The Impact of Cancer-Associated Fibroblasts on the Biology and Progression of Colorectal Carcinomas.

(1) Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally. Cancer-associated fibroblasts (CAFs) are major components of CRC's tumour microenvironment (TME), but their biological background and interplay with the TME remain poorly understood. This study investigates CAF biology and its impact on CRC progression. (2) The cohort comprises 155 cases, including CRC, with diverse localizations, adenomas, inflammations, and controls. Digital gene expression analysis examines genes associated with signalling pathways (MAPK, PI3K/Akt, TGF-ß, WNT, p53), while next-generation sequencing (NGS) determines CRC mutational profiles. Immunohistochemical FAP scoring assesses CAF density and activity. (3) FAP expression is found in 81 of 150 samples, prevalent in CRC (98.4%), adenomas (27.5%), and inflammatory disease (38.9%). Several key genes show significant associations with FAP-positive fibroblasts. Gene set enrichment analysis (GSEA) highlights PI3K and MAPK pathway enrichment alongside the activation of immune response pathways like natural killer (NK)-cell-mediated cytotoxicity via CAFs. (4) The findings suggest an interplay between CAFs and cancer cells, influencing growth, invasiveness, angiogenesis, and immunogenicity. Notably, TGF-ß, CDKs, and the Wnt pathway are affected. In conclusion, CAFs play a significant role in CRC and impact the TME throughout development.

Tumor cell cytoplasmic metallothionein expression associates with differential tumor immunogenicity and prognostic outcome in high-grade serous ovarian carcinoma.

Background: The underlying mechanism of high T-cell presence as a favorable prognostic factor in high-grade serous ovarian carcinoma (HGSOC) is not yet understood. In addition to immune cells, various cofactors are essential for immune processes. One of those are metallothioneins (MTs), metal-binding proteins comprising various isoforms. MTs play a role in tumor development and drug resistance. Moreover, MTs influence inflammatory processes by regulating zinc homeostasis. In particular, T-cell function and polarization are particularly susceptible to changes in zinc status. The aim of the present study was to investigate a possible role of MT-mediated immune response and its association with prognostic outcome in ovarian cancer. Methods: A retrospective study was conducted on a clinically well-characterized cohort of 24 patients with HGSOC treated at the University Hospital of Essen. Gene expression patterns for anti-cancer immunogenicity-related targets were performed using the NanoString nCounter platform for digital gene expression analysis with the appurtenant PanCancer Immune Profiling panel, consisting of 770 targets and 30 reference genes. Tumor-associated immunohistochemical MT protein expression was evaluated using a semi-quantitative four-tier Immunohistochemistry (IHC) scoring. Results: MT immunoexpression was detected in 43% (10/23) of all HGSOC samples. MT immunoexpression levels showed a significant association to survival, leading to prolonged progression-free and overall survival in positively stained tumors. Furthermore, T-cell receptor signaling gene signature showed a strong activation in MT-positive tumors. Activated downstream signaling cascades resulting in elevated interferon-gamma expression with a shift in the balance between T helper cells (TH1 and TH2) could be observed in the MT-positive subgroup. In addition, a higher expression pattern of perforin and several granzymes could be detected, overall suggestive of acute, targeted anti-cancer immune response in MT-positive samples. Conclusion: This is the first study combining broad, digital mRNA screening of anti-tumor immune response-associated genes and their relation to MT-I/II in ovarian cancer. MT overexpression is associated with molecular characteristics of an anti-cancer immune response and is a strong prognostic marker in ovarian HGSOC. The observed immune cell activation associated with tumor MT expression comprises but is not limited to T cells and natural killer cells.

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.

CAF-Associated Paracrine Signaling Worsens Outcome and Potentially Contributes to Chemoresistance in Epithelial Ovarian Cancer.

