Facoltà di Medicina e Chirurgia
Scuola di Specializzazione in Oncologia Medica
Direttore : Professor Alfredo Falcone
PD-1 and PD-L1 expression in molecularly
selected non-small-cell lung cancer patients
Specializzando:
Relatore:
Dott. Gabriele Minuti
Prof. Alfredo Falcone
Abstract
Background: Agents targeting programmed death-1 receptor (PD-1) and its ligand (PD-L1) are showing
promising results in non-small-cell lung cancer (NSCLC). It is unknown whether PD-1/PD-L1 are differently expressed in oncogene-addicted NSCLC.
Methods: We analyzed a cohort of 125 NSCLC patients, including 56 EGFR mutated, 29 KRAS mutated, 10
ALK translocated and 30 EGFR/KRAS/ALK wild-type. PD-L1 and PD-1 expression were assessed by
immunohistochemistry (IHC). All cases with moderate or strong staining (2+/3+) in >5% of cells were considered as positive.
Results: PD-1 positive (+) was significantly associated with current smoking status (p=0.02) and with
presence of KRAS mutations (P=0.006), while PD-L1+ was significantly associated to adenocarcinoma histology (P=0.005) and with presence of EGFR mutations (P=0.001). In patients treated with EGFR tyrosine kinase inhibitors (N=95), sensitivity to gefitinib or erlotinib was higher in PD-L1+ versus PD-L1 negative in terms of response rate (RR: P=0.01) time to progression (TTP: P<0.0001) and survival (OS: P=0.09), with no difference in PD1+ versus PD-1 negative. In the subset of 54 EGFR mutated patients, TTP was significantly longer in PD-L1+ than in PD-L1 negative (P=0.01).
Conclusion: PD-1 and PD-L1 are differentially expressed in oncogene-addicted NSCLC supporting further
investigation of specific checkpoint inhibitors in combination with targeted therapies.
SUMMARY
1.0 BACKGROUNG
1.1 Non-Small-Cell Lung Cancer
1.2 Immunotherapy for NSCLC
1.3 PD-1 and PD-L1 pathway
1.4 Anti-PD-1 and –PD-L1 monoclonal antibodies
1.5 Biomarkers expression and anti-PD-1 or anti-PD-L1 strategies
2.0 PROPOSAL
3.0 MATERIALS AND METHODS
3.1 Patient selection
3.2 Immunohistochemistry
3.3 Statistical Analyses
4.0 RESULTS
4.1 Patient Characteristics
4.2 PD-1/PD-L1 expression and patient characteristics
4.3 PD-1/PD-L1 expression and outcome of patients treated with EGFR-TKIs
5.0 DISCUSSION
6.0 REFERENCES
7.0 TABLES
1.0 BACKGROUNG
1.1 Non-Small-Cell Lung Cancer
Lung cancer is the leading cause of cancer-related mortality, accounting for 1.37 million deaths annually and for 18.2% of cancer mortality (Siegel et al, 2013). Non-small cell lung cancer (NSCLC) represents the 75% of all lung cancer cases and includes different subtypes, underlying relevant biological differences. The most part of patients with NSCLC are diagnosed in advanced stage, with a 5-year survival rate inferior to 15% (U.S. National Institutes of Health SEER Cancer Statistics Review, 1975-2010).
Nowadays, NSCLC treatment is based on accurate patient stratification according to tumor histology and, most importantly, on biological characteristics. In the NSCLC scenario, adenocarcinoma (40% of all lung cancer) is certainly the most studied histological type in terms of molecular characterization. Several studies have analyzed genomic expression, mutational and proteomic profile of this subtype, succeeding to identify the presence of driver mutations in more than 50% of cases (Sequist et al, 2012). The frequency of genetic alterations is closely correlated to clinical features including smoking habit and patients race (Govindan et al. 2012).
As a consequence of genetic alterations, the tumor can become highly dependent (“addicted”) on the function of a single oncogene, defined as a “driver oncogene”. In 2013, the largest retrospective study evaluating the molecular profile of more than 10.000 NSCLC specimens (Barlesi et al, 2013) confirmed that, in unselected NSCLC patients, the most frequent event is the presence of KRAS mutations (26.9%) followed by epidermal growth factor receptor (EGFR) mutations (9.4%) and anaplastic lymphoma kinase (ALK) translocation (4.0%). Others molecular events, such as ROS1 and RET translocations, HER2, BRAF and PI3K mutations, had a frequency ≤ 3%. The majority of these genetic alterations (95%) resulted mutually exclusive.
During the last years, tyrosine kinase inhibitors (TKIs) targeting EGFR, such as gefitinib, erlotinib or afatinib and drugs targeting ALK kinase, such as crizotinib, have radically change the possibility of treatment, and consequently the outcome, of EGFR-mutated or ALK-translocated patients (Mok et al, 2009; Han et al, 2012¸ Mitsudomi et al, 2010; Maemondo et al, 2010; Zhou et al, 2011; Rosell et al, 2012; Sequist et al, 2013; Wu et al, 2014; Shaw et al, 2013).Currently,in this molecularly selected NSCLC population, EGFR- or ALK-TKIs represent the best therapeutic option that we can offer to our patients, shifting treatment from a classical platinum-based chemotherapy to a molecularly tailored approach.
Nevertheless, no patient with a metastatic NSCLC, also in these cases, can obtain a definitive cure with target agents. Inevitably, due to the emerging of resistant cell clones with reduced or no dependence on therapeutics target, disease progression occurs after a median of 8-12 months, highlighting the high adaptability of cancer cells and the urgent need for additional and more effective strategies.
1.2 Immunotherapy for NSCLC
During the clinical curse of human cancers, genetic and epigenetic aberrations commonly occur and expose the immune system to a large variety of tumor antigens. Cancer epitopes can be selectively recognized by T-cells, activating adaptive and defensive mechanisms. To generate efficient anti-tumor immune responses, while maintaining self-tolerance, host reactions are tightly regulated through a combination of stimulatory and inhibitory signals. Therefore, between the host and the tumor exists a dynamic interaction. The possibility of cancer to evade immune activation can deeply impact on its natural history, favoring proliferation, spreading and progression (Drake et al, 2006; Pardoll, 2012).
For many years several investigators evaluated the possibility to modulate the immune system for treating lung cancer, with disappointing results. First generation immunotherapy, such as cancer vaccines, largely demonstrated minimal activity, substantial toxicity, and no predictive biomarkers to select patients. (Topalian et al, 2011; Hall et al, 2013).Lack of efficacy was probably related to the ability of the tumor to activate several distinct pathways to escape host immune response, including endogenous “immune checkpoints”. These immunologic nodes are key pathways hijacked by tumors to suppress immune control.
Cancer immunotherapy has entered into a new phase from the discovery of drugs able to interfere with these specific immune checkpoints.
Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) was the first investigated critical checkpoint pathway target. CTLA-4 is an inhibitory receptor that limits the initial stage of T-cell activation. Antagonist monoclonal antibodies targeting CTLA-4 demonstrated anti-tumor activity, mostly in melanoma (Klein et al, 2009). In 2013 Ipilimumab (Yervoy, Bristol-Myres Squibb), an anti-CTLA-4 antibody, was approved for the treatment of patients with advanced melanoma on the basis of the demonstrated survival improvement in a phase III trial (Hodi et al, 2010).
More recently, the programmed death-1(PD-1) T-cell co-receptor and its ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC), emerged as a novel immune checkpoint with a pivotal role in cancer tolerance, inhibiting the last step of cytotoxic T-lymphocytes (CTLs) activation and preventing killing of cancer cells (Chen et al,
2012). Encouraging early results from clinical blockade of this pathway have highlighted its role as a new target for cancer immunotherapy.
1.3 PD-1 and PD-L1 pathway
PD-1 (CD279) receptor, is a 288 amino acid cell surface protein of the CD28 family, expressed on activated T-cell, B cells, dendritic cells, and macrophages. PD-1 engagement inhibits function in these immune cell population (Keir et al, 2007). PD-1 has two know ligands, PD-L1/B7-H1 and PD-L2/B7-DC, which are members of the B7 family. Several pre-clinical and clinical evidences suggest the significance of the PD-L1/PD-1 axis in inhibiting effective anti-tumor immunity, considering that this pathway is a critical modulator of immune responses (Keir et al, 2008).
Different studies showed that cancer microenvironment manipulates the PD-L1/PD-1 pathway. The induction of PD-L1 expression on tumor cells leads to the inhibition of immune responses against cancer, permitting progression and metastasis (Drake et al, 2006; Pardoll, 2012). Spranger and colleagues demonstrated in a murine mechanistic experiments that the induction of PD-L1 in melanoma microenvironment is mediated by IFN-y produced by T cells that are antigen experienced and therefore express PD-1 (Spranger et al, 2013). Indeed, in tumors expressing PD-L1, the engagement with the inhibitory receptor PD-1, expressed on T-cell upon activation, can render CTLs non-functional or inactivated through the reduction of cytokine production and the inhibition of T-cells proliferation (Carter et al, 2002; Freeman et al, 2000). From this point of view, PD-L1 expression represents a regulatory mechanism that protect cells against CTLs killing. This pathway, for example, plays a critical role during viral infections in order to dampen chronic immune responses (Keir et
al, 2007). Cancer is a chronic and frequently inflammation-provoking disease that potentially, through PD-L1
up-regulation, can exploit this protective mechanism during disease progression, to evade the host immune response. Moreover, PD-L1 can play an immunosuppressive role, dumping immune response, through its interaction with other surface protein, such as B7.1. This mechanism can potentially block B7.1 ability to active T-cells, mediated by CD28 binding (Butte et al, 2007).
PD-L1 is the primary ligand for PD-1 and is broadly expressed on different tissues, including T and B cells, dendritic cells and macrophages. High PD-L1 expression is frequently found in several types of tumors. The up-regulation of PD-L1 expression can result by the activation of relevant oncogenic pathway, including PI3K and MAPK, and mostly by interferon (IFN)-Ɣ, produced by activated T-cell in cancer microenvironment (Parsa et al, 2007). The PD-L1 expression has been correlated with poor outcomes in human cancers, such as melanoma, lung cancer, breast cancer, bladder cancer, ovarian cancer, pancreatic
cancers, oesophagus adenocarcinoma, kidney tumors as well as in hematopoietic malignancies (Zou and Chen, 2008).However, other reports indicated a lack of association between PD-L1 expression and outcome (Konishi et al, 2004; Mischinger et al, 2010) or that PD-L1 expression was associated with improved survival and increased TILs (Taube et al, 2012).
Recent studies showed that PD-L1 expression may be found in almost a half of NSCLC biopsies, irrespective of histology (Grosso et al, 2013). At European Lung Cancer Conference (ECC) in 2013, Soria and colleagues reported new data about positive PD-L1 staining in NSCLC, showing a prevalence of approximately 45% and 50% respectively for adenocarcinoma and squamous cell carcinoma (Soria et al, 2013).
PD-L2 has a similar role to PD-L1 in damping T-cell responses through PD-1 engagement, even though its expression is restricted principally on dendritic cell, macrophages and mast cell derived from bone marrow. Compared to the broad expression of PD-L1 and its general role in immune-tolerance, the restricted expression of PD-L2 exclusively on antigen-presenting cells correlates with its function in regulating only T-cell priming or polarization. Similarly, PD-L2 expression on cancer cells, across different tumor types, is less frequent than PD-L1 (Chen et al, 2012).
1.4 Anti-PD-1 and –PD-L1 monoclonal antibodies
The evidence that PD-L1 is frequently up-regulated in NSCLC and that PD-1 is expressed on the majority of tumor infiltrating lymphocytes (TILs), represented the rationale for development of monoclonal antibodies against PD-L1 or PD-1, and several agents are currently under investigation (Grosso et al, 2013; Topalian et al, 2012).
Topalian and colleagues in a phase I study investigated the safety and the activity of Nivolumab (BMS-936558, Bristol-Mayers Squibb), a fully humanized monoclonal antibody (IgG4) anti-PD1 in 296 patients with advanced or metastatic solid tumors treated with at last one prior line of treatment (47% of patients were heavily pre-treated with ≥ 3 previous regimens). The study enrolled 122 patients with non– small-cell lung cancer, 104 with melanoma, 34 with renal-cell cancer, 19 with colorectal cancer and 17 with castration-resistant prostate cancer. Nivolumab was administered at a dose of 0.1 to 10.0 mg per kilogram of body weight intravenous (iv) every 2 weeks, with each cycle define as four administration of the drug over an 8-weeks period. The efficacy analysis was performed in 236 patients evaluable for response by RECIST criteria (version 1.0), 76 of whom were NSCLC patients. Common drug-related adverse events (AEs) were fatigue, rash, diarrhea, anorexia, nausea and pruritus. Grade 3 or 4 adverse reactions occurred in 14% of
patients with three deaths for pulmonary toxicity. Objective responses were observed in melanoma, in NSCLC and in kidney cancer patients, with antitumor activity observed at all doses tested. The overall response rate (ORR) in NSCLC patients was 18% (14 patients: 7 individuals with non-squamous histology, 6 with squamous cell carcinoma and 1 with unknown type), 28% in patients with melanoma and 27% in patients with renal cancer. Responses were usually durable and 8 of the 14 NSCLC patients maintained response for at last 24 weeks from the beginning of treatment. Among NSCLC patients treated, no stable disease were observed in individuals with squamous cell histology, whereas approximately 10% of patients with non-squamous cell tumors had disease stabilization lasting more than 24 weeks (Topalian et al, 2012).
Brahmer and colleagues in a similar phase I study in terms of design and objectives, investigated immunotherapy with BMS-936559, a fully humanized monoclonal antibody (IgG4) direct against PD-L1, in 207 patients with solid tumors (75 with NSCLC, 55 with melanoma, 17 with renal cancer, 17 with ovarian cancer, 14 with pancreatic cancer, 7 with gastric cancer and 4 with breast cancer). BMS-936559 was administered in a dose-escalation accelerated tritation design at dose of 0.3, 1, 3 and 10 mg per kilogram of body weight. AEs of any grade were reported in 91% of patients. The most common drug-related adverse events, commonly of low grade, were fatigue, infusion reactions, diarrhea, arthralgia, rush, nausea, prutitus and headache, while grade 3 or 4 drug-related toxic effects occurred in 9% of patients. Immune-related AEs were observed in 81 of 207 patients (39%), mostly including rash, hypothyroidism and hepatitis. Among patients evaluable for response, a complete or partial response was observed in 9 of 52 patients with melanoma, 2 of 17 with kidney cancer, 5 of 49 with NSCLC, and 1 of 17 with ovarian cancer. Responses lasted for 1 year or more in 8 of 16 patients with at least 1 year of follow-up. The median duration of therapy was 12 weeks (Brahmer et al, 2012).
