Evolutionary trajectories of small cell lung cancer under therapy.

The evolutionary processes that underlie the marked sensitivity of small cell lung cancer (SCLC) to chemotherapy and rapid relapse are unknown1-3. Here we determined tumour phylogenies at diagnosis and throughout chemotherapy and immunotherapy by multiregion sequencing of 160 tumours from 65 patients. Treatment-naive SCLC exhibited clonal homogeneity at distinct tumour sites, whereas first-line platinum-based chemotherapy led to a burst in genomic intratumour heterogeneity and spatial clonal diversity. We observed branched evolution and a shift to ancestral clones underlying tumour relapse. Effective radio- or immunotherapy induced a re-expansion of founder clones with acquired genomic damage from first-line chemotherapy. Whereas TP53 and RB1 alterations were exclusively part of the common ancestor, MYC family amplifications were frequently not constituents of the founder clone. At relapse, emerging subclonal mutations affected key genes associated with SCLC biology, and tumours harbouring clonal CREBBP/EP300 alterations underwent genome duplications. Gene-damaging TP53 alterations and co-alterations of TP53 missense mutations with TP73, CREBBP/EP300 or FMN2 were significantly associated with shorter disease relapse following chemotherapy. In summary, we uncover key processes of the genomic evolution of SCLC under therapy, identify the common ancestor as the source of clonal diversity at relapse and show central genomic patterns associated with sensitivity and resistance to chemotherapy.

Farnesyl-transferase inhibitors show synergistic anticancer effects in combination with novel KRAS-G12C inhibitors.

BACKGROUND: Inhibition of mutant KRAS challenged cancer research for decades. Recently, allele-specific inhibitors were approved for the treatment of KRAS-G12C mutant lung cancer. However, de novo and acquired resistance limit their efficacy and several combinations are in clinical development. Our study shows the potential of combining G12C inhibitors with farnesyl-transferase inhibitors. METHODS: Combinations of clinically approved farnesyl-transferase inhibitors and KRAS G12C inhibitors are tested on human lung, colorectal and pancreatic adenocarcinoma cells in vitro in 2D, 3D and subcutaneous xenograft models of lung adenocarcinoma. Treatment effects on migration, proliferation, apoptosis, farnesylation and RAS signaling were measured by histopathological analyses, videomicroscopy, cell cycle analyses, immunoblot, immunofluorescence and RAS pulldown. RESULTS: Combination of tipifarnib with sotorasib shows synergistic inhibitory effects on lung adenocarcinoma cells in vitro in 2D and 3D. Mechanistically, we present antiproliferative effect of the combination and interference with compensatory HRAS activation and RHEB and lamin farnesylation. Enhanced efficacy of sotorasib in combination with tipifarnib is recapitulated in the subcutaneous xenograft model of lung adenocarcinoma. Finally, combination of additional KRAS G1C and farnesyl-transferase inhibitors also shows synergism in lung, colorectal and pancreatic adenocarcinoma cellular models. DISCUSSION: Our findings warrant the clinical exploration of KRAS-G12C inhibitors in combination with farnesyl-transferase inhibitors.

Krebs von den Lungen 6 (KL-6) is a novel diagnostic and prognostic biomarker in pleural mesothelioma.

OBJECTIVES: Pleural mesothelioma (PM) is a rare disease with dismal outcome. Systemic treatment options include chemotherapy and immunotherapy, but biomarkers for treatment personalization are missing. The only FDA-approved diagnostic biomarker is the soluble mesothelin-related protein (SMRP). Krebs von den Lungen-6 (KL-6) is a human mucin 1 (MUC1) glycoprotein, which has shown diagnostic and prognostic value as a biomarker in other malignancies. The present study investigated whether KL-6 can serve as a diagnostic and/or prognostic biomarker in PM. MATERIALS AND METHODS: Using a fully-automated chemiluminescence enzyme immunoassay (CLEIA) for KL-6 and SMRP, pleural effusion samples from 87 consecutive patients with PM and 25 patients with non-malignant pleural disorders were studied. In addition, KL-6 and SMRP levels were determined in corresponding patient sera, and in an independent validation cohort (n = 122). MUC1 mRNA and protein expression, and KL-6 levels in cell line supernatants were investigated in PM primary cell lines in vitro. RESULTS: PM patients had significantly higher KL-6 levels in pleural effusion than non-malignant controls (AUC 0.78, p < 0.0001). Among PM patients, levels were highest in those with epithelioid or biphasic histologies. There was a strong positive correlation between pleural effusion levels of KL-6 and SMRP (p < 0.0001). KL-6 levels in sera similarly associated with diagnosis of PM, however, to a lesser extent (AUC 0.71, p = 0.008). PM patients with high pleural effusion KL-6 levels (≥303 IU/mL) had significantly better overall survival (OS) compared to those with low KL-6 levels (HR 0.51, p = 0.004). Congruently, high tumor cell MUC1 mRNA expression in primary cell lines associated with prolonged corresponding patient OS (HR 0.35, p = 0.004). These findings were confirmed in an independent validation cohort. CONCLUSION: This is the first study demonstrating KL-6 as a potential novel liquid-based diagnostic and prognostic biomarker in PM.

