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Made available in DSpace on 2020-12-12T01:44:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-11-19 Following the information obtained by a rational design study, a cyclic and helical-stabilized analogue of the peptide Cm-p5 was synthetized. The cyclic monomer showed an increased activity in vitro against Candida albicans and Candida parapsilosis, compared to Cm-p5. Initially, 14 mutants of Cm-p5 were synthesized following a rational design to improve the antifungal activity and pharmacological properties. Antimicrobial testing showed that the activity was lost in each of these 14 analogues, suggesting, as a main conclusion, that a Glu-His salt bridge could stabilize Cm-p5 helical conformation during the interaction with the plasma membrane. A derivative, obtained by substitution of Glu and His for Cys, was synthesized and oxidized with the generation of a cyclic monomer with improved antifungal activity. In addition, two dimers were generated during the oxidation procedure, a parallel and antiparallel one. The dimers showed a helical secondary structure in water, whereas the cyclic monomer only showed this conformation in SDS. Molecular dynamic simulations confirmed the helical stabilizations for all of them, therefore indicating the possible essential role of the Glu-His salt bridge. In addition, the antiparallel dimer showed a moderate activity against Pseudomonas aeruginosa and a significant activity against Listeria monocytogenes. Neither the cyclic monomer nor the dimers were toxic against macrophages or THP-1 human cells. Due to its increased capacity for fungal control compared to fluconazole, its low cytotoxicity, together with a stabilized α-helix and disulfide bridges, that may advance its metabolic stability, and in vivo activity, the new cyclic Cm-p5 monomer represents a potential systemic antifungal therapeutic candidate. General Chemistry Department Faculty of Chemistry University of Havana, Zapata y G Center for Natural Products Research Faculty of Chemistry University of Havana, Zapata y G Center for Protein Studies Faculty of Biology University of Havana 25 and i Synthetic Peptides Group Center for Genetic Engineering and Biotechnology, P.O. Box 6162 Institute of Medical Microbiology and Hygiene University Clinic of Ulm, Robert Koch Str. 8 Department of Inorganic Chemistry Analytical and Physical Chemistry Facultad de Ciencias Exactas y Naturales Universidad de Buenos Aires Center of Excellence for Research in Sustainable Chemistry (CERSusChem) Department of Chemistry Federal University of São Carlos-UFSCar Laboratory of Pharmaceutical Biotechnology Department of Drugs and Medicines School of Pharmaceutical Sciences UNESP Core Facility for Functional Peptidomics Ulm Peptide Pharmaceuticals (U-PEP) University Ulm Faculty of Medicine Ulm University Institute of Pharmaceutical Biotechnology Ulm University, James-Frank-Ring N27 Laboratory of Pharmaceutical Biotechnology Department of Drugs and Medicines School of Pharmaceutical Sciences UNESP

Anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancers (NSCLC) have the best prognosis among metastatic pulmonary malignancies, with a median patient survival currently exceeding 5 years. While this is definitely a major therapeutic success for thoracic oncology, it may not be entirely attributable to rapid drug development and the strenuous clinical efforts. At the genetic level, ALK+ disease is also unique, distinguished by the lowest tumor mutational burden (mean below 3 mutations/Mbp), the lowest frequency of TP53 mutations (20-25%) and very few other co-mutations compared to other NSCLC. The relative simplicity and stability of the genetic landscape not only contribute to the relatively favourable clinical course, but also make study of the effects from individual molecular features easier. EML4-ALK fusion variant 3 (E6;A20) and TP53 mutations were recently identified as main molecular determinants of adverse outcome: they occur in about 30-40% and 20-25% of newly-diagnosed cases, respectively, have possibly synergistic effects and are independently associated with more aggressive disease, shorter progression-free survival under treatment with ALK inhibitors and worse overall survival. Secondary detection of TP53 mutations at disease progression in previously negative patients defines another subset (about 20%) with similarly poor outcome, while detection of ALK resistance mutations guides next-line therapy. As our biological understanding deepens, additional molecular risk factors will be identified and refine our concepts further. The translation of clinical risk at the molecular level and the ability to predict early events are of key importance for individualized patient management and preclinical modeling in order to advance therapeutic options.