Background: High-grade serous ovarian cancer (HGSOC) is the predominant and deadliest form of ovarian cancer. Some of its histological subtypes can be distinguished by frequent occurrence of cancer-associated myofibroblasts (CAFs) and desmoplastic stroma reaction (DSR). In this study, we want to explore the relationship between therapy outcome and the activity of CAF-associated signaling pathways in a homogeneous HGSOC patient collective. Furthermore, we want to validate these findings in a general Epithelial ovarian cancer (EOC) cohort. Methods: The investigation cohort consists of 24 HGSOC patients. All of them were treated with platinum-based components and clinical follow-up was available. The validation cohort was comprised of 303 patients. Sequencing data (whole transcriptome) and clinical data were extracted from The Cancer Genome Atlas (TCGA). RNA of HGSOC patients was isolated using a Maxwell RSC instrument and the appropriate RNA isolation kit. For digital expression analysis a custom-designed gene panel was employed. All genes were linked to various DSR- and CAF- associated pathways. Expression analysis was performed on the NanoString nCounter platform. Finally, data were explored using the R programming environment (v. 4.0.3). Result: In total, 15 CAF-associated genes were associated with patients' survival. More specifically, 6 genes (MMP13, CGA, EPHA3, PSMD9, PITX2, PHLPP1) were linked to poor therapy outcome. Though a variety of different pathways appeared to be associated with therapy failure, many were related to CAF paracrine signaling, including MAPK, Ras and TGF-ß pathways. Similar results were obtained from the validation cohort. Discussion: In this study, we could successfully link CAF-associated pathways, as shown by increased Ras, MAPK and PI3K-Akt signaling to therapy failure (chemotherapy) in HGSOC and EOCs in general. As platinum-based chemotherapy has been the state-of-the-art therapy to treat HGSOC for decades, it is necessary to unveil the reasons behind resistance developments and poor outcome. In this work, CAF-associated signaling is shown to compromise therapy response. In the validation cohort, CAF-associated signaling is also associated with therapy failure in general EOC, possibly hinting towards a conserved mechanism. Therefore, it may be helpful to stratify HGSOC patients for CAF activity and consider alternative treatment options.

Bortezomib sensitivity is tissue dependent and high expression of the 20S proteasome precludes good response in malignant pleural mesothelioma.

BACKGROUND: Bortezomib is an approved proteasome inhibitor for the treatment of certain lymphoma subtypes. Two clinical trials investigated bortezomib in patients with malignant pleural mesothelioma (MPM) and failed to improve outcome. We present a potential explanation for this event. METHODS: 171 patients with MPM were analyzed for their mRNA expression of proteasomal subunits PSMA1, PSMA5, PSMB1, PSMB2, PSMB4 and PSMB5 via qPCR (n=84) or sequencing (n=87 TCGA/cBioPortal data set "Mesothelioma"). Outcome and subunit expression were correlated. Four mesothelial and one fibroblast cell line were treated with bortezomib and cisplatin. Cellular response was measured after 0, 6, 12, 24, 48 and 72 hrs. Enzyme activity of proteasomal subunits was assessed via functional enzyme activity assays. RESULTS: Patients with MPM presented with elevated expression of proteasomal subunits compared to benign controls (p<0.001). PSMB4 correlated with outcome (Cox propotiortional-hazards model (COXPH): p<0.0175, TCGA/cBioPortal data). In cell lines, apoptosis was the main event with a peak after 48 hr incubation for bortezomib or cisplatin. Only two cell lines with comparably low proteasome activity (PSMB2 and PSMB5) responded to 50 nM and 100 nM bortezomib better than to cisplatin (MRC-5, NCI-H2052). MSTO-211H responded to cisplatin only, whereas the other two cell lines were considered therapy resistant (Met-5A, NCI-H2452). INTERPRETATION: Two clinical trials testing bortezomib in MPM failed, although MPM presents with high proteasome expression, which predicts bortezomib sensitivity in several tumors. Bortezomib induced apoptosis in MPM cell lines with low proteasome activity only. Bortezomib is not suitable for the treatment of MPM, and biomarker-based stratification could have improved both clinical trials. TRIAL REGISTRATION: NCT00513877 and NCT00458913.

miRNA regulation is important for DNA damage repair and recognition in malignant pleural mesothelioma.

