Activity of afatinib in patients with NSCLC harboring novel uncommon EGFR mutations with or without co-mutations: a case report.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) represent first-line standard of care in unresectable EGFR mutation-positive (EGFRm+) non-small cell lung cancer (NSCLC). However, 10-20% of patients with EGFRm+ NSCLC have uncommon EGFR variants, defined as mutations other than L858R substitutions or exon 19 deletions. NSCLC harboring uncommon EGFR mutations may demonstrate lower sensitivity to targeted agents than NSCLC with L858R or exon 19 deletion mutations. Prospective clinical trial data in patients with NSCLC uncommon EGFR mutations are lacking. Afatinib is a second-generation TKI and the only Food and Drug Administration-approved drug for some of the more prevalent uncommon EGFR mutations. We present a series of seven case reports describing clinical outcomes in afatinib-treated patients with NSCLC harboring a diverse range of extremely rare mutations with or without co-mutations affecting other genes. EGFR alterations included compound mutations, P-loop αC-helix compressing mutations, and novel substitution mutations. We also present a case with NSCLC harboring a novel EGFR::CCDC6 gene fusion. Overall, the patients responded well to afatinib, including radiologic partial responses in six patients during treatment. Responses were durable for three patients. The cases presented are in line with a growing body of clinical and preclinical evidence that indicating that NSCLC with various uncommon EGFR mutations, with or without co-mutations, may be sensitive to afatinib.

Evaluation of the Prognostic Impact of SP263-Evaluated PD-L1 Expression in Patients with Stage III Non-Small Cell Lung Cancer (NSLC) Treated with Radio-Chemotherapy.

BACKGROUND: The PACIFIC study showed that after radio-chemotherapy, patients with NSCLC derived a benefit in PFS and OS when treated with durvalumab. This effect was limited to patients with a PD-L1 expression of >1%, partly because the outcome in the observational control arm was surprisingly favorable. Thus, it could be speculated that a lack of PD-L1 expression confers a favorable outcome for patients with stage III NSCLC. METHODS: Clinical data, PD-L1 expression, predictive blood markers, and the outcomes of 99 homogeneously treated patients with stage III NSCLC were retrospectively captured. Statistical analyses using the log rank test were performed. RESULTS: The median OS of patients with an expression of PD-L1 < 1% was 20 months (CI 10.5-29.5) and the median OS of patients with an expression of PD-L1 ≥ 1% was 28 months (CI 16.5-39.2) (p = 0.734). The median PFS of patients with an expression of PD-L1 < 1% was 9 months (CI 6.3-11.6) and the median PFS of patients with an expression of PD-L1 ≥ 1% was 12 months (CI 9.8-14.2) (p = 0.112). CONCLUSIONS: The assumption that the lack of PD-L1 expression represents a favorable prognostic factor after radio-chemotherapy vs. PD-L1 expression > 1% was not confirmed.

The Combined Therapy of Cabozantinib, Crizotinib, and Osimertinib in a Lung Cancer Patient with Acquired MET Amplification and Resistance Mutations.

EGFR-mutant lung cancers develop a wide range of potential resistance alterations under therapy with the third-generation EGFR tyrosine kinase inhibitor osimertinib. MET amplification ranks among the most common acquired resistance alterations and is currently being investigated as a therapeutic target in several studies. Nevertheless, targeted therapy of MET might similarly result in acquired resistance by point mutations in MET, which further expands therapeutic and diagnostic challenges. Here, we report a 50-year-old male patient with EGFR-mutant lung adenocarcinoma and stepwise acquired resistance by a focal amplification of MET followed by D1246N (D1228N), D1246H (D1228H), and L1213V (L1195V) point mutations in MET, all detected by NGS. The patient successfully responded to the combined and sequential treatment of osimertinib, osimertinib/crizotinib, and third-line osimertinib/cabozantinib. This case highlights the importance of well-designed, sequential molecular diagnostic analyses and the personalized treatment of patients with acquired resistance.

Fluctuation of Acquired Resistance Mutations and Re-Challenge with EGFR TKI in Metastatic NSCLC: A Case Report.

