The neo-open reading frame peptides that comprise the tumor framome are a rich source of neoantigens for cancer immunotherapy.

Identification of immunogenic cancer neoantigens as targets for therapy is challenging. Here, we integrate cancer whole genome and long-read transcript sequencing to identify the collection of novel open reading frame peptides (NOPs) expressed in tumors, termed the framome. NOPs represent tumor-specific peptides that are different from wild-type proteins and may be strongly immunogenic. We describe an uncharacterized class of hidden NOPs, which derive from structural genomic variants involving an upstream protein coding gene driving expression and translation of non-coding regions of the genome downstream of a rearrangement breakpoint. NOPs represent a vast amount of possible neoantigens particularly in tumors with many (complex) structural genomic variants and a low number of missense mutations. We show that NOPs are immunogenic and epitopes derived from NOPs can bind to MHC class I molecules. Finally, we provide evidence for the presence of memory T-cells specific for hidden NOPs in lung cancer patient peripheral blood.

Blood platelet RNA profiles do not enable for nivolumab response prediction at baseline in patients with non-small cell lung cancer.

BACKGROUND: Anti-PD-(L)1 immunotherapy has emerged as a promising treatment approach for non-small cell lung cancer (NSCLC), though the response rates remain low. Pre-treatment response prediction may improve patient allocation for immunotherapy. Blood platelets act as active immune-like cells, thereby constraining T-cell activity, propagating cancer metastasis, and adjusting their spliced mRNA content. OBJECTIVE: We investigated whether platelet RNA profiles before start of nivolumab anti-PD1 immunotherapy may predict treatment responses. METHODS: We performed RNA-sequencing of platelet RNA samples isolated from stage III-IV NSCLC patients before treatment with nivolumab. Treatment response was scored by the RECIST-criteria. Data were analyzed using a predefined thromboSeq analysis including a particle-swarm-enhanced support vector machine (PSO/SVM) classification algorithm. RESULTS: We collected and processed a 286-samples cohort, separated into a training/evaluation and validation series and subjected those to training of the PSO/SVM-classification algorithm. We observed only low classification accuracy in the 107-samples validation series (area under the curve (AUC) training series: 0.73 (95% -CI: 0.63-0.84, n = 88 samples), AUC evaluation series: 0.64 (95% -CI: 0.51-0.76, n = 91 samples), AUC validation series: 0.58 (95% -CI: 0.45-0.70, n = 107 samples)), employing a five-RNAs biomarker panel. CONCLUSIONS: We concluded that platelet RNA may have minimally discriminative capacity for anti-PD1 nivolumab response prediction, with which the current methodology is insufficient for diagnostic application.

A comprehensive overview of the heterogeneity of EGFR exon 20 variants in NSCLC and (pre)clinical activity to currently available treatments.

Activating EGFR mutations are commonly observed in non-small cell lung cancer (NSCLC). About 4-10 % of all activating epidermal growth factor receptor (EGFR) mutations are heterogenous in-frame deletion and/or insertion mutations clustering within exon 20 (EGFRex20+). NSCLC patients with EGFRex20+ mutations are treated as a single disease entity, irrespective of the type and location of the mutation. Here, we provide a comprehensive assessment of the literature reporting both in vitro and clinical drug sensitivity across different EGFRex20+ mutations. The activating A763_Y764insFQEA mutation has a better tumor response in comparison with mutations in the near- and far regions directly following the C-helix and should therefore be treated differently. For other EGFRex20+ mutations marked differences in treatment responses have been reported indicating the need for a classification beyond the exon-based classification. A further classification can be achieved using a structure-function modeling approach and experimental data using patient-derived cell lines. The detailed overview of TKI responses for each EGFRex20+ mutation can assist treating physicians to select the most optimal drug for individual NSCLC patients.

Detection and Monitoring of Tumor-Derived Mutations in Circulating Tumor DNA Using the UltraSEEK Lung Panel on the MassARRAY System in Metastatic Non-Small Cell Lung Cancer Patients.