The great interest in immunotherapy for cancer treatment leads to the development, by at last 7 Companies, of several PD-1 checkpoints inhibitors, that are in different phase of investigation.
In 2013 at ECC Soria and colleagues presented the preliminary results of the engineered antibody anti-PD-L1 MPDL3280A (Genentech). Patients received the monoclonal antibody by iv infusion every 3 weeks for a median of approximately 100 days. This phase I study included a cohort of 85 NSCLC patients (65 non-squamous and 20 squamous cell carcinoma). A total of 37 patients were evaluable for efficacy. Study patients were heavily pretreated and more than one-half (55%) had received at least three prior therapies. A large number of patients (84%) were smokers or former smokers. The median duration of therapy was 48 weeks. The ORR was 24% in patients with NSCLC. Among responders, 17% of patients remained stable over 24 weeks. The 24- week progression-free survival (PFS) rate was 44% in patients with
squamous cell NSCLC and 46% in patients with non-squamous cell NSCLC. MPDL3280A treatment was well tolerated, AEs of any grade occurred in 66% of patients, but he majority were of low grade and did not require intervention. No severe pneumonitis were detected, while immune-related Grade 3/4 AEs were observed only in one patient (Soria et al, 2013).
During the last American Society of Clinical Oncology (ASCO) annual meeting in 2014 novel data, concerning safety, tolerability and activity, of different anti-PD-1 or anti-PD-L1 agents, were reported.
MK-3475 (Merck & Co) is a selective anti-PD-1 antibody that demonstrated a good safety profile and a robust antitumor activity in a cohort of over 200 previously-treated PD-L1 positive NSCLC patients. This compound demonstrated an ORR of 16% and 24%, respectively, when response evaluation was performed using RECIST 1.1 criteria or immune-related response criteria (irRC) (Garon et al, 2014). Moreover, MK-3475 demonstrated an interesting activity in NSCLC patients as initial treatment. In a phase I trial restricted to PD-L1 positive patients, Rizvi and colleagues showed an ORR, assessed by irRC, of 36% among the first 45 patients treated into the study (Rizvi et al, 2014).
MEDI4736 is a human IgG1 monoclonal antibody which binds specifically to PD-L1, preventing binding to PD-1 and CD80. With evidence of clinical activity and acceptable safety in phase I, an expansion study was initiated in multiple cancer types including NSCLC, melanoma, gastroesophageal, hepatocellular carcinoma, pancreatic, head and neck and triple negative breast cancer. The compound was administered at 10 mg/kg IV every 2 weeks for 12 months with the possibility of retreatment at the time of progression was permitted after 12 months of therapy. Preliminary results demonstrated that drug-related AEs occurred in 33% of patients, with severe events in 7%. The most frequently observed AEs were fatigue (13%), nausea (8%), rash (6%), vomiting (5%), and pyrexia (5%). Response evaluation was performed by RECIST 1.1 and with a median follow-up of 6 weeks, tumor shrinkage was already detectable in different cancer types such as melanoma, pancreatic, head and neck, and gastroesophageal cancer. The expansion cohort is currently enrolling patients with evidence of activity in NSCLC. Further evaluation of MEDI4736 as monotherapy and in combination with a variety of immune-modulators and targeted agents is ongoing (Segal et al, 2014).
To date, Nivolumab, MK-3475, Pidilizumab (CureTech), MPDL3280A and MEDI-4736 (Astra Zeneca/Medimmune) are tested, as single agents, for NSCLC treatment in phase II or III trials and results are strongly awaited. In addition, an attractive filed of research is the possibility of combined strategies with anti-PD-1 or -PD-L1 agents and chemotherapy or other target agents, in order to increase disease control and patients outcome.
1.5 Biomarkers expression and anti-PD-1 or anti-PD-L1 strategies
Preliminary results suggested that PD-L1 positivity may correlate with response to treatment with PD-1 pathway inhibitors.
In NSCLC patients, Soria and colleagues, in the phase I trial with MPDL3280A, showed that individuals with PD-L1 2+/3+ and 3+ staining by immunohistochemestry (IHC) achieved an ORR of 46% and 83%, respectively while, in the population unselected for the biomarker, the ORR was of 23% (Soria et al, 2013). An increasing number of data, derived from the evaluation of monoclonal antibodies efficacy (MK-3475, Nivolumab, MEDI4736) in phase I trials according to PD-L1 expression, suggest that PD-L1 positivity is a potential predictive factor in cancer treatment with anti-PD-1 or -PD-L1 agents (Brahmer et al, 2013; Garon et al 2014). In addition, same phase III ongoing trials are evaluating the activity of these compounds only in a selected population of PD-L1 positive NSCLC. These preliminary evidences, concerning a significant correlation between the biomarker expression and the tumor response, have to be confirmed and validated in prospective studies. Even though, to date, same key issues are not solved, including the variability in tissue collection timing, the cell sampling, the monoclonal antibodies used for staining, the IHC criteria used in each studies to asses PD-L1 positive cases and the evidences that also PD-L1 negative patients can benefit from the treatment.
Recent data suggested that checkpoint inhibitors could be more effective in smokers (Soria et al, 2013) where somatic gene mutations are frequent. The presence of an high mutational rate may correlate with an increase tumor immunogenicity, considering that any given mutations provide a chance to generate a new antigen, which can be recognized as foreign by T cells. Immunogenic tumors may develop different mechanisms to evade immune response, underling a potential different expression of PD-1/PD-L1 in presence of specific molecular events, including KRAS mutations.
In addition, a recent pre-clinical study demonstrated that the mutation-driven activation of the EGFR pathway in bronchial epithelial cells induce PD-L1 expression and other immunosuppressive factors to evade the host anti-tumor response. This role of EGFR signaling seems to be independent of the classic role on cell proliferation and survival, suggesting an active role of EGFR in modulate microenvironment. The study also demonstrated that, in NSCLC cell lines with EGFR mutation, PD-L1 expression was reduced with EGFR-TKIs (Akbay et al, 2013).
2.0 PROPOSAL
Based on these premises we supposed that PD-1/PD-L1 expression could differ according to the molecular phenotype of the tumor. The aim of the present study was to assess whether PD-1/PD-L1 expression was differently expressed in NSCLC patients according to presence or absence of EGFR mutations, ALK translocations, or KRAS mutations.
3.0 MATERIALS AND METHODS
3.1 Patient selection
This retrospective study was conducted in a cohort of 125 metastatic NSCLC patients followed in 3 Italian centers. We selected two cohorts of patients (EGFR mutated and EGFR wild-type) with availability of additional tumor tissue from the same tumor sample previously used for EGFR assessment. In addition, we included onto the study only cases evaluated for KRAS and ALK status, with full clinical data including previous therapies and survival. EGFR mutations and KRAS mutations were evaluated using Polymerase Chain Reaction (PCR) and direct sequencing, while presence of ALK translocations were detected using fluorescence in situ hybridization (FISH). All tests were performed locally as a part of clinical practice. The study was approved by the local Ethics Committee and was conducted in accordance with ethical principles stated in the most recent version of the Declaration of Helsinki or the applicable guidelines on good clinical practice, whichever represented the greater protection of the individuals.