Osimertinib has become the preferred first-line therapy for epidermal growth factor receptor (EGFR) mutation-positive metastatic non-small cell lung cancer (NSCLC) in recent years. Originally, it was approved for second-line treatment after epidermal growth factor receptor EGFR tyrosine kinase inhibitors (TKIs) of the first and second generations had failed and EGFR T790M had emerged as a mode of resistance. Osimertinib itself provokes a wide array of on- and off-target molecular alterations that can limit therapeutic success. Liquid biopsy ctDNA (circulating tumor DNA) analysis by hybrid capture (HC) next-generation sequencing (NGS) can help to identify alterations in a minimally invasive way and allows for the detection of common as well as rare resistance alterations. We describe a young female patient who was initially diagnosed with metastatic EGFR L858R-positive NSCLC. She received EGFR TKI therapy at different timepoints during the course of the disease and developed sequential EGFR resistance alterations (EGFR T790M and C797S). In the course of her disease, resistance alteration became undetectable, and the tumor was successfully rechallenged with the original first-generation EGFR TKI as well as osimertinib and altogether showed prolonged response despite a prognostically negative TP53 alteration. To date, the patient has been alive for more than seven years, though initially diagnosed with a heavy metastatic burden.

Development of the NOGGO GIS v1 Assay, a Comprehensive Hybrid-Capture-Based NGS Assay for Therapeutic Stratification of Homologous Repair Deficiency Driven Tumors and Clinical Validation.

The worldwide approval of the combination maintenance therapy of olaparib and bevacizumab in advanced high-grade serous ovarian cancer requires complex molecular diagnostic assays that are sufficiently robust for the routine detection of driver mutations in homologous recombination repair (HRR) genes and genomic instability (GI), employing formalin-fixed (FFPE) paraffin-embedded tumor samples without matched normal tissue. We therefore established a DNA-based hybrid capture NGS assay and an associated bioinformatic pipeline that fulfils our institution's specific needs. The assay´s target regions cover the full exonic territory of relevant cancer-related genes and HRR genes and more than 20,000 evenly distributed single nucleotide polymorphism (SNP) loci to allow for the detection of genome-wide allele specific copy number alterations (CNA). To determine GI status, we implemented an %CNA score that is robust across a broad range of tumor cell content (25-85%) often found in routine FFPE samples. The assay was established using high-grade serous ovarian cancer samples for which BRCA1 and BRCA2 mutation status as well as Myriad MyChoice homologous repair deficiency (HRD) status was known. The NOGGO (Northeastern German Society for Gynecologic Oncology) GIS (GI-Score) v1 assay was clinically validated on more than 400 samples of the ENGOT PAOLA-1 clinical trial as part of the European Network for Gynaecological Oncological Trial groups (ENGOT) HRD European Initiative. The "NOGGO GIS v1 assay" performed using highly robust hazard ratios for progression-free survival (PFS) and overall survival (OS), as well a significantly lower dropout rate than the Myriad MyChoice clinical trial assay supporting the clinical utility of the assay. We also provide proof of a modular and scalable routine diagnostic method, that can be flexibly adapted and adjusted to meet future clinical needs, emerging biomarkers, and further tumor entities.

Response of an HER2-Mutated NSCLC Patient to Trastuzumab Deruxtecan and Monitoring of Plasma ctDNA Levels by Liquid Biopsy.

HER2-targeted therapy is currently the subject of several studies in lung cancer and other solid tumors using either tyrosine kinase inhibitors (TKI) or targeted-antibody-drug conjugates. We describe a 61-year-old female patient with HER2 mutated adenocarcinoma of the lungs who received chemo-immunotherapy, followed by trastuzumab deruxtecan (T-DXd) and third-line Ramucirumab/Docetaxel at disease progression. Plasma ctDNA monitoring was obtained at 12 timepoints during therapy and revealed HER2 mutation allele frequencies that corresponded to the clinical course of disease. HER2-targeted T-DXd therapy resulted in a profound clinical response and may be an option for NSCLC patients carrying an activated HER2 mutation. Longitudinal liquid biopsy quantification of the underlying driver alteration can serve as a powerful diagnostic tool to monitor course of therapy.