Induction of metallothionein expression by supplementation of zinc induces resistance against platinum-based treatment in malignant pleural mesothelioma.

Background: Malignant pleural mesothelioma (MPM) is an aggressive tumor with a dismal prognosis. Currently, multimodality treatment including chemotherapy with cisplatin or carboplatin in combination with pemetrexed offers the best options. Detoxification of heavy metals in the cell by metallothioneins (MT) is associated with early failure to platin-based chemotherapy. The induction of MTs gene expression or its enzyme results in saturation by exposure to metal ions such as zinc or cadmium. Its therapeutically effect is still not analyzed in depth. Methods: In our study, we investigated three MPM cell lines and one fibroblast cell line in the course of cisplatin treatment and supplementation of zinc. Cell state analyses via an enzyme-activity based assay were performed. With this, we were able to analyze apoptosis, necrosis and viability of cells. Additionally, we tested treated cells for changes in metallothionein IIA (MT2A) expression by using quantitative realtime polymerase chain reaction. Results: Zinc supplementation induces gene expression of MT2A. Overall, a zinc dose-dependent induction of apoptosis under platin-based treatment could be observed. This effect could be verified in all analyzed cell lines in varying intensity. Conclusions: MT expression is induced by zinc in a dose-dependent manner and inhibits a successful cisplatin therapy. Therefore, heavy metal exposure during cisplatin therapy, e.g., via cigarette smoke, might be an important factor. This should be considered in further therapeutic approaches.

Early Detection of Lung Cancer Using Small RNAs.

INTRODUCTION: Lung cancer remains the deadliest cancer in the world, and lung cancer survival is heavily dependent on tumor stage at the time of detection. Low-dose computed tomography screening can reduce mortality; however, annual screening is limited by low adherence in the United States of America and still not broadly implemented in Europe. As a result, less than 10% of lung cancers are detected through existing programs. Thus, there is a great need for additional screening tests, such as a blood test, that could be deployed in the primary care setting. METHODS: We prospectively recruited 1384 individuals meeting the National Lung Screening Trial demographic eligibility criteria for lung cancer and collected stabilized whole blood to enable the pipetting-free collection of material, thus minimizing preanalytical noise. Ultra-deep small RNA sequencing (20 million reads per sample) was performed with the addition of a method to remove highly abundant erythroid RNAs, and thus open bandwidth for the detection of less abundant species originating from the plasma or the immune cellular compartment. We used 100 random data splits to train and evaluate an ensemble of logistic regression classifiers using small RNA expression of 943 individuals, discovered an 18-small RNA feature consensus signature (miLung), and validated this signature in an independent cohort (441 individuals). Blood cell sorting and tumor tissue sequencing were performed to deconvolve small RNAs into their source of origin. RESULTS: We generated diagnostic models and report a median receiver-operating characteristic area under the curve of 0.86 (95% confidence interval [CI]: 0.84-0.86) in the discovery cohort and generalized performance of 0.83 in the validation cohort. Diagnostic performance increased in a stage-dependent manner ranging from 0.73 (95% CI: 0.71-0.76) for stage I to 0.90 (95% CI: 0.89-0.90) for stage IV in the discovery cohort and from 0.76 to 0.86 in the validation cohort. We identified a tumor-shed, plasma-bound ribosomal RNA fragment of the L1 stalk as a dominant predictor of lung cancer. The fragment is decreased after surgery with curative intent. In additional experiments, results of dried blood spot collection and sequencing revealed that small RNA analysis could potentially be conducted through home sampling. CONCLUSIONS: These data suggest the potential of a small RNA-based blood test as a viable alternative to low-dose computed tomography screening for early detection of smoking-associated lung cancer.

Laser ablation-inductively coupled plasma-mass spectrometry analysis reveals differences in chemotherapeutic drug distribution in surgically resected pleural mesothelioma.