Analysis of circulating tumor DNA (ctDNA) is a potential minimally invasive molecular tool to guide treatment decision-making and disease monitoring. A suitable diagnostic-grade platform is required for the detection of tumor-specific mutations with high sensitivity in the circulating cell-free DNA (ccfDNA) of cancer patients. In this multicenter study, the ccfDNA of 72 patients treated for advanced-stage non-small cell lung cancer (NSCLC) was evaluated using the UltraSEEK® Lung Panel on the MassARRAY® System, covering 73 hotspot mutations in EGFR, KRAS, BRAF, ERBB2, and PIK3CA against mutation-specific droplet digital PCR (ddPCR) and routine tumor tissue NGS. Variant detection accuracy at primary diagnosis and during disease progression, and ctDNA dynamics as a marker of treatment efficacy, were analyzed. A multicenter evaluation using reference material demonstrated an overall detection rate of over 90% for variant allele frequencies (VAFs) > 0.5%, irrespective of ccfDNA input. A comparison of UltraSEEK® and ddPCR analyses revealed a 90% concordance. An 80% concordance between therapeutically targetable mutations detected in tumor tissue NGS and ccfDNA UltraSEEK® analysis at baseline was observed. Nine of 84 (11%) tumor tissue mutations were not covered by UltraSEEK®. A decrease in ctDNA levels at 4-6 weeks after treatment initiation detected with UltraSEEK® correlated with prolonged median PFS (46 vs. 6 weeks; p < 0.05) and OS (145 vs. 30 weeks; p < 0.01). Using plasma-derived ccfDNA, the UltraSEEK® Lung Panel with a mid-density set of the most common predictive markers for NSCLC is an alternative tool to detect mutations both at diagnosis and during disease progression and to monitor treatment response.

The additional diagnostic value of virtual bronchoscopy navigation in patients with pulmonary nodules - The NAVIGATOR study.

BACKGROUND: The number of solitary pulmonary nodules to be evaluated is expected to increase and therefore we need to improve diagnostic and therapeutic tools to approach these nodules. To prevent patients from futile invasive procedures and receiving treatment without histological confirmation of cancer, we evaluated the value of virtual bronchoscopy navigation to obtain a diagnosis of the solitary pulmonary nodule in a real-world clinical setting. METHODS: In the NAVIGATOR single center, prospective, observational cohort study patients underwent a virtual bronchoscopy navigation procedure with or without guide sheet tunnelling to assess a solitary pulmonary nodule. Nodules were considered not accessible if a diagnosis could not be obtained by either by CT-guided transthoracic biopsy or conventional bronchoscopy. RESULTS: Between February 2021 and January 2022 35 patients underwent the virtual bronchoscopy navigation procedure. The overall diagnostic yield was 77% and was dependent on size of the nodule and chosen path, with highest yield in lesions with an airway path. Adverse events were few and manageable. CONCLUSION: Virtual bronchoscopy navigation with or without sheet tunnelling is a new technique with a good diagnostic yield, also in patients in whom previously performed procedures failed to establish a diagnosis and/or alternative procedures are considered not feasible based on expected yield and/or safety. Preventing futile or more invasive procedures like surgery or transthoracic punctures with a higher complication rate is beneficial for patients, and allowed treatment adaptation in two-third of the analyzed patient population.

Treatment Characteristics and Real-World Progression-Free Survival in Patients With Unresectable Stage III NSCLC Who Received Durvalumab After Chemoradiotherapy: Findings From the PACIFIC-R Study.