BACKGROUND: Liquid rebiopsies can detect resistance mutations to guide therapy of anaplastic lymphoma kinase-rearranged (ALK(+)) non-small-cell lung cancer (NSCLC) failing tyrosine kinase inhibitors (TKI). Here, we analyze how their results relate to the anatomical pattern of disease progression and patient outcome. METHODS: Clinical, molecular, and radiologic characteristics of consecutive TKI-treated ALK(+) NSCLC patients were analyzed using prospectively collected plasma samples and the 17-gene targeted AVENIO kit, which covers oncogenic drivers and all TP53 exons. RESULTS: In 56 patients, 139 instances of radiologic changes were analyzed, of which 133 corresponded to disease progression. Circulating tumor DNA (ctDNA) alterations were identified in most instances of extracranial progression (58/94 or 62%), especially if concomitant intracranial progression was also present (89%, P

Computed tomography (CT) scans are the gold standard to measure treatment success of non-small cell lung cancer (NSCLC) therapies. Here, we investigated the very early tumor response of patients receiving chemotherapy or targeted therapies using a panel of already established and explorative liquid biomarkers. Blood samples from 50 patients were taken at baseline and at three early time points after therapy initiation. DNA mutations, a panel of 17 microRNAs, glycodelin, glutathione disulfide, glutathione, soluble caspase-cleaved cytokeratin 18 (M30 antigen), and soluble cytokeratin 18 (M65 antigen) were measured in serum and plasma samples. Baseline and first follow-up CT scans were evaluated and correlated with biomarker data. The detection rate of the individual biomarkers was between 56% and 100%. While only keratin 18 correlated with the tumor load at baseline, we found several individual markers correlating with the tumor response to treatment for each of the three time points of blood draws. A combination of the five best markers at each time point resulted in highly significant marker panels indicating therapeutic response (R2 = 0.78, R2 = 0.71, and R2 = 0.71). Our study demonstrates that an early measurement of biomarkers immediately after therapy start can assess tumor response to treatment and might support an adaptation of treatment to improve patients&rsquo, outcome.

Lung adenocarcinoma (ADC) is the most prevalent subtype of lung cancer and characterized by considerable morphological and mutational heterogeneity. However, little is known about the epigenomic intratumor variability between spatially separated histological growth patterns of ADC. In order to reconstruct the clonal evolution of histomorphological patterns, we performed global DNA methylation profiling of 27 primary tumor regions, seven matched normal tissues and six lymph node metastases from seven ADC cases. Additionally, we investigated the methylation data from 369 samples of the TCGA ADC cohort. All regions showed varying degrees of methylation changes between segments of different, but also of the same growth patterns. Similarly, copy number variations were seen between spatially distinct segments of each patient. Hierarchical clustering of promoter methylation revealed extensive heterogeneity within and between the cases. Intratumor DNA methylation heterogeneity demonstrated a branched clonal evolution of ADC regions driven by genomic instability with subclonal copy number changes. Notably, methylation profiles within tumors were not more similar to each other than to those from other individuals. In two cases, different tumor regions of the same individuals were represented in distant clusters of the TCGA cohort, illustrating the extensive epigenomic intratumor heterogeneity of ADCs. We found no evidence for the lymph node metastases to be derived from a common growth pattern. Instead, they had evolved early and separately from a particular pattern in each primary tumor. Our results suggest that extensive variation of epigenomic features contributes to the molecular and phenotypic heterogeneity of primary ADCs and lymph node metastases.

Recently, many genome-wide profiling studies provided insights into the molecular make-up of major cancer types. The deeper understanding of these genetic alterations and their functional consequences led to the discovery of novel therapeutic opportunities improving clinical management of cancer patients. While tissue-based molecular patient stratification is the gold standard for precision medicine, it has certain limitations: Tissue biopsies are invasive sampling procedures carrying the risk of complications and may not represent the entire tumor due to underlying genetic heterogeneity. In this context, complementary characterization of genetic information in the blood of cancer patients can serve as minimal-invasive 'liquid biopsy'. Fragments of circulating cell-free DNA (cfDNA) are released from tissues of healthy individuals as well as cancer patients. The fraction of cfDNA that is released from primary tumors or metastases (i.e. circulating tumor DNA, ctDNA) represents genetic aberrations in cancer cells, which are a potential source for diagnostic, prognostic, and predictive biomarkers. Recent studies have demonstrated technical feasibility and clinical applications including detection of drug targets and resistance mutations as well as longitudinal monitoring of tumors under therapy. To this end, a variety of pre-analytical procedures for blood processing, isolation and quantification of cfDNA are being employed and several analytical methods and technologies ranging from PCR-based single locus assays to genome-wide approaches are available, which considerably differ in sensitivity, specificity, and throughput. However, broad implementation of ctDNA analysis in daily clinical practice requires a thorough understanding of theoretical, technical, and biological concepts and necessitates standardization and validation of pre-analytical and analytical procedures across different technologies. Here, we review the pertinent literature and discuss the advantages and limitations of available methodologies and their potential applications in molecular diagnostics.