Acquired G2032R Resistance Mutation in ROS1 to Lorlatinib Therapy Detected with Liquid Biopsy.

Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK)/receptor tyrosine kinase inhibitor (ROS1), demonstrated efficacy in ROS1 positive (ROS1+) non-small cell lung cancer (NSCLC), although approval is currently limited to the treatment of ALK+ patients. However, lorlatinib-induced resistance mechanisms, and its efficacy against the resistance mutation G2032R in ROS1, respectively, have not yet been fully understood. Furthermore, concomitant tumor suppressor gene p53 (TP53) mutations occur in driver alteration positive NSCLC, but their prognostic contribution in the context of ROS1 inhibition remains unclear. Here we report a ROS1+ NSCLC patient who developed an on target G2032R resistance mutation during second-line lorlatinib treatment, indicating the lack of activity of lorlatinib against ROS1 G2032R. The resistance mutation was detected in plasma-derived ctDNA, signifying the clinical utility of liquid biopsies.

Hypothermia Induced Impairment of Platelets: Assessment With Multiplate vs. ROTEM-An In Vitro Study.

Introduction: This experimental in vitro study aimed to identify and characterize hypothermia-associated coagulopathy and to compare changes in mild to severe hypothermia with the quantitative measurement of rotational thromboelastometry (ROTEM) and multiple-electrode aggregometry (MULTIPLATE). Methods: Whole blood samples from 18 healthy volunteers were analyzed at the target temperatures of 37, 32, 24, 18, and 13.7°C with ROTEM (ExTEM, InTEM and FibTEM) and MULTIPLATE using the arachidonic acid 0.5 mM (ASPI), thrombin receptor-activating peptide-6 32 µM (TRAP) and adenosine diphosphate 6.4 µM (ADP) tests at the corresponding incubating temperatures for coagulation assessment. Results: Compared to baseline (37°C) values ROTEM measurements of clotting time (CT) was prolonged by 98% (at 18°C), clot formation time (CFT) was prolonged by 205% and the alpha angle dropped to 76% at 13.7°C (p < 0.001). At 24.0°C CT was prolonged by 56% and CFT by 53%. Maximum clot firmness was only slightly reduced by ≤2% at 13.7°C. Platelet function measured by MULTIPLATE was reduced with decreasing temperature (p < 0.001): AUC at 13.7°C -96% (ADP), -92% (ASPI) and -91% (TRAP). Conclusion: Hypothermia impairs coagulation by prolonging coagulation clotting time and by decreasing the velocity of clot formation in ROTEM measurements. MULTIPLATE testing confirms a linear decrease in platelet function with decreasing temperatures, but ROTEM fails to adequately detect hypothermia induced impairment of platelets.

TP53 co-mutations as an independent prognostic factor in 2nd and further line therapy-EGFR mutated non-small cell lung cancer IV patients treated with osimertinib.

BACKGROUND: The negative prognostic and predictive value of TP53 co-mutations (TP53 mt+) in EGFR mutated (EGFR mt+) non-small cell lung cancer (NSCLC) is increasingly being acknowledged. Data consistently show that TP53 mt+ impact negatively on 1st line objective response rate (ORR), progression free survival (PFS) and overall survival (OS) with 1st and 2nd generation tyrosine kinase inhibitors (TKI). However, a negative predictive impact has not been shown for the 3rd generation TKI Osimertinib. Therefore, we investigated the impact of TP53 mt+ in EGFR mt+ NSCLC carrying a T790M resistance mutation and treated in 2nd/further lines with Osimertinib. METHODS: A total of 77 EGFR mt+ NSCLC IV patients carrying a T790M resistance mutation from two institutions were analyzed for TP53 mt+. Clinical data including sex, age, presence of CNS metastases, etc., as well as types of EGFR and TP53 mt+ were captured. PFS and OS were calculated from the start of Osimertinib. RESULTS: TP53 mt+ were found in 32/77 patients (42%). TP53 mt+ was a statistically significant independent negative predictive factor for PFS and OS. PFS for TP53 mt+ patients were 9 months vs. 14 months for patients with TP53 wild-type (TP53WT) (P<0.008). OS for TP53 mt+ patients was 16 months vs. 24 months patients with TP53WT (P<0.025). CONCLUSIONS: TP53 mt+ have a negative impact on PFS and OS in a group of patients carrying a sensitizing EGFR mt+ and a T790M resistance mutation treated with Osimertinib. These data, together with the data for 1st/2nd generation TKI in 1st line treatment call for additional therapeutic and management concepts for this subgroup of patients.

