CC BY
12UDC: 616.24.006.
Nishanov Donier Anorbaevich
MD, DSC., Head of Department of Pathomorphology of the Republican specialized Scientific and Practical Medical Center of Oncology and Radiology of the
Ministry of Health of the Republic of Uzbekistan, Tashkent DOI: 10.24411/2520-6990-2019-11178
INCREASED TUMOR-INFILTRATING LYMPHOCYTES IN PATIENTS WITH SCLC AND NEUROLOGIC PARANEOPLASTIC SYNDROMES
Abstract
Background: Approximately 10% of patients with SCLC develop a paraneoplastic syndrome (PNS). Neurologic PNS are thought to improve prognosis, which we hypothesized is related to increased tumor-infiltrating lymphocytes and immune recognition.
Methods: We queried 2,512,042 medical records from a single institution to identify patients who have SCLC with and without PNS and performed manual, retrospective chart review. We then performed multiplexed fluorescence immunohistochemistry and automated quantitative analysis (AQUA Technology) on tumors to assess CD3, CD4, and CD8 T cell infiltrates and programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) interactions. T cell infiltrates and PD-1/PD-L1 interaction scores were compared among patients with neurologic PNS, endocrinologic PNS, and a control group without PNS. Clinical outcomes were analyzed using the Kaplan-Meier method and Cox proportional hazards models.
Results: We evaluated 145 SCLC patients: 55 with PNS (25 neurologic and 30 endocrinologic) and 90 controls. Patients with neurologic PNS experienced improved overall survival compared to patients with endocrino-logic PNS and controls (median overall survival of 24 months versus 12 months versus 13 months, respectively). Of the 145 patients, we identified tumor tissue from 34 patients that Journal of Thoracic Oncology Vol. 14 No. 11: 1970-1981 was adequate for AQUA analysis. Among 37 specimens from these 34 patients, patients with neurologic PNS had increased T cell infiltrates (p % 0.033) and PD-1/PD-L1 interaction (p % 0.014) compared to tumors from patients with endocrinologic PNS or controls.
Conclusions: Tumor tissue from patients with SCLC with neurologic PNS showed increased tumor-infiltrating lymphocytes and PD-1/PD-L1 interaction consistent with an inflamed tumor microenvironment.
Аннотация
Введение: приблизительно у 10% пациентов с SCLC развивается паранеопластический синдром (ПНС). Считается, что неврологические ПНСулучшают прогноз, который, как мы предположили, связан с увеличением количества инфильтрирующих опухоль лимфоцитов и распознаванием иммунитета.
Методы: мы запросили 2 512 042 медицинских карт из одного учреждения, чтобы определить пациентов, у которых был SCLC с и без ПНС, и выполнили ручной ретроспективный обзор. Затем мы провели мультиплексную флуоресцентную иммуногистохимию и автоматический количественный анализ (технология AQUA) опухолей для оценки инфильтратов CD3, CD4 и CD8 Т-клеток и запрограммированных взаимодействий смерти 1 (PD-1) /запрограммированной смерти лиганда 1 (PD-L1). Т-клеточные инфильтраты и оценки взаимодействия PD-1 / PD-L1 сравнивались у пациентов с неврологическим PNS, эндокринологическим PNS и контрольной группой без PNS. Клинические результаты были проанализированы с использованием метода Каплана-Мейера и моделей пропорциональных рисков Кокса.
Результаты: Мы оценили 145 пациентов с SCLC: 55 с ПНС (25 неврологических и 30 эндокринологических) и 90 контрольных. У пациентов с неврологическим ПНС общая выживаемость улучшилась по сравнению с пациентами с эндокринологическим ПНС и контрольной группой (медиана общей выживаемости 24 месяца против 12 месяцев против 13 месяцев соответственно). Из 145 пациентов мы определили опухолевую ткань у 34 пациентов, которые опубликовали в журнале Thoracic Oncology Vol. 14 № 11: 1970-1981 было достаточно для анализа AQUA. Среди 37 образцов от этих 34 пациентов, у пациентов с неврологическим PNS наблюдалось увеличение инфильтратов Т-клеток (p 0,033) и взаимодействия PD-1 / PD-L1 (p 0,014) по сравнению с опухолями пациентов с эндокринологическим PNS или контролем.