The availability of tyrosine kinase inhibitors (TKI) during the past ten years has led to improved response and overall survival of patients suffering from metastatic clear cell renal cell carcinoma (ccRCC). However, most of these tumors will eventually progress due to resistance evolving under therapy. The objective of this pilot study was to determine whether molecular alterations in ccRCC tissues sampled over the course of the disease might be suggestive of potential therapies. We performed whole exome sequencing of nine samples from four patients in the MORE (Molecular Renal Cancer Evolution) trial. We analyzed the mutational patterns in the tissues at baseline and compared them to those detectable in biopsy samples after progression under TKI therapy. We found limited genetic concordance between primary and secondary tumor sites with private mutations in FLT4, MTOR, ITGA5, SETD2, PBRM1, and BRCA1 on progression. One patient who showed an increased mutational load in the metastasis responded to nivolumab treatment. Our data provide evidence for clonal evolution and diverse pathways leading to acquired TKI resistance of ccRCC. Acquired resistance to TKI in metastatic ccRCC is due to intra-tumor heterogeneity and clonal evolution of resistant subclones. Mutations occurring under progression might be informative for alternative targeted therapies.

The pathogenic yeast Candida auris has received increasing attention due to its ability to cause fatal infections, its resistance toward important fungicides, and its ability to persist on surfaces including medical devices in hospitals. To brace health care systems for this considerable risk, alternative therapeutic approaches such as antifungal peptides are urgently needed. In clinical wound care, a significant focus has been directed toward novel surgical (wound) dressings as first defense lines against C. auris. Inspired by Cerberus the Greek mythological “hound of Hades” that prevents the living from entering and the dead from leaving hell, the preparation of a gatekeeper hybrid hydrogel is reported featuring lectin-mediated high-affinity immobilization of C. auris cells from a collagen gel as a model substratum in combination with a release of an antifungal peptide drug to kill the trapped cells. The vision is an efficient and safe two-layer medical composite hydrogel for the treatment of severe wound infections that typically occur in hospitals. Providing this new armament to the repertoire of possibilities for wound care in critical (intensive care) units may open new routes to shield and defend patients from infections and clinical facilities from spreading and invasion of C. auris and probably other fungal pathogens., publishedVersion

Background Targeted therapies (TKI) have improved the prognosis of ALK-rearranged lung cancer (ALK+ NSCLC), but clinical courses vary widely. Early identification and molecular characterisation of treatment failure have key importance for subsequent therapies. We performed copy number variation (CNV) profiling and targeted panel sequencing from cell-free DNA (cfDNA) to monitor ALK+ NSCLC. Methods 271 longitudinal plasma DNA samples from 73 patients with TKI-treated metastatic ALK+ NSCLC were analysed by capture-based targeted (average coverage 4,100x), and shallow whole genome sequencing (sWGS, 0.5x). Mutations were called using standard algorithms. CNVs were quantified using the trimmed median absolute deviation from copy number neutrality (t-MAD). Findings cfDNA mutations were identified in 58% of patients. They included several potentially actionable alterations, e.g. in the genes BRAF, ERBB2, and KIT. sWGS detected CNVs in 18% of samples, compared to 6% using targeted sequencing. Several of the CNVs included potentially druggable targets, such as regions harboring EGFR, ERBB2, and MET. Circulating tumour DNA (ctDNA) mutations and t-MAD scores increased during treatment, correlated with markers of higher molecular risk, such as the EML4-ALK variant 3 and/or TP53 mutations, and were associated with shorter patient survival. Importantly, t-MAD scores reflected the tumour remission status in serial samples similar to mutant ctDNA allele frequencies, and increased with disease progression in 79% (34/43) of cases, including those without detectable single nucleotide variant (SNV). Interpretation Combined copy number and targeted mutation profiling could improve monitoring of ALK+ NSCLC. Potential advantages include the identification of treatment failure, in particular for patients without detectable mutations, and broader detection of genomic changes acquired during therapy, especially in later treatment lines and in high-risk patients. Funding This work was supported by the German Center for Lung Research (DZL), by the German Cancer Consortium (DKTK), by the Heidelberg Center for Personalized Oncology at the German Cancer Research Center (DKFZ-HIPO), and by Roche Sequencing Solutions (Pleasanton, CA, USA).

