First-site-metastasis pattern in patients with inoperable stage III NSCLC treated with concurrent chemoradiotherapy with or without immune check-point inhibition: a retrospective analysis.

PURPOSE: The aim of this study was to investigate a first-site-metastasis pattern (FSMP) in unresectable stage III NSCLC after concurrent chemoradiotherapy (cCRT) with or without immune checkpoint inhibition (ICI). METHODS: We defined three patient subgroups according to the year of initial multimodal treatment: A (2011-2014), B (2015-2017) and C (2018-2020). Different treatment-related parameters were analyzed. Observed outcome parameters were brain metastasis-free survival (BMFS), extracranial distant metastasis-free survival (ecDMFS) and distant metastasis-free survival (DMFS). RESULTS: 136 patients treated between 2011 and 2020 were included with ≥ 60.0 Gy total dose and concurrent chemotherapy (cCRT); thirty-six (26%) received ICI. Median follow-up was 49.7 (range:0.7-126.1), median OS 31.2 (95% CI:16.4-30.3) months (23.4 for non-ICI vs not reached for ICI patients, p = 0.001). Median BMFS/ecDMFS/DMFS in subgroups A, B and C was 14.9/16.3/14.7 months, 20.6/12.9/12.7 months and not reached (NR)/NR/36.4 months (p = 0.004/0.001/0.016). For cCRT+ICI median BMFS was 53.1 vs. 19.1 months for cCRT alone (p = 0.005). Median ecDMFS achieved 55.2 vs. 17.9 (p = 0.003) and median DMFS 29.5 (95% CI: 1.4-57.6) vs 14.93 (95% CI:10.8-19.0) months (p = 0.031), respectively. Multivariate analysis showed that age over 65 (HR:1.629; p = 0.036), GTV ≥ 78 cc (HR: 2.100; p = 0.002) and V20 ≥ 30 (HR: 2.400; p = 0.002) were negative prognosticators for BMFS and GTV ≥ 78 cc for ecDMFS (HR: 1.739; p = 0.027). After onset of brain metastasis (BM), patients survived 13.3 (95% CI: 6.4-20.2) months and 8.6 months (95% CI: 1.6-15.5) after extracranial-distant-metastasis (ecDM). Patients with ecDM as FSMP reached significantly worse overall survival of 22.1 (range:14.4-29.8) vs. 40.1 (range:18.7-61.3) months (p = 0.034) in the rest of cohort. In contrast, BM as FSMP had no impact on OS. CONCLUSION: This retrospective analysis of inoperable stage III NSCLC patients revealed that age over 65, V20 ≥ 30 and GTV ≥ 78 cc were prognosticators for BMFS and GTV ≥ 78 cc for ecDMFS. ICI treatment led to a significant improvement of BMFS, ecDMFS and DMFS. ecDM as FSMP was associated with significant deterioration of OS, whereas BM as FSMP was not.

Concurrent/sequential versus sequential immune checkpoint inhibition in inoperable large stage III non-small cell lung cancer patients treated with chemoradiotherapy: a prospective observational study.

PURPOSE/AIM: The international standard for patients with large inoperable stage III NSCLC is durvalumab consolidation after concurrent chemoradiotherapy (CRT). In this single centre observational study based on individual data, we prospectively evaluated the role of concurrent/sequential versus sequential immune checkpoint inhibition (ICI). METHODS AND PATIENTS: In total, 39 stage III NSCLC patients were prospectively enrolled, 11 (28%) patients were treated with simultaneous and consolidation therapy with PD-1 inhibition (nivolumab) (SIM-cohort) and 28 (72%) patients received PD-L1 inhibition (durvalumab) as consolidation treatment up to 12 months after the end of CRT (SEQ-cohort). RESULTS: For the entire cohort, median progression-free survival (PFS) was 26.3 months and median survival (OS), locoregional recurrence-free survival and distant metastasis-free survival were not reached. For the SIM-cohort, median OS was not reached and PFS was 22.8 months, respectively. In the SEQ-cohort, neither median PFS nor OS were reached. After propensity score matching, PFS at 12/24 months were 82/44% in the SIM-cohort and 57/57% in the SEQ-cohort (p = 0.714), respectively. In the SIM-cohort, 36.4/18.2% of patients showed grade II/III pneumonitis; in the SEQ-cohort 18.2/13.6% after PSM (p = 0.258, p = 0.55). CONCLUSION: Both concurrent/sequential and sequential ICI show a favorable side effect profile and promising survival in treated patients with inoperable large stage III NSCLC. Concurrent ICI showed a numerical non-significant improvement regarding 6- and 12-months PFS and distant control compared to sequential approach in this small study. However, concurrent ICI to CRT was associated with a non-significant moderate increase in grade II/III pneumonitis.