Outcome of Ordinary Polymorphous Adenocarcinomas of the Salivary Glands in Comparison With Papillary and Cribriform Subtypes.

BACKGROUND/AIM: Polymorphous adenocarcinoma (PAC) is a low-grade salivary gland malignancy in contrast to variants with papillary (PAP) or cribriform (CASG) architecture and confers the second most common malignancy of minor salivary glands. Our study aimed to identify prognostic factors and to evaluate histomorphological and molecular diagnostic criteria of PACs. PATIENTS AND METHODS: A series of 155 PACs, including 10 PAPs and 12 CASGs from the population-based Cancer Registry of North Rhine-Westphalia (LKR-NRW) and the Hamburg Salivary Gland Reference Centre (HRC) were analyzed. RESULTS: One fifth of the tumors were located in the major salivary glands and PACS/CASGS invariably lacked p40 expression. Fifty-two percent of PACs showed a PRKD1 E710D mutation. Ordinary PACs had a disease-specific 10-year survival probability of 97% compared to 90% when combining PAPs and CASGs. T-stage at diagnosis was a prognostic factor with 98% for stages T1/T2 versus 75% for T3/T4. CONCLUSION: Diagnostic algorithms for the PAC/CASG spectrum of tumors need to be improved and should include molecular markers.

CPR with restricted patient access using alternative rescuer positions: a randomised cross-over manikin study simulating the CPR scenario after avalanche burial.

BACKGROUND: The aim of this manikin study was to evaluate the quality of cardiopulmonary resuscitation (CPR) with restricted patient access during simulated avalanche rescue using over-the-head and straddle position as compared to standard position. METHODS: In this prospective, randomised cross-over study, 25 medical students (64% male, mean age 24) performed single-rescuer CPR with restricted patient access in over-the-head and straddle position using mouth-to-mouth ventilation or pocket mask ventilation. Chest compression depth, rate, hand position, recoil, compression/decompression ratio, hands-off times, tidal volume of ventilation and gastric insufflation were compared to CPR with unrestricted patient access in standard position. RESULTS: Only 28% of all tidal volumes conformed to the guidelines (400-800 ml), 59% were below 400 ml and 13% were above 800 ml. There was no significant difference in ventilation parameters when comparing standard to atypical rescuer positions. Participants performed sufficient chest compressions depth in 98.1%, a minimum rate in 94.7%, correct compression recoil in 43.8% and correct hand position in 97.3% with no difference between standard and atypical rescuer positions. In 36.9% hands-off times were longer than 9 s. CONCLUSIONS: Efficacy of CPR from an atypical rescuer position with restricted patient access is comparable to CPR in standard rescuer position. Our data suggest to start basic life-support before complete extrication in order to reduce the duration of untreated cardiac arrest in avalanche rescue. Ventilation quality provided by lay rescuers may be a limiting factor in resuscitation situations where rescue ventilation is considered essential.

Mucoepidermoid carcinoma of the salivary glands revisited with special reference to histologic grading and CRTC1/3-MAML2 genotyping.