Lung cancer is the number one cause of cancer-related mortality worldwide. To improve disease outcome, it is crucial to implement biomarkers into the clinics which assist physicians in their decisions regarding diagnosis, prognosis, as well as prediction of treatment response. Liquid biopsy offers an opportunity to obtain such biomarkers in a minimal invasive manner by retrieving tumor-derived material from body fluids of the patient. The abundance of circulating microRNAs is known to be altered in disease and has therefore been studied extensively as a cancer biomarker. Circulating microRNAs present a variety of favorable characteristics for application as liquid biopsy-based biomarkers, including their high stability, relatively high abundance, and presence is nearly all body fluids. Although the application of circulating microRNAs for the management of lung cancer has not entered the clinics yet, several studies showed their utility for diagnosis, prognosis, and efficacy prediction of various treatment strategies, including surgery, radio-/chemotherapy, as well as targeted therapy. To compensate for their limited tumor specificity, several microRNAs are frequently combined into microRNA panels. Moreover, the possibility to combine single microRNAs or microRNA panels with tumor imaging or other cancer-specific biomarkers has the potential to increase specificity and sensitivity and could lead to the clinical application of novel multi-marker combinations.

Lung cancer is the number one cause of cancer-related mortality worldwide. To improve disease outcome, it is crucial to implement biomarkers into the clinics which assist physicians in their decisions regarding diagnosis, prognosis, as well as prediction of treatment response. Liquid biopsy offers an opportunity to obtain such biomarkers in a minimal invasive manner by retrieving tumor-derived material from body fluids of the patient. The abundance of circulating microRNAs is known to be altered in disease and has therefore been studied extensively as a cancer biomarker. Circulating microRNAs present a variety of favorable characteristics for application as liquid biopsy-based biomarkers, including their high stability, relatively high abundance, and presence is nearly all body fluids. Although the application of circulating microRNAs for the management of lung cancer has not entered the clinics yet, several studies showed their utility for diagnosis, prognosis, and efficacy prediction of various treatment strategies, including surgery, radio-/chemotherapy, as well as targeted therapy. To compensate for their limited tumor specificity, several microRNAs are frequently combined into microRNA panels. Moreover, the possibility to combine single microRNAs or microRNA panels with tumor imaging or other cancer-specific biomarkers has the potential to increase specificity and sensitivity and could lead to the clinical application of novel multi-marker combinations.

Genetic rearrangements involving the anaplastic lymphoma kinase (ALK) gene confer sensitivity to ALK tyrosine kinase inhibitors (TKIs) and superior outcome in non-small-cell lung cancer (NSCLC). However, clinical courses vary widely, and recent studies suggest that molecular profiling of ALK+ NSCLC can provide additional predictors of therapy response that could assist further individualization of patient management. As repeated tissue biopsies often pose technical difficulties and significant procedural risk, analysis of tumor constituents circulating in the blood, including ctDNA and various proteins, is increasingly recognized as an alternative method of tumor sampling (“liquid biopsy”). Here, we report the case of a KLC1–ALK-rearranged NSCLC patient responding to crizotinib treatment and demonstrate how analysis of plasma and serum biomarkers can be used to identify the ALK fusion partner and monitor therapy over time. Results of ctDNA sequencing and copy-number alteration profiling as well as serum protein concentrations at various time points during therapy reflected the current remission status and could predict the subsequent clinical course. At the time of disease progression, we identified four distinct secondary mutations in the ALK gene in ctDNA potentially causing treatment failure, accompanied by rising levels of CEA and CYFRA 21–1. Moreover, several copy-number variations were detected at the end of the treatment, including an amplification of a region on Chromosome 12 encompassing the TP53 regulator MDM2. In summary, our findings illustrate the utility of noninvasive longitudinal molecular profiling for assessing remission status, exploring mechanisms of treatment failure, predicting subsequent clinical course, and dissecting dynamics of drug-resistant clones in ALK+ lung cancer.