INTRODUCTION: The phase 3 PACIFIC trial established consolidation therapy with durvalumab as standard of care for patients with unresectable, stage III NSCLC and no disease progression after definitive chemoradiotherapy (CRT). The observational PACIFIC-R study assesses the real-world effectiveness of durvalumab in patients from an early access program. Here, we report treatment characteristics and a preplanned analysis of real-world progression-free survival (rwPFS). METHODS: PACIFIC-R (NCT03798535) is an ongoing, international, retrospective study of patients who started durvalumab (intravenously; 10 mg/kg every 2 wk) within an early access program between September 2017 and December 2018. The primary end points are investigator-assessed rwPFS and overall survival (analyzed by Kaplan-Meier method). RESULTS: As of November 30, 2020, the full analysis set comprised 1399 patients from 11 countries (median follow-up duration, 23.5 mo). Patients received durvalumab for a median of 11.0 months. Median rwPFS was 21.7 months (95% confidence interval: 19.1-24.5). RwPFS was numerically longer among patients who received concurrent versus sequential CRT (median, 23.7 versus 19.3 mo) and among patients with programmed cell death-ligand 1 expression greater than or equal to 1% versus less than 1% (22.4 versus 15.6 mo). Overall, 16.5% of the patients had adverse events leading to treatment discontinuation; 9.5% of all patients discontinued because of pneumonitis or interstitial lung disease. CONCLUSIONS: Consolidation durvalumab after definitive CRT was well tolerated and effective in this large, real-world cohort study of patients with unresectable, stage III NSCLC. As expected, rwPFS was longer among patients who received concurrent versus sequential CRT and patients with higher programmed cell death-ligand 1 expression. Nevertheless, favorable rwPFS outcomes were observed regardless of these factors.

Randomised controlled trial of first-line tyrosine-kinase inhibitor (TKI) versus intercalated TKI with chemotherapy for EGFR-mutated nonsmall cell lung cancer.

Introduction: Previous studies have shown interference between epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors and chemotherapy in the cell cycle, thus reducing efficacy. In this randomised controlled trial we investigated whether intercalated erlotinib with chemotherapy was superior compared to erlotinib alone in untreated advanced EGFR-mutated nonsmall cell lung cancer (NSCLC). Materials and methods: Treatment-naïve patients with an activating EGFR mutation, ECOG performance score of 0-3 and adequate organ function were randomly assigned 1:1 to either four cycles of cisplatin-pemetrexed with intercalated erlotinib (day 2-16 out of 21 days per cycle) followed by pemetrexed and erlotinib maintenance (CPE) or erlotinib monotherapy. The primary end-point was progression-free survival (PFS). Secondary end-points were overall survival, objective response rate (ORR) and toxicity. Results: Between April 2014 and September 2016, 22 patients were randomised equally into both arms; the study was stopped due to slow accrual. Median follow-up was 64 months. Median PFS was 13.7 months (95% CI 5.2-18.8) for CPE and 10.3 months (95% CI 7.1-15.5; hazard ratio (HR) 0.62, 95% CI 0.25-1.57) for erlotinib monotherapy; when compensating for number of days receiving erlotinib, PFS of the CPE arm was superior (HR 0.24, 95% CI 0.07-0.83; p=0.02). ORR was 64% for CPE versus 55% for erlotinib monotherapy. Median overall survival was 31.7 months (95% CI 21.8-61.9 months) for CPE compared to 17.2 months (95% CI 11.5-45.5 months) for erlotinib monotherapy (HR 0.58, 95% CI 0.22-1.41 months). Patients treated with CPE had higher rates of treatment-related fatigue, anorexia, weight loss and renal toxicity. Conclusion: Intercalating erlotinib with cisplatin-pemetrexed provides a longer PFS compared to erlotinib alone in EGFR-mutated NSCLC at the expense of more toxicity.

Detection and localization of early- and late-stage cancers using platelet RNA.

Cancer patients benefit from early tumor detection since treatment outcomes are more favorable for less advanced cancers. Platelets are involved in cancer progression and are considered a promising biosource for cancer detection, as they alter their RNA content upon local and systemic cues. We show that tumor-educated platelet (TEP) RNA-based blood tests enable the detection of 18 cancer types. With 99% specificity in asymptomatic controls, thromboSeq correctly detected the presence of cancer in two-thirds of 1,096 blood samples from stage I-IV cancer patients and in half of 352 stage I-III tumors. Symptomatic controls, including inflammatory and cardiovascular diseases, and benign tumors had increased false-positive test results with an average specificity of 78%. Moreover, thromboSeq determined the tumor site of origin in five different tumor types correctly in over 80% of the cancer patients. These results highlight the potential properties of TEP-derived RNA panels to supplement current approaches for blood-based cancer screening.