Treatment patterns and prognosis of patients with inoperable stage III NSCLC after completion of concurrent chemoradiotherapy ± immune checkpoint inhibition: a decade-long single-center historical analysis.

PURPOSE: To investigate the impact of treatment time and patterns in inoperable stage III non-small cell lung cancer (NSCLC) following concurrent chemoradiotherapy (cCRT) ± immune checkpoint inhibitors (ICIs). METHODS: Patients were stratified by treatment year: A (2011-2014), B (2015-2017) and C (2018-2020). Tumor- and treatment-related characteristics regarding locoregional recurrence-free survival (LRRFS), progression-free survival (PFS) and overall survival (OS) were investigated. RESULTS: One hundred and thirty-six consecutive patients were analyzed. All patients completed thoracic radiotherapy (TRT) to a total dose ≥ 60.0 Gy; 36 (26%) patients received ICI. Median PFS in subgroups A, B and C was 8.0, 8.2 and 26.3 months (p = 0.007). Median OS was 19.9 months, 23.4 months and not reached (NR), respectively. In group C, median LRRFS and PFS were 27.2 vs. NR; and 14.2 vs. 26.3 months in patients treated with and without ICI. On multivariate analysis planning target volume (PTV) ≥ 700 cc was a negative prognosticator of LRRFS (HR 2.194; p = 0.001), PFS (HR 1.522; p = 0.042) and OS (HR 2.883; p = 0.001); ICI was a predictor of LRRFS (HR 0.497; p = 0.062), PFS (HR 0.571; p = 0.071) and OS (HR 0.447; p = 0.1). In the non-ICI cohort, multivariate analyses revealed PTV ≥ 700 cc (p = 0.047) and a maximum standardized uptake value (SUVmax) ≥ 13.75 (p = 0.012) were predictors of PFS; PTV ≥ 700 cc (p = 0.017), SUVmax ≥ 13.75 (p = 0.002) and a total lung V20 ≥ 30% (V20 ≥ 30) (p < 0.05) were predictors of OS. CONCLUSIONS: Patients treated after 2018 had improved survival regardless of ICI use. Implementation of ICI resulted in further significant increase of all tested survival endpoints. PTV ≥ 700 cc and ICI were only prognosticators for LRRFS, PFS and OS in the analyzed cohort.

Case report: Immunovirotherapy as a novel add-on treatment in a patient with thoracic NUT carcinoma.

NUT carcinoma (NC) is a rare and extremely aggressive form of cancer, usually presenting with intrathoracic or neck manifestations in adolescents and young adults. With no established standard therapy regimen and a median overall survival of only 6.5 months, there is a huge need for innovative treatment options. As NC is genetically driven by a single aberrant fusion oncoprotein, it is generally characterized by a low tumor mutational burden, thus making it immunologically cold and insusceptible to conventional immunotherapy. Recently, we have demonstrated that oncolytic viruses (OVs) are able to specifically infect and lyse NC cells, thereby turning an immunologically cold tumor microenvironment into a hot one. Here, we report an intensive multimodal treatment approach employing for the first time an OV (talimogene laherparepvec (T-VEC); IMLYGIC®) together with the immune checkpoint inhibitor pembrolizumab as an add-on to a basic NC therapy (cytostatic chemotherapy, radiation therapy, epigenetic therapy) in a patient suffering from a large thoracic NC tumor which exhibits an aberrant, unique BRD3:NUTM1 fusion. This case demonstrates for the first time the feasibility of this innovative add-on immunovirotherapy regimen with a profound, repetitive and durable replication of T-VEC that is instrumental in achieving tumor stabilization and improvement in the patient´s quality of life. Further, a previously unknown BRD3:NUTM1 fusion gene was discovered that lacks the extraterminal domain of BRD3.