3.2 Immunohistochemistry
Four-micron sections of 125 primary or metastatic NSCLC samples were used throughout this study. Standard indirect immunoperoxidase procedures were used for immunohistochemistry (IHC; ABC-Elite, Vector Laboratories, Burlingame, CA). We tested previously two different commercial antibodies for PD-L1 in colon carcinoma (Droeser et al, 2013), and we found comparable results. Briefly, slides were dewaxed and rehydrated in distilled water. Endogenous peroxidase activity was blocked using 0.5% H2O2. The sections were treated with 10% normal goat serum (DakoCytomation, Carpinteria, CA) for 20 min and incubated with primary antibodies PD-L1 (CD274) ab58810 (Abcam) and PD-1 760-4448 (Ventana) at room temperature. Sections were further incubated with peroxidase-labelled secondary antibody (DakoCytomation) for 30 min at room temperature. For visualization of the antigen, the sections were immersed in 3-amino-9-ethylcarbazole plus substrate-chromogen (DakoCytomation) for 30 min, and counterstained with Gill’s hematoxylin.
Two well-experienced pathologists (M.A. and L.T.) examined the immunohistochemical slides without any prior information on clinicopathological features of the patient samples.
Percentages of PD-L1 and PD-1 positive tumor cells and staining intensity were evaluated for each sample. Staining intensity was scored considering 0 as negative or trace, 1 as weak, 2 as moderate and 3 as high. In absence of any standardized score system, all cases with staining intensity ≥ 2 in more than 5% of tumor cells were considered as positive, similarly to previous studies (Antonia et al, 2013; Grosso
et al, 2013; Soria et al, 2013 ). Moreover, a semi-quantitative approach was used to generate a score for
each tissue core. The percentage of stained cells (0% to 100%) was multiplied by the dominant intensity pattern of staining ranging from 0 to 3. Therefore, the overall semiquantitative score ranged from 0 to 300.
3.3 Statistical Analyses
Sample size was computed assuming a difference of 25% in PD-1 or PD-L1 expression in EGFR mutated versus EGFR wild-type. With a power of 80% and a significance level of 0.05 (1-tailed test), a sample size of at least 49 patients was required for each group. Statistical analyses were performed to compare differences between patients with and without PD-1 and PD-L1 expression according to presence or absence of a specific biomarker. Clinical characteristics and associations with biomarkers were examined comparing the differences by χ2 test or Fisher’s exact test as appropriate. Comparison between the two groups with or without PD-1 or PD-L1 expression was performed by log rank test. A multivariable analysis was performed using a logistic regression model in order to explore the association of PD-1/PD-L1 expression with patient characteristics (smoke, histology, EGFR, KRAS and ALK). A step-down procedure method was selected. The criterion for variable removal was the likelihood ratio statistic based on the maximum partial likelihood estimates (default P-value of 0.10 for removal from the model). Differences between median score was performed by U-Mann Witney test. Correlative analyses of PD-1 or PD-L1 expression and outcome of patients according to a specific therapy were evaluated. Time to Progression (TTP) was calculated from date of therapy start to progression or last follow-up date; Overall Survival (OS) was calculated from the date of therapy start to death or last follow-up date, with 95% confidence intervals calculated using the Kaplan-Meier method (Kaplan and Meier P, 1985). The significance level for all analyses was set at p<0.05 and all P-values were two-sided. Statistical analysis was performed by using IBM-SPSS Statistics version 20.
4.0 RESULTS
4.1 Patient Characteristics
A total of 125 metastatic NSCLC patients were included in the study. A total of 98 (78.4%) samples were obtained from primary tumor and 17 (13.6%) from metastatic sites. This study include 99 cases treated with EGFR-TKIs (gefitinib: N=30, 30.3%; erlotinib: N=69, 69.7%) in first (N=29, 29.3%) or subsequent lines of treatment (N=70, 70.7%). The median age was 64 years (range: 41-84 years). The majority of patients were male (N=67, 53.6%), former (N=58, 46.4%) or current smokers (N=17, 13.6%) and mostly presented two metastatic sites (N=43, 34.4%). The most frequent metastatic sites were lung (N=72, 57.6%), followed by lymph nodes (N=55, 44.0%), bone (N=37, 29.6%), brain (N=18, 14.4%), liver (N=13, 10.4%) and adrenal glands (N=8, 6.4%). Adenocarcinoma was the most frequent histology (N=83, 66.4%). All patients were analyzed for presence of EGFR and KRAS mutation and for ALK translocation: this analysis included 56 (44.8%) EGFR mutated, 29 (23.2%) KRAS mutated, 10 (8.0%) ALK translocated and 30 (24.0%)
EGFR/KRAS/ALK wild-type, defined as triple negative. Exon 19 deletion (N=30, 24.0%) and codon 12
mutation (N=26, 20.8%), were the most frequent EGFR and KRAS alterations, respectively (Table 1). In this study, because of the criteria for patient selection, incidence of EGFR mutations, KRAS mutations and ALK translocations was not representative of a standard Caucasian population.
4.2 PD-1/PD-L1 expression and patient characteristics
PD-1 was successfully evaluated in 122 specimens. Median PD-1 expression was 30. As illustrated in Figure 3, median PD-1 expression resulted high in male, in current smokers, in adenocarcinoma histology, in EGFR wild-type, in ALK negative and in patients harboring KRAS mutations. A total of 43 cases (35.2%) had moderate (2+) or strong (3+) staining in at least 5% of cells and were considered as PD1+ as illustrated in Figure 1 and in Figure 2. As reported in Table 2, PD-1 positive (+) patients were more frequently male with adenocarcinoma histology, even if the association was not statistically significant. PD-1 positivity was significantly associated with current smoking status (P=0.02) and with presence of KRAS mutations (P=0.006), while no significant association was observed with presence of EGFR mutations or ALK translocations. A multivariable analysis confirmed the significant association between PD-1 and KRAS mutations (P=0.05) (Table 4).
PD-L1 was successfully evaluated in 123 specimens, with a median expression level of 75. As illustrated in Figure 4 A-F, median PD-L1 expression was high in female, in never/former smokers, in
adenocarcinoma histology, in patients harboring EGFR mutations, and in patients with ALK translocations. A score of 2+ or 3+ in >5% of tumor cells (PD-L1+) was observed in 68 cases (55.3%), as illustrated in Figure 2. As reported in Table 2, PD-L1+ status was significantly associated to adenocarcinoma histology (P=0.005) and more frequently observed in female and in never/former smokers, even if the association was not statistically significant. Importantly, presence of EGFR mutations was significantly associated with PD-L1 positive status (P=0.001). Multivariable analysis confirmed the significant association between PD-L1 and
EGFR mutations (P=0.002) and between PD-L1 and adenocarcinoma histology (P=0.10) (Table 4) .
A formal comparison between primary tumors and metastasis was limited by the low number of samples (only 17 cases form metastatic sites). In order to clarify whether our results could be influenced by the simultaneous presence of primary and metastatic tumor samples, we reanalyzed the data by excluding all metastatic cases. In this analysis we confirmed that PD-1 positivity was significantly associated with current smoking status (P=0.05) and with presence of KRAS mutations (P=0.02) and that PD-L1 positivity status was significantly associated with adenocarcinoma histology (P=0.007) and with presence of EGFR mutations (P <0.001) (Table 3).