AIMS: Pleural mesothelioma (PM) is a highly aggressive thoracic tumour with poor prognosis. Although reduced tissue drug accumulation is one of the key features of platinum (Pt) resistance, little is known about Pt distribution in human PM. METHODS: We assessed Pt levels of blood samples and surgically resected specimens from 25 PM patients who had received neoadjuvant Pt-based chemotherapy (CHT). Pt levels and tissue distributions were measured by laser ablation-inductively coupled plasma-mass spectrometry and correlated with clinicopathological features. RESULTS: In surgically resected PM specimens, mean Pt levels of nontumourous (fibrotic) areas were significantly higher (vs tumourous regions, P = 0.0031). No major heterogeneity of Pt distribution was seen within the tumourous areas. Pt levels correlated neither with the microvessel area nor with apoptosis rate in the tumourous or nontumourous regions. A significant positive correlation was found between serum and both full tissue section and tumourous area mean Pt levels (r = 0.532, P = 0.006, 95% confidence interval [95% CI] 0.161-0.771 and r = 0.415, P = 0.039, 95% CI 0.011-0.702, respectively). Furthermore, a significant negative correlation was detected between serum Pt concentrations and elapsed time from the last cycle of CHT (r = -0.474, P = 0.017, 95% CI -0.738--0.084). Serum Pt levels correlated negatively with overall survival (OS) (P = 0.029). CONCLUSIONS: There are major differences in drug distribution between tumourous and nontumourous areas of PM specimens. Serum Pt levels significantly correlate with full section and tumourous area average Pt levels, elapsed time from the last CHT cycle, and OS. Further studies investigating clinicopathological factors that modulate tissue Pt concentration and distribution are warranted.

Circulating FGF18 is decreased in pleural mesothelioma but not correlated with disease prognosis.

BACKGROUND: Pleural mesothelioma (PM) is a relatively rare malignancy with limited treatment options and dismal prognosis. We have previously found elevated FGF18 expression in PM tissue specimens compared with normal mesothelium. The objective of the current study was to further explore the role of FGF18 in PM and evaluate its suitability as a circulating biomarker. METHODS: FGF18 mRNA expression was analyzed by real-time PCR in cell lines and in silico in datasets from the Cancer Genome Atlas (TCGA). Cell lines overexpressing FGF18 were generated by retroviral transduction and cell behavior was investigated by clonogenic growth and transwell assays. Plasma was collected from 40 PM patients, six patients with pleural fibrosis, and 40 healthy controls. Circulating FGF18 was measured by ELISA and correlated to clinicopathological parameters. RESULTS: FGF18 showed high mRNA expression in PM and PM-derived cell lines. PM patients with high FGF18 mRNA expression showed a trend toward longer overall survival (OS) in the TCGA dataset. In PM cells with low endogenous FGF18 expression, forced overexpression of FGF18 resulted in reduced growth but increased migration. Surprisingly, despite the high FGF18 mRNA levels observed in PM, circulating FGF18 protein was significantly lower in PM patients and patients with pleural fibrosis than in healthy controls. No significant association of circulating FGF18 with OS or other disease parameters of PM patients was observed. CONCLUSIONS: FGF18 is not a prognostic biomarker in PM. Its role in PM tumor biology and the clinical significance of decreased plasma FGF18 in PM patients warrant further investigation.

One Third of Malignant Pleural Mesothelioma Shows High Immunohistochemical Expression of MSLN or CXCR4 Which Indicates Potent Candidates for Endo-Radiotherapy.

Malignant pleural mesothelioma (MPM) is a mainly asbestos-related tumour associated with a very poor prognosis. Therapeutic approaches include multimodal therapy and chemotherapeutics, with cisplatin being the drug of choice, but response rates of only up to 14% indicate very poor outcomes. Effective treatment options are lacking. Besides the diagnostic usage of radioligands in positron emission tomography (PET)/computed tomography (CT), the endo-radioligand therapy with Lu177 has been proven as a powerful tool in cancer therapy. Mesothelin (MSLN) and C-XC chemokine receptor 4 (CXCR4) are membrane-bound proteins, expressed in certain cancers, and thus are promising targets for endo-radiotherapy. A significant portion of high MSLN- or CXCR4-expressing tumors within the MPM may open the field for this sophisticated treatment approach in the near future. Formalin-fixed, paraffin-embedded (FFPE) tumour specimens from 105 patients suffering from MPM and treated at the Lung Cancer Centre of Essen and at the Helios Klinikum Emil von Behring Berlin were screened. The tumour samples were arranged in tissue microarrays. We immunohistochemically stained the tumour samples against MSLN and CXCR4. The protein expressions of the stainings were scored by a pathologist by using a semiquantitative method. The data obtained were correlated with the clinical outcome. Overall, 77.1% of the analysed tumours showed CXCR4 protein expression (25.7% of them at high expression level (Score 3)). 48.6% of all samples showed an overall strong staining (Score ≥ 2), 59% of the investigated tumours showed MSLN protein expression (10.5% of them at high expression (Score 3)), and 36.2% of all samples showed an overall strong staining (Score ≥ 2). Our results show significant tissue expression levels, for both CXCR4 and MSLN protein, in a major portion of clinical MPM samples. One-third of patients showed outstanding immunoexpression of at least one of these markers, making them interesting candidates for radioligand-based PET/CT diagnostics and follow-up and furthermore may profit from endo-radiotherapy.

TROP2 expression and SN38 antitumor activity in malignant pleural mesothelioma cells provide a rationale for antibody-drug conjugate therapy.

OBJECTIVES: Malignant pleural mesothelioma (MPM) is an aggressive cancer which at large is not amenable to curative surgery. Despite the recent approval of immune checkpoint inhibitor therapy, the response rates and survival following systemic therapy is still limited. Sacituzumab govitecan is an antibody-drug conjugate targeting the topoisomerase I inhibitor SN38 to trophoblast cell-surface antigen 2 (TROP-2)-positive cells. Here we have explored the therapeutic potential of sacituzumab govitecan in MPM models. MATERIALS AND METHODS: TROP2 expression was analyzed in a panel of two well established and 15 pleural effusion derived novel lines by RT-QPCR and immunoblotting, TROP2 membrane-localization was studied by flow cytometry and immunohistochemistry. Cultured mesothelial cells and pneumothorax pleura served as controls. The sensitivity of MPM cell lines to irinotecan and SN38 was studied using cell viability, cell cycle, apoptosis and DNA damage assays. Drug sensitivity of cell lines was correlated with RNA expression of DNA repair genes. Drug sensitivity was defined as an IC50 below 5 nM in the cell viability assay. RESULTS: TROP2 expression was detected at RNA and protein level in 6 of the 17 MPM cell lines, but not in in cultured mesothelial control cells or in the mesothelial layer of the pleura. TROP2 was detectable on the cell membrane in 5 MPM lines and was present in the nucleus in 6 cell models. Ten of 17 MPM cell lines showed sensitivity to SN38 treatment, among those 4 expressed TROP2. High AURKA RNA expression and high proliferation rate correlated with sensitivity to SN38-induced cell death, DNA damage response, cell cycle arrest and cell death. Sacituzumab govitecan treatment effectively induced cell cycle arrest and cell death in TROP2-positive MPM cells. CONCLUSION: TROP2 expression and sensitivity to SN38 in MPM cell lines support biomarker-selected clinical exploration of sacituzumab govitecan in patients with MPM.

Preoperative PET-SUVmax and volume based PET parameters of the primary tumor fail to predict nodal upstaging in early-stage lung cancer.

OBJECTIVES: Accurate nodal staging is of utmost importance in patients with lung cancer. FDG-PET/CT imaging is now part of the routine staging. Despite thorough preoperative staging nodal upstaging still occurs in early-stage lung cancer. However, the predictive value of preoperative PET metrics of the primary tumor on nodal upstaging remains to be unexplored. Our aim was to assess the association of these preoperative PET-parameters with nodal upstaging in histologically confirmed lung adenocarcinoma and squamous cell carcinoma. METHODS: From January 2016 to November 2018, 500 patients with pT1-T2/cN0 lung cancer received an anatomical resection with curative intent. 171 patients with adenocarcinoma and squamous cell carcinoma and available PET-CTs were retrospectively included. We analyzed the the association of nodal upstaging with preoperative PET-SUVmax and metabolic PET metrics including total lesion glycolysis (TLG) and metabolic tumor volume (MTV) with different defined thresholds. RESULTS: High values of preoperative PET-SUVmax of the primary tumor were associated with squamous cell carcinoma (p < 0.0001) and with larger tumors (p < 0.0001). Increased preoperative C-reactive protein levels (<1mg/dL) correlated significantly with high preoperative PET-SUVmax values (p < 0.0001). No significant relationship between PET-SUVmax and lactate dehydrogenase activity (p = 0.6818), white blood cell count (p = 0.7681), gender (p = 0.1115) or age (p = 0.9284) was observed. Nodal upstaging rate was 14.0 % with 8.8 % N1 and 5.3 % N2 upstaging. Tumor size (p = 0.0468) and number of removed lymph nodes (p = 0.0461) were significant predictors of nodal upstaging but no significant association was found with histology or PET parameters. Of note, increased MTV - regardless of the threshold - tended to associate with nodal upstaging. CONCLUSION: Early-stage lung cancer patients with squamous histology and T2 tumors presented increased preoperative PET-SUVmax values. Nevertheless, beyond tumor size and number of removed lymph nodes neither SUVmax nor metabolic PET parameters MTV and TLG were significant predictors of nodal upstaging.