Planning target volume as a predictor of disease progression in inoperable stage III non-small cell lung cancer patients treated with chemoradiotherapy and concurrent and/or sequential immune checkpoint inhibition.

BACKGROUND: The present study evaluates outcome after chemoradiotherapy (CRT) with concurrent and/or sequential Programmed Cell Death 1 (PD-1) or Ligand 1 (PD-L1) immune checkpoint inhibition (CPI) for inoperable stage III NSCLC patients depending on planning target volume (PTV). METHOD AND PATIENTS: Prospective data of thirty-three consecutive patients with inoperable stage III NSCLC treated with CRT and sequential durvalumab (67%, 22 patients) or concurrent and sequential nivolumab (33%, 11 patients) were analyzed. Different PTV cut offs and PTV as a continuous variable were evaluated for their association with progression-free (PFS), local-regional progression-free (LRPFS), extracranial distant metastasis-free (eMFS) and brain-metastasis free-survival (BMFS). RESULTS: All patients were treated with conventionally fractionated thoracic radiotherapy (TRT); 93% to a total dose of at least 60 Gy, 97% of patients received two cycles of concurrent platinum-based chemotherapy. Median follow-up for the entire cohort was 19.9 (range: 6.0-42.4) months; median overall survival (OS), LRFS, BMFS and eMFS were not reached. Median PFS was 22.8 (95% CI: 10.7-34.8) months. Patients with PTV ≥ 900ccm had a significantly shorter PFS (6.9 vs 22.8 months, p = 0.020) and eMFS (8.1 months vs. not reached, p = 0.003). Furthermore, patients with PTV ≥ 900ccm and stage IIIC disease (UICC-TNM Classification 8th Edition) achieved a very poor outcome with a median PFS and eMFS of 3.6 vs 22.8 months (p < 0.001) and 3.6 months vs. not reached (p = 0.001), respectively. PTV as a continuous variable also had a significant impact on eMFS (p = 0.048). However, no significant association of different PTV cut-offs or PTV as a continuous variable with LRPFS and BMFS could be shown. The multivariate analysis that was performed for PTV ≥ 900ccm and age (≥ 65 years), gender (male), histology (non-ACC) as well as T- and N-stage (T4, N3) as covariates also revealed PTV ≥ 900ccm as the only factor that had a significant correlation with PFS (HR: 5.383 (95% CI:1.263-22.942, p = 0.023)). CONCLUSION: In this prospective analysis of inoperable stage III NSCLC patients treated with definitive CRT combined with concurrent and/or sequential CPI, significantly shorter PFS and eMFS were observed in patients with initial PTV ≥ 900ccm.

Durvalumab after Chemoradiotherapy for PD-L1 Expressing Inoperable Stage III NSCLC Leads to Significant Improvement of Local-Regional Control and Overall Survival in the Real-World Setting.

Concurrent chemoradiotherapy (CRT) followed by maintenance treatment with the PD-L1 inhibitor durvalumab is a new standard of care for inoperable stage III NSCLC. The present study compares the oncological outcome of patients treated with CRT to those treated with CRT and durvalumab (CRT-IO) in the real-world setting. The analysis was performed based on the retro- and prospectively collected data of 144 consecutive inoperable stage III NSCLC patients treated between 2011-2020. Local-regional-progression-free-survival (LRPFS-defined as progression in the mediastinum, hilum and/or supraclavicular region at both sites and the involved lung), progression-free survival (PFS), and overall survival (OS) were evaluated from the last day of thoracic radiotherapy (TRT). Median follow-up for the entire cohort was 33.1 months (range: 6.3-111.8) and median overall survival was 27.2 (95% CI: 19.5-34.9) months. In the CRT-IO cohort after a median follow-up of 20.9 (range: 6.3-27.4) months, median PFS was not reached, LRPFS (p = 0.002), PFS (p = 0.018), and OS (p = 0.005) were significantly improved vs. the historical cohort of conventional CRT patients. After propensity-score matching (PSM) analysis with age, gender, histology, tumor volume, and treatment mode, and exact matching for T-and N-stage, 22 CRT-IO patients were matched 1:2 to 44 CRT patients. Twelve-month LRPFS, PFS, and OS rates in the CRT-IO vs. CRT cohort were 78.9 vs. 45.5% (p = 0.002), 60.0 vs. 31.8% (p = 0.007), and 100 vs. 70.5% (p = 0.003), respectively. This real-world analysis demonstrated that durvalumab after CRT led to significant improvement of local-regional control, PFS, and OS in PD-L1 expressing inoperable stage III NSCLC patients compared to a historical cohort.

Real-world prospective analysis of treatment patterns in durvalumab maintenance after chemoradiotherapy in unresectable, locally advanced NSCLC patients.

The aim of this prospective study is to evaluate the clinical use and real-world efficacy of durvalumab maintenance treatment after chemoradiotherapy (CRT) in unresectable stage, locally advanced non-small cell lung cancer (NSCLC). All consecutive patients with unresectable, locally advanced NSCLC and PD-L1 expression (≥1%) treated after October 2018 were included. Regular follow up, including physical examination, PET/CT and/or contrast-enhanced CT-Thorax/Abdomen were performed every three months after CRT. Descriptive treatment pattern analyses, including reasons of discontinuation and salvage treatment, were undertaken. Statistics were calculated from the last day of thoracic irradiation (TRT). Twenty-six patients were included. Median follow up achieved 20.6 months (range: 1.9-30.6). Durvalumab was initiated after a median of 25 (range: 13-103) days after completion of CRT. In median 14 (range: 2-24) cycles of durvalumab were applied within 6.4 (range 1-12.7) months. Six patients (23%) are still in treatment and seven (27%) have completed treatment with 24 cycles. Maintenance treatment was discontinued in 13 (50%) patients: 4 (15%) patients developed grade 3 pneumonitis according to CTCAE v5 after a median of 3.9 (range: 0.5-11.6) months and 7 (range: 2-17) cycles of durvalumab. Four (15%) patients developed grade 2 skin toxicity. One (4%) patient has discontinued treatment due to incompliance. Six and 12- month progression-free survival (PFS) rates were 82% and 62%, median PFS was not reached. No case of hyperprogression was documented. Eight (31%) patients have relapsed during maintenance treatment after a median of 4.8 (range: 2.2-11.3) months and 11 (range: 6-17) durvalumab cycles. Two patients (9%) developed a local-regional recurrence after 14 and 17 cycles of durvalumab. Extracranial distant metastases and brain metastases as first site of failure were detected in 4 (15%) and 2 (8%) patients, respectively. Three (13%) patients presented with symptomatic relapse. Our prospective study confirmed a favourable safety profile of durvalumab maintenance treatment after completion of CRT in unresectable stage, locally advanced NSCLC in a real-world setting. In a median follow-up time of 20.6 months, durvalumab was discontinued in 27% of all patients due to progressive disease. All patients with progressive disease were eligible for second-line treatment.

Targeted Natural Killer Cell-Based Adoptive Immunotherapy for the Treatment of Patients with NSCLC after Radiochemotherapy: A Randomized Phase II Clinical Trial.

PURPOSE: Non-small cell lung cancer (NSCLC) is a fatal disease with poor prognosis. A membrane-bound form of Hsp70 (mHsp70) which is selectively expressed on high-risk tumors serves as a target for mHsp70-targeting natural killer (NK) cells. Patients with advanced mHsp70-positive NSCLC may therefore benefit from a therapeutic intervention involving mHsp70-targeting NK cells. The randomized phase II clinical trial (EudraCT2008-002130-30) explores tolerability and efficacy of ex vivo-activated NK cells in patients with NSCLC after radiochemotherapy (RCT). PATIENTS AND METHODS: Patients with unresectable, mHsp70-positive NSCLC (stage IIIa/b) received 4 cycles of autologous NK cells activated ex vivo with TKD/IL2 [interventional arm (INT)] after RCT (60-70 Gy, platinum-based chemotherapy) or RCT alone [control arm (CTRL)]. The primary objective was progression-free survival (PFS), and secondary objectives were the assessment of quality of life (QoL, QLQ-LC13), toxicity, and immunobiological responses. RESULTS: The NK-cell therapy after RCT was well tolerated, and no differences in QoL parameters between the two study arms were detected. Estimated 1-year probabilities for PFS were 67% [95% confidence interval (CI), 19%-90%] for the INT arm and 33% (95% CI, 5%-68%) for the CTRL arm (P = 0.36, 1-sided log-rank test). Clinical responses in the INT group were associated with an increase in the prevalence of activated NK cells in their peripheral blood. CONCLUSIONS: Ex vivo TKD/IL2-activated, autologous NK cells are well tolerated and deliver positive clinical responses in patients with advanced NSCLC after RCT.

VOC pattern recognition of lung cancer: a comparative evaluation of different dog- and eNose-based strategies using different sampling materials.

Background: It has been reported that canine scent tests offer the possibility to screen for cancer. Assuming that breath samples can be collected with carrier materials, we tested the practicability of different carrier materials to be presented to dogs and validated and compared results with an electronic nose (eNose). Moreover, we hypothesized that cancer detection ability of dogs differs according to their working experience. Methods: In a methodological approach, two dog teams participated, one using experienced working dogs and the other ordinary household dogs to find the most qualified dogs and training method. To find best carrier material for breath sampling we compared charcoal containing glass tubes and fleece masks. In a second validating part, experienced working dogs were trained with improved training strategies. For breath sampling, two different, previously successfully tested fleece-based carrier materials were used: one was used with the dog team and both materials were compared with eNose. Results: In the methodological approach, it turned out that the charcoal-based sampling strategy qualified not sufficiently for VOC-detection. Moreover, we could determine that using experienced working dogs provided several advantages. Overall results of dogs in the validating part regarding specificity were 83%, regarding sensitivity 56%, but with great variability among dogs. Using eNose for breath analysis collected with both fleece carrier materials, specificity was 97% and sensitivity 89-100%. Conclusion: Our data confirmed that the diagnostic accuracy of dogs depended on the type of dog training and on the carrier materials. A comparison of breath samples analysis with an eNose achieved better results for both, sensitivity and specificity than for dogs. The use of fleece masks or fleeces in glass tubes as a sampling material can be recommended as successful VOC carriers, encouraging their use for clinical screenings.

Pleurectomy/decortication and hyperthermic intrathoracic chemoperfusion using cisplatin and doxorubicin for malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive malignancy with few long-term survivors. Despite the dismal prognosis, hyperthermic intrathoracic chemoperfusion (HITHOC) was shown to improve survival in a selective group of patients. We analyzed the influence of HITHOC following pleurectomy and decortication on postoperative morbidity and overall survival for patients suffering from localized mesothelioma. METHODS: From 2009 until 2013, 71 patients with localized pleural mesothelioma underwent pleurectomy and decortication followed by HITHOC with cisplatin and doxorubicin. We analyzed postoperative morbidity, age, overall survival and influence of macroscopic resection on survival. RESULTS: Median patient age was 70 years (range, 65-73 years). Patients having the sarcomatoid subtype of mesothelioma showed a poor median survival of 9.2 months. In contrast, patients having the epithelioid subtype had a median survival of 17.9 months. Patients following macroscopic complete resection had a significantly better survival with 28.2 months compared to 13.1 months in patients with incomplete resection of the mesothelioma (P<0.0001). HITHOC was performed in all patients after tumor resection using cisplatin and doxorubicin. CONCLUSIONS: Taken together, HITHOC following pleurectomy and decortication is supposed to be a safe therapeutic option for selected patients with localized epithelial pleural mesothelioma.

[Follow-Up and Surveillance after Lung Cancer Treatment]. / Die Nachsorge des Lungenkarzinoms.

The aim of this article is to present the current guideline recommendations for the follow-up and surveillance of lung cancer patients and to outline an evidence-based approach to planing individualised follow-up care.

Frequency and clinical relevance of EGFR mutations and EML4-ALK translocations in octogenarians with non-small cell lung cancer.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) have improved response rates in some patients with non-small cell lung cancer (NSCLC), and testing for EGFR mutation and ALK translocation is recommended for all patients with advanced lung adenocarcinoma. The frequency of driver mutations in elderly and very elderly patients has not been described. PATIENTS AND METHODS: We reviewed EGFR and ALK in patients over the age of 70 years diagnosed and treated at our center in 2015 (subgroups: 70-74, 75-79 and >80 years). We then assessed a second cohort, including all patients with lung cancer over the age of 80 years diagnosed in 2014. We also analyzed smoking history, treatment and response. RESULTS: In the 2015 cohort of 179 patients, 16 were 80 years or older at diagnosis. Six of eight (75%) octogenarians with non-squamous NSCLC were EGFR or ALK positive. The 2014 cohort confirmed the high rate of driver alterations in octogenarians. Of 334 patients, 32 were 80 years or older and, of these, 10 had non-squamous histology and were tested for driver alterations (four of 10 [40%] EGFR or ALK positive). Rates of genetic drivers were somewhat lower in patients with non-squamous NSCLC aged 70-74 years (27.0%) and 75-79 years (26.7%). When treated with a TKI, octogenarians had high response rates and progression-free survival. Most octogenarians with lung adenocarcinoma were never smokers, with an inverse correlation of pack-years smoked to age at diagnosis. CONCLUSION: Very elderly patients with non-squamous NSCLC show high rates of driver alterations in EGFR and ALK. This, often frail and comorbid, population may not be fit for treatment with cytotoxic chemotherapy and may benefit from targeted treatments. Testing for EGFR and ALK alterations should not be restricted to younger patients. The biology of lung cancer in the very elderly may differ from that of moderately elderly patients, as longevity may select for individuals more resistant to, or with little exposure to, environmental carcinogens.

Metformin Triggers Autophagy to Attenuate Drug-Induced Apoptosis in NSCLC Cells, with Minor Effects on Tumors of Diabetic Patients.

The biologic plausibility of an association between type 2 diabetes mellitus (T2D) and lung cancer has received increasing attention, but the results of investigations remain largely inconclusive. In the present study we investigated the influence of the anti-diabetic drug metformin on the cytotoxic effects of EGFR targeted therapy and chemotherapy in 7 non-small cell lung cancer (NSCLC) cell lines and a cohort of lung cancer patients with/without T2D. In vitro cell viability assays indicated that metformin didn't potentiate the growth inhibitory effects of erlotinib at different doses in cell lines that are of distinct genetic background. EGFR downstream signaling evaluation further demonstrated that metformin, at its IC50 value, modified apoptosis caused in erlotinib or chemotherapeutic agent-treated cells via AKT activation and the inhibition of caspase 3 and PARP cleavages. These regulations were driven independently from EGFR, LKB1, KRAS, PTEN and p53 status. Metformin triggered autophagy (LC3B expression) was identified to interplay with apoptosis to attenuate the drug effect and postpone cancer cell death. In the retrospective study of 8 NSCLC patients, the administration of metformin did not induce statistically significant changes as assessed by immunohistochemical staining of pERK, pAKT and cleaved PARP. Consequently, the application of metformin for T2D NSCLC patients receiving chemo or EGFR targeted therapy should be considered with caution.

EWSR1-fusion-negative, SMARCB1-deficient primary pulmonary myxoid sarcoma.

Primary pulmonary myxoid sarcoma (PPMS) is a recently defined rare neoplasm with histological and molecular similarity to extraskeletal myxoid chondrosarcoma. To date, 20 cases have been reported. A 48-year-old man presented with a huge mass filling the right hemithorax and extending into the tracheobronchial system. Histological findings were consistent with PPMS. Immunohistochemistry was positive for vimentin, CD10, and EMA, but other lineage-specific markers were negative. SMARCB1 (INI1) expression was lost in the tumour cells. FISH analysis (EWSR1, FUS, NR4A3, and SMARCB1) revealed no abnormalities. This case suggests SMARCB1 loss as a possible alternative molecular event driving EWSR1-negative PPMS.

Genetic changes of non-small cell lung cancer under neoadjuvant therapy.

BACKGROUND: Large scale sequencing efforts defined common molecular alterations in non-small cell lung cancer (NSCLC) and revealed potentially druggable mutations. Yet, systematic data on the changes of the respective molecular profiles under standard therapy in NSCLC are limited. RESULTS: 14 out of 68 observed coding mutations (21%) and 6 out of 33 (18%) copy number variations (CNV) were lost or gained during therapy. Mutational and CNV changes clustered in 6/37 (16%) and 3/37 (8%) patients. Changes in clinically relevant mutations were rare but present in single cases for genes such as BRAF and PIK3CA. The type of radiochemotherapy but not the duration of therapy impacted on the frequency of mutational changes. METHODS: We established a lung cancer specific next-generation sequencing panel covering ~7500 hotspots of 41 genes frequently mutated in NSCLC and performed ultradeep multigene sequencing of 37 corresponding pre- and post-therapeutic formalin fixed paraffin-embedded specimens to discover mutational changes and copy number variations under neo-adjuvant radio- (RTX) and/or chemotherapy (CTX). CONCLUSION: We unraveled changes in common driver gene candidates in NSCLC under neo-adjuvant therapy. Our data shed first light on the genetic changes of NSCLC under conventional therapy and might be taken into account when the relevance of sequential biopsy approaches is discussed.

Heat Shock Protein 70 (Hsp70) Peptide Activated Natural Killer (NK) Cells for the Treatment of Patients with Non-Small Cell Lung Cancer (NSCLC) after Radiochemotherapy (RCTx) - From Preclinical Studies to a Clinical Phase II Trial.

Heat shock protein 70 (Hsp70) is frequently overexpressed in tumor cells. An unusual cell surface localization could be demonstrated on a large variety of solid tumors including lung, colorectal, breast, squamous cell carcinomas of the head and neck, prostate and pancreatic carcinomas, glioblastomas, sarcomas and hematological malignancies, but not on corresponding normal tissues. A membrane (m)Hsp70-positive phenotype can be determined either directly on single cell suspensions of tumor biopsies by flow cytometry using cmHsp70.1 monoclonal antibody or indirectly in the serum of patients using a novel lipHsp70 ELISA. A mHsp70-positive tumor phenotype has been associated with highly aggressive tumors, causing invasion and metastases and resistance to cell death. However, natural killer (NK), but not T cells were found to kill mHsp70-positive tumor cells after activation with a naturally occurring Hsp70 peptide (TKD) plus low dose IL-2 (TKD/IL-2). Safety and tolerability of ex vivo TKD/IL-2 stimulated, autologous NK cells has been demonstrated in patients with metastasized colorectal and non-small cell lung cancer (NSCLC) in a phase I clinical trial. Based on promising clinical results of the previous study, a phase II randomized clinical study was initiated in 2014. The primary objective of this multicenter proof-of-concept trial is to examine whether an adjuvant treatment of NSCLC patients after platinum-based radiochemotherapy (RCTx) with TKD/IL-2 activated, autologous NK cells is clinically effective. As a mHsp70-positive tumor phenotype is associated with poor clinical outcome only mHsp70-positive tumor patients will be recruited into the trial. The primary endpoint of this study will be the comparison of the progression-free survival of patients treated with ex vivo activated NK cells compared to patients who were treated with RCTx alone. As secondary endpoints overall survival, toxicity, quality-of-life, and biological responses will be determined in both study groups.

Post-study therapy as a source of confounding in survival analysis of first-line studies in patients with advanced non-small-cell lung cancer.

Clinical trials exploring the long-term effects of first-line therapy in patients with advanced non-small-cell lung cancer generally disregard subsequent treatment although most patients receive second and third-line therapies. The choice of further therapy depends on critical intermediate events such as disease progression and it is usually left at the physician's discretion. Time-dependent confounding may then arise with standard survival analyses producing biased effect estimates, even in randomized trials. Herein we describe the concept of time-dependent confounding in detail and discuss whether the response to first-line treatment may be a potential time-dependent confounding factor for survival in the context of subsequent therapy. A prospective observational study of 406 patients with advanced non-small-cell lung cancer served as an example base. There is evidence that time-dependent confounding may occur in multivariate survival analysis after first-line therapy when disregarding subsequent treatment. In the light of this important but underestimated aspect some of the large and meaningful recent clinical first-line lung cancer studies are discussed, focussing on subsequent treatment and its potential impact on the survival of the study patients. No recently performed lung cancer trial applied adequate statistical analyses despite the frequent use of subsequent therapies. In conclusion, effect estimates from standard survival analysis may be biased even in randomized controlled trials because of time-dependent confounding. To adequately assess treatment effects on long-term outcomes appropriate statistical analyses need to take subsequent treatment into account.

Clinical relevance of thymidine kinase for the diagnosis, therapy monitoring and prognosis of non-operable lung cancer.