Platin-containing regimes are currently considered as state-of-the-art therapies in malignant pleural mesotheliomas (MPM) but show dissatisfying response rates ranging from 6 to 16% only. Still, the reasons for the rather poor efficacy remain largely unknown. A clear stratification of patients based on new biomarkers seems to be a promising approach to enhance clinical management, which would be a long-needed improvement for MPM patients but does not seem likely soon unless new biomarkers can be validated. Twenty-four formalin-fixed, paraffin-embedded (FFPE) tumour specimens were subjected to a miRNA expression screening of 800 important miRNAs using digital quantification via the nCounter technique (NanoString). We defined a small subset of miRNAs regulating the key enzymes involved in the repair of platin-associated DNA damage. Particularly, the TP53 pathway network for DNA damage recognition as well as genes related to the term "BRCAness" are the main miRNA targets within this context. The TP53 pathway network for DNA damage recognition as well as genes related to the term "BRCAness" are the main players for risk stratification in patients suffering from this severe disease. Taking the specific molecular profile of the tumour into account can help to enhance the clinical management prospectively and to smooth the way to better response prediction.

Folic acid phenotype (FAP) is a superior biomarker predicting response to pemetrexed-based chemotherapy in malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a rare tumor linked to a dismal prognosis. Even the most effective chemotherapeutical regime of pemetrexed combined with cisplatin leads to a remission-rate of only about 40%. The reasons for the rather poor efficacy remain largely unknown. RESULTS: Phenotypes were significantly associated with progression (p=0.0279) and remission (p=0.0262). Cox-regression revealed significant associations between SLC19A1/TYMS-ratio (p=0.0076) as well as FPGS/TYMS-ratio (p=0.0026) and OS. For differentiation by risk-groups, COXPH identified a strong correlation (p=0.0008). METHODS: 56 MPM specimens from patients treated with pemetrexed were used for qPCR analysis. Phenotypes and risk groups were defined by their expression levels of members of the folic acid metabolism and correlated to survival and objective response. CONCLUSION: Our results indicate that the balance between folic acid uptake, activation and metabolism plays a crucial role in response to pemetrexed-based chemotherapy and the prognosis of MPM patients. Implementing this marker profile in MPM stratification may help to individualize MPM-therapy more efficiently.

MiR-200b and miR-155 as predictive biomarkers for the efficacy of chemoradiation in locally advanced head and neck squamous cell carcinoma.

BACKGROUND: The predictive value of microRNAs (miRNAs) in tumour cells and infiltrating immune cells for the efficacy of chemoradiation (CRTX) in locally advanced head and neck squamous cell carcinoma (HNSCC) was evaluated. METHODS: Formalin-fixed, paraffin-embedded tumour material was collected from patients with locally advanced HNSCC treated within the ARO-0401 phase III trial with radiotherapy in combination with either 5-fluorouracil/cisplatin (CDDP-CRTX) or 5-fluorouracil/mitomycin C (MMC-CRTX). MiRNA and immune profiles were established in a test cohort of 48 oropharyngeal carcinoma (OPSCC) cases by Affymetrix miRNA microarrays and the nanoString PanCancer Immune Panel, respectively. Expression of miRNA candidates was measured in 149 HNSCC patients by real-time PCR. Interference of miRNA profiles with CRTX efficacy was determined by Kaplan-Meier and Cox regression analysis. RESULTS: Expression levels of five miRNAs (miR-27b, -130b, -200b, -451 and -532-5p) were significantly associated with overall survival after MMC-CRTX. Six different miRNAs (miR-125b, -146a, -150, -155, -187 and -342-5p) were correlated with overall survival after CDDP-CRTX. Validation by real-time PCR confirmed the predictive value of miR-200b and miR-155 in OPSCC, which was absent in hypopharyngeal carcinomas. MiR-146a was revealed as a prognostic marker for both CRTX regimens. MiR-200b expression was mainly associated with distant metastasis, whereas miR-155 correlated with local recurrence. MiR-155 and miR-146a were identified as surrogate markers for tumour-infiltrating lymphocytes. CONCLUSIONS: MiR-200b and miR-155 were established as potential markers for personalised treatment selection of two standard regimens of CRTX. The predictive role of miR-155 deserves further investigation, especially within the framework of clinical trials of CRTX/immune checkpoint inhibitor combinations.