Mucoepidermoid carcinoma (MEC) is the most common carcinoma of the salivary glands. Here, we have used two large patient cohorts with MECs comprising 551 tumors to study clinical, histological, and molecular predictors of survival. One cohort (n = 167), with known CRCT1/3-MAML2 fusion status, was derived from the Hamburg Reference Centre (HRC; graded with the AFIP and Brandwein systems) and the other (n = 384) was derived from the population-based Cancer Registry of North Rhine-Westphalia (LKR-NRW; graded with the AFIP system). The reliability of both the AFIP and Brandwein grading systems was excellent (n = 155). The weighted kappa for inter-rater agreement was 0.81 (95% CI 0.65-0.97) and 0.83 (95% CI 0.71-0.96) for the AFIP and Brandwein systems, respectively. The 5-year relative survival was 79.7% (95% CI 73.2-86.2%). Although the Brandwein system resulted in a higher rate of G3-MECs, survival in G3-tumors (AFIP or Brandwein grading) was markedly worse than in G1/G2-tumors. Survival in > T2 tumors was markedly worse than in those with lower T-stage. Also, fusion-negative MECs had a worse 5-year progression-free survival. The frequency of fusion-positive MECs in the HRC cohort was 78.4%, of which the majority (86.7%) was G1/G2-tumors. In conclusion, the AFIP and Brandwein systems are useful in estimating prognosis and to guide therapy for G3-MECs. However, their significance regarding young age (≤ 30 years) and location-dependent heterogeneity of in particular G2-tumors is more questionable. We conclude that CRTC1/3-MAML2 testing is a useful adjunct to histologic scoring of MECs and for pinpointing tumors with poor prognosis with higher precision, thus avoiding overtreatment.

Comparison of Resistance Spectra after First and Second Line Osimertinib Treatment Detected by Liquid Biopsy.

Since 2009, several first, second, and third generation EGFR tyrosine kinase inhibitors (TKI) have been approved for targeted treatment of EGFR mutated metastatic non-small lung cancer (NSCLC). A vast majority of patients is improving quickly on treatment; however, resistance is inevitable and typically occurs after one year for TKI of the first and second generation. Osimertinib, a third generation TKI, has recently been approved for first line treatment in the palliative setting and is expected to become approved for the adjuvant setting as well. Progression-free survival (PFS) under osimertinib is superior to its predecessors but its spectrum of resistance alterations appears significantly more diverse compared to first and second generation EGFR TKI. As resistance mechanisms to osimertinib are therapeutically targetable in some cases, it is important to comprehensively test for molecular alterations in the relapse scenario. Liquid biopsy may be advantageous over tissue analysis as it has the potential to represent tumor heterogeneity and clonal diversification. We have previously shown high concordance of hybrid capture (HC) based next generation sequencing (NGS) in liquid biopsy versus solid tumor biopsies. In this study, we now present real-word data from 56 patients with metastatic NSCLC that were tested by liquid biopsy at the time of disease progression on mostly second line treated osimertinib treatment. We present examples of single and multiple TKI resistance mechanisms, including mutations in multiple pathways, copy number changes and rare fusions of RET, ALK, FGFR3 and BRAF. In addition, we present the added value of HC based NGS to reveal polyclonal resistance development at the DNA level encoding multiple EGFR C797S and PIK3CA mutations.

BRAF mutations and BRAF mutation functional class have no negative impact on the clinical outcome of advanced NSCLC and associate with susceptibility to immunotherapy.

OBJECTIVE: BRAF mutations have been subtyped in three functional classes with different oncogenic modes of action. The clinical impact of BRAF mutational subtypes in non-small-cell lung cancer (NSCLC) remains to be defined. So far, ambiguous results were reported from analyses of heterogeneous patient cohorts. METHODS: We studied patients with metastatic or recurrent NSCLC who were sequentially enrolled in precision oncology programs at two large German lung cancer centres from 2009 to 2019. The study period allowed evaluating the specific impact of BRAF V600E-targeting. RESULTS: In a cohort of 72 patients, BRAF mutation subtyping revealed p.V600E mutations in 31 cases (43%), whereas 41 cases (57%) harboured 18 different BRAF mutational subtypes of functional classes II/III. Functionally relevant comutations were observed in 6.4% of class I, and 24.4% of class II/III BRAF mutations. Most patients were treated with chemotherapy. Targeted therapy was administered in 11 patients with a response rate of 72.7%. PD-1/PD-L1-immunotherapy was given in 14 patients with a response rate of 28.6%. Overall survival of patients with BRAF-mutated NSCLC was inferior (HR 1.38, p = 0.048) as compared to patients with BRAF wild-type cancers. Median time-to-treatment-failure with BRAF-targeting agents was shorter as compared to approved targeted therapy of other oncogenic drivers (HR 1.97, p = 0.05). Survival outcomes were not impacted by BRAF mutation subtype functional class. CONCLUSIONS: Patients with BRAF-mutated NSCLC have an inferior prognosis, which is not determined by BRAF mutation functional class. In contrast to NSCLC with other tractable driver mutations, BRAF-mutated NSCLC exhibit high susceptibility to immune checkpoint inhibitors.