PD1/PD-L1 expression was evaluable in 29 triple negative cases. In such subgroup, median PD-1 expression was 40, a result that was similar to the median PD-1 expression observed in EGFR mutants (median 20), KRAS mutants (median 60) or ALK translocated (median 15), as reported in Table 2. In the same subgroup of triple negative patients, median PD-L1 was 20, lower than the median PD-L1 levels observed in EGFR mutants (median 120) or in ALK translocated (median 115) and similar to KRAS mutants (median 55). As reported in Table 2, when compared to KRAS mutants, triple negative patients were more frequently PD-1 negative (P=0.01). When compared to patients with EGFR mutations, or with KRAS mutations or with ALK translocations, triple negative individuals were more frequently PD-L1 negative, with a
P-value of <0.001, 0.02 and 0.06, respectively.
4.3 PD-1/PD-L1 expression and outcome of patients treated with EGFR-TKIs
Because of the strong association of PD-1/PD-L1 expression and KRAS or EGFR mutations, we further investigated the potential effect of such biomarkers on sensitivity to anti-EGFR agents. With such purpose we analysed the outcome of 99 patients treated with gefitinib or erlotinib. EGFR-TKIs were given as front-line therapy in 29 patients including 26 EGFR mutated patients, and in second or subsequent lines of therapy in 70 individuals, including 29 EGFR mutated, 16 KRAS mutated and 25 triple negative. EGFR-TKIs
were offered in second or further lines of treatment in patients with KRAS mutations or in triple negative according to clinical practice (Kim et al, 2008, Shepherd et al, 2009; Cappuzzo et al, 2010). In the group of patients treated with erlotinib or gefitinib response rate (RR) was 47.5%, median TTP 7.9 months and median OS 18.0 months.
PD-1 was assessed in 96 specimens and a positive expression was observed in 28 (29.2%) cases. Among the 93 patients evaluable for response, no differences in terms of RR (46.2% versus 50.7%, P=0.70), TTP (7.0 months versus 8.0 months, P=0.97) and OS (17.8 months versus 18.9 months, P=0.82) were identified in PD-1 positive (N=26, 28.0%) versus PD-1 negative (N=67, 72.0%) cases, as illustrated in Figure 5 A-B.
PD-L1 was successfully evaluated in 98 specimens and PD-L1 positive expression was observed in 52 (53.1%) cases. Among the 95 patients treated with gefitinib or erlotinib and evaluable for response, PD-L1 positive (N=49, 51.6%) patients had significantly higher RR (61.2% versus 34.8%, P=0.01), significant longer TTP (11.7 months versus 5.7 months, P<0.0001) and longer OS (21.9 months versus 12.5 months,
P=0.09) than PD-L1 negative (N=46, 48.4%) patients, as illustrated in Figure 5 C-D.
In order to explore whether the higher sensitivity to PD-L1 positive patients was related only to the concomitant presence of EGFR mutations or whether PD-L1 positive patients had a more indolent disease outcome when treated with EGFR-TKIs in presence of EGFR mutations, we further analysed the outcome of 54 EGFR mutated patients treated with gefitinib or erlotinib and evaluable for response. In this subgroup, although no difference in RR was detected (76.3% versus 75.0%, P=1.00), PD-L1 positive (N=38, 70.4%) patients had a significantly longer TTP (13.1 versus 8.5 months, P=0.01) and a non significantly longer OS (29.5 versus 21.1 months, P=0.75) than PD-L1 negative (N=16, 29.6%) patients, as reported in Figure 6 A-B.
5.0 DISCUSSION
In the present study, the first specifically conducted in a cohort of molecularly selected NSCLC, we showed that expression of immune checkpoints differs according to tumor biology and patient characteristics. Patients harboring KRAS mutations had higher levels of PD-1 expression when compared to the KRAS wild-type population and presence of EGFR mutations or ALK translocations were associated with increased PD-L1 protein levels. Moreover, the clinical profile of patients expressing PD-1 was different from the profile of individuals expressing PD-L1. As a consequence of association with molecular characteristics, patients expressing PD-1 were more frequently male, smokers with adenocarcinoma, while patients
expressing PD-L1 were more frequently female, never/former smokers with adenocarcinoma, the typical clinical features of KRAS mutants or EGFR mutants, respectively.
During the last few years, a growing interest surrounded immunotherapy and several agents targeting molecules involved in regulation of the immune system are under development. Agents targeting PD-1 and PD-L1 showed promising results in patients with NSCLC, particularly in individuals with high PD-L1 expression. PD-L1 is frequently found highly expressed in many human cancer types including NSCLC, being up-regulated in tumors by activation of key oncogenic pathways, such as the PI3KCA-AKT and the RAS-RAF-MAPK pathways (Parsa et al, 2007).These findings suggested that high mutational rates may contribute to increased immunogenicity (Chen et al, 2012) indirectly predicting sensitivity to checkpoint inhibitors (Soria et al, 2013).These findings are in agreement with our data where PD-L1 or PD-1 levels resulted higher in individuals with mutations. In a recent study, Antonia et al. showed that PD-L1 levels were numerically higher in individuals with EGFR mutations (Antonia et al, 2013). Nevertheless, the very low number of patients with EGFR mutations included in this study, only 4 cases, precluded any firm conclusion.
Importantly, we described for the first time PD-1 expression on tumor cells. This finding could have therapeutic implications and need additional confirmation. The lack of additional tumor tissue precluded the possibility to perform further experiments.
Interestingly, it seems that mechanisms of immune system deregulation differ according to the type of mutation, with potential therapeutic implications. PD-1 expression was significantly associated with presence of KRAS mutations, while PD-L1 was strongly associated with presence of EGFR mutations, potentially modulating sensitivity to anti-EGFR agents. In a recent study, Akbay et al. showed that activation of the EGFR pathway induces PD-L1 expression to facilitate evasion of the host anti-tumor immune response (Akbay et al, 2013).This role of EGFR signaling was independent of its effects on cell proliferation and survival, suggesting an active role for the EGFR oncogene in remodeling the immune microenvironment. Moreover, pharmacological blockade of the PD-1 pathway using EGFR-TKIs reduced PD-L1 expression, leading to tumor reduction, with a positive impact on overall survival (Akbay et al, 2013).The same effect was observed in our study. Patients harboring EGFR mutations with high PD-L1 expression resulted more sensitive to gefitinib or erlotinib probably because of PD-L1 downregulation induced by the EGFR inhibition. The evidence that PD-L1 expression is a potential negative prognostic factor in several cancers further supports the hypothesis that the observed improvement in TTP was related to an indirect effect of the therapy on checkpoint expression (Zou and Chen, 2008). Overall, our and other data suggest that
combination of PD-1 blockade with EGFR TKIs may be a promising therapeutic strategy to extend the duration of treatment response and delay development of resistance (Akbay et al, 2013).
Another interesting finding was the evidence that levels of PD-L1 were higher in patients with ALK translocations. Although the association was not statistically significant and although we were not able to evaluate whether a different checkpoint expression impacted on efficacy of anti-ALK agents, median PD-L1 levels were 5 times higher in ALK positive when compared to triple negative patients. Similarly to EGFR mutations, a potential effect of ALK translocation on checkpoint expression cannot be excluded and additional studies are recommended in this particular NSCLC subpopulation.