Massive parallel sequencing and digital gene expression analysis reveals potential mechanisms to overcome therapy resistance in pulmonary neuroendocrine tumors.

Background: Lung cancer is the leading cause of cancer-related deaths worldwide. 25% show neuroendocrine differentiation (typical/atypical carcinoids, large-/small-cell neuroendocrine carcinomas). Carcinoids present with long survival rates, but metastatic carcinoids correlate with decreased survival and are commonly insensitive to standard chemotherapy or radiation. Therefore, novel therapeutic strategies are urgently needed. Material and methods: 70 representative tumor specimens were used for next-generation sequencing analysis of 14 genes related to therapy response. Additionally, mRNA-expression profiles of 60 matching samples were determined for 13 selected drug targets by using the NanoString nCounter technology. Results: A number of features known to sensitize tumors for different targeted therapies could be identified, which hopefully improve the clinical management of this subgroup of lung neoplasias. In particular, EGFR expression was observed in the investigated tumors in a noteworthy manner. Additionally, MDM2 was strongly expressed in the majority of all samples whereas the expression of its physiological inhibitor, CDKN2A, was nearly absent in all low-grade tumors. TP53 showed a high frequency of variants in high-grade tumors but mutations were rare in carcinoids. Conclusion: Based on our results, therapeutic approaches with MDM2-inhibitors and monoclonal anti-EGFR antibodies may be promising in pulmonary carcinoid tumors.

ACTB, CDKN1B, GAPDH, GRB2, RHOA and SDCBP Were Identified as Reference Genes in Neuroendocrine Lung Cancer via the nCounter Technology.

BACKGROUND: Neuroendocrine lung cancer (NELC) represents 25% of all lung cancer cases and large patient collectives exist as formalin-fixed, paraffin-embedded (FFPE) tissue only. FFPE is controversially discussed as source for molecular biological analyses and reference genes for NELC are poorly establishes. MATERIAL AND METHODS: Forty-three representative FFPE-specimens were used for mRNA expression analysis using the digital nCounter technology (NanoString). Based on recent literature, a total of 91 mRNA targets were investigated as potential tumor markers or reference genes. The geNorm, NormFinder algorithms and coefficient of correlation were used to identify the most stable reference genes. Statistical analysis was performed by using the R programming environment (version 3.1.1). RESULTS: RNA integrity (RIN) ranged from 1.8 to 2.6 and concentrations from 34 to 2,109 ng/µl. However, the nCounter technology gave evaluable results for all samples tested. ACTB, CDKN1B, GAPDH, GRB2, RHOA and SDCBP were identified as constantly expressed genes with high stability (M-)values according to geNorm, NormFinder and coefficients of correlation. CONCLUSION: FFPE-derived mRNA is suitable for molecular biological investigations via the nCounter technology, although it is highly degraded. ACTB, CDKN1B, GAPDH, GRB2, RHOA and SDCBP are potent reference genes in neuroendocrine tumors of the lung.