BACKGROUND: Recent findings indicate that the currently approved predictive biomarker for immune checkpoint blockade (ICB), i.e. PD-L1 expression, is imperfect. Hence, additional biomarkers are warranted, and ICB-related gene expression profiling (GEP) was advocated as a promising approach enabling comprehensive interrogation of the ICB-effector compartment. Here, we assessed inter- and intra-tumor heterogeneity (ITH) of the immunological microenvironment using a comprehensive gene expression profiling approach (770 genes) to further elucidate the biological basis for new diagnostic applications. METHODS: All formalin-fixed paraffin-embedded tissue specimens used for this study were derived from surgically resected lung adenocarinomas at the Thoraxklinik at Heidelberg University Hospital and diagnosed (according to 2015 WHO Classification of lung tumors) at the Institute of Pathology at Heidelberg University Hospital. To evaluate the significance of ITH on gene expression profiling, multi-regional gene expression analysis (2-4 samples per tumor) was performed for 24 ADC using the NanoString IO 360 Panel. ANOVA was used to compare inter-tumor variance to intra-tumor variance of mRNA expression. The F statistics was calculated as ratio of these quantities. A list of genes with significantly higher inter-tumor variance was isolated using the F-test. Multiple testing was addressed using the Benjamini-Hochberg method to control the false discovery rate (FDR). RESULTS: In an unsupervised hierarchical clustering analysis of the entire set of 770 genes, the two to four segments of each of the tumors clustered together. For 752 of the 770 genes (97.7%) the inter-tumor variance was significantly higher than the intra-tumor variance (FDR CONCLUSION: In our preliminary analysis of ICB-GEP, variance of expression levels between tumors was substantial and exceeded ITH for the majority of analyzed genes. In-depth analyses are ongoing to further delineate the influence on gene signatures and ICB related pathways. Citation Format: Daniel Kazdal, Jan Budczies, Martina Kirchner, Klaus Kluck, Michael Allgäuer, Olaf Neumann, Regine Brandt, Eugen Rempel, Carolin Ploeger, Moritz von Winterfeld, Marc Schneider, Steffen Dietz, Hauke Winter, Thomas Muley, Holger Sülmann, Michael Meister, Felix Herth, Claus Heussel, Roland Penzel, Volker Endris, Peter Schirmacher, Michael Thomas, Petros Christopoulos, Albrecht Stenzinger. Inter- and intra- tumorheterogeneity of the microenvironment in pulmonary adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1571.

Non-small cell lung cancer (NSCLC) patients with ALK rearrangements are routinely treated with tyrosine kinase inhibitors (TKIs), leading to improved survival. However, clinical courses vary widely and the disease remains incurable. Therefore, early detection and molecular characterization of treatment failure is important for therapy guidance. To identify indicators of therapy response and resistance, we performed genotyping of circulating cell-free DNA (cfDNA), including panel-based deep sequencing (>4,200x coverage) and shallow whole-genome sequencing (sWGS; 0.5x coverage) from serial plasma samples (n=282) of 73 patients with ALK rearrangements. Variable mutation levels were marked in all patients and correlated with clinical features. At progression, individual dynamics of resistance mutations were seen: while some mutations became undetectable upon therapy switch, variant fractions of other alterations further increased. We also found mutated TP53 at the time of progression in patients with initially TP53 wild-type tumors. The progression-free survival of patients with acquired TP53 mutations was comparable to that of primarily TP53 mutated and shorter than that of persistently TP53 wild-type cases. Shallow WGS of cfDNA identified copy number variations (CNVs), some of which might contribute to disease progression. To quantitatively assess CNVs, trimmed median absolute deviation from copy number neutrality (t-MAD) scores were calculated from sWGS data. Longitudinal t-MAD assessment reflected the kinetics of mutant allele frequencies in cfDNA and allowed disease monitoring with increasing t-MAD and mutation levels at tumor progression. Moreover, EML4-ALK fusion variant 3 (V3) and TP53 mutations define molecular determinants of adverse outcome in ALK+ NSCLC. Shallow WGS of cfDNA identified increased t-MAD levels in patients with EML4-ALK V3 compared to those with fusion V1. Similarly, patients with TP53 mutations showed a higher t-MAD than TP53 wild-type cases. In summary, overall survival of patients with detectable mutations in plasma DNA at any time point during clinical follow-up was shorter than that of patients without detectable mutations in cfDNA. Similarly, overall survival time from the first detection of mutations cfDNA until death/end of follow-up was shorter than in cases without detectable mutations. Our data suggest that liquid biopsies could help to improve the care of ALK+ NSCLC patients through early detection of disease progression and tailored treatment of resistant tumors. Although individual courses were seen, rising mutation levels could indicate the need of radiologic reassessment earlier than scheduled. Detection of CNVs as surrogate for genomic instability and acquired TP53 mutations in liquid identified high-risk cases and suggests the potential clinical utility of cfDNA monitoring for this disease beyond profiling of ALK resistance mutations. Citation Format: Steffen Dietz, Petros Christopoulos, Lisa Gu, Volker Endris, Zhao Yuan, Simon J. Ogrodnik, Tomasz Zemojtel, Marc A. Schneider, Anna-Lena Volckmar, Michael Meister, Thomas Muley, Martin Reck, Matthias Schlesner, Michael Thomas, Albrecht Stenzinger, Holger Sültmann. Longitudinal analysis of cell-free DNA for therapy monitoring of ALK-positive non-small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A21.

