferol in multiple scle-rosis. Neurology . 2016;86(4):382 390. doi:10.1212/wnl.000000000 0002316 31. Joshi S, Pantalena LC, Liu XK, et al. 1,25dihydroxyvitamin D(3) ameliorates Th17 autoimmunity via transcriptional modulation of interleukin 17A. Mol Cell Biol . 2011;31(17):3653 3669. doi:10.1128/ mcb.05020 11 32. Tarhini AA, Zahoor H, Lin Y, et al. Baseline circulating IL 17 predicts toxicity while TGF  1 and IL 10 are prognostic of relapse in ipili-mumab neoadjuvant therapy of melanoma. J Immunother Cancer .2015;3(1):39. doi:10.1186/s40425 015 0081 133. Stucci S, Palmirotta R, Passarelli A, et al. Immune related adverse events during anticancer immunotherapy: pathogenesis and man-agement. Oncol Lett . 2017;14(5):5671 5680. 34. Grover S, Dougan M, Tyan K, et al. Vitamin D intake is associated with decreased risk of immune checkpoint inhibitor induced colitis.

Cancer . 2020;126(16):3758 3767. doi:10.1002/cncr.32966 35. Walker DD, Reeves TD, de Costa AM, Schuyler C, Young MR. Immunological modulation by 1 ,25 dihydroxyvitamin D3 in pa-tients with squamous cell carcinoma of the head and neck. Cytokine .2012;58(3):448 454. doi:10.1016/j.cyto.2012.03.002 36. Kulbersh JS, Day TA, Gillespie MB, Young MR. 1 alpha,25 Dihy-droxyvitamin D(3) to skew intratumoral levels of immune inhibitory CD34( +) progenitor cells into dendritic cells. Otolaryngol Head Neck Surg . 2009;140(2):235 240. doi:10.1016/j.otohns.2008.11.011 37. Larkin J, Chiarion Sileni V, Gonzalez R, et al. Combined nivolumab and ipilimumab or monotherapy in untreated melanoma. N Engl J Med . 2015;373(1):23 34. doi:10.1056/nejmoa1504030 38. Dimitrov V, Bouttier M, Boukhaled G, et al. Hormonal vitamin D up

regulates tissue specific PD L1 and PD L2 surface glycoprotein expression in humans but not mice. J Biol Chem . 2017;292(50): 20657 20668. doi:10.1074/jbc.m117.793885 39. Bendix M, Greisen S, Dige A, et al. Vitamin D increases programmed death receptor 1 expression in Crohn's disease. Oncotarget .2017;8(15):24177 24186. doi:10.18632/oncotarget.15489 40. Cusato J, Genova C, Tomasello C, et al. Influence of vitamin D in advanced non small cell lung cancer patients treated with nivolu-mab. Cancers . 2019;11:125. doi:10.3390/cancers11010125 41. Koolen SLW, Basak EA, Hurkmans et al. Correlation between nivo-lumab exposure and treatment outcome in NSCLC. In Proceedings of the 2018 ASCO Annual Meeting, Chicago, IL, USA , 15 June 2018. 42. Broyna A, Hoffman R, Slominski A. Relevance of vitamin D in melanoma development, progression and therapy. Anticancer Res .2020;40(1):473 489. doi:10.21873/anticanres.13976 43. Holick MF, Binkley NC, Gordon CM, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab . 2011;96(7):1911 1930. doi:10.1210/jc.2011 0385 44. Pudowski P, Karczmarewicz E, Bayer M, et al. Practical guidelines for the supplementation of vitamin D and the treatment of deficits in Central Europe  recommended vitamin D intakes in the general population and groups at risk of vitamin D deficiency. Endokrynol Pol .2013;64(4):319 327. doi:10.5603/ep.2013.0012

How to cite this article: Galus , Michalak M, Lorenz M, et al. Vitamin D supplementation increases objective response rate and prolongs progression free time in patients with advanced melanoma undergoing antiPD 1 therapy. Cancer .2023;129(13):2047 2055. doi:10.1002/cncr.34718

GALUS ET AL . - 2055 10970142, 2023, 13, Downloaded from https://acsjournals.onlinelibrary.wiley.com/doi/10.1002/cncr.34718 by Test, Wiley Online Library on [23/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Transcript for:Galus 2023. Vitamin D supplementation increases objective response rate and prolongs progression‐free time in patients with advanced melanoma undergoing anti–PD‐1 therapy.

Transcript for:
Galus 2023. Vitamin D supplementation increases objective response rate and prolongs progression‐free time in patients with advanced melanoma undergoing anti–PD‐1 therapy.