The Presence of EGFR T790M in TKI-Naïve Lung Cancer Samples of Patients Who Developed a T790M-Positive Relapse on First or Second Generation TKI Is Rare.

EGFR-mutated non-small cell lung cancer (NSCLC) patients can be effectively treated with tyrosine kinase inhibitors (TKI) but frequently present with an EGFR T790M resistance mutation at relapse. We aimed to screen for T790M in pre-treatment formalin-fixed and paraffin-embedded (FFPE) tissue samples of patients with a confirmed T790M mutation at progression. We analyzed 33 pre-treatment DNA samples of NSCLC patients who progressed upon TKI between 2013 to 2019. To establish storage-time dependent formalin fixation-induced background levels for C>T mutations, we analyzed DNA isolated from archival (stored >1 year, n = 22) and recently generated (stored <1 month, n = 11) FFPE samples and included DNA isolated from white blood cells (WBC) (n = 24) as controls. DNA samples were analyzed by droplet digital (dd)PCR, and positivity was defined by outlier detection according to Grubb's criterion. The T790M background allele frequency levels were 0.160% in DNA isolated from archival-FFPE, 0.100% in fresh FFPE, and 0.035% in WBC. Progression-free survival (PFS) time of the single T790M positive patient was 9 months, while T790M negative patients had a median PFS of 10 months (range 2−27). Proper storage time matched FFPE control samples are essential for reliable detection of T790M mutation at low VAF. The presence of EGFR T790M mutations in pre-TKI samples is rare, even in patients who progressed with EGFR T790M mutations.

High dose osimertinib in patients with advanced stage EGFR exon 20 mutation-positive NSCLC: Results from the phase 2 multicenter POSITION20 trial.

INTRODUCTION: Patients with life-threatening advanced non-small cell lung cancer (NSCLC) who harbor an exon 20 deletion and/or insertion mutation (EGFRex20 + ) have limited effective treatment options. The high dose 3rd generation tyrosine kinase inhibitor (TKI) osimertinib shows promising in vitro activity in EGFRex20 + NSCLC tumors. METHODS: The POSITION20 is a single arm phase II, multicenter study investigating 160 mg osimertinib in patients with EGFRex20+, T790M negative NSCLC. We allowed patients to be treatment naïve and to have asymptomatic brain metastases. The primary endpoint was overall response rate (ORR). Secondary outcomes were duration of response (DoR), progression free survival (PFS), overall survival (OS), and treatment related adverse events (trAEs). RESULTS: From June 2018 to October 2021, 25 patients were enrolled across five centers in the Netherlands. The median age was 70 years (range, 47-87), 20 patients (80%) were women, and the median number of previous lines of therapy was 1 (range, 0-3). The exon 20 mutations were clustered between A763 and L777. The most common exon 20 mutations were p.(N771_H773dup) (n = 3) and p.(A767_V769dup) (n = 3). The ORR was 28% (95% CI, 12-49%), including seven partial responses, with a median DoR of 5.3 months (range, 2.7-27.6). The median PFS was 6.8 months (95% CI, 4.6-9.1) and the median OS was 15.2 months (95% CI, 14.3-16.0). The most common trAEs were diarrhea (72%), dry skin (44%), and fatigue (44%). The primary reason for discontinuation was progressive disease in 14 patients (56%). CONCLUSION: The POSITION20 study showed modest antitumor activity in patients with EGFRex20 + NSCLC treated with 160 mg osimertinib, with a confirmed ORR of 28% and acceptable toxicity.

Actionability of on-target ALK Resistance Mutations in Patients With Non-Small Cell Lung Cancer: Local Experience and Review of the Literature.