Integration of Tumor Mutation Burden and PD-L1 Testing in Routine Laboratory Diagnostics in Non-Small Cell Lung Cancer.

In recent years, Non-small cell lung cancer (NSCLC) has evolved into a prime example for precision oncology with multiple FDA-approved "precision" drugs. For the majority of NSCLC lacking targetable genetic alterations, immune checkpoint inhibition (ICI) has become standard of care in first-line treatment or beyond. PD-L1 tumor expression represents the only approved predictive biomarker for PD-L1/PD-1 checkpoint inhibition by therapeutic antibodies. Since PD-L1-negative or low-expressing tumors may also respond to ICI, additional factors are likely to contribute in addition to PD-L1 expression. Tumor mutation burden (TMB) has emerged as a potential candidate; however, it is the most complex biomarker so far and might represent a challenge for routine diagnostics. We therefore established a hybrid capture (HC) next-generation sequencing (NGS) assay that covers all oncogenic driver alterations as well as TMB and validated TMB values by correlation with the assay (F1CDx) used for the CheckMate 227 study. Results of the first consecutive 417 patients analyzed in a routine clinical setting are presented. Data show that fast reliable comprehensive diagnostics including TMB and targetable alterations are obtained with a short turn-around time. Thus, even complex biomarkers can easily be implemented in routine practice to optimize treatment decisions for advanced NSCLC.

Mutational spectrum of acquired resistance to reversible versus irreversible EGFR tyrosine kinase inhibitors.

BACKGROUND: Over the past years, EGFR tyrosine kinase inhibitors (TKI) revolutionized treatment response. 1st-generation (reversible) EGFR TKI and later the 2nd -generation irreversible EGFR TKI Afatinib were aimed to improve treatment response. Nevertheless, diverse resistance mechanisms develop within the first year of therapy. Here, we evaluate the prevalence of acquired resistance mechanisms towards reversible and irreversible EGFR TKI. METHODS: Rebiopsies of patients after progression to EGFR TKI therapy (> 6 months) were targeted to histological and molecular analysis. Multiplexed targeted sequencing (NGS) was conducted to identify acquired resistance mutations (e.g. EGFR p.T790M). Further, Fluorescence in situ hybridisation (FISH) was applied to investigate the status of bypass mechanisms like, MET or HER2 amplification. RESULTS: One hundred twenty-three rebiopsy samples of patients that underwent first-line EGFR TKI therapy (PFS ≥6 months) were histologically and molecularly profiled upon clinical progression. The EGFR p.T790M mutation is the major mechanism of acquired resistance in patients treated with reversible as well as irreversible EGFR TKI. Nevertheless a statistically significant difference for the acquisition of T790M mutation has been identified: 45% of afatinib- vs 65% of reversible EGFR TKI treated patients developed a T790M mutation (p-value 0.02). Progression free survival (PFS) was comparable in patients treated with irreversible EGFR irrespective of the sensitising primary mutation or the acquisition of p.T790M. CONCLUSIONS: The EGFR p.T790M mutation is the most prominent mechanism of resistance to reversible and irreversible EGFR TKI therapy. Nevertheless there is a statistically significant difference of p.T790M acquisition between the two types of TKI, which might be of importance for clinical therapy decision.

TP53 co-mutations in EGFR mutated patients in NSCLC stage IV: A strong predictive factor of ORR, PFS and OS in EGFR mt+ NSCLC.

