Mariaelena CaponepersName, Diana GiannarellipersName, Domenico MallardopersName, Gabriele MadonnapersName, Lucia FestinopersName, Antonio Maria GrimaldipersName, Vito VanellapersName, Ester SimeonepersName, Miriam PaonepersName, Giuseppe PalmieripersName, Ernesta CavalcantipersName, Corrado CaracòpersName, Paolo Antonio AsciertopersName (2018date). “Baseline neutrophil-to-lymphocyte ratio (NLR) and derived NLR could predict overall survival in patients with advanced melanoma treated with nivolumab.” Journal for ImmunoTherapy of Cancertitle·j.
Object of study —
Research question — whether baseline NLRterm·def and derived NLRterm·def predict nivolumabterm outcomes in stage-IV melanoma
Contribution — baseline NLRterm·def and dNLRterm·def as inexpensive, available survival biomarkers
phenomenon · claim-type · cross-domain-transfer · mechanism · claim-relation · replication-status · limitation — grounded in the full text| phenomenon | baseline inflammatory blood ratios and survival during nivolumab treatment for advanced melanoma — “This retrospective study investigates the role of the NLR and dNLR as predictive markers of response to treatment with nivolumab in patients with advanced melanoma.” | |
| claim-type | existence — “NLR and dNLR are simple, inexpensive and readily available biomarkers that could be used to help predict response to immunotherapy in patients with advanced melanoma.” | |
| method — evidence | — “This was a retrospective analysis of 97 consecutive patients with stage IV melanoma who were treated with nivolumab.” | |
| research question — evidence | — “This retrospective study investigates the role of the NLR and dNLR as predictive markers of response to treatment with nivolumab in patients with advanced melanoma.” | |
| contribution — evidence | — “NLR and dNLR are simple, inexpensive and readily available biomarkers that could be used to help predict response to immunotherapy in patients with advanced melanoma.” | |
| mechanism | Engagement of PD-1 with its ligands PD-L1 or PD-L2 expressed on antigen-presenting cells and on tumor cells results in inhibition of T-cell proliferation and cytokine secretion, potentiating T-cell responses. — “Engagement of PD-1 with its ligands PD-L1 or PD-L2 expressed on antigen-presenting cells and on tumor cells results in inhibition of T-cell proliferation and cytokine secretion, potentiating T-cell responses.” | mechanisms[0] |
| mechanism | In recent years, it has become evident that cancer-related inflammation responses, such as increased and defective myelopoiesis, and both local and systemic inflammation, have an important role in tumorigenesis, disease progression and patient prognosis [ 6 , 7 ]. — “In recent years, it has become evident that cancer-related inflammation responses, such as increased and defective myelopoiesis, and both local and systemic inflammation, have an important role in tumorigenesis, disease progression and patient prognosis [ 6 , 7 ].” | mechanisms[1] |
| mechanism | Systemic inflammation is associated with alterations in peripheral blood leukocytes that can be captured by the neutrophil-to-lymphocyte ratio (NLR) [ 8 ]. — “Systemic inflammation is associated with alterations in peripheral blood leukocytes that can be captured by the neutrophil-to-lymphocyte ratio (NLR) [ 8 ].” | mechanisms[2] |
| mechanism | These results suggest that the systemic inflammatory response is a potent stimulator of cancer progression in established disease. — “These results suggest that the systemic inflammatory response is a potent stimulator of cancer progression in established disease.” | mechanisms[3] |
| mechanism | Elevated NLR suggests a systemic inflammatory state and can be indicative of neutrophilia, lymphopenia, or a combination of both. — “Elevated NLR suggests a systemic inflammatory state and can be indicative of neutrophilia, lymphopenia, or a combination of both.” | mechanisms[4] |
| mechanism | Although many mechanisms are unclear, neutrophils are thought to primarily propagate a pro-tumor environment promoting angiogenesis and tumor growth, degrading the extracellular matrix and providing favorable conditions for metastasis, and potentiating genome instability and tumor evolution [ 27 ]. — “Although many mechanisms are unclear, neutrophils are thought to primarily propagate a pro-tumor environment promoting angiogenesis and tumor growth, degrading the extracellular matrix and providing favorable conditions for metastasis, and potentiating genome instability and tumor evolution [ 27 ].” | mechanisms[5] |
| mechanism | A recent study also demonstrated that the expression of PD-L1 on tumor infiltrating neutrophils could functionally inhibit the activation of T cells [ 28 ]. — “A recent study also demonstrated that the expression of PD-L1 on tumor infiltrating neutrophils could functionally inhibit the activation of T cells [ 28 ].” | mechanisms[6] |
| mechanism | For these reasons, whether due to neutrophilia or lymphopenia, elevated NLR physiologically suggests an inability of the immune system to suppress cancer progression, and thus it can be an index for OS and PFS. — “For these reasons, whether due to neutrophilia or lymphopenia, elevated NLR physiologically suggests an inability of the immune system to suppress cancer progression, and thus it can be an index for OS and PFS.” | mechanisms[7] |
| supports | previous LDH-response and survival studies [23-25] — “Lower baseline LDH also correlated with a better response and survival, consistent with several previous studies [ 23 – 25 ], which was maintained in multivariate analysis.” | claim_relations[0] |
| sense | the retrospective association is a prognostic result requiring prospective validation before predictive clinical use — “The biggest limitation of this study is the low number of patients that we have analyzed and validation is required in larger prospective studies.” | senses[0] |
| control | Methods This was a retrospective analysis of 97 consecutive patients with stage IV melanoma who were treated with nivolumab. — rules out hand-selection of responders — “This was a retrospective analysis of 97 consecutive patients with stage IV melanoma who were treated with nivolumab.” | controls[0] |
| control | For all patients, clinical data (white blood cell [WBC] count, absolute lymphocyte count [ALC], absolute neutrophil count [ANC], serum lactate dehydrogenase [LDH], and other clinical data, including BRAF status, brain metastasis, lines of prior treatment, and response) were collected before starting nivolumab treatment and until last follow-up. — rules out post-treatment biomarker measurement — “For all patients, clinical data (white blood cell [WBC] count, absolute lymphocyte count [ALC], absolute neutrophil count [ANC], serum lactate dehydrogenase [LDH], and other clinical data, including BRAF status, brain metastasis, lines of prior treatment, and response) were collected before starting nivolumab treatment and until last follow-up.” | controls[1] |
| control | Tumor assessment was performed at baseline, at week 12, and every 12 weeks thereafter, and clinical response were classified according to response evaluation criteria in solid tumors (RECIST) [ 14 ]. — rules out inconsistent response-assessment timing — “Tumor assessment was performed at baseline, at week 12, and every 12 weeks thereafter, and clinical response were classified according to response evaluation criteria in solid tumors (RECIST) [ 14 ].” | controls[2] |
| control | Multivariate Cox proportional hazard models were used to investigate associations of NLR with survival, adjusted for baseline characteristics. — rules out unadjusted baseline prognostic imbalance — “Multivariate Cox proportional hazard models were used to investigate associations of NLR with survival, adjusted for baseline characteristics.” | controls[3] |
| control | The best cutoff values for NLR and dNLR were derived using Cutoff Finder software based on an R routine which optimized the significance of the split between Kaplan-Meier survival curves. — rules out arbitrary optimized-threshold selection — “The best cutoff values for NLR and dNLR were derived using Cutoff Finder software based on an R routine which optimized the significance of the split between Kaplan-Meier survival curves.” | controls[4] |
| control | The performance of an NLR cut-off value of 4.7 was overlapping with 5 as cut-off value, Median PFS and OS were also significantly longer in patients with dNLR < 3.8 compared to ≥3.8 (see Additional files 1 and 2 ). — rules out dependence on one unbenchmarked cutoff — “The performance of an NLR cut-off value of 4.7 was overlapping with 5 as cut-off value, Median PFS and OS were also significantly longer in patients with dNLR < 3.8 compared to ≥3.8 (see Additional files 1 and 2 ).” | controls[5] |
| note | systemic inflammation —operationalised-as→ neutrophil-to-lymphocyte ratio — The paper flatly says peripheral leukocyte alterations from systemic inflammation can be captured by NLR. — “Systemic inflammation is associated with alterations in peripheral blood leukocytes that can be captured by the neutrophil-to-lymphocyte ratio (NLR) [ 8 ].” | concept_edges[0] |
| weight | systemic inflammation —operationalised-as→ neutrophil-to-lymphocyte ratio — 0.9 — “Systemic inflammation is associated with alterations in peripheral blood leukocytes that can be captured by the neutrophil-to-lymphocyte ratio (NLR) [ 8 ].” | concept_edges[0] |
| note | progression-free survival —distinct-from→ overall survival — The endpoints have necessary, explicit event and censoring definitions; their quantified distinction earns 1.0. — “Progression-free survival (PFS) was determined from the first cycle of treatment to disease progression documented by imaging, or death (event), or last follow-up (censored). Overall survival (OS) was calculated from the first cycle of treatment to death (event) or last follow-up (censored).” | concept_edges[2] |
| no type for this | nivolumab → PD-1 — — “Nivolumab (Opdivo®, Bristol-Myers Squibb) is a fully human IgG4 monoclonal antibody that blocks the PD-1 receptor, a negative regulator of T-cell activity that has been shown to be involved in the control of T-cell immune responses.” | unhoused_relations[0] |
| no type for this | neutrophil-to-lymphocyte ratio → overall survival — — “In univariate analysis, increasing absolute neutrophil count (ANC), NLR, dNLR and lactate dehydrogenase (LDH) (continuous variables) were all significantly associated with OS. Only NLR (hazard ratio [HR] = 2.85; 95% CI 1.60–5.08; p < 0.0001) and LDH (HR = 2.51; 95% CI 1.36–4.64; p < 0.0001) maintained a significant association with OS in multivariate analysis.” | unhoused_relations[1] |
| no type for this | derived NLR → overall survival — — “In univariate analysis, increasing absolute neutrophil count (ANC), NLR, dNLR and lactate dehydrogenase (LDH) (continuous variables) were all significantly associated with OS. Only NLR (hazard ratio [HR] = 2.85; 95% CI 1.60–5.08; p < 0.0001) and LDH (HR = 2.51; 95% CI 1.36–4.64; p < 0.0001) maintained a significant association with OS in multivariate analysis.” | unhoused_relations[2] |
| no type for this | neutrophil-to-lymphocyte ratio → progression-free survival — — “Patients with baseline NLR ≥5 had significantly worse OS and PFS than patients with NLR < 5, as did patients with baseline dNLR ≥3 versus < 3.” | unhoused_relations[3] |
| no type for this | derived NLR → progression-free survival — — “Patients with baseline NLR ≥5 had significantly worse OS and PFS than patients with NLR < 5, as did patients with baseline dNLR ≥3 versus < 3.” | unhoused_relations[4] |
| limitation | Nevertheless, a significant portion of patients do not benefit from nivolumab treatment. — “Nevertheless, a significant portion of patients do not benefit from nivolumab treatment.” | limitations[0] |
| limitation | The median PFS was 4 months (range 0–26) and median OS was 6 months (range 2–26); about 10% of patients had started therapy no more than 6 months earlier. — “The median PFS was 4 months (range 0–26) and median OS was 6 months (range 2–26); about 10% of patients had started therapy no more than 6 months earlier.” | limitations[1] |
| limitation | In this study, which is the first to report an association between NLR and outcomes in patients with advanced melanoma treated with nivolumab, we analyzed the predictive power of baseline NLR and dNLR in a small retrospective cohort of patients. — “In this study, which is the first to report an association between NLR and outcomes in patients with advanced melanoma treated with nivolumab, we analyzed the predictive power of baseline NLR and dNLR in a small retrospective cohort of patients.” | limitations[2] |
| limitation | The biggest limitation of this study is the low number of patients that we have analyzed and validation is required in larger prospective studies. — “The biggest limitation of this study is the low number of patients that we have analyzed and validation is required in larger prospective studies.” | limitations[3] |
| limitation | The biggest limitation of this study is the low number of patients that we have analyzed and validation is required in larger prospective studies. — “The biggest limitation of this study is the low number of patients that we have analyzed and validation is required in larger prospective studies.” | limitations[4] |
| limitation | Although many mechanisms are unclear, neutrophils are thought to primarily propagate a pro-tumor environment promoting angiogenesis and tumor growth, degrading the extracellular matrix and providing favorable conditions for metastasis, and potentiating genome instability and tumor evolution [ 27 ]. — “Although many mechanisms are unclear, neutrophils are thought to primarily propagate a pro-tumor environment promoting angiogenesis and tumor growth, degrading the extracellular matrix and providing favorable conditions for metastasis, and potentiating genome instability and tumor evolution [ 27 ].” | limitations[5] |
finding · quantity · variable · definition · key-term — grounded in the full text| finding | Only NLR (hazard ratio [HR] = 2.85; 95% CI 1.60–5.08; p < 0.0001) and LDH (HR = 2.51; 95% CI 1.36–4.64; p < 0.0001) maintained a significant association with OS in multivariate analysis. — “Only NLR (hazard ratio [HR] = 2.85; 95% CI 1.60–5.08; p < 0.0001) and LDH (HR = 2.51; 95% CI 1.36–4.64; p < 0.0001) maintained a significant association with OS in multivariate analysis.” | content_tags.findings[0] |
| finding | Patients with baseline NLR ≥5 had significantly worse OS and PFS than patients with NLR < 5, as did patients with baseline dNLR ≥3 versus < 3. — “Patients with baseline NLR ≥5 had significantly worse OS and PFS than patients with NLR < 5, as did patients with baseline dNLR ≥3 versus < 3.” | content_tags.findings[1] |
| finding | In the subgroup of 27 patients with brain metastases, NLR < 4.7 and dNLR < 3.8 were both significantly associated with improved OS and PFS in univariate analysis. — “In the subgroup of 27 patients with brain metastases, NLR < 4.7 and dNLR < 3.8 were both significantly associated with improved OS and PFS in univariate analysis.” | content_tags.findings[2] |
| finding | In multivariate analysis, only NLR (HR = 2.85; 95% CI: 1.60–5.08; P < 0.0001) and LDH (HR = 2.51; 95% CI: 1.36–4.64; P < 0.0001) maintained a significant association with OS (Table 2 ). — “In multivariate analysis, only NLR (HR = 2.85; 95% CI: 1.60–5.08; P < 0.0001) and LDH (HR = 2.51; 95% CI: 1.36–4.64; P < 0.0001) maintained a significant association with OS (Table 2 ).” | content_tags.findings[3] |
| finding | NLR was predictive of survival independent of BRAF status (data not shown). — “NLR was predictive of survival independent of BRAF status (data not shown).” | content_tags.findings[4] |
| finding | Lower baseline LDH also correlated with a better response and survival, consistent with several previous studies [ 23 – 25 ], which was maintained in multivariate analysis. — “Lower baseline LDH also correlated with a better response and survival, consistent with several previous studies [ 23 – 25 ], which was maintained in multivariate analysis.” | content_tags.findings[5] |
| finding | Despite the small number of patients, both NLR and dNLR were good predictors of survival when baseline levels were lower than cut-off values. — “Despite the small number of patients, both NLR and dNLR were good predictors of survival when baseline levels were lower than cut-off values.” | content_tags.findings[6] |
| quantity | Nivolumab has demonstrated clinical activity in previously treated and treatment-naïve patients with advanced melanoma, with overall response rates (ORR) ranging between 20 and 40% in patients treated at various dose levels [ 3 – 5 ]. — “Nivolumab has demonstrated clinical activity in previously treated and treatment-naïve patients with advanced melanoma, with overall response rates (ORR) ranging between 20 and 40% in patients treated at various dose levels [ 3 – 5 ].” | content_tags.quantities[0] |
| quantity | The median age at first treatment was 61 years (range 21–85) and 56.7% of patients were male. — “The median age at first treatment was 61 years (range 21–85) and 56.7% of patients were male.” | content_tags.quantities[1] |
| quantity | BRAF-mutated melanomas were present in 38 patients (39.1%); 54 (55.7%) had BRAF wild-type tumors. — “BRAF-mutated melanomas were present in 38 patients (39.1%); 54 (55.7%) had BRAF wild-type tumors.” | content_tags.quantities[2] |
| quantity | The median PFS was 4 months (range 0–26) and median OS was 6 months (range 2–26); about 10% of patients had started therapy no more than 6 months earlier. — “The median PFS was 4 months (range 0–26) and median OS was 6 months (range 2–26); about 10% of patients had started therapy no more than 6 months earlier.” | content_tags.quantities[3] |
| quantity | The median PFS was 4 months (range 0–26) and median OS was 6 months (range 2–26); about 10% of patients had started therapy no more than 6 months earlier. — “The median PFS was 4 months (range 0–26) and median OS was 6 months (range 2–26); about 10% of patients had started therapy no more than 6 months earlier.” | content_tags.quantities[4] |
| quantity | Overall, 57 patients (58.8%) had progressive disease (PD), 20 patients (20.6%) had a partial response (PR) and 20 patients (20.6%) had stable disease (SD). — “Overall, 57 patients (58.8%) had progressive disease (PD), 20 patients (20.6%) had a partial response (PR) and 20 patients (20.6%) had stable disease (SD).” | content_tags.quantities[5] |
| quantity | NLR and dNLR At baseline, 27 (27.8%) patients had a NLR ≥5 and 31 (32%) patients had a dNLR ≥3, the cut-offs most often indicated in the literature. — “NLR and dNLR At baseline, 27 (27.8%) patients had a NLR ≥5 and 31 (32%) patients had a dNLR ≥3, the cut-offs most often indicated in the literature.” | content_tags.quantities[6] |
| quantity | In order to better investigate the role of NLR and dNLR, the optimal cutoffs were derived by maximizing the significance assessed by the log-rank test; these values were 4.7 for NLR and 3.8 for dNLR. — “In order to better investigate the role of NLR and dNLR, the optimal cutoffs were derived by maximizing the significance assessed by the log-rank test; these values were 4.7 for NLR and 3.8 for dNLR.” | content_tags.quantities[7] |
| quantity | In multivariate analysis, only NLR (HR = 2.85; 95% CI: 1.60–5.08; P < 0.0001) and LDH (HR = 2.51; 95% CI: 1.36–4.64; P < 0.0001) maintained a significant association with OS (Table 2 ). — “In multivariate analysis, only NLR (HR = 2.85; 95% CI: 1.60–5.08; P < 0.0001) and LDH (HR = 2.51; 95% CI: 1.36–4.64; P < 0.0001) maintained a significant association with OS (Table 2 ).” | content_tags.quantities[8] |
| definition | NLR — “NLR was calculated as NLR = ANC/ALC and dNLR was calculated as dNLR = ANC/(WBC − ALC).” | content_tags.definitions[0] |
| definition | dNLR — “NLR was calculated as NLR = ANC/ALC and dNLR was calculated as dNLR = ANC/(WBC − ALC).” | content_tags.definitions[1] |
| definition | progression-free survival — “Progression-free survival (PFS) was determined from the first cycle of treatment to disease progression documented by imaging, or death (event), or last follow-up (censored).” | content_tags.definitions[2] |
| definition | overall survival — “Overall survival (OS) was calculated from the first cycle of treatment to death (event) or last follow-up (censored).” | content_tags.definitions[3] |
| concept | neutrophil-to-lymphocyte ratio — “NLR was calculated as NLR = ANC/ALC and dNLR was calculated as dNLR = ANC/(WBC − ALC).” | concepts[0] |
| concept | progression-free survival — “Progression-free survival (PFS) was determined from the first cycle of treatment to disease progression documented by imaging, or death (event), or last follow-up (censored).” | concepts[1] |
| concept | overall survival — “Overall survival (OS) was calculated from the first cycle of treatment to death (event) or last follow-up (censored).” | concepts[2] |
| concept | nivolumab — “Nivolumab (Opdivo®, Bristol-Myers Squibb) is a fully human IgG4 monoclonal antibody that blocks the PD-1 receptor, a negative regulator of T-cell activity that has been shown to be involved in the control of T-cell immune responses.” | concepts[3] |
| concept | PD-1 — “Nivolumab (Opdivo®, Bristol-Myers Squibb) is a fully human IgG4 monoclonal antibody that blocks the PD-1 receptor, a negative regulator of T-cell activity that has been shown to be involved in the control of T-cell immune responses.” | concepts[4] |
| concept | Cutoff Finder — “The best cutoff was derived using Cutoff Finder software [ 15 ] based on an R routine which optimized the significance of the split between Kaplan-Meier survival curves measured by the log-rank test.” | concepts[5] |
| concept | RECIST — “Tumor assessment was performed at baseline, at week 12, and every 12 weeks thereafter, and clinical response were classified according to response evaluation criteria in solid tumors (RECIST) [ 14 ].” | concepts[6] |
| concept | systemic inflammation — “Systemic inflammation is associated with alterations in peripheral blood leukocytes that can be captured by the neutrophil-to-lymphocyte ratio (NLR) [ 8 ].” | concepts[7] |
Full text — tagged
The reference list is not part of the text we hold for this paper. Our copy was extracted from the publisher's page and the bibliography did not survive that extraction, so the article below ends before its references. Nothing has been removed by us.
Abstract. Background Previous studies have suggested that elevated neutrophil-to-lymphocyte ratioterm (NLRterm·def) is prognostic for worse outcomes in patients with a variety of solid cancers, including those treated with immune checkpoint inhibitors. Methods This was a retrospective analysis of 97 consecutive patients with stage IV melanoma who were treated with nivolumabterm. Baseline NLRterm·def and derived (d) NLRterm·def were calculated and, along with other characteristics, correlated with progression-free survivalterm (PFS) and overall survivalterm (OS) in univariate and multivariate analyses. The best cutoff values for NLRterm·def and dNLRterm·def were derived using Cutoff Finderterm software based on an R routine which optimized the significance of the split between Kaplan-Meier survival curves. Results In univariate analysis, increasing absolute neutrophil count (ANC), NLRterm·def, dNLRterm·def and lactate dehydrogenase (LDH) (continuous variables) were all significantly associated with OS. Only NLRterm·def (hazard ratio [HR] = 2.85; 95% CI 1.60–5.08; p < 0.0001) and LDH (HR = 2.51; 95% CI 1.36–4.64; p < 0.0001) maintained a significant association with OS in multivariate analysis. Patients with baseline NLRterm·def ≥5 had significantly worse OS and PFS than patients with NLRterm·def < 5, as did patients with baseline dNLRterm·def ≥3 versus < 3. Optimal cut-off values were ≥ 4.7 for NLRterm·def and ≥ 3.8 for dNLRterm·def. Using this ≥4.7 cut-off for NLRterm·def, the values for OS and PFS were overlapping to the canonical cut-off for values, and dNLRterm·def< 3.8 was also associated with better OS and PFS. Conclusion Both Neutrophil-to-lymphocyte ratioterm (NLRterm·def) and derived (d) NLRterm·def were associated with improved survival when baseline levels were lower than cut-off values. NLRterm·def and dNLRterm·def are simple, inexpensive and readily available biomarkers that could be used to help predict response to immunotherapy in patients with advanced melanoma. Electronic supplementary material The online version of this article (10.1186/s40425-018-0383-1) contains supplementary material, which is available to authorized users. Background Improved understanding of cancer and the role of the immune response has resulted in the development of new therapies, including immune checkpoint inhibitors targeting the cytotoxic T-lymphocyte-associated protein (CTLA)-4 (e.g. ipilimumab) and programmed death (PD)-1 receptors (e.g. nivolumabterm and pembrolizumab). These agents have revolutionized the treatment and outcomes of various cancers, in particular melanoma, with improved long-term disease control and prolonged patient survival [ 1 , 2 ]. Nivolumabterm (Opdivo®, Bristol-Myers Squibb) is a fully human IgG4 monoclonal antibody that blocks the PD-1term receptor, a negative regulator of T-cell activity that has been shown to be involved in the control of T-cell immune responses. Engagement of PD-1term with its ligands PD-L1 or PD-L2 expressed on antigen-presenting cells and on tumor cells results in inhibition of T-cell proliferation and cytokine secretion, potentiating T-cell responses. Nivolumabterm has demonstrated clinical activity in previously treated and treatment-naïve patients with advanced melanoma, with overall response rates (ORR) ranging between 20 and 40% in patients treated at various dose levels [ 3 – 5 ]. Nevertheless, a significant portion of patients do not benefit from nivolumabterm treatment. Given that immunotherapy can be associated with significant toxicity as well as high treatment costs, it is important that those patients most likely to respond to treatment are better identified. To achieve this involves the recognition and validation of novel biomarkers predictive of treatment outcomes. Any such biomarkers should ideally be simple and easy-to-use, inexpensive and readily available. In recent years, it has become evident that cancer-related inflammation responses, such as increased and defective myelopoiesis, and both local and systemic inflammationterm, have an important role in tumorigenesis, disease progression and patient prognosis [ 6 , 7 ]. Therefore, predictive biomarkers that reflect the inflammatory response to treatment may help clinical decision making in the management of patients with melanoma. Systemic inflammationterm is associated with alterations in peripheral blood leukocytes that can be captured by the neutrophil-to-lymphocyte ratioterm (NLRterm·def) [ 8 ]. Several studies have demonstrated that NLRterm·def predicts outcomes in patients with a variety of solid cancers [ 9 , 10 ]. In melanoma, raised leukocyte, neutrophil and monocyte counts, as well as high NLRterm·def have been associated with poor prognosis in patients with advanced melanoma, most of whom were receiving immunotherapies [ 11 , 12 ]. Furthermore, in a recent Italian study, elevated NLRterm·def and derived (d) NLRterm·def were independently and significantly associated with an increased risk of death and disease progression in melanoma patients receiving ipilimumab [ 13 ]. This retrospective study investigates the role of the NLRterm·def and dNLRterm·def as predictive markers of response to treatment with nivolumabterm in patients with advanced melanoma. Methods Patients selection A total of 97 consecutive patients with stage IV melanoma who were treated with nivolumabterm 3 mg/kg in a 60-min intravenous (IV) infusion every 2 weeks at the NCI Fondazione “G. Pascale” of Napoli between February 2015 and February 2017 had available hematological values and were included in this analysis. For all patients, clinical data (white blood cell [WBC] count, absolute lymphocyte count [ALC], absolute neutrophil count [ANC], serum lactate dehydrogenase [LDH], and other clinical data, including BRAF status, brain metastasis, lines of prior treatment, and response) were collected before starting nivolumabterm treatment and until last follow-up. Patients were treated with nivolumabterm until disease progression, intolerable toxicity and/or the investigator’s decision. Tumor assessment was performed at baseline, at week 12, and every 12 weeks thereafter, and clinical response were classified according to response evaluation criteria in solid tumors (RECISTterm) [ 14 ]. Statistical analysis Descriptive statistics were used to summarize patient and treatment characteristics. NLRterm·def was calculated as NLRterm·def = ANC/ALC and dNLRterm·def was calculated as dNLRterm·def = ANC/(WBC − ALC). Progression-free survivalterm (PFS) was determined from the first cycle of treatment to disease progression documented by imaging, or death (event), or last follow-up (censored). Overall survivalterm (OS) was calculated from the first cycle of treatment to death (event) or last follow-up (censored). Multivariate Cox proportional hazard models were used to investigate associations of NLRterm·def with survival, adjusted for baseline characteristics. Results were presented as hazard ratios (HR) with 95% confidence intervals (CIs). The best cutoff was derived using Cutoff Finderterm software [ 15 ] based on an R routine which optimized the significance of the split between Kaplan-Meier survival curves measured by the log-rank test. Statistical analyses were performed with IBM-SPSS version 21.0. Results Baseline patient characteristics are summarized in Table 1 . The median age at first treatment was 61 years (range 21–85) and 56.7% of patients were male. A total of 27 patients (27.8%) had CNS metastases. BRAF-mutated melanomas were present in 38 patients (39.1%); 54 (55.7%) had BRAF wild-type tumors. Twenty-five patients (25.8%) had received one previous line of therapy, 48 (49.5%) had two lines and 24 (24.7%) had ≥3 lines. Table 1 Baseline patient characteristics N = 97 Gender, N (%), Female/Male 42 (43.2) / 55 (56.7) Age, years, median (range) 61 (21–85) Number of previous therapies, N (%) 1 25 (25.8) 2 48 (49.5) ≥ 3 24 (24.7) Central nervous system (CNS) metastasis 27 (27.8) Baseline lactate dehydrogenase, N (%) ≤ upper limit of normal (ULN) 50 (51.5) > ULN 45 (46.4) Unknown 2 (2.1) B-RAF, N (%): Mutation 38 (39.1) Wild-Type 54 (55.7) Unknown 5 (5.2) As of May 2017, 44 patients (45.4%) were still alive. The median PFS was 4 months (range 0–26) and median OS was 6 months (range 2–26); about 10% of patients had started therapy no more than 6 months earlier. There was no significant difference in OS by BRAF status ( P = 0.65). Overall, 57 patients (58.8%) had progressive disease (PD), 20 patients (20.6%) had a partial response (PR) and 20 patients (20.6%) had stable disease (SD). The disease control rate (DCR) was 41.2%. At baseline, median WBC was 8620/μL cells in patients who had PD, 7540/μL in patients with SD and 6645/μL in patients with PR (median 7.000/μL in patients with disease control). A total of 22 patients (22.7%) had neutrophilia (ANC > 7.5) before the start of treatment. ANC In univariate analysis, elevated ANC (> 8000) was associated with poor prognosis, with a median of OS of 2.6 months compared to 16.0 months in patients with normal ANC ( P < 0.0001). Using median ANC as cut-off value, median OS was 16 months (95% CI: 7.6–24.4) in patients with ANC < 5.4 compared to 5.7 months (95% CI: 2.6–8.8) in patients with ANC ≥5.4 (HR 2.04; 95% CI: 1.17–3.57; P = 0.01). Median PFS was also increased in patients with ANC < 5.4 compared with ANC ≥5.4 (8 months [95% CI: 1.6–14.4] versus 3 months [95% CI:1.5–4.5]) but this was not statistically significant (HR 1.60; 95% CI: 0.98–2.61; P = 0.06) (See Additional files 1 and 2 ). NLRterm·def and dNLRterm·def At baseline, 27 (27.8%) patients had a NLRterm·def ≥5 and 31 (32%) patients had a dNLRterm·def ≥3, the cut-offs most often indicated in the literature. Using Harrell’s c-index to determine the prognostic power of NLRterm·def and dNLRterm·def, we obtained a good prognostic value for both, with c = 0.72 (95% CI: 0.67–0.77) for NLRterm·def and c = 0.70 (95% CI: 0.65–0.75) for dNLRterm·def. PFS When a baseline NLRterm·def value of 5 was used as the cut-off, patients with NLRterm·def < 5 ( n = 70; 72.2%;) had a significantly longer median PFS of 9 months (95% CI: 2.4–15.6) compared to patients with NLRterm·def ≥5 (median PFS of 2 months, 95% CI: 1.0–3.0; P < 0.0001) (Fig. 1a ). Patients with baseline dNLRterm·def < 3 ( n = 66; 68%) had a median PFS of 9 months (95% CI: 2.1–15.9) while patients with dNLRterm·def ≥3 ( n = 31; 32%) had a median PFS of 3 months (95% CI: 1.8–4.2) ( P = 0.001) (Fig. 1b ). Fig. 1 Kaplan-Maier PFS curves of melanoma patient treated with nivolumabterm. a Patients stratified according baseline neutrophils-to lymphocyte ratio (NLRterm·def). Green line: NLRterm·def ≥ 5; Blue line: NLRterm·def < 5. b Patients stratified according baseline derived neutrophils-to lymphocyte ratio (dNLRterm·def). Green line: dNLRterm·def≥3; Blue line: dNLRterm·def< 3 OS Median OS was 2.9 months (95% CI: 1.5–4.3) for patients with baseline NLRterm·def ≥5 compared with 16 months for patients with NLRterm·def < 5 (95% CI: 7.5–24.5) ( P < 0.0001) ( Fig. 2a ). Using dNLRterm·def ≥3 as cut-off the difference was significant, with a median OS of 16.0 months (95% CI: 8.2–23.7) for patients with dNLRterm·def < 3 and 3.1 months (95% CI: 1.4–4.8) for patients with dNLRterm·def ≥3 ( P < 0.0001) (Fig. 2b ). Fig. 2 Kaplan-Maier overall survivalterm curves of melanoma patient treated with nivolumabterm. a Patients stratified according baseline neutrophils-to lymphocyte ratio (NLRterm·def). Green line: NLRterm·def ≥ 5; Blue line: NLRterm·def < 5. b Patients stratified according baseline derived neutrophils-to lymphocyte ratio (dNLRterm·def). Green line: dNLRterm·def≥3; Blue line: dNLRterm·def< 3 Response The proportion of patients with an NLRterm·def ≥5 was 20% (4/20) in patients with a PR, 5% (1/20) in patients with with SD and 38.6% (22/57) in patients with PD. In order to better investigate the role of NLRterm·def and dNLRterm·def, the optimal cutoffs were derived by maximizing the significance assessed by the log-rank test; these values were 4.7 for NLRterm·def and 3.8 for dNLRterm·def. The performance of an NLRterm·def cut-off value of 4.7 was overlapping with 5 as cut-off value, Median PFS and OS were also significantly longer in patients with dNLRterm·def < 3.8 compared to ≥3.8 (see Additional files 1 and 2 ). In the subgroup of 27 patients with brain metastases, NLRterm·def < 4.7 and dNLRterm·def < 3.8 were both significantly associated with improved OS and PFS in univariate analysis. However, in multivariate analysis, only NLRterm·def < 4.7 was associated with significantly longer OS (HR 3.22, 95% CI: 0.99–10.51) and PFS (HR 2.55, 95% CI: 0.98–6.65) (See Additional files 1 and 2 ). Univariate analysis using a Cox model was performed using median values as cut-off for WBC, ANC, and LDH; these three variables together with NLRterm·def and dNLRterm·def were all significantly associated with OS, while there was no significant association with sex, age, number of lines of treatment, presence of brain metastases or BRAF mutation status. In multivariate analysis, only NLRterm·def (HR = 2.85; 95% CI: 1.60–5.08; P < 0.0001) and LDH (HR = 2.51; 95% CI: 1.36–4.64; P < 0.0001) maintained a significant association with OS (Table 2 ). When considering PFS, LDH, NLRterm·def and dNLRterm·def showed a prognostic role in univariate analysis while, in multivariate analysis, associations were only observed for NLRterm·def (HR = 2.10; 95% CI: 1.23–3.59; P = 0.007) and LDH (HR = 1.74; 95% CI: 1.03–2.94; P = 0.04) (Table 2 ). Table 2 Cox regression models on overall survivalterm and progression-free survivalterm Overall survivalterm Progression-free survivalterm Factors Univariate HR, 95% CI, P -value Multivariate HR, 95% CI, P -value Univariate HR, 95% CI, P -value Multivariate HR, 95% CI, P -value Sex (M vs F) 1.71 (0.96–3.05), P = 0.07 1.35 (0.82–2.21) P = 0.24 Age (years) 1.00 (0.98–1.02), P = 0.72 1.00 (0.98–1.01) P = 0.74 Lines of treatment: P = 0.55 P = 0.63 2 vs 1 1.23 (0.57–2.64) 1.06 (0.56–1.99) ≥ 3 vs 1 1.56 (0.69–3.55) 1.35 (0.67–2.73) BRAF (mutant vs wild-type) 1.14 (0.64–2.04), P = 0.65 1.32 (0.80–2.19) P = 0.28 Brain metastases (yes vs no) 1.03 (0.56–1.87), P = 0.93 1.30 (0.77–2.18) P = 0.32 Lactate dehydrogenase (≥454 vs < 454) 3.06 (1.69–5.57), P < 0.0001 2.51 (1.36–4.64), P = 0.003 2.16 (1.18–3.99) P = 0.01 1.74 (1.03–2.94) P = 0.04 Absolute neutrophil count (≥5.4 vs < 5.4) 2.04 (1.17–3.57), P = 0.01 1.60 (0.98–2.61) P = 0.06 White blood cell count (≥7.6 vs < 7.6) 1.57 (0.91–2.73), P = 0.11 1.55 (0.95–2.54) P = 0.08 Neutrophil-to-lymphocyte ratioterm (≥5.0 vs < 5.0) 3.53 (2.02–6.16), P < 0.0001 2.85 (1.60–5.08), P < 0.0001 2.53 (1.53–4.18) P < 0.0001 2.10 (1.23–3.59) P = 0.007 Derived neutrophil-to-lymphocyte ratioterm (≥3.0 vs < 3.0) 2.70 (1.55–4.69), P < 0.0001 2.50 (1.48–4.23) P = 0.001 These two factors can be used to stratify patients into three groups: both elevated NLRterm·def (≥5.0) and elevated LDH (≥455 mL/U) with a median OS of 2.3 months (95% CI: 1.3–3.2); either elevated NLRterm·def (≥5.0) or elevated LDH (≥455 mL/U) with a median OS of 6.9 months (95% CI: 3.9–9.9) and normal NLRterm·def and LDH (median OS is not yet reached, 1-year OS 72.5%) (Fig. 3 ). Fig. 3 Kaplan-Maier OS curves of melanoma patients treated with nivolumabterm and stratified according to LDH and NLRterm·def (LDH cutoff = 455 mL/U; NLRterm·def cutoff = 5). Green line: only one factor below cutoffs; Yellow line: both factors over cutoffs; Blue line: Both factors below cutoffs Discussion Melanoma is an aggressive cancer with frequent relapse and a high potential for metastasis. Nevertheless, in recent years, the range of therapeutic options for melanoma has been greatly improved with the introduction of immunomodulatory antibodies as well as molecular targeted drugs [ 16 ]. These agents have shown promising results, but not all patients benefit and toxicity can be a significant concern. Because of this, it is important to be able to identify which patients are most likely to benefit from treatment. In recent years, NLRterm·def has been extensively studied as a prognostic marker in patients with solid tumors on the basis that it reflects the inflammatory response to cancer. In patients with advanced cancer, there is usually a change in peripheral blood cell composition that is suggestive of an expansion in the myeloid component (neutrophils and monocytes) and a reduction in the lymphoid compartment. A number of studies have shown that systemic inflammationterm markers are associated with the outcome of malignant melanoma patients, including those treated with ipilimumab [ 13 , 17 , 18 ]. In 69 patients with metastatic melanoma, pre-treatment NLRterm·def was identified as an independent marker for response in multivariate analyses, with patients with baseline NLRterm·def ≥5 having significantly worse PFS and OS compared with those with a NLRterm·def < 5 [ 18 ]. Similarly, a significant association between elevated NLRterm·def and worse survival was significant after adjustment for potential confounders in an analysis of 58 patients treated with ipilimumab [ 19 ]. In another study of patients with unresectable stage III-IV melanoma, elevated NLRterm·def (≥5) was associated with worse OS, PFS, and clinical response [ 20 ]. In addition, increasing NLRterm·def from baseline during treatment was correlated with worse OS and PFS in patients treated with ipilimumab but not those treated with vemurafenib, suggestive of a predictive value for response to immunotherapy but not targeted drugs. In another recent study, in 162 melanoma patients, 22.5% receiving ipilimumab, elevated baseline dNLRterm·def (≥3) was independently and significantly associated with an increased risk of death and disease progression [ 13 ]. Elevated baseline NLRterm·def ≥ 5 has also been associated with worse outcomes in patients treated with nivolumabterm, although this was in non-small-cell lung cancer (NSCLC) rather than melanoma [ 21 ]. In a retrospective analysis of 175 patients with previously treated advanced NSCLC, NLRterm·def ≥5 was independently associated with reduced OS (median 5.5 vs. 8.4 months; HR 2.07, 95% CI: 1.3–3.3; p = 0.002) and PFS (median 1.9 vs. 2.8 months; HR 1.43, 95% CI: 1.02–2.0; p = 0.04). Data from the Italian nivolumabterm extended access program (EAP) has also reported that NLRterm·def (≥3) was a prognostic factor for OS in patients with metastatic renal cell cancer treated with nivolumabterm [ 22 ]. In this study, which is the first to report an association between NLRterm·def and outcomes in patients with advanced melanoma treated with nivolumabterm, we analyzed the predictive power of baseline NLRterm·def and dNLRterm·def in a small retrospective cohort of patients. Despite the small number of patients, both NLRterm·def and dNLRterm·def were good predictors of survival when baseline levels were lower than cut-off values. The optimal cut-off values were shown to be ≥4.7 for NLRterm·def and ≥ 3.8 for dNLRterm·def, with the NLRterm·def cut-off value a more robust predictor than the dNLRterm·def value. NLRterm·def was predictive of survival independent of BRAF status (data not shown). Lower baseline LDH also correlated with a better response and survival, consistent with several previous studies [ 23 – 25 ], which was maintained in multivariate analysis. Patients with two or three risk factors of elevated NLRterm·def, ANC and LDH had worse survival than patients with none or just one of these factors alone. These results suggest that the systemic inflammatory response is a potent stimulator of cancer progression in established disease. Elevated NLRterm·def suggests a systemic inflammatory state and can be indicative of neutrophilia, lymphopenia, or a combination of both. The biggest limitation of this study is the low number of patients that we have analyzed and validation is required in larger prospective studies. The benefits that can be gained with the validation of these parameters may be significant because they offer the potential for a low-cost, non-invasive and easy-to-read test that can be used to help define the patient’s status, prognosis, and response to therapeutic treatment. Conclusion Neutrophilia can occur in cancer patients at a peripheral level, but neutrophils can also localize to the tumor due to multiple factors, including general inflammatory signals such as IL-1 and TNF-α [ 26 ]. Although many mechanisms are unclear, neutrophils are thought to primarily propagate a pro-tumor environment promoting angiogenesis and tumor growth, degrading the extracellular matrix and providing favorable conditions for metastasis, and potentiating genome instability and tumor evolution [ 27 ]. A recent study also demonstrated that the expression of PD-L1 on tumor infiltrating neutrophils could functionally inhibit the activation of T cells [ 28 ]. Lymphopenia in the context of cancer also suggests more aggressive disease progression. For these reasons, whether due to neutrophilia or lymphopenia, elevated NLRterm·def physiologically suggests an inability of the immune system to suppress cancer progression, and thus it can be an index for OS and PFS. Because the NLRterm·def and dNLRterm·def are obtained from complete blood counts, their use does not involve additional procedures for patients or extra resources and associated costs for healthcare providers. If it can be shown that NLRterm·def and dNLRterm·def are predictors of response to immunotherapy as well as being prognostic markers, their use in clinical practice could allow better patient selection and optimal clinical management. Additional files Additional file 1: Table S1A. Cox regression models on overall survivalterm in patients with brain metastases (DOCX 15 kb) Additional file 2: Figure S1. Kaplan-Maier OS and PFS curves of melanoma patient treated with nivolumabterm. (A) Patients stratified according baseline median ANC as cutoff . Green line: ANC≥5.4; Blue line: ANC<5.4. (B) Patients stratified for PFS according baseline median ANC as cut-off . Green line: ANC≥5.4; Blue line: ANC<5.4. Figure S2. Kaplan-Maier OS and PFS curves of melanoma patient treated with nivolumabterm using optimal cutoff for derived neutrophils-to lymphocyte ratio (dNLRterm·def). (A)) Patients stratified according baseline dNLRterm·def. Green line: dNLRterm·def≥3.8; Blue line: dNLRterm·def<3.8. (DOCX 168 kb)