INTRODUCTION: Non-small cell lung cancer (NSCLC) patients with Anaplastic Lymphoma Kinase (ALK) gene fusions respond well to ALK inhibitors but commonly develop on-target resistance mutations. The objective of this study is to collect clinical evidence for subsequent treatment with ALK inhibitors. PATIENTS AND METHODS: Local experience with on-target ALK resistance mutations and review of the literature identified 387 patients with ALK inhibitor resistance mutations. Clinical benefit of mutation-inhibitor combinations was assessed based on reported response, progression-free survival and duration of treatment. Furthermore, this clinical evidence was compared to previously reported in vitro sensitivity of mutations to the inhibitors. RESULTS: Of the pooled population of 387 patients in this analysis, 239 (62%) received at least 1 additional line of ALK inhibition after developing on-target resistance to ALK inhibitor therapy. Clinical benefit was reported for 177 (68%) patients, but differed for each mutation-inhibitor combination. Agreement between in vitro predicted sensitivity of 6 published models and observed clinical benefit ranged from 69% to 89%. The observed clinical evidence for highest probability of response in the context of specific on-target ALK inhibitor resistance mutations is presented. CONCLUSION: Molecular diagnostics performed on tissue samples that are refractive to ALK inhibitor therapy can reveal new options for targeted therapy for NSCLC patients. Our comprehensive overview of clinical evidence of drug actionability of ALK on-target resistance mechanisms may serve as a practical guide to select the most optimal drug for individual patients.

Leukapheresis increases circulating tumour cell yield in non-small cell lung cancer, counts related to tumour response and survival.

BACKGROUND: Circulating tumour cells (CTCs) can be used to monitor cancer longitudinally, but their use in non-small cell lung cancer (NSCLC) is limited due to low numbers in the peripheral blood. Through diagnostic leukapheresis (DLA) CTCs can be obtained from larger blood volumes. METHODS: Patients with all stages of NSCLC were selected. One total body blood volume was screened by DLA before and after treatment. Peripheral blood was drawn pre- and post DLA for CTC enumeration by CellSearch. CTCs were detected in the DLA product (volume equalling 2 × 108 leucocytes) and after leucocyte depletion (RosetteSep, 9 mL DLA product). Single-cell, whole-genome sequencing was performed on isolated CTCs. RESULTS: Fifty-six patients were included. Before treatment, CTCs were more often detected in DLA (32/55, 58%) than in the peripheral blood (pre-DLA: 18/55, 33%; post DLA: 13/55, 23%, both at p < 0.01). CTCs per 7.5 mL DLA product were median 9.2 times (interquartile range = 5.6-24.0) higher than CTCs in 7.5 mL blood. RosetteSEP did not significantly improve CTC detection (pretreatment: 34/55, 62%, post treatment: 16/34, 47%) and CTCs per mL even decreased compared to DLA (p = 0.04).. Patients with advanced-stage disease with DLA-CTC after treatment showed fewer tumour responses and shorter progression-free survival (PFS) than those without DLA-CTC (median PFS, 2.0 vs 12.0 months, p < 0.01). DLA-CTC persistence after treatment was independent of clinical factors associated with shorter PFS (hazard ratio (HR) = 5.8, 95% confidence interval (CI), 1.4-35.5, p = 0.02). All evaluable CTCs showed aneuploidy. CONCLUSIONS: DLA detected nine times more CTCs than in the peripheral blood. The sustained presence of CTCs in DLA after treatment was associated with therapy failure and shortened PFS. TRIAL REGISTRATION: The study was approved by the Medical Ethical Committee (NL55754.042.15) and was registered in the Dutch trial register (NL5423).

mRNA-1273 COVID-19 vaccination in patients receiving chemotherapy, immunotherapy, or chemoimmunotherapy for solid tumours: a prospective, multicentre, non-inferiority trial.