INTRODUCTION: The impact of TP53 co-mutations in EGFR mutated patients on PFS and OS is controversial. Different classifications of TP53 mutations with respect to functional and potential clinical impact have been published. Therefore, we retrospectively analyzed the impact of TP53 co-mutations on ORR, PFS and OS in a cohort of EGFR mutated NSCLC IV patients (UICC 7) using different classifications of TP53 mutations. METHODS: 75 EGFR mutated NSCLC IV patients homogeneously treated with 1st line EGFR TKI were analyzed for TP53 co-mutations. TP53 mutations were classified according to three different types of classifications. The endpoints ORR, PFS and OS were investigated. RESULTS: TP53 co-mutations were found in 29/59 patients (49.2%). TP53 co-mutations were a statistically significant independent negative predictive factor for ORR, PFS and OS. TP53 co-mutations were associated with inferior mPFS and mOS: mPFS/mOS 12 vs. 18/24 vs. 42 months for non-disruptive/disruptive mutations vs. WT (p < 0.004)/(p < 0.009), 11 vs. 17/23 vs. 42 months for pathogenic vs. non-pathogenic/WT (p < 0.001)/(p < 0.001), and 7 vs. 12 vs. 18/12 vs. 28 vs. 42 months for exon 8 vs. non-exon 8 vs. WT (p < 0.001)/(p < 0.002). CONCLUSIONS: TP53 co-mutations are frequent in EGFR mt+ NSCLC and have a strong negative impact on all clinical endpoints of TKI therapy.

Determination of PD-L1 Expression in Circulating Tumor Cells of NSCLC Patients and Correlation with Response to PD-1/PD-L1 Inhibitors.

Circulating tumor cells (CTCs) hold great potential to answer key questions of how non-small cell lung cancer (NSCLC) evolves and develops resistance upon anti-PD-1/PD-L1 treatment. Currently, their clinical utility in NSCLC is compromised by a low detection rate with the established, Food and Drug Administration (FDA)-approved, EpCAM-based CellSearch® System. We tested an epitope-independent method (ParsortixTM system) and utilized it to assess PD-L1 expression of CTCs from NSCLC patients. We prospectively collected 127 samples, 97 of which were analyzed with the epitope-independent system in comparison to the CellSearch system. CTCs were determined by immunocytochemistry as intact, nucleated, CD45-, pankeratins (K)+ cells. PD-L1 status of CTCs was evaluated from 89 samples. With the epitope-independent system, ≥1 CTC per blood sample was detected in 59 samples (61%) compared to 31 samples (32%) with the EpCAM-based system. Upon PD-L1 staining, 47% of patients harbored only PD-L1+CTCs, 47% had PD-L1+ and PD-L1-CTCs, and only 7% displayed exclusively PD-L1-CTCs. The percentage of PD-L1+CTCs did not correlate with the percentage of PD-L1+ in biopsies determined by immunohistochemistry (p = 0.179). Upon disease progression, all patients showed an increase in PD-L1+CTCs, while no change or a decrease in PD-L1+CTCs was observed in responding patients (n = 11; p = 0.001). Our data show a considerable heterogeneity in the PD-L1 status of CTCs from NSCLC patients. An increase of PD-L1+CTCs holds potential to predict resistance to PD-1/PD-L1 inhibitors.

P14 methylation: an epigenetic signature of salivary gland mucoepidermoid carcinoma in the Serbian population.

OBJECTIVE: To investigate the prevalence of p16INK4 a, p14ARF, tumor protein p53 (TP53), and human telomerase reverse transcriptase (hTERT) promoter hypermethylation in mucoepidermoid carcinomas (MECs) and search for a possible association between methylation status and clinicopathological parameters. STUDY DESIGN: DNA extracted from 35 formalin-fixed and paraffin-embedded MEC samples and 10 normal salivary gland (NSG) tissue samples was analyzed for the presence of promoter hypermethylation using methylation-specific polymerase chain reaction testing. RESULTS: The percentages of gene hypermethylation in MECs versus NSGs were the following: p14: 100% versus 20% (P<.001); p16: 60% versus 20% (P = .035); hTERT: 54.3% versus 20% (P = .078); and TP53: 31.4% versus 30% (P = .981). Multiple sites were found to be methylated in 86% of MECs compared with 10% in NSGs (P< .001). TP53 and hTERT were more often methylated in lower clinical stages (P = .033 and P = .005, respectively). CONCLUSIONS: Hypermethylation of p14 appears to be an important event in the development of mucoepidermoid carcinoma. High frequency of gene hypermethylation and high incidence of methylation at multiple sites point to the importance of epigenetic phenomena in the pathogenesis of MECs, although with modest impact on clinical parameters.