[Farnesyltransferase inhibitors have antitumoral effects in mutant KRAS containing cancer cells in preclinical models]. / Farnezil-transzferáz-inhibitorok (FTI) szinergista módon potencírozzák a mutáns KRAS inhibitorai daganatellenes hatásait preklinikai modellekben.

In silico studies raised the possibility that farnesyltransferase inhibitors (FTIs) may have antitumoral effects on KRAS mutant cancer cells. Accordingly, we have tested FTIs (tipifarnib and lonafarnib) in G12C mutant human cancer cell lines in vitro and in vivo. We have discovered that the combination of the two drugs has a synergistic antitumoral effect. Next, we have tested FTIs on G12D mutant human cancer cell lines and found that the combination has antitumoral effect in various preclinical cancer models. At last, we have also tested FTIs on G12V mutant human cancer cells and again we have detected antitumoral effects. We suggest that FTIs may have clinical relevance outside the HRAS mutant cancers.

Malignant pleural mesothelioma nodules remodel their surroundings to vascularize and grow.

Background: The microanatomical steps of malignant pleural mesothelioma (MPM) vascularization and the resistance mechanisms to anti-angiogenic drugs in MPM are unclear. Methods: We investigated the vascularization of intrapleurally implanted human P31 and SPC111 MPM cells. We also assessed MPM cell's motility, invasion and interaction with endothelial cells in vitro. Results: P31 cells exhibited significantly higher two-dimensional (2D) motility and three-dimensional (3D) invasion than SPC111 cells in vitro. In co-cultures of MPM and endothelial cells, P31 spheroids permitted endothelial sprouting (ES) with minimal spatial distortion, whereas SPC111 spheroids repealed endothelial sprouts. Both MPM lines induced the early onset of submesothelial microvascular plexuses covering large pleural areas including regions distant from tumor colonies. The development of these microvascular networks occurred due to both intussusceptive angiogenesis (IA) and ES and was accelerated by vascular endothelial growth factor A (VEGF-A)-overexpression. Notably, SPC111 colonies showed different behavior to P31 cells. P31 nodules incorporated tumor-induced capillary plexuses from the earliest stages of tumor formation. P31 cells deposited a collagenous matrix of human origin which provided "space" for further intratumoral angiogenesis. In contrast, SPC111 colonies pushed the capillary plexuses away and thus remained avascular for weeks. The key event in SPC111 vascularization was the development of a desmoplastic matrix of mouse origin. Continuously invaded by SPC111 cells, this matrix transformed into intratumoral connective tissue trunks, providing a route for ES from the diaphragm. Conclusions: Here, we report two distinct growth patterns of orthotopically implanted human MPM xenografts. In the invasive pattern, MPM cells invade and thus co-opt peritumoral capillary plexuses. In the pushing/desmoplastic pattern, MPM cells induce a desmoplastic response within the underlying tissue which allows the ingrowth of a nutritive vasculature from the pleura.

The clinical utility of cfRNA for disease detection and surveillance: A proof of concept study in non-small cell lung cancer.

BACKGROUND: CT scans are used in routine clinical practice for the diagnosis and treatment surveillance of non-small cell lung cancer (NSCLC). However, more sensitive methods are desirable. Liquid biopsy analyses of RNA and DNA can offer more sensitive diagnostic approaches. Cell-free RNA (cfRNA) has been described in several malignancies, but its clinical utility has not previously been explored. METHODS: We evaluated the clinical utility of cfRNA for early detection and surveillance of tumor disease in a proof-of-concept study. Using real-time-droplet digital polymerase chain reaction we characterized a candidate transcript (MORF4L2) in plasma samples from 41 advanced stage, 38 early stage NSCLC and 39 healthy samples. We compared its diagnostic performance with tumor markers and evaluated its utility for disease monitoring. RESULTS: MORF4L2 cfRNA was more abundant in patients than in healthy donors (p < 0.0001). Using the Youden index approach (cutoff value of 537 copies/ml was established) with a sensitivity of 0.73 (95% CI: 0.61-0.82) and a specificity of 0.87 (95% CI: 0.73-0.96). Positive and negative predictive values of 0.92 (95% CI: 0.83-0.95) and 0.59 (95% CI: 0.47-0.83) were achieved. Combination of cfRNA and Cyfra21-1 improved its predictive value from 89.5% to 94.7%. Low baseline MORF4L2 levels were associated with better overall survival (HR:0.25, 95% CI: 0.09-0.7, p = 0.009) and progression-free survival for patients treated with tyrosine kinase inhibitors (p = 0.011) and chemotherapy (p = 0.019). MORF4L2 profile between baseline and follow-up mirrored radiological response and tumor dynamics better than tumor markers. cfRNA transcripts allowed monitoring tumor dynamics in patients without tumor-reported genetic alterations. CONCLUSION: Our data support clinical utility of cfRNA for detection and surveillance of NSCLC. Further studies with larger cohorts are required.

Prognostic Factors for Leiomyosarcoma with Isolated Metastases to the Lungs: Impact of Metastasectomy.

BACKGROUND: Leiomyosarcoma (LMS) most frequently metastasizes to the lung. Metastatic LMS is considered incurable. Selected patients may benefit from pulmonary metastasectomy (PM) within multimodal therapy. This study analyzed the prognostic relevance of clinicopathologic factors in these patients. METHODS: Patients with metastatic LMS to the lung treated in our center from 2004 to 2020 were included in this single-center retrospective study. Overall survival (OS), progression-free survival (PFS), and prognostic factors were analyzed. RESULTS: The study had 64 patients (33 males, 52%) with metastatic LMS to the lung. The 5-year OS was 55% after the diagnosis of pulmonary metastases. Age older than 60 years at the primary tumor diagnosis, primary tumor larger than 70 mm, and five or more lung metastases were associated with poorer OS. Of the 64 patients, 44 underwent PM. The postoperative mortality rate was 0%. The patients selected for PM were younger and had smaller primary tumors, fewer metastases, and metastases that more often were metachronous. Metastasis grade (G1 vs. G2/3) and size (20-mm cutoff) were significant prognostic factors for OS (p = 0.05) and PFS (p = 0.028) after PM, respectively. The 44 patients who underwent PM had a survival benefit compared with the patients who were selected but did not undergo PM (n = 6) and the patients who were not selected for PM (n = 14). Three patients (7%) were alive and free of disease at the last follow-up visit respectively 5.5, 9, and 12 years after PM. CONCLUSIONS: For patients with leiomyosarcoma, PM is safe. Despite aggressive multimodal treatment, most patients will experience recurrence and eventually die of their disease. However, a small subgroup of patients could potentially be cured after PM.

Nintedanib and Dasatinib Treatments Induce Protective Autophagy as a Potential Resistance Mechanism in MPM Cells.

Malignant pleural mesothelioma (MPM) is a rare type of cancer with a grim prognosis. So far, no targetable oncogenic mutation was identified in MPM and biomarkers with predictive value toward drug sensitivity or resistance are also lacking. Nintedanib (BIBF1120) is a small-molecule tyrosine kinase inhibitor that showed promising efficacy preclinically and in phase II trial in MPM as an angiogenesis inhibitor combined with chemotherapy. However, the extended phase III trial failed. In this study, we investigated the effect of nintedanib on one of its targets, the SRC kinase, in two commercial and six novel MPM cell lines. Surprisingly, nintedanib treatment did not inhibit SRC activation in MPM cells and even increased phosphorylation of SRC in several cell lines. Combination treatment with the SRC inhibitor dasatinib could reverse this effect in all cell lines, however, the cellular response was dependent on the drug sensitivity of the cells. In 2 cell lines, with high sensitivity to both nintedanib and dasatinib, the drug combination had no synergistic effect but cell death was initiated. In 2 cell lines insensitive to nintedanib combination treatment reduced cell viability synergisticaly without cell death. In contrast, in these cells both treatments increased the autophagic flux assessed by degradation of the autophagy substrate p62 and increased presence of LC3B-II, increased number of GFP-LC3 puncta and decreased readings of the HiBiT-LC3 reporter. Additionaly, autophagy was synergistically promoted by the combined treatment. At the transcriptional level, analysis of lysosomal biogenesis regulator Transcription Factor EB (TFEB) showed that in all cell lines treated with nintedanib and to a lesser extent, with dasatinib, it became dephosphorylated and accumulated in the nucleus. Interestingly, the expression of certain known TFEB target genes implicated in autophagy or lysosomal biogenesis were significantly modified only in 1 cell line. Finally, we showed that autophagy induction in our MPM cell lines panel by nintedanib and dasatinib is independent of the AKT/mTOR and the ERK pathways. Our study reveals that autophagy can serve as a cytoprotective mechanism following nintedanib or dasatinib treatments in MPM cells.

The Prognostic Relevance of PMCA4 Expression in Melanoma: Gender Specificity and Implications for Immune Checkpoint Inhibition.

PMCA4 is a critical regulator of Ca2+ homeostasis in mammalian cells. While its biological and prognostic relevance in several cancer types has already been demonstrated, only preclinical investigations suggested a metastasis suppressor function in melanoma. Therefore, we studied the expression pattern of PMCA4 in human skin, nevus, as well as in primary and metastatic melanoma using immunohistochemistry. Furthermore, we analyzed the prognostic power of PMCA4 mRNA levels in cutaneous melanoma both at the non-metastatic stage as well as after PD-1 blockade in advanced disease. PMCA4 localizes to the plasma membrane in a differentiation dependent manner in human skin and mucosa, while nevus cells showed no plasma membrane staining. In contrast, primary cutaneous, choroidal and conjunctival melanoma cells showed specific plasma membrane localization of PMCA4 with a wide range of intensities. Analyzing the TCGA cohort, PMCA4 mRNA levels showed a gender specific prognostic impact in stage I-III melanoma. Female patients with high transcript levels had a significantly longer progression-free survival. Melanoma cell specific PMCA4 protein expression is associated with anaplasticity in melanoma lung metastasis but had no impact on survival after lung metastasectomy. Importantly, high PMCA4 transcript levels derived from RNA-seq of cutaneous melanoma are associated with significantly longer overall survival after PD-1 blockade. In summary, we demonstrated that human melanoma cells express PMCA4 and PMCA4 transcript levels carry prognostic information in a gender specific manner.

Metabolic synthetic lethality by targeting NOP56 and mTOR in KRAS-mutant lung cancer.

BACKGROUND: Oncogenic KRAS mutations are prevalent in human cancers, but effective treatment of KRAS-mutant malignancies remains a major challenge in the clinic. Increasing evidence suggests that aberrant metabolism plays a central role in KRAS-driven oncogenic transformation. The aim of this study is to identify selective metabolic dependency induced by mutant KRAS and to exploit it for the treatment of the disease. METHOD: We performed an integrated analysis of RNAi- and CRISPR-based functional genomic datasets (n = 5) to identify novel genes selectively required for KRAS-mutant cancer. We further screened a customized library of chemical inhibitors for candidates that are synthetic lethal with NOP56 depletion. Functional studies were carried out by genetic knockdown using siRNAs and shRNAs, knockout using CRISPR/Cas9, and/or pharmacological inhibition, followed by cell viability and apoptotic assays. Protein expression was determined by Western blot. Metabolic ROS was measured by flow cytometry-based quantification. RESULTS: We demonstrated that nucleolar protein 5A (NOP56), a core component of small nucleolar ribonucleoprotein complexes (snoRNPs) with an essential role in ribosome biogenesis, confers a metabolic dependency by regulating ROS homeostasis in KRAS-mutant lung cancer cells and that NOP56 depletion causes synthetic lethal susceptibility to inhibition of mTOR. Mechanistically, cancer cells with reduced NOP56 are subjected to higher levels of ROS and rely on mTOR signaling to balance oxidative stress and survive. We also discovered that IRE1α-mediated unfolded protein response (UPR) regulates this process by activating mTOR through p38 MAPK. Consequently, co-targeting of NOP56 and mTOR profoundly enhances KRAS-mutant tumor cell death in vitro and in vivo. CONCLUSIONS: Our findings reveal a previously unrecognized mechanism in which NOP56 and mTOR cooperate to play a homeostatic role in the response to oxidative stress and suggest a new rationale for the treatment of KRAS-mutant cancers.

Combined multimodal ctDNA analysis and radiological imaging for tumor surveillance in Non-small cell lung cancer.