In conclusion, our study showed that immune checkpoints are differently expressed in oncogene-addicted NSCLC potentially modulating sensitivity to targeted agents. Our findings represent the rationale to choose a different checkpoint inhibitor according to the tumor driver and to combine anti-PD-L1 or anti-PD-1 agents with targeted therapies. Considering the retrospective nature of our investigation, additional prospective studies are warranted.
Acknowledgements: This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) [IG 2012-13157], Fondazione Ricerca Traslazionale (FoRT) and Istituto Toscano Tumori (ITT), [Project F13/16].
6.0 REFERENCES
Akbay EA, Koyama S, Carretero J, et al. (2013). Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors. Cancer Discov 3: 1355-1363, doi: 10.1158/2159-8290.CD-13-0310.
Antonia S, Grosso J, Horak C, et al. (2013). Association of tumor PD-L1 expression and immune biomarkers with clinical activity in patients with non-small cell lung cancer treated with nivolumab. J Thorac Oncol 8 (suppl; abstract P2.11-035).
Barlesi F, Blons H, Beau-Faller M et al. (2013) Biomarkers (BM) France: Results of routine EGFR, HER2, KRAS, BRAF, PI3KCA mutations detection and EML4-ALK gene fusion assessment on the first 10,000 non-small cell lung cancer (NSCLC) patients (pts). ASCO meeting 2013. J Clin Oncol 31, 2013 (suppl; abstr 8000).
Brahmer JR, Tykodi SS, Chow LQ, et al. (2012). Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med 366(26):2455-65. doi: 10.1056/NEJMoa1200694.
Butte MJ, Keir ME, Phamduy TB, et al. (2007). Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses. Immunity 27:111–22.
Cappuzzo F, Ciuleanu T, Stelmakh L, et al.(2010). Erlotinib as maintenance treatment in advanced non-small-cell lung cancer: a multicentre, randomised, placebo-controlled phase 3 study. Lancet Oncol 11:521-529.
Carter L, Fouser LA, Jussif J, et al. (2002). PD-1:PD-L inhibitory pathway affects both CD4(þ) and CD8(þ) T cells and is overcome by IL-2. Eur J Immunol 32:634–43.
Chen DS, Irving BA, Hodi FS (2012). Molecular pathways: next-generation immunotherapy-inhibiting programmed death-ligand 1 and programmed death-1. Clin Cancer Res 18: 6580-6587, doi: 10.1158/1078-0432.CCR-12-1362.
Drake CG, Jaffee E, Pardoll DM (2006). Mechanisms of immune evasion by tumors. Adv Immunol 90: 51-81.
Droeser RA, Hirt C, Viehl CT, et al. (2013). Clinical impact of programmed cell death ligand 1 expression in colorectal cancer. Eur J Cancer 49: 2233-2242. doi: 10.1016/j.ejca.2013.02.015.
immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med 192:1027–34.
Garon EB, Leighl NB, Rizvi NA, et al. (2014). Safety and clinical activity of MK-3475 in previously treated patients (pts) with non-small cell lung cancer (NSCLC). 2014 ASCO Annual Meeting, J Clin Oncol 32:5s, (suppl; abstr 8020).
Goldstraw P, Crowley J, Chansky K, et al. (2007). The IASLC Lung Cancer Staging Project: proposals for the revision of the TNM stage groupings in the forthcoming (seventh) edition of the TNM Classification of malignant tumours. J Thorac Oncol 2: 706-714.
Govindan R, Ding L, Griffith M et al. (2012) Genomic landscape non-small cell lung cancer in smokers and never-smokers. Cell 150:1121-34.
Grosso J, Horak CE, Inzunza D, et al. (2013). Association of tumor PD-L1 expression and immune biomarkers with clinical activity in patients (pts) with advanced solid tumors treated with nivolumab (anti-PD-1; BMS-936558; ONO-4538). J Clin Oncol 31 (suppl; abstract 3016).
Keir ME, Francisco LM, Sharpe AH.(2007). PD-1 and its ligands in T-cell immunity. Curr Opin Immunol 19:309–14.
Keir ME, Butte MJ, Freeman GJ, et al. (2008). PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol 26:677-704. doi: 10.1146/annurev. Kim ES, Hirsh V, Mok T, et al. (2008). Gefitinib versus docetaxel in previously treated
non-small-cell lung cancer (INTEREST): a randomised phase III trial. Lancet 372:1809-1818. Klein O, Ebert LM, Nicholaou T, Browning J, et al. (2009). Melan-A-specific cytotoxic T cells
are associated with tumor regression and autoimmunity following treatment with anti-CTLA-4. Clin Cancer Res 15: 2507-13. doi: 10.1158/1078-0432.
Hall RD, Gray JE, Chiappori AA. (2013). Beyond the standard of care: a review of novel immunotherapy trials for the treatment of lung cancer. Cancer Control 20: 22-31.
Han JY, Park K, Kim SW, et al. (2012). First-SIGNAL: first-line single-agent iressa versus gemcitabine and cisplatin trial in never-smokers with adenocarcinoma of the lung. J Clin Oncol 30: 1122-1128, doi: 10.1200/JCO.2011.36.8456.
Hodi FS, O'Day SJ, McDermott DF, et al. (2010). Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363:711-23. doi: 10.1056/NEJMoa1003466.
Kaplan EL, Meier P. (1985). Nonparametric estimation from incomplete observations. J Am Stat Ass 53: 457-481.
Maemondo M, Inoue A, Kobayashi K, et al. (2010). Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med 362: 2380-2388, doi: 10.1056/NEJMoa0909530.
Mitsudomi T, Morita S, Yatabe Y, et al. (2010). Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial. Lancet Oncol 11:121-128, doi: 10.1016/S1470-2045(09)70364-X.
Mok TS, Wu YL, Thongprasert S, et al. (2009). Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med 361: 947-957, doi: 10.1056/NEJMoa0810699. Pardoll DM. (2012). The blockade of immune checkpoints in cancer immunotherapy. Nat
Rev Cancer 12: 252-264, doi: 10.1038/nrc3239.
Parsa AT, Waldron JS, Panner A, et al. (2007). Loss of tumor suppressor PTEN function increases B7-H1 expression and immunoresistance in glioma. Nat Med 13: 84-88.
Rizvi NA, Garon EB, Patnaik A, et al. (2014). Safety and clinical activity of MK-3475 as initial therapy in patients with advanced non-small cell lung cancer (NSCLC). J Clin Oncol 32:5s, (suppl; abstr 8007).
Rosell R, Carcereny E, Gervais R, et al. (2012). Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial. Lancet Oncol 13: 239-246, doi: 10.1016/S1470-2045(11)70393-X.
Segal NH, Antonia SJ,. Brahmer JR, et al. (2014). Preliminary data from a multi-arm expansion study of MEDI4736, an anti-PD-L1 antibody. 2014 ASCO Annual Meeting. J Clin Oncol 32:5s, (suppl; abstr 3002^).
Sequist LV, Yang JC, Yamamoto N, et al. (2013). Phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations. J Clin Oncol 31: 3327-3334, doi: 10.1200/JCO.2012.44.2806.
Sequist LV, Heist RS, Shaw AT et al. (2011). Implementing multiplex genotyping of non-small-cell lung cancers into routine clinical practice. Ann Oncol 22:2616-24.