BACKGROUND: Patients with cancer have an increased risk of complications from SARS-CoV-2 infection. Vaccination to prevent COVID-19 is recommended, but data on the immunogenicity and safety of COVID-19 vaccines for patients with solid tumours receiving systemic cancer treatment are scarce. Therefore, we aimed to assess the impact of immunotherapy, chemotherapy, and chemoimmunotherapy on the immunogenicity and safety of the mRNA-1273 (Moderna Biotech, Madrid, Spain) COVID-19 vaccine as part of the Vaccination Against COVID in Cancer (VOICE) trial. METHODS: This prospective, multicentre, non-inferiority trial was done across three centres in the Netherlands. Individuals aged 18 years or older with a life expectancy of more than 12 months were enrolled into four cohorts: individuals without cancer (cohort A [control cohort]), and patients with solid tumours, regardless of stage and histology, treated with immunotherapy (cohort B), chemotherapy (cohort C), or chemoimmunotherapy (cohort D). Participants received two mRNA-1273 vaccinations of 100 µg in 0·5 mL intramuscularly, 28 days apart. The primary endpoint, analysed per protocol (excluding patients with a positive baseline sample [>10 binding antibody units (BAU)/mL], indicating previous SARS-CoV-2 infection), was defined as the SARS-CoV-2 spike S1-specific IgG serum antibody response (ie, SARS-CoV-2-binding antibody concentration of >10 BAU/mL) 28 days after the second vaccination. For the primary endpoint analysis, a non-inferiority design with a margin of 10% was used. We also assessed adverse events in all patients who received at least one vaccination, and recorded solicited adverse events in participants who received at least one vaccination but excluding those who already had seroconversion (>10 BAU/mL) at baseline. This study is ongoing and is registered with ClinicalTrials.gov, NCT04715438. FINDINGS: Between Feb 17 and March 12, 2021, 791 participants were enrolled and followed up for a median of 122 days (IQR 118 to 128). A SARS-CoV-2-binding antibody response was found in 240 (100%; 95% CI 98 to 100) of 240 evaluable participants in cohort A, 130 (99%; 96 to >99) of 131 evaluable patients in cohort B, 223 (97%; 94 to 99) of 229 evaluable patients in cohort C, and 143 (100%; 97 to 100) of 143 evaluable patients in cohort D. The SARS-CoV-2-binding antibody response in each patient cohort was non-inferior compared with cohort A. No new safety signals were observed. Grade 3 or worse serious adverse events occurred in no participants in cohort A, three (2%) of 137 patients in cohort B, six (2%) of 244 patients in cohort C, and one (1%) of 163 patients in cohort D, with four events (two of fever, and one each of diarrhoea and febrile neutropenia) potentially related to the vaccination. There were no vaccine-related deaths. INTERPRETATION: Most patients with cancer develop, while receiving chemotherapy, immunotherapy, or both for a solid tumour, an adequate antibody response to vaccination with the mRNA-1273 COVID-19 vaccine. The vaccine is also safe in these patients. The minority of patients with an inadequate response after two vaccinations might benefit from a third vaccination. FUNDING: ZonMw, The Netherlands Organisation for Health Research and Development.

Circulating tumor DNA as a biomarker for monitoring early treatment responses of patients with advanced lung adenocarcinoma receiving immune checkpoint inhibitors.

Immunotherapy for metastasized non-small-cell lung cancer (NSCLC) can show long-lasting clinical responses. Selection of patients based on programmed death-ligand 1 (PD-L1) expression shows limited predictive value for durable clinical benefit (DCB). We investigated whether early treatment effects as measured by a change in circulating tumor DNA (ctDNA) level is a proxy of early tumor response to immunotherapy according to response evaluation criteria in solid tumors v1.1 criteria, progression-free survival (PFS), DCB, and overall survival (OS). To this aim, blood tubes were collected from advanced-stage lung adenocarcinoma patients (n = 100) receiving immune checkpoint inhibitors (ICI) at baseline (t0 ) and prior to first treatment evaluation (4-6 weeks; t1 ). Nontargetable (driver) mutations detected in the pretreatment tumor biopsy were used to quantify tumor-specific ctDNA levels using droplet digital PCR. We found that changes in ctDNA levels were strongly associated with tumor response. A > 30% decrease in ctDNA at t1 correlated with a longer PFS and OS. In total, 80% of patients with a DCB of ≥ 26 weeks displayed a > 30% decrease in ctDNA levels. For patients with a PD-L1 tumor proportion score of ≥ 1%, decreasing ctDNA levels were associated with a higher frequency a DCB (80%) and a prolonged median PFS (85 weeks) and OS (101 weeks) compared with patients with no decrease in ctDNA (34%; 11 and 39 weeks, respectively). This study shows that monitoring of ctDNA dynamics is an easy-to-use and promising tool for assessing PFS, DCB, and OS for ICI-treated NSCLC patients.

EPAC-lung: European pooled analysis of the prognostic value of circulating tumour cells in small cell lung cancer.

BACKGROUND: Circulating tumour cell (CTC) number is an independent prognostic factor in patients with small cell lung cancer (SCLC) but there is no consensus on the CTC threshold for prognostic significance. We undertook a pooled analysis of individual patient data to clinically validate CTC enumeration and threshold for prognostication. METHODS: Four European cancer centres, experienced in CellSearch CTC enumeration for SCLC provided pseudo anonymised data for patients who had undergone pre-treatment CTC count. Data was collated, and Cox regression models, stratified by centre, explored the relationship between CTC count and survival. The added value of incorporating CTCs into clinico-pathological models was investigated using likelihood ratio tests. RESULTS: A total of 367 patient records were evaluated. A one-unit increase in log-transformed CTC counts corresponded to an estimated hazard ratio (HR) of 1.24 (95% CI: 1.19-1.29, P<0.0001) for progression free survival (PFS) and 1.23 (95% CI: 1.18-1.28, P<0.0001) for overall survival (OS). CTC count of ≥15 or ≥50 was significantly associated with an increased risk of progression (CTC ≥15: HR 3.20, 95% CI: 2.50-4.09, P<0.001; CTC ≥50: HR 2.56, 95% CI: 2.01-3.25, P<0.001) and an increased risk of death (CTC ≥15: HR 2.90, 95% CI: 2.28-3.70, P<0.001; CTC ≥50: HR 2.47, 95% CI: 1.95-3.13, P<0.001). There was no significant inter-centre heterogeneity observed. Addition of CTC count to clinico-pathological models as a continuous log-transformed variable, offers further prognostic value (both likelihood ratio P<0.001 for OS and PFS). CONCLUSIONS: Higher pre-treatment CTC counts are a negative independent prognostic factor in SCLC when considered as a continuous variable or dichotomised counts of ≥15 or ≥50. Incorporating CTC counts, as a continuous variable, improves clinic-pathological prognostic models.

A unique small cell lung carcinoma disease progression model shows progressive accumulation of cancer stem cell properties and CD44 as a potential diagnostic marker.

OBJECTIVES: Cancer stem cells (CSCs) have been implicated in disease progression of aggressive cancers including small cell lung carcinoma (SCLC). Here, we have examined the possible contribution of CSCs to SCLC progression and aggressiveness. MATERIALS AND METHODS: GLC-14, GLC-16 and GLC-19 SCLC cell lines derived from one patient, representing increasing progressive stages of disease were used. CSC marker expressions was determined by RT-qPCR and western blotting analyses, and heterogeneity was studied by CSC marker expression by immunofluorescence microscopy and flow cytometry. Colony formation assays were used to assess stem cell properties and therapy sensitivity. RESULTS: Increasing expression of stem cell markers MYC, SOX2 and particularly CD44 were found in association with advancing disease. Single and overlapping expression of these markers indicated the presence of different CSC populations. The accumulation of more homogeneous double- and triple-positive CSC populations evolved with disease progression. Functional characterization of CSC properties affirmed higher proficiency of colony forming ability and increased resistance to γ-irradiation in GLC-16 and GLC-19 compared to GLC-14. GLC-19 colony formation was significantly inhibited by a human anti-CD44 antibody. CONCLUSION: The progressive increase of MYC, SOX2 and particularly CD44 expression that was accompanied with enhanced colony forming capacity and resistance in the in vitro GLC disease progression model, supports the potential clinical relevance of CSC populations in malignancy and disease relapse of SCLC.