Screening of Pleural Mesotheliomas for DNA-damage Repair Players by Digital Gene Expression Analysis Can Enhance Clinical Management of Patients Receiving Platin-Based Chemotherapy.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a rare, predominantly asbestos-related and biologically highly aggressive tumour leading to a dismal prognosis. Multimodality therapy consisting of platinum-based chemotherapy is the treatment of choice. The reasons for the rather poor efficacy of platinum compounds remain largely unknown. MATERIAL AND METHODS: For this exploratory mRNA study, 24 FFPE tumour specimens were screened by digital gene expression analysis. Based on data from preliminary experiments and recent literature, a total of 366 mRNAs were investigated using a Custom CodeSet from NanoString. All statistical analyses were calculated with the R i386 statistical programming environment. RESULTS: CDC25A and PARP1 gene expression were correlated with lymph node spread, BRCA1 and TP73 expression levels with higher IMIG stage. NTHL1 and XRCC3 expression was associated with TNM stage. CHECK1 as well as XRCC2 expression levels were correlated with tumour progression in the overall cohort of patients. CDKN2A and MLH1 gene expression influenced overall survival in this collective. In the adjuvant treated cohort only, CDKN2A, CHEK1 as well as ERCC1 were significantly associated with overall survival. Furthermore, TP73 expression was associated with progression in this subgroup. CONCLUSION: DNA-damage response plays a crucial role in response to platin-based chemotherapeutic regimes. In particular, CHEK1, XRCC2 and TP73 are strongly associated with tumour progression. ERCC1, MLH1, CDKN2A and most promising CHEK1 are prognostic markers for OS in MPM. TP73, CDKN2A, CHEK1 and ERCC1 seem to be also predictive markers in adjuvant treated MPMs. After a prospective validation, these markers may improve clinical and pathological practice, finally leading to a patients' benefit by an enhanced clinical management.

Folic-acid metabolism and DNA-repair phenotypes differ between neuroendocrine lung tumors and associate with aggressive subtypes, therapy resistance and outcome.

PURPOSE: 25% of all lung cancer cases are neuroendocrine (NELC) including typical (TC) and atypical carcinoid (AC), large-cell neuroendocrine (LCNEC) and small cell lung cancer (SCLC). Prognostic and predictive biomarkers are lacking. EXPERIMENTAL DESIGN: Sixty patients were used for nCounter mRNA expression analysis of the folic-acid metabolism (ATIC, DHFR, FOLR1, FPGS, GART, GGT1, SLC19A1, TYMS) and DNA-repair (ERCC1, MLH1, MSH2, MSH6, XRCC1). Phenotypic classification classified tumors (either below or above the median expression level) with respect to the folic acid metabolism or DNA repair. RESULTS: Expression of FOLR1, FPGS, MLH1 and TYMS (each p<0.0001) differed significantly between all four tumor types. FOLR1 and FPGS associated with tumor differentiation (both p<0.0001), spread to regional lymph nodes (FOLR1 p=0.0001 and FPGS p=0.0038), OS and PFS (FOLR1 p<0.0050 for both and FPGS p<0.0004 for OS). Phenotypic sorting revealed the Ft-phenotype to be the most prominent expression profile in carcinoids, whereas SCLC presented nearly univocal with the fT and LCNEC with fT or ft. These results were significant for tumor subtype (p<0.0001). CONCLUSIONS: The assessed biomarkers and phenotypes allow for risk stratification (OS, PFS), diagnostic classification and enhance the biological understanding of the different subtypes of neuroendocrine tumors revealing potential new therapy options and clarifying known resistance mechanisms.

microRNAs are differentially regulated between MDM2-positive and negative malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a highly aggressive tumour first-line treated with a combination of cisplatin and pemetrexed. MDM2 and P14/ARF (CDKN2A) are upstream regulators of TP53 and may contribute to its inactivation. In the present study, we now aimed to define the impact of miRNA expression on this mechanism. MATERIAL AND METHODS: 24 formalin-fixed paraffin-embedded (FFPE) tumour specimens were used for miRNA expression analysis of the 800 most important miRNAs using the nCounter technique (NanoString). Significantly deregulated miRNAs were identified before a KEGG-pathway analysis was performed. RESULTS: 17 miRNAs regulating TP53, 18 miRNAs regulating MDM2, and 11 miRNAs directly regulating CDKN2A are significantly downregulated in MDM2-expressing mesotheliomas. TP53 is downregulated in MDM2-negative tumours through miRNAs with a miSVR prediction score of 11.67, RB1 with a prediction score of 8.02, MDM2 with a prediction score of 4.50 and CDKN2A with a prediction score of 1.27. CONCLUSION: MDM2 expression seems to impact miRNA expression levels in MPM. Especially, miRNAs involved in TP53-signaling are strongly decreased in MDM2-positive mesotheliomas. A better understanding of its tumour biology may open the chance for new therapeutic approaches and thereby augment patients' outcome.