Выводы. Опухолевая ткань от пациентов с SCLC с неврологическим PNS показала увеличение инфильтрирующих опухоль лимфоцитов и взаимодействие PD-1 / PD-L1 в соответствии с микроокружением воспаленной опухоли.
Key words: Paraneoplastic syndromes; Tumor infiltrating lymphocytes; Immunotherapy; Tumor microenvironment.
Ключевые слова: Паранеопластические синдромы; Опухолевые инфильтрирующие лимфоциты; иммунотерапия; Опухолевая микросреда.
Introduction
SCLC accounts for 15% of lung cancers and 30,000 deaths in the United States annually. [1] The median overall survival (OS) for SCLC patients is approximately 8 to 12 months for patients with extensive-
stage SCLC disease and 12 to 20 months for patients with limited-stage disease (LS-SCLC). [1] The combination of elusive pathophysiology, poor prognosis, and minimal therapeutic improvement for several decades has led the National Cancer Institute to designate SCLC
as a recalcitrant cancer. [2]
Among patients with SCLC, approximately 10% develop a paraneoplastic syndrome (PNS), such as Lambert-Eaton myasthenic syndrome (LEMS), syndrome of inappropriate antidiuretic hormone (SIADH), or Cushing syndrome. [3-5] LEMS is a neurologic PNS, which is thought to be an immune-mediated phenomenon. [6] In contrast, endocrinologic PNS, such as SIADH and Cushing syndrome, reflect ectopic tumor hormonal secretion. [6,7]
Prognostic differences have been observed in small case series among patients with SCLC with en-docrinologic PNS, neurologic PNS, or no PNS. Patients with SCLC with endocrinologic PNS, especially Cush-ing syndrome, have shown a worse prognosis than patients with SCLC with no PNS.8 Similarly, patients with SCLC with neurologic PNS have improved OS compared to patients with SCLC with no PNS.[9-11] We sought to extend these observations in one of the largest series reported in patients with SCLC with neurologic PNS to date.
Furthermore, we evaluated tumor immune micro-environmental factors that contribute to unique outcomes in patients with SCLC with neurologic PNS by investigating both lymphocytic tumor infiltrates and programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) interactions. Blocking negative regulatory immune checkpoints, such as PD-1 and PD-L1, has revolutionized cancer care in many tumor subtypes, and these treatments have shown promise in patients with SCLC.[12-15] However, in unselected populations of patients with SCLC, less than 20% have greater than 1% tumor PD-L1 positivity, and better tumor immune recognition biomarkers are needed in this population. [13] We hypothesized that in tumors from patients with SCLC with neurologic PNS, a surrogate for adequate tumor immune recognition, tumor-infiltrating lymphocytes are increased and PD-1 and PD-L1 are up-regulated. To our knowledge, this is the first study to evaluate tumor PD-L1 staining in patients with SCLC with neurologic PNS, and it is the first to directly compare tumor tissue from patients with SCLC with neurologic PNS to tumor tissue from patients with SCLC with endocrinologic PNS.
Methods
Study Design and Patients An automated search algorithm of 2,512,042 patients in the Vanderbilt University Medical Center
(VUMC) electronic medical record was performed to identify cases with SCLC with neurologic or endocrin-ologic PNS, as well as control patients with SCLC without a PNS. There are no dedicated billing codes for SCLC or PNS. As a result, our search algorithm used free-text search of all clinical notes in the electronic medical record to flag potential patients, cross-referenced with cancer registry-designated SCLC cases.