Anaplastic lymphoma kinase (ALK) sequencing can identify resistance mechanisms and guide next-line therapy in ALK+ non-small-cell lung cancer (NSCLC), but the clinical significance of other rebiopsy findings remains unclear. We analysed all stage-IV ALK+ NSCLC patients with longitudinally assessable TP53 status treated in our institutions (n = 62). Patients with TP53 mutations at baseline (TP53mutbas, n = 23) had worse overall survival (OS) than patients with initially wild-type tumours (TP53wtbas, n = 39, 44 vs. 62 months in median, p = 0.018). Within the generally favourable TP53wtbas group, detection of TP53 mutations at progression defined a “converted” subgroup (TP53mutconv, n = 9) with inferior OS, similar to that of TP53mutbas and shorter than that of patients remaining TP53 wild-type (TP53wtprogr, 45 vs. 94 months, p = 0.043). Progression-free survival (PFS) under treatment with tyrosine kinase inhibitors (TKI) for TP53mutconv was comparable to that of TP53mutbas and also shorter than that of TP53wtprogr cases (5 and 8 vs. 13 months, p = 0.0039). Fewer TP53wtprogr than TP53mutbas or TP53mutconv cases presented with metastatic disease at diagnosis (67% vs. 91% or 100%, p < 0.05). Thus, acquisition of TP53 mutations at progression is associated with more aggressive disease, shorter TKI responses and inferior OS in ALK+ NSCLC, comparable to primary TP53 mutated cases.

Lung adenocarcinoma (ADC) is the most prevalent subtype of lung cancer and characterized by considerable morphological and mutational heterogeneity. However, little is known about the epigenomic intratumor variability between spatially separated histological growth patterns of ADC. In order to reconstruct the clonal evolution of histomorphological patterns, we performed global DNA methylation profiling of 27 primary tumor regions, seven matched normal tissues and six lymph node metastases from seven ADC cases. Additionally, we investigated the methylation data from 369 samples of the TCGA ADC cohort. All regions showed varying degrees of methylation changes between segments of different, but also of the same growth patterns. Similarly, copy number variations were seen between spatially distinct segments of each patient. Hierarchical clustering of promoter methylation revealed extensive heterogeneity within and between the cases. Intratumor DNA methylation heterogeneity demonstrated a branched clonal evolution of ADC regions driven by genomic instability with subclonal copy number changes. Notably, methylation profiles within tumors were not more similar to each other than to those from other individuals. In two cases, different tumor regions of the same individuals were represented in distant clusters of the TCGA cohort, illustrating the extensive epigenomic intratumor heterogeneity of ADCs. We found no evidence for the lymph node metastases to be derived from a common growth pattern. Instead, they had evolved early and separately from a particular pattern in each primary tumor. Our results suggest that extensive variation of epigenomic features contributes to the molecular and phenotypic heterogeneity of primary ADCs and lymph node metastases.