Shaw AT, Kim DW, Nakagawa K, et al. (2013). Crizotinib versus chemotherapy in advanced ALK-positive lung cancer. N Engl J Med 368: 2385-2394, doi: 10.1056/NEJMoa1214886. Shepherd FA, Douillard J, Fukuoka M, et al. (2009). Comparison of gefitinib and docetaxel
in patients with pretreated advanced non-small cell lung cancer (NSCLC): Meta-analysis from four clinical trials. J Clin Oncol 27(suppl; abstr 8011).
Siegel R, Naishadham D, Jemal A. (2013). Cancer statistics. CA Cancer J Clin 63: 11-30, doi: 10.3322/caac.21166.
Soria JC, Cruz C, Bahleda R, et al. (2013). Clinical activity, safety and biomarkers of PD-L1 blockade in non-small cell lung cancer (NSCLC): additional analyses from a clinical study of the engineered antibody MPDL3280A (anti-PDL1). Eur J Cancer 49 (suppl; abstract 3408). Spranger S, Spaapen RM, Zha Y, Williams J, et al. (2013). Up-regulation of PD-L1, IDO, and
T(regs) in the melanoma tumor microenvironment is driven by CD8(+) T cells. Sci Transl Med 5: 200ra116. doi: 10.1126/scitranslmed.3006504.
Taube JM, Anders RA, Young GD, et al. (2012). Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Sci Transl Med 4: 127ra37. doi: 10.1126/scitranslmed.3003689.
Topalian SL, Weiner GJ, Pardoll DM. (2011). Cancer immunotherapy comes of age. J Clin Oncol 29: 4828-4836, doi: 10.1200/JCO.2011.38.0899.
Topalian SL, Hodi FS, Brahmer JR, et al. (2012). Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366: 2443-2454, doi: 10.1056/NEJMoa1200690. U.S. National Institutes of Health. National Cancer Institute. SEER Cancer Statistics Review,
1975-2010.
Wu YL, Zhou C, Hu CP, et al. (2014). Afatinib versus cisplatin plus gemcitabine for first-line treatment of Asian patients with advanced non-small-cell lung cancer harbouring EGFR mutations (LUX-Lung 6): an open-label, randomised phase 3 trial. Lancet Oncol 15: 213-222, doi: 10.1016/S1470-2045(13)70604-1.
Zhou C, Wu YL, Chen G, et al. (2011). Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study. Lancet Oncol 12: 735-742, doi: 10.1016/S1470-2045(11)70184-X.
Zou W, Chen L. (2008). Inhibitory B7-family molecules in the tumor microenvironment. Nat Rev Immunol 8: 467-477, doi: 10.1038/nri2326.
Table 1: Clinical and biological characteristic in the whole population
Characteristic Total (No) % Total number of patients 125 100
Median age (years - range) 64 41-84
Gender Male 67 53.6 Female 58 46.4 Histology Adenocarcinoma 83 66.4 Squamous-cell carcinoma 23 18.4 Othera 19 15.2 Smoking history Never 37 29.6 Former 58 46.4 Current 17 13.6 Unknown 13 10.4 Sites of metastasis Lung 72 57.6 Lymph nodes 55 44.0 Bone 37 29.6 Brain 18 14.4 Liver 13 10.4 Adrenal glands 8 6.4 EGFR status Mutatedb 56 44.8 Wild type 69 55.2 KRAS status Mutatedc 29 23.2 Wild type 96 76.8 ALK status Translocated 10 8.0 Wild type 115 92.0 Triple negatived 30 24.0
Abbreviations: NAS= not otherwise specified; EGFR= epidermal growth factor receptor; KRAS=Kirsten rat sarcoma viral oncogene homolog; ALK=anaplastic lymphoma kinase; PD-L1= programmed death-ligand ; PD-1=programmed death-1.
Footnotes: aOther histologies included: large cell=4 (3.2%), NAS=3 (2.4%), mixed histology= 2 (1.6%), unknown= 10 (8.0%). bEGFR mutations included: exon 18=3 (2.4%); exon 19=30 (24.0%); exon 20=4
(3.2%); exon 21=14 (11.2%); other=5 (4.0%). cKRAS mutations included: codon 12=26 (20.8%); codon 13=2
Table 2: PD-1/PD-L1 expression and patient characteristics Characteristic PD-1+ (No/%) PD-1 – (No/%) P-value median PD-1 levels PD-L1+ (No/%) PD-L1 – (No/%) P-value median PD-L1 levels Gender Male 24/55.8 41/51.9 0.68 60 36/52.9 30/54.5 0.86 60 Female 19/44.2 38/48.1 20 32/47.1 25/45.5 80 Smoking history Never/Former smokers 27/73.0 65/90.3 0.02 20 53/86.9 41/82.0 0.48 60 Current smokers 10/27.0 7/9.7 60 8/13.1 9/18.0 30 Histology Adenocarcinoma 29/85.3 52/75.4 0.25 40 52/88.1 30/65.2 0.005 100
Squamous cell carcinoma 5/14.7 17/24.6 0 7/11.9 16/34.8 0
EGFR status EGFR mutated 17/39.5 38/48.1 0.36 20 40/58.8 16/29.1 0.001 120
EGFR wild type 26/60.5 41/51.9 40 28/41.2 39/70.9 20
KRAS status KRAS mutated 16/37.2 12/15.2 0.006 60 15/22.1 13/23.6 0.84 55
KRAS wild type 27/62.8 67/84.8 25 53/77.9 42/76.4 80
ALK status ALK translocated 3/7.0 7/8.9 1.00 15 6/8.8 4/7.3 1.00 115
ALK wild type 40/93.0 72/91.1 35 62/91.2 51/92.7 70
EGFR statusa EGFR mutated 17/70.8 38/63.3 0.51 20 40/85.1 16/42.1 < 0.001 120 Triple negativeb 7/29.2 22/36.7 40 7/14.9 22/57.9 20 KRAS statusa KRAS mutated 16/69.6 12/35.3 0.01 60 15/68.2 13/37.1 0.02 55 Triple negativeb 7/30.4 22/64.7 40 7/31.8 22/62.9 20 ALK statusa ALK translocated 3/30.0 7/24.1 0.70 15 6/46.2 4/15.4 0.06 115 Triple negativeb 7/70.0 22/75.9 40 7/53.8 22/84.6 20
Abbreviations: PD-L1=programmed death-ligand 1; PD-1= programmed death-1; EGFR=epidermal growth factor receptor; KRAS=Kirsten rat sarcoma viral oncogene homolog; ALK= anaplastic lymphoma kinase; PD-1+ = PD-1 positive; PD-1–= PD-1 negative; PD-LPD-1+= PD-L1 positive; PD-L1–= PD-L1 negative.
Footnotes: a Analysis performed versus triple negative cases. b Triple negative included EGFR/KRAS/ALK wild-type patients.