BACKGROUND: Whether thymidine kinase (TK) is considered a new diagnostic biomarker in lung cancer depends on it being superior to or adding further information to already established tumor markers. Here, we investigated its relevance in diagnosis, therapy monitoring and prognosis of patients with diverse forms of lung cancer. PATIENTS AND METHODS: Pretherapeutic TK concentrations were analyzed by radioimmunoassay in serum of 181 patients with advanced lung cancer (53 small cell lung cancer (SCLC), 128 non-small cell lung cancer (NSCLC)), 40 with benign lung diseases, 44 with benign non-lung-related diseases and 29 healthy controls. Diagnostic power of TK was compared with that of established lung cancer markers carcinoembryonic antigen (CEA), cytokeratin 19-fragments (CYFRA 21-1), neuron-specific enolase (NSE) and progastrin-releasing peptide (ProGRP). Furthermore, TK courses of 29 NSCLC patients during cytotoxic chemotherapy were recorded and prognostic relevance of pretherapeutic TK levels was tested in 128 NSCLC patients. RESULTS: While healthy controls had low TK serum levels (median 2.5 U/l, 95th percentile 8.8 U/l), they were significantly higher in patients with lung cancer (median 4.2 U/l, p=0.014) and also in patients with benign lung diseases (median 5.7 U/l; p=0.002). Patients with lung cancer and benign lung diseases could not be separated by TK values. No noticeable difference of TK concentrations was further found in NSCLC (median 4.3 U/l) as compared with SCLC patients (median 3.7 U/l) neither in adeno cell carcinomas (median 5.4 U/l) and squamous cell carcinomas (median 3.0 U/l). In NSCLC, the best diagnostic capacity versus benign lung diseases was found for CYFRA 21-1 (AUC 88.2%), NSE (AUC 86.4%), and CEA (AUC 82.9%), while TK reached only an AUC of 45.7%. The best diagnostic profile in SCLC versus benign lung diseases was observed for NSE (AUC 93.9%) and ProGRP (AUC 85.4%), while TK did not have any diagnostic power (AUC 46.6%). Concerning therapy monitoring, TK was unable to discriminate between the various response groups, neither pretherapeutically, nor before therapy cycles 2 and 3. However, pretherapeutic TK levels showed high prognostic value for overall survival in NSCLC patients: While median survival in patients with TK levels >or=20 U/l was only 3.1 months, it was 9.0 months in patients with TK levels <20 U/l. In multivariate analyses, TK remained an independent prognostic marker, along with the clinical variables stage and performance score. CONCLUSION: Although the performance of serum TK for diagnosis and therapy monitoring of advanced lung cancer was poor, it has a promising prognostic relevance which will have to be further validated.

Delineating Rearrangements in Single Yeast Artificial Chromosomes by Quantitative DNA Fiber Mapping.

Cloning of large chunks of human genomic DNA in recombinant systems such as yeast or bacterial artificial chromosomes has greatly facilitated the construction of physical maps, the positional cloning of disease genes or the preparation of patient-specific DNA probes for diagnostic purposes. For this process to work efficiently, the DNA cloning process and subsequent clone propagation need to maintain stable inserts that are neither deleted nor otherwise rearranged. Some regions of the human genome; however, appear to have a higher propensity than others to rearrange in any host system. Thus, techniques to detect and accurately characterize such rearrangements need to be developed. We developed a technique termed 'Quantitative DNA Fiber Mapping (QDFM)' that allows accurate tagging of sequence elements of interest with near kilobase accuracy and optimized it for delineation of rearrangements in recombinant DNA clones. This paper demonstrates the power of this microscopic approach by investigating YAC rearrangements. In our examples, high-resolution physical maps for regions within the immunoglobulin lambda variant gene cluster were constructed for three different YAC clones carrying deletions of 95 kb and more. Rearrangements within YACs could be demonstrated unambiguously by pairwise mapping of cosmids along YAC DNA molecules. When coverage by YAC clones was not available, distances between cosmid clones were estimated by hybridization of cosmids onto DNA fibers prepared from human genomic DNA. In addition, the QDFM technology provides essential information about clone stability facilitating closure of the maps of the human genome as well as those of model organisms.