Purpose Matrix assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-MSI) is a powerful tool to analyze the spatial distribution of peptides in tissues. Digital PCR (dPCR) is a method to reliably detect genetic mutations. Biopsy material is often limited due to minimally invasive techniques, but information on diagnosis, prognosis, and prediction is required for subsequent clinical decision making. Thus, saving tissue material during diagnostic workup is highly warranted for best patient care. The possibility to combine proteomic analysis by MALDI-MSI and mutational analysis by dPCR from the same tissue section is evaluated. Experimental design Ten 0.5 × 0.5 cm formalin-fixed paraffin embedded tissue samples of pulmonary adenocarcinomas with known EGFR or KRAS mutations are analyzed by MALDI-MSI. Subsequently, DNA is extracted from the analyzed tissue material and tested for the respective driver mutation by dPCR. Results Detection of driver gene mutations after MALDI MSI analysis is successful in all analyzed samples. Determined mutant allele frequencies are in good agreement with values assessed from untreated serial tissue sections with a mean absolute deviation of 0.16. Conclusion and clinical relevance It has been demonstrated that MALDI-MSI can be combined with genetic analysis, like dPCR. Workflows enabling the subsequent analysis of proteomic and genetic markers are particularly promising for the analysis of limited sample material such as biopsy specimen.

Multiregional analysis provided first indications for morphological and molecular heterogeneity in lung adenocarcinomas (ADCs), but comprehensive morpho-molecular comparisons are still lacking. The purpose of our study was to investigate the spatial distribution of EGFR and KRAS alterations systematically throughout whole tumor cross-sections in correlation with the tumor cell content and the histopathological patterns. Central sections of 19 ADCs were subdivided into 467 segments of 5 mm × 5 mm. We determined the predominant histological growth pattern and the allele frequencies of driver gene mutations by digital PCR in every segment. We further quantified the absolute cell counts and proportions of tumor and non-neoplastic cells in all segments to normalize the mutant allele frequencies. Driver gene mutations could be detected in99% of the tumor containing segments, with high levels of inter- and intratumor heterogeneity regarding the mutant allele frequency (range: 0.04-19.36). Different patterns for the distribution of the variant allele frequency within a tumor were recognizable. While some cases showed ubiquitously low or high levels, others revealed regions with focally elevated frequencies. Differences between KRAS and EGFR alterations were not significant. The great majority of the analyzed tumor sections (16/19) exhibited two or more morphological growth patterns. Mutant allele frequencies were significantly higher in segments with a predominant solid pattern compared to all other histologies (p 0.01). Our data indicate that driver gene mutations are present with high levels of inter- and intratumor heterogeneity throughout the whole tumor, with a correlation between the allele frequencies and histological growth patterns.

Genetic rearrangements involving the anaplastic lymphoma kinase (ALK) gene confer sensitivity to ALK tyrosine kinase inhibitors (TKIs) and superior outcome in non-small-cell lung cancer (NSCLC). However, clinical courses vary widely, and recent studies suggest that molecular profiling of ALK+ NSCLC can provide additional predictors of therapy response that could assist further individualization of patient management. As repeated tissue biopsies often pose technical difficulties and significant procedural risk, analysis of tumor constituents circulating in the blood, including ctDNA and various proteins, is increasingly recognized as an alternative method of tumor sampling (“liquid biopsy”). Here, we report the case of a KLC1–ALK-rearranged NSCLC patient responding to crizotinib treatment and demonstrate how analysis of plasma and serum biomarkers can be used to identify the ALK fusion partner and monitor therapy over time. Results of ctDNA sequencing and copy-number alteration profiling as well as serum protein concentrations at various time points during therapy reflected the current remission status and could predict the subsequent clinical course. At the time of disease progression, we identified four distinct secondary mutations in the ALK gene in ctDNA potentially causing treatment failure, accompanied by rising levels of CEA and CYFRA 21–1. Moreover, several copy-number variations were detected at the end of the treatment, including an amplification of a region on Chromosome 12 encompassing the TP53 regulator MDM2. In summary, our findings illustrate the utility of noninvasive longitudinal molecular profiling for assessing remission status, exploring mechanisms of treatment failure, predicting subsequent clinical course, and dissecting dynamics of drug-resistant clones in ALK+ lung cancer.

Circulating DNA released from cancerous tissues has been found to harbour tumour-associated alterations and to represent the molecular composition of the tumour. Recent advances in technologies, especially in next-generation sequencing, enable the analysis of low amounts of circulating DNA from body fluids. These “liquid biopsies” allow tumour genotyping and therapy monitoring without invasive procedures and offer the potential for early cancer detection.

Long-lasting success in lung cancer therapy using tyrosine kinase inhibitors (TKIs) is rare since the tumors develop resistance due to the occurrence of molecularly altered subclones. The aim of this study was to monitor tumors over time based on the quantity of mutant plasma DNA and to identify early indications for therapy response and tumor progression. Serial plasma samples from lung adenocarcinoma patients treated with TKIs were used to quantify EGFR and KRAS mutations in circulating DNA by digital PCR. Mutant DNA levels were compared with the courses of responses to treatment with TKIs, conventional chemotherapy, radiotherapy, or combinations thereof. Variations in plasma DNA mutation levels over time were found in 15 patients. We categorize three major courses: First, signs of therapy response are associated with a fast clearing of plasma DNA mutations within a few days. Second, periods of stable disease are accompanied by either absence of mutations or fluctuation at low levels. Finally, dramatic increase of mutational load is followed by rapid tumor progression and poor patient survival. In summary, the serial assessment of EGFR mutations in the plasma of NSCLC patients allows conclusions about controlled disease and tumor progression earlier than currently available methods.

Circulating cell-free DNA (cfDNA) released from cancerous tissues has been found to harbor tumor-associated alterations and to represent the molecular composition of the tumor. Recent advances in technologies, especially in next-generation sequencing, enable the analysis of low amounts of cfDNA from body fluids. We analyzed the exomes of tumor tissue and matched serum samples to investigate the molecular representation of the tumor exome in cfDNA. To this end, we implemented a workflow for sequencing of cfDNA from low serum volumes (200 μl) and performed whole-exome sequencing (WES) of serum and matched tumor tissue samples from six non-small cell lung cancer (NSCLC) patients and two control sera. Exomes, including untranslated regions (UTRs) of cfDNA were sequenced with an average coverage of 68.5x. Enrichment efficiency, target coverage, and sequencing depth of cfDNA reads were comparable to those from matched tissues. Discovered variants were compared between serum and tissue as well as to the COSMIC database of known mutations. Although not all tissue variants could be confirmed in the matched serum, up to 57% of the tumor variants were reflected in matched cfDNA with mutations in PIK3CA, ALK, and PTEN as well as variants at COSMIC annotated sites in all six patients analyzed. Moreover, cfDNA revealed a mutation in MTOR, which was not detected in the matched tissue, potentially from an untested region of the heterogeneous primary tumor or from a distant metastatic clone. WES of cfDNA may provide additional complementary molecular information about clinically relevant mutations and the clonal heterogeneity of the tumors.

Proton-coupled monocarboxylate transporters (MCTs) mediate the exchange of high energy metabolites like lactate between different cells and tissues. We have reported previously that carbonic anhydrase II augments transport activity of MCT1 and MCT4 by a noncatalytic mechanism, while leaving transport activity of MCT2 unaltered. In the present study, we combined electrophysiological measurements in Xenopus oocytes and pulldown experiments to analyze the direct interaction between carbonic anhydrase II (CAII) and MCT1, MCT2, and MCT4, respectively. Transport activity of MCT2-WT, which lacks a putative CAII-binding site, is not augmented by CAII. However, introduction of a CAII-binding site into the C terminus of MCT2 resulted in CAII-mediated facilitation of MCT2 transport activity. Interestingly, introduction of three glutamic acid residues alone was not sufficient to establish a direct interaction between MCT2 and CAII, but the cluster had to be arranged in a fashion that allowed access to the binding moiety in CAII. We further demonstrate that functional interaction between MCT4 and CAII requires direct binding of the enzyme to the acidic cluster (431)EEE in the C terminus of MCT4 in a similar fashion as previously shown for binding of CAII to the cluster (489)EEE in the C terminus of MCT1. In CAII, binding to MCT1 and MCT4 is mediated by a histidine residue at position 64. Taken together, our results suggest that facilitation of MCT transport activity by CAII requires direct binding between histidine 64 in CAII and a cluster of glutamic acid residues in the C terminus of the transporter that has to be positioned in surroundings that allow access to CAII.