Patient Cohort Validation Through manual chart review we validated 145 patients with SCLC over a 16-year time span, from September 1998 to May 2014 under institutional review board (IRB) - approved protocol #141343. Case validation was based on the following definitions. The
diagnosis of SIADH was defined by serum sodium of less than 135 mmol/L refractory to intravenous fluid administration with urine osmolarity greater than 100 and congruent clinical documentation noting SIADH. The diagnosis of Cushing syndrome was defined by an elevated adrenocorticotropic hormone with hypokale-mia and congruent clinical documentation noting Cush-ing syndrome. The laboratory diagnosis of a neurologic PNS involved serologic, and rarely cerebrospinal fluid testing, for the presence of antineuronal nuclear antibodies type 1 (also known as anti-Hu antibody), 2, and 3, Purkinje cell cytoplasmic antibodies types 1, 2, and Tr, striational antibody, N-type calcium channel binding antibody, P/Q type calcium channel antibody, voltage gated calcium channel antibody, voltage gated potassium channel antibody, CRMP-5 IgG antibody, ace-tylcholine receptor (muscle) antibody, acetylcholine receptor (ganglionic) neuronal antibody, and am-phiphysin antibody. If patients had one positive serology for a paraneoplastic autoantibody in conjunction with a neurology consultation diagnosis or, in five cases, a neurologic consultation without a positive se-rology, they were classified as having neurologic PNS. Seven patients had both neurologic PNS and SIADH, all seven had serum paraneoplastic autoantibodies assessed and six of seven were positive; these seven patients were included only in the neurologic PNS group as our primary hypothesis centered on the presence or absence of unique tumor immune recognition in the setting of any neurologic PNS. Any patients meeting the aforementioned criteria for a neurologic or endocrinologic PNS with histologic confirmation of SCLC were included. Control patients were defined as those patients with a histologically confirmed diagnosis of SCLC, no criteria for an endocrinologic or neurologic PNS, and clinical Abstract recording treatment modality, disease progression, and date of death. Patients were only excluded if they lacked sufficient histologic documentation of SCLC, diagnosis of a PNS as defined above, or, in control patients, sufficient clinical Abstract as described above.
Multiplexed Fluorescence Immunohistochemistry
Formalin-fixed, paraffin-embedded samples were obtained from the VUMC pathology archives for patients with a neurologic PNS, endocrinologic PNS, or no PNS under an IRB-approved protocol (IRB# 160769). We performed multiplexed fluorescence im-munohistochemistry combined with automated quantitative analysis (AQUA Technology, Navigate Bio-Pharma Services, Inc., Carlsbad, California) to assess CD3, CD4, CD8, PD-1, and PD-L1 expression as previously described.16 Two slides were used, one to assess CD3, CD4, and CD8, and one to assess PD-1 and PD-L1. Staining for CD3, CD4, and CD8 was excluded for cytology specimens. The following primary antibodies were used: rabbit anti-CD3 (EP41, dilution 1:200, Bio-care Medical), mouse anti-CD4 (4B12, dilution 1:50, DAKO), mouse anti-CD8 (C8/144B, 1:400, DAKO), 0.5 mg/mL mouse anti-PD-1 (NAT105, Biocare), 3.6 mg/mL rabbit anti-PD-L1 (E1L3N, Cell Signaling Technology), and mouse anti-TTF1 (8G7G31, 1:500, DAKO). The following secondary antibodies were
used: anti-mouse Envision HRP (DAKO) and anti-rabbit Envision HRP (DAKO), plus 40,6-diamidino-2phenylindole (DAPI). The following reagents were used to detect secondary antibodies: TSA-Cy3.5 (Per-kin Elmer), TSA-Cy5 (Perkin Elmer), Opa 520 (Perkin Elmer), and TSA-Cy3 (Perkin Elmer).
Fluorescence Immunohistochemistry Image Analysis
Forty high-power (20) fluorescence images for each sample (one slide per sample/per test as described above) were acquired with the Vectra 2 Intelligent Slide Analysis System using Vectra software version 2.0.8 (Perkin Elmer) and quantified with AQUAnalysis software as previously described.[16 ] DAPI was used to identify all cells, and transcription termination factor 1 (TTF1) expression, present in 70% to 90% of SCLC tumors, was used to identify tumor cells.[17] Each pixel in the image was identified as either positive or negative for the signal of interest to create a binary mask for each cell type. Similarly, the CD3, CD4, CD8, PD-1, and PD-L1, expression co-localized with DAPI-positive or TTF1positive cells was used to create binary masks of all cells expressing these biomarkers of interest, respectively. Percent positivity was calculated from the total area, measured in pixels, for the cell type of interest divided by the total area, measured in pixels, of the corresponding cells of interest (all cells, tumor cells, or T cells [CD3 positive]). Tumor scoring of T cell infiltrates and PD-1/PD-L1 was completed by pathologists blinded to patient clinical characteristics. T cell subtypes (CD4- or CD8-positive) were defined by double-positivity for CD3/ CD4 or CD3/CD8. The PD-1/PD-L1 interaction score was calculated by measuring the total area of PD-1-positive cells within the proximity of PD-L1-positive cells. This area was then divided by the total area of all nontumor nucleated cells in the image and multiplied by a factor of 10,000. The interaction score provides a numerical reflection of the overall proportion of PD-1-positive cells within an approximately one-cell distance to PD-L1- positive cells. The interaction score algorithm was designed based on the hypothesis that coexistence of two markers will be a better measure of immunosuppression than either PD1 or PD-L1 alone. This novel algorithm was shown to predict response to anti-PD-L1 therapy in metastatic melanoma akin to the PD1/L1 proximity score reported by an independent study. [18,19] Similarly, the utility of interaction score to predict resistance to chimeric antigen receptor T cell (CAR-T) therapy in a prospective diffuse large b cell lymphoma (DLBCL) study and response to chemotherapy in a randomized NSCLC cohort have been reported. [20,21] Maximum PD-L1 score was defined as the PD-L1 score for the field of view with the highest percentage of tumor cells expressing PD-L1 out of all the 20 fields of view measured per sample.
Statistical Analysis
Patient demographic and clinical characteristics were summarized using median with interquartile range for continuous variables or frequency with percentages for categorical variables. The standardized CD3, CD4, Table 1
CD8 cell counts (of all cells) and PD-1/PD-L1 interaction scores were compared using a Kruskal-Wallis test among endocrinologic-, neurologic- and non-PNS groups and Mann-Whitney tests for pairwise comparisons. Progression-free survival (PFS) was defined as radiographic disease growth on surveillance computed tomography or death, and OS was graphically represented using the Kaplan-Meier method and compared using a Log rank test. Multivariable Cox proportional hazards models were used to assess the associations of PNS type on survival outcomes while adjusting for age and disease stage. Hazard ratios (HRs) and 95% confidence intervals (CIs) were reported. Statistical significance was considered with a two-sided alpha less than 0.05. Statistical analyses were performed using R software (version 3.4.3).
Results
Patient Demographics
A total of 145 SCLC patients were identified, 55 with a PNS (25 neurologic and 30 endocrinologic) and 90 control patients without a PNS. The date range of SCLC diagnosis was similar among the groups, with each containing patients diagnosed from 1998 to 2014. The demographic and treatment information for the three patient cohorts is listed in Table 1. A greater proportion of patients with neurologic PNS (56%) had LSSCLC compared to patients with endocrinologic PNS (37%), but this difference was not statistically significant (p V 0.152). Patients were similar in age at diagnosis and extent of smoking history, although there was a notably higher proportion of patients in the neurologic PNS group (24%) that received no systemic therapy for SCLC compared to the endocrinologic PNS (7%) and control (5%) patient cohorts (p V 0.007) (Table 1).
Description of Paraneoplastic Syndromes
Serologic evaluation of SCLC patients who presented with a neurologic disorder was completed using a clinical autoantibody panel. Among the 25 patients with a neurologic PNS, 20 (80%) had a detectable serum paraneoplastic autoantibody. Autoantibody tests from 3 patients were negative, but all 3 patients were clinically diagnosed with a neurologic PNS through consultation with the neurology service. Two patients were not tested for serum paraneoplastic autoantibodies but were clinically documented as having a neurologic PNS upon consultation with a neurologist: one case of autonomic neuropathy and the other with pseudoacha-lasia (Table 2). The most commonly identified serum paraneoplastic autoantibodies were P/Q-type calcium channel antibodies (7 patients) and anti-Hu antibodies (6 patients). The manifestation of a PNS predated the diagnosis of SCLC in nine patients (36%) with neurologic and three patients (10%) with endocrinologic PNS. The longest lag time between PNS development and identification of the underlying SCLC was 12 months in a patient with a neurologic PNS and 6 months in a patient with an endocrinologic PNS. The majority of patients with PNS that manifested before SCLC were diagnosed with SCLC within 1 month.
Baseline Demographics of SCLC Patients with Neurologic PNS, endocrinologist PNS, and Control Patients With no PNS
Neurologic Endocrine Control _PNS (n % 25) PNS (n % 30) (n % 90)
Age at diagnosis (range), years 64 (57-67) 64 (56-73) 61 (54-69)
Male
9 (36)
11 (37%)
47 (52%)
Female 16 (64) 19 (63%) 43 (48%)
Race
Caucasian 25 (100) 30 (100%) 73 (81%)
African American 0 (0) 0 (0%) 10 (11%)
Asian 0 (0) 0 (0%) 1 (1%)
Unspecified 0 (0) 0 (0%) 6 (7%)
Smoking pack-years 55 (48-78) 50 (40-68) 45 (30-70)
Stage at diagnosis
Limited-stage SCLC 14 (56) 11 (37%) 40 (44%)
Extensive-stage SCLC 11 (44) 19 (63%) 50 (56%)
Sites of metastasis Hepatic 3 (12) 11 (37%) 30 (33%)
Brain parenchymal 3 (12) 1 (3%) 12 (13%)
Osseous 2 (8) 12 (40%) 16 (18%)
Pleural 6 (24) 9 (30%) 16 (18%)
Adrenal 2 (8) 6 (20%) 10 (11%)
Mechanism of diagnosis of PNS
Lab with serum paraneoplastic autoantibody 20 (80) 0 (0%) 0 (0%)
Lab without paraneoplastic autoantibody 0 (0) 30 (100%) 0 (0%)
Clinical (neurology consultation) 5 (20) 0 (0%) 0 (0%)
First-line cytotoxic chemotherapy Platinum-based with etoposide Platinum-based with irinotecan Other
No systemic therapy
18 (72) 1 (4) 0 (0) 6 (24)
22 (73%) 2 (7%) 4 (13%) 2 (7%)
83 (92%)
3 (3%) 0 (0%)
4 (5%)
Prophylactic cranial irradiation 14 (56) 15 (50%) 49 (54%)
Concurrent chemoradiation (% of limited stage patients)_
10 (72)
8 (73%)
34 (85%)
Values shown are n (%) unless otherwise stated. PNS, paraneoplastic syndrome.
Parenthetical ranges indicate the lower and upper quartiles for patient age and smoking history, and percent of total for each grouping for the remaining variables.
Clinical Outcomes Most patients in all three groups were treated with first-line platinum plus etoposide (Table 1), and no patients were treated with surgical resection. Six patients in the neurologic PNS cohort did not receive systemic therapy for SCLC: four were lost to follow-up, one died nearly 3 months after SCLC diagnosis; and one died 4.5 months after SCLC diagnosis (Table 1). Including patients who did not receive systemic therapy for SCLC, patients with neurologic PNS experienced a doubling of their OS compared to patients with endocrinologic PNS and controls (median OS 24 months versus 12 months versus 13 months, respectively) (Fig. 1A, Table 2). When evaluating PFS from the date of diagnosis, patients with neurologic PNS experienced a more than two-fold improvement in disease control compared to patients with endocrinologic PNS and controls (median
PFS 14 months versus 6 months versus 7 months, respectively) (Fig. 1B, Table 2). When limited to only patients who received systemic therapy for their SCLC, the differences were further accentuated (OS HR % 0.24, 95% CI: 0.10-0.55, p % 0.007; PFS HR % 0.41, 95% CI: 0.20-0.85, p % 0.015) (Figs. 1C and D). Finally, when controlling for age and stage between patients with SCLC with neurologic PNS and SCLC with endocrinologic PNS, these findings remained statistically significant for both
OS (HR % 0.23, 95% CI: 0.10-0.54, p % 0.006) (Figs. 1A and B) and PFS (HR % 0.43, 95% CI: 0.210.87, p % 0.019) (Figs. 1C and D). There was no statistically significant difference in OS or PFS when patients with endocrinologic PNS or no PNS were compared.
Tumor Tissue CD3/CD4/CD8 and PD-1/PD-L1
Staining
The clinical data described above show that the presence of a neurologic PNS more than doubled median OS and PFS in our cohort. As improved tumor recognition by the host immune system is an increas-
ingly appreciated mechanism of cancer control, we hypothesized that improved clinical outcomes in patients with SCLC with neurologic PNS are related to increased tumorinfiltrating lymphocytes and PD-1/PD-L1 interactions in tumors from SCLC patients with neurologic PNS. We tested this hypothesis through examination of formalinfixed, paraffin-embedded SCLC tumor samples using automated quantitative analysis (AQUA technology) to assess infiltrating CD3-, CD4-, and CD8-positive lymphocytes as well as PD-1/PD-L1 interactions. T cell markers and PD-1/PD-L1 interaction scores were compared among patients with neurologic PNS, endocrinologic PNS, and a control group without PNS.
We evaluated 105 potential tumor specimens from the 145 SCLC patients. Of these potential specimens, 37 specimens from 34 unique patients were adequate for AQUA analysis. No surgically resected tumors were available for analysis, and most tumor specimens were obtained before chemotherapy administration (n V 31, 84%). Of the 37 specimens, 11 (30%) were from nodal metastases, 19 (51%) were from the primary tumor, and 7 (19%) were from other metastatic sites (3 bone, 2 liver, 2 pleural). The maximum tumor PD-L1 positivity observed among 11 specimens from patients with SCLC with endocrinologic PNS was 8%, compared to a maximum tumor PD-L1 expression of 79% among 10 specimens from patients with SCLC with neurologic PNS. Furthermore, among the 10 specimens from SCLC patients with neurologic PNS, only 2 (20%) exhibited tumor PD-L1 of 0%, compared to 4 of 16 (25%) specimens from SCLC patients with no PNS and 6 of 11 (55%) specimens from SCLC patients with endocrinologic PNS. For the aggregate analysis, one specimen (sample ID #21) from a patient with SCLC with a neurologic PNS was excluded due to low TTF1 staining and questionable tumor content. As a group, tumors from patients with SCLC with neurologic PNS (n V 9) had an elevated PD-1/PD-L1 interaction score (p V 0.014) (Fig. 2A) compared to tumors from patients
with SCLC with endocrinologic PNS (n V 11) or no PNS (n V 16). In a pairwise comparison between the neurologic PNS and control group there was no statistically significant difference in PD-1/PD-L1 interaction score (p V 0.963); however, there was a statistically significant enrichment in PD-1/PD-L1 interaction in control samples compared to the endocrinologic PNS group (p V 0.007). When comparing PD-1/PD-L1 interaction scores between the neurologic PNS and endo-crinologic PNS, there was a significant increase in the neurologic PNS group (p V 0.016). Limiting to only core biopsy specimens due to improved staining repro-ducibility, tumors from patients with SCLC with neurologic PNS (n V 8) had a trend toward increased CD4-and CD8-positive T cell infiltrates (p V 0.08 and p V 0.09, respectively) (Figs. 2B and C), and a statistically significant increase in CD3-positive T cell infiltrates compared to tumors from patients with SCLC with en-docrinologic PNS (n V 3) or no PNS (n V 17, p V 0.033) (Fig. 2D). In a pairwise comparison, core biopsy samples from patients with neurologic PNS exhibited a statistically significant increase in CD3-positive T cells compared to controls (p V 0.034) but not in CD4- or CD8-positive T cells (p V 0.147 and p V 0.184, respectively). There were no statistically significant differences in T cell infiltrates between core biopsy samples from patients with endocrinologic PNS and controls, but there were significant increases in both CD3- and CD4-positive T cell infiltrates comparing the neurologic PNS and endocrinologic PNS groups (p V 0.048 and p V 0.048, respectively) and a marginally significant difference in CD8-positive T cell infiltrate (p V 0.052). Only one of three (33%) core biopsy specimens from patients with SCLC with endocrinologic PNS had greater than 1% CD3-positive T cell infiltration, compared to seven of eight (88%) core biopsy specimens from patients with SCLC with neurologic PNS and 10 of 16 (63%) core biopsy specimens from patients with SCLC with no PNS.
Figure 1. Progression-free survival (PFS) and overall survival (OS) according to the presence and type of paraneoplastic syndrome (PNS) in patients with SCLC.
Kaplan-Meier estimates of (A) OS and (B) PFS in patients with SCLC according to the absence or presence of a PNS. Kaplan-Meier estimates of (C) OS and (D) PFS limited to only patients who received systemic therapy for SCLC.
Table 2
Comparison of OS and PFS From Diagnosis in Patients With SCLC and neurologic PNS, SCLC and Endocrinologic PNS, or SCLC and no PNS
SCLC and Neurologic PNS_SCLC and endocrinologic PNS_SCLC and no PNS
OS 24 months, 12 months, 13 months,
(16.4 - not reached) (8.3 - 15.5) (12.2 - 16.0)
PFS 14 months, 6 months, (4.6 - 9.5) 7 months, (6.6 - 8.1)
(9.3 - not reached)
Data displayed indicates median and 95% confidence interval in months. OS, overall survival; PFS, progression-free survival, PNS, paraneoplastic syndrome.
Discussion
To our knowledge, this is the most comprehensive report to interrogate both clinical outcomes and tumor immune microenvironment in patients with SCLC and
PNS. Our study extends prior retrospective data that patients with SCLC with neurologic PNS have improved OS compared to patients with SCLC with endocrinologic PNS or SCLC and no PNS, despite nearly 25% of our patients with SCLC with neurologic PNS receiving no systemic therapy. Furthermore, our results show that increased tumor-infiltrating lymphocytes, increased PD-L1 expression, and increased PD-1/PD-L1 interactions are apparent in tumors from patients with SCLC
with neurologic PNS. These findings suggest that differences in tumor immune recognition and cellular immunity contribute to the improved tumor control observed in patients with SCLC with neurologic PNS.
These findings are especially relevant in patients with SCLC because initial clinical trials using checkpoint inhibitors have shown that 80% of SCLC tumors have less than 1% PD-L1 staining (versus 30% in patients with SCLC with neurologic PNS in our co-hort).[13] This is inconsistent with the high tumor mutational burden in SCLC, and it implies that SCLC tumors use immune evasion strategies independent of PD-1/PD-L1.[22, 24] Herein we have shown that both tumor T cell infiltration and PD-1/PD-L1 interactions are enriched in patients with SCLC with neurologic PNS. This suggests that regardless of the outcome of checkpoint inhibitor clinical trials in large cohorts of patients with SCLC, patients with SCLC with neurologic PNS may be an ideal target population for this treatment. [13-15,23] However, as neurologic PNS can be debilitating independent of tumor progression, caution must be used in balancing the antitumor effects of immunotherapy with exacerbation of the neurologic PNS.[6] One patient with a neurologic PNS in our cohort received nivolumab, but response could not be assessed as the patient developed pneumonitis and disease progression after four doses.
The primary limitation of this study is the inclusion of both primary tumor and nodal metastases. This
to accurately assess T cell infiltrates in nodal metastases. This may have biased our T cell infiltrate analysis, as more nodal tissue was evaluated in the neurologic PNS group compared to the endocrinologic PNS group and control group. Furthermore, it is possible that tumor immune infiltrates change over time and sampling the tumor at the time of onset of a neurologic PNS, which is not always simultaneous with SCLC diagnosis, may show the most accurate pathophysiologic link. Also, we included patients with no detectable serum paraneoplastic autoantibody (20% of patients with neurologic PNS), which may have biased the neurologic PNS cohort against an improved prognosis, as these patients may have had pathophysiology similar to controls. Finally, a higher proportion of patients with neurologic PNS had LS-SCLC compared to patients with endocrinologic PNS or controls.
The current study provides a key link between decades-old clinical observations in patients with SCLC with neurologic PNS and the current immunomodulation paradigm in oncology focusing on tumor-infiltrating lymphocytes and PD-1/PD-L1 interactions. These hypothesis-generating data are expected to lead to studies evaluating the full spectrum of circulating antitumor antibodies in patients with SCLC, with the hope of eventually identifying new disease-specific drug targets. Further study should also investigate the application of checkpoint inhibitors in patients with SCLC with neurologic PNS and the full spectrum of immune
introduces potential confounding because it is difficult evasion strategies used in SCLC tumors.
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Figure 2. Overall comparison of CD3, CD4, CD8, and programmed death 1 (PD-1) , programmed death ligand 1 (PD-L1) interaction scores.
Tumors from patients with SCLC with neurologic paraneoplastic syndromes (PNS) had significantly increased PD-1/PD-L1 interaction scores (A) compared to tumors from patients with SCLC with endocrino-logic PNS and tumors from patients with SCLC and no paraneoplastic syndrome (control). Tumors from patients with SCLC with neurologic PNS had a trend to-
wards increased CD4 (B) and CD8 (C) infiltrates compared to tumors from patients with SCLC with endo-crinologic PNS and tumors from patients with SCLC and no PNS (control). Tumors from patients with SCLC with neurologic PNS had significantly increased CD3 (D) infiltrates compared to tumors from patients with SCLC with endocrinologic PNS and tumors from patients with SCLC and no PNS (control).
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Figure 3. Immunologic correlates by automated quantitative analysis with accompanying computed tomography (CT). (A) 4 and 20, 40,6-diamidino-2-phenylindole (DAPI) in blue, transcription termination factor 1 (TTF1) in green, programmed death 1 (PD-1) in yellow, and programmed death ligand 1 (PD-L1) in red. (B) 20, DAPI in blue, CD3 in yellow, CD4 in red, and CD8 in green (first image with CD3 present, second image with CD3 removed). (C) CT scan shows the primary tumor at diagnosis (left) followed by progression of disease (right, red arrow indicates primary tumor in both panels).
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