Table 3: PD-1/PD- and patients characteristics in primary tumors Characteristic PD-1+ (No/%) PD-1 – (No/%) P-value PD-L1+ ( No/%) PD-L1 – ( No/%) P-value Gender Male 14/53.8 37/52.9 0.93 25/51.0 27/56.2 0.61 Female 12/46.2 33/47.1 24/49.0 21/43.8 Smoking history Never/Former smokers 17/70.8 60/89.6 0.05 42/89.4 37/80.4 0.23 Current smokers 7/29.2 7/10.4 5/10.6 9/19.6 Histology Adenocarcinoma 18/81.8 47/73.4 0.43 40/87.0 26/61.9 0.007 Squamous cell carcinoma 4/18.2 17/26.6 6/13.0 16/38.1
EGFR status
EGFR mutated 11/42.3 36/51.4
0.43
33/67.3 14/29.2
<0.001
EGFR wild type
15/57.7 34/48.6 16/32.7 34/70.8 KRAS status KRAS mutated 10/38.5 11/15.7 0.02 8/16.3 13/27.1 0.20
KRAS wild type 16/61.5 59/84.3 41/83.7 35/72.9
ALK status ALK translocated 0/0 2/2.9 1.00 1/2.0 1/2.1 1.00
ALK wild type
26/100.0 68/97.1 48/98.0 47/97.9 EGFR statusa EGFR mutated 11/68.8 36/63.2 0.68 33/82.5 14/41.2 < 0.001 Triple negative 5/31.2 21/36.8 7/17.5 20/58.8 KRAS statusa KRAS mutated 10/66.7 11/34.4 0.038 8/53.3 13/39.4 0.37 Triple negative 5/33.3 21/65.6 7/46.7 20/60.6 ALK statusa ALK translocated 0/0 2/8.7 1.00 1/12.5 1/4.8 0.48 Triple negative 5/100.0 21/91.3 7/87.5 20/95.2
Abbreviations: PD-L1=programmed death-ligand 1; PD-1= programmed death-1; EGFR=epidermal growth factor receptor; KRAS=Kirsten rat sarcoma viral oncogene homolog; ALK= anaplastic lymphoma kinase; PD-1+ = PD-1 positive; PD-1–= PD-1 negative; PD-LPD-1+= PD-L1 positive; PD-L1–= PD-L1 negative.
Footnotes: a Analysis performed versus triple negative cases. b Triple negative included EGFR/KRAS/ALK wild-type patients.
Table 4: PD-1 and PD-L1 Multivariable Analysis
PD-1 Multivariable
aPD-L1 Multivariable
aVariable
Category
OR
95% CI
P-value
OR
95% CI
P-value
Sex
Male/Female
1.43
0.56-3.63
0.5
0.91
0.37-2.28
0.8
Smoke
Current/Never
2.37
0.69-8.18
0.18
0.90
0.26-3.15
0.9
Histology
Adenocarcinoma/
Other
0.50
0.15-1.71
0.27
0.40
0.13-1.20
0.10
bEGFR
Mutated/Wild type
1.28
0.37-4.47
0.7
6.23
1.90-20.37
0.002
b
KRAS
Mutated/Wild type
3.71
0.99-13.83
0.05
b
2.62
0.74-9.30
0.14
ALK
Traslocated/Wild type
0
-
1.0
1.01
Abbreviations: OR= Odds Ratio; EGFR= epidermal growth factor receptor; KRAS=Kirsten
rat sarcoma viral oncogene homolog; ALK=anaplastic lymphoma kinase; PD-L1=
programmed death-ligand 1 ; PD-1=programmed death-1
Footnotes
aSex, smoking status, histology, KRAS and ALK were removed from the model
at the last step of multivariable analysis.
bStatistically significant at the last step of
8.0 FIGURES
Figure 5: Kaplan-Meier curve of time to progression (A-C) and overall survival (B-D) in patients treated with EGFR-TKIs
Figure 6: Kaplan-Meier curve of time to progression (A) and overall survival (B) in PD-L1 positive versus PD-L1 negative EGFR mutated patients treated with EGFR-TKIs
TITLES AND LEGENDS TO FIGURES
Figure 1: PD-1 and PD-L1 immunohistochemistry analysis
This figure illustrates 4 cases of PD-1 IHC analysis (A-D) and 4 cases of PD-L1 IHC analysis (E-H). Specifically, this picture showed: a PD-1 negative case (A), a PD-1 1+ case in 60% of tumor cells (B), a PD-1 2+ case in 80% of tumor cells (C), a PD-1 3+ in 95% of tumor cells (D), a PD-L1 negative case (E), a PD-L1 1+ case in 10% of tumor cells (F), a PD-L1 2+ case in 50% of tumor cells (G) and a PD-L1 3+ case in 70% of tumor cells (H). The magnification used for the images was 20x.
Figure 2: PD-1 positive cases by immunohistochemistry analysis
This figure includes 4 additional cases (A-D) showing PD-1 expression on tumor cells, evaluated by immunohistochemistry. Specifically, this picture showed a PD-1 3+ case in 95% of tumor cells (A), a PD-1 3+ case in 90% of tumor cells (B), a PD-1 2+ case in 80% of tumor cells (C), a PD-1 3+ in 90% of tumor cells (D). The magnification used for the images was 20x.
Figure 3: Levels of PD-1 expression and patient characteristics
This picture illustrates changes in the levels of PD-1 expression according to clinical (A-C) and biological (D-F) characteristics. Median levels of PD-1 score and interquartile ranges are showed.
Median levels of PD-1 expression were higher in male (median score 60 versus 20) than in female (A), in current (median score 60 versus 20) than in never/former smokers (B), in adenocarcinoma (median score 40 versus 0) than in squamous-cell carcinoma histology (C), in EGFR wild-type (median score 40 versus 20) than in EGFR mutated (D), in KRAS mutated (median score 60 versus 25) than in KRAS wild-type (E) and in
ALK wild-type (median score 35 versus 15) than in ALK translocated (F) patients.
Figure 4: Levels of PD-L1 expression and patient characteristics
This picture illustrates changes in the levels of PD-L1 expression according to clinical (A-C) and biological (D-F) characteristics. Median levels of PD-1 score and interquartile ranges are showed.
Median levels of PD-L1 expression were higher in female (median score 80 versus 60) than in male (A), in never/former (median score 60 versus 30) than in current smokers (B), in adenocarcinoma (median score 100 versus 0) than in squamous-cell carcinoma histology (C), in EGFR mutated (median score 120 versus 20) than in EGFR wild-type (D), in KRAS wild-type (median score 80 versus 55) than in KRAS mutated (E) and in ALK translocated (median score 115 versus 70) than in ALK wild-type (F).
Figure 5: Kaplan-Meier curve of time to progression (A-C) and overall survival (B-D) in patients treated with EGFR-TKIs
In the subgroup of patients (N=99) treated with EGFR-TKIs no differences in terms of TTP (7.0 months versus 8.0 months, P=0.97) (A) and OS (17.8 months versus 18.9 months, P=0.82) (B) were identified in PD-1 positive (N=26, 28.0%) versus PD-PD-1 negative (N=67, 72.0%) cases.
PD-L1 positive (N=49, 51.6%) patients had a significant longer TTP (11.7 months versus 5.7 months,
P<0.0001) (C) and a longer OS (21.9 months versus 12.5 months, P=0.09) (D) than PD-L1 negative (N=46,
48.4%) patients.
Figure 6: Kaplan-Meier curve of time to progression (A) and overall survival (B) in PD-L1 positive versus PD-L1 negative EGFR mutated patients treated with EGFR-TKIs.
In EGFR mutated patients (N=54) treated with EGFR-TKIs, PD-L1 positive (N=38, 70.4%) cases had a significantly longer TTP (13.1 versus 8.5 months, P=0.01) (A) and a non significantly longer OS (29.5 versus 21.1 months, P=0.75) (B) than PD-L1 negative (N=16, 29.6%) cases.