BACKGROUND: Radiology is the current standard for monitoring treatment responses in lung cancer. Limited sensitivity, exposure to ionizing radiations and related sequelae constitute some of its major limitation. Non-invasive and highly sensitive methods for early detection of treatment failures and resistance-associated disease progression would have additional clinical utility. METHODS: We analyzed serially collected plasma and paired tumor samples from lung cancer patients (61 with stage IV, 48 with stages I-III disease) and 61 healthy samples by means of next-generation sequencing, radiological imaging and droplet digital polymerase chain reaction (ddPCR) mutation and methylation assays. RESULTS: A 62% variant concordance between tumor-reported and circulating-free DNA (cfDNA) sequencing was observed between baseline liquid and tissue biopsies in stage IV patients. Interestingly, ctDNA sequencing allowed for the identification of resistance-mediating p.T790M mutations in baseline plasma samples for which no such mutation was observed in the corresponding tissue. Serial circulating tumor DNA (ctDNA) mutation analysis by means of ddPCR revealed a general decrease in ctDNA loads between baseline and first reassessment. Additionally, serial ctDNA analyses only recapitulated computed tomography (CT) -monitored tumor dynamics of some, but not all lesions within the same patient. To complement ctDNA variant analysis we devised a ctDNA methylation assay (methcfDNA) based on methylation-sensitive restriction enzymes. cfDNA methylation showed and area under the curve (AUC) of > 0.90 in early and late stage cases. A decrease in methcfDNA between baseline and first reassessment was reflected by a decrease in CT-derive tumor surface area, irrespective of tumor mutational status. CONCLUSION: Taken together, our data support the use of cfDNA sequencing for unbiased characterization of the molecular tumor architecture, highlights the impact of tumor architectural heterogeneity on ctDNA-based tumor surveillance and the added value of complementary approaches such as cfDNA methylation for early detection and monitoring.

Bone-Specific Metastasis Pattern of Advanced-Stage Lung Adenocarcinoma According to the Localization of the Primary Tumor.

Background: Patients with advanced-stage lung adenocarcinoma (LADC) often develop distant metastases in the skeletal system. Yet, the bone-specific metastasis pattern is still controversial. We, therefore, aimed to examine how the primary tumor location affects bone specificity and survival in LADC patients diagnosed with skeletal metastases. Methods: In total, 209 bone-metastatic Caucasian LADC patients from two thoracic centers were included in this study. Focusing on the specific location of primary tumors and bone metastatic sites, clinicopathological variables were included in a common database and analyzed retrospectively. Skeletal metastases were diagnosed according to the contemporary diagnostic guidelines and confirmed by bone scintigraphy. Besides region- and side-specific localization, primary tumors were also classified as central or peripheral tumors based on their bronchoscopic visibility. Results: The most common sites for metastasis were the spine (n = 103) and the ribs (n = 60), followed by the pelvis (n = 36) and the femur (n = 22). Importantly, femoral (p = 0.022) and rib (p = 0.012) metastases were more frequently associated with peripheral tumors, whereas centrally located LADCs were associated with humeral metastases (p = 0.018). Moreover, we deduced that left-sided tumors give rise to skull metastases more often than right-sided primary tumors (p = 0.018). Of note, however, the localization of the primary tumor did not significantly influence the type of affected bones. Multivariate Cox regression analysis adjusted for clinical parameters demonstrated that central localization of the primary tumor was an independent negative prognostic factor for overall survival (OS). Additionally, as expected, both chemotherapy and bisphosphonate therapy conferred a significant benefit for OS. Conclusion: The present study demonstrates unique bone-specific metastasis patterns concerning primary tumor location. Peripherally located LADCs are associated with rib and femoral metastases and improved survival outcomes. Our findings might contribute to the development of individualized follow-up strategies in bone-metastatic LADC patients and warrant further clinical investigations on a larger sample size.

HER2 mediates clinical resistance to the KRASG12C inhibitor sotorasib, which is overcome by co-targeting SHP2.

INTRODUCTION: Mutant RAS guanosine triphosphate hydrolases (GTPases) are key oncogenic drivers in many cancers. The KRASG12C variant has recently become targetable by a new drug class specifically locking KRASG12C in its inactive guanosine diphosphate (GDP)-bound state. Clinical activity was demonstrated in patients with advanced lung cancers harbouring KRASG12C mutations but was limited by the development of resistance. METHODS: A biopsy from progressing lung cancer of a patient treated with the KRASG12C inhibitor sotorasib was obtained, and the underlying resistance factors were analysed. Mechanistic studies were performed in vitro and in vivo to uncover strategies to overcome resistance to KRASG12C inhibition. RESULTS: We demonstrated acquisition of HER2 copy number gain and KRASG12C mutation retention in the post-progression biopsy. To explore HER2 gain as the relevant resistance mechanism, we generated KRASG12C lung cancer models overexpressing HER2. MAPK pathway signalling remained active despite KRASG12C inhibitor treatment. Combined pharmacological inhibition of KRASG12C and SHP2 synergistically overcame HER2-mediated resistance in vitro and in vivo. CONCLUSIONS: These findings establish HER2 copy number gain as a clinically relevant mechanism of resistance to pharmacological KRASG12C inhibition that can be overcome by co-targeting SHP2.