Mutational analysis of pulmonary tumours with neuroendocrine features using targeted massive parallel sequencing: a comparison of a neglected tumour group.

BACKGROUND: Lung cancer is the leading cause of cancer-related deaths worldwide. The typical and atypical carcinoid (TC and AC), the large-cell neuroendocrine carcinoma (LCNEC) and the small-cell lung cancers (SCLC) are subgroups of pulmonary tumours that show neuroendocrine differentiations. With the rising impact of molecular pathology in routine diagnostics the interest for reliable biomarkers, which can help to differentiate these subgroups and may enable a more personalised treatment of patients, grows. METHODS: A collective of 70 formalin-fixed, paraffin-embedded (FFPE) pulmonary neuroendocrine tumours (17 TCs, 17 ACs, 19 LCNECs and 17 SCLCs) was used to identify biomarkers by high-throughput sequencing. Using the Illumina TruSeq Amplicon-Cancer Panel on the MiSeq instrument, the samples were screened for alterations in 221 mutation hot spots of 48 tumour-relevant genes. RESULTS: After filtering >26 000 detected variants by applying strict algorithms, a total of 130 mutations were found in 29 genes and 49 patients. Mutations in JAK3, NRAS, RB1 and VHL1 were exclusively found in SCLCs, whereas the FGFR2 mutation was detected in LCNEC only. KIT, PTEN, HNF1A and SMO were altered in ACs. The SMAD4 mutation corresponded to the TC subtype. We prove that the frequency of mutations increased with the malignancy of tumour type. Interestingly, four out of five ATM-mutated patients showed an additional alteration in TP53, which was by far the most frequently altered gene (28 out of 130; 22%). We found correlations between tumour type and IASLC grade for ATM- (P=0.022; P=0.008) and TP53-mutated patients (P<0.001). Both mutated genes were also associated with lymph node invasion and distant metastasis (P⩽0.005). Furthermore, PIK3CA-mutated patients with high-grade tumours showed a reduced overall survival (P=0.040) and the mutation frequency of APC and ATM in high-grade neuroendocrine lung cancer patients was associated with progression-free survival (PFS) (P=0.020). CONCLUSIONS: The implementation of high-throughput sequencing for the analysis of the neuroendocrine lung tumours has revealed that, even if these tumours encompass several subtypes with varying clinical aggressiveness, they share a number of molecular features. An improved understanding of the biology of neuroendocrine tumours will offer the opportunity for novel approaches in clinical management, resulting in a better prognosis and prediction of therapeutic response.

Comprehensive Analysis of Disease-Related Genes in Chronic Lymphocytic Leukemia by Multiplex PCR-Based Next Generation Sequencing.

BACKGROUND: High resolution molecular studies have demonstrated that the clonal acquisition of gene mutations is an important mechanism that may promote rapid disease progression and drug resistance in chronic lymphocytic leukemia (CLL). Therefore, the early and sensitive detection of such mutations is an important prerequisite for future predictive CLL diagnostics in the clinical setting. MATERIAL & METHODS: Here, we describe a novel, target-specific next generation sequencing (NGS) approach, which combines multiplex PCR-based target enrichment and library generation with ultra-deep high-throughput parallel sequencing using a MiSeq platform. We designed a CLL specific target panel, covering hotspots or complete coding regions of 15 genes known to be recurrently mutated and/or related to B-cell receptor signaling. RESULTS: High-throughput sequencing was performed using as little as 40 ng of peripheral blood B-cell DNA from 136 CLL patients and a dilution series of two ATM- or TP53-mutated cell lines, the latter of which demonstrated a limit of mutation detection below 5%. Using a stringent functional assessment algorithm, 102 mutations in 8 genes were identified in CLL patients, including hotspot regions of TP53, SF3B1, NOTCH1, ATM, XPO1, MYD88, DDX3X and the B-cell receptor signaling regulator PTPN6. The presence of mutations was significantly associated with an advanced disease status und molecular markers of an inferior prognosis, such as an unmutated IGHV mutation status or positivity for ZAP70 by flow cytometry. CONCLUSION: In summary, targeted sequencing using an amplicon based library technology allows a resource-efficient and sensitive mutation analysis for diagnostic or exploratory purposes and facilitates molecular subtyping of patient sets with adverse prognosis.

Identification of deregulation of apoptosis and cell cycle in neuroendocrine tumors of the lung via NanoString nCounter expression analysis.

BACKGROUND: Neuroendocrine tumors of the lung comprise typical (TC) and atypical carcinoids (AC), large-cell neuroendocrine cancer (LCNEC) and small-cell lung cancer (SCLC). Cell cycle and apoptosis are key pathways of multicellular homeostasis and deregulation of these pathways is associated with cancerogenesis. MATERIALS AND METHODS: Sixty representative FFPE-specimens (16 TC, 13 AC, 16 LCNEC and 15 SCLC) were used for mRNA expression analysis using the NanoString technique. Eight genes related to apoptosis and ten genes regulating key points of cell cycle were investigated. RESULTS: ASCL1, BCL2, CASP8, CCNE1, CDK1, CDK2, CDKN1A and CDKN2A showed lower expression in carcinoids compared to carcinomas. In contrast, CCNE1 and CDK6 showed elevated expression in carcinoids compared to carcinomas. The calculated BCL2/BAX ratio showed increasing values from TC to SCLC. Between SCLC and LCNEC CDK2, CDKN1B, CDKN2A and PNN expression was significantly different with higher expression in SCLC. CONCLUSION: Carcinoids have increased CDK4/6 and CCND1 expression controlling RB1 phosphorylation via this signaling cascade. CDK2 and CCNE1 were increased in carcinomas showing that these use the opposite way to control RB1. BAX and BCL2 are antagonists in regulating apoptosis. BCL2 expression increased over BAX expression with increasing malignancy of the tumor from TC to SCLC.

SOX4, SOX11 and PAX6 mRNA expression was identified as a (prognostic) marker for the aggressiveness of neuroendocrine tumors of the lung by using next-generation expression analysis (NanoString).

BACKGROUND: Neuroendocrine tumors of the lung (NELC) account for 25% of all lung cancer cases and transcription factors may drive dedifferentiation of these tumors. This study was conducted to identify supportive diagnostic and prognostic biomarkers. MATERIALS & METHODS: A total of 16 TC, 13 AC, 16 large cell neuroendocrine carcinomas and 15 small cell lung cancer were investigated for the mRNA expression of 11 transcription factors and related genes (MYB, MYBBP1A, OCT4, PAX6, PCDHB, RBP1, SDCBP, SOX2, SOX4, SOX11, TEAD2). RESULTS: SOX4 (p = 0.0002), SOX11 (p < 0.0001) and PAX6 (p = 0.0002) were significant for tumor type. Elevated PAX6 and SOX11 expression correlated with poor outcome in large cell neuroendocrine carcinomas and small cell lung cancer (p < 0.0001 and p = 0.0232, respectively) based on survival data of 34 patients (57%). CONCLUSION: Aggressiveness of NELC correlated with increasing expression of transcription factors. SOX11 seems to be a highly valuable diagnostic and prognostic marker for aggressive NELC.

Gene Expression Analysis of the 26S Proteasome Subunit PSMB4 Reveals Significant Upregulation, Different Expression and Association with Proliferation in Human Pulmonary Neuroendocrine Tumours.

BACKGROUND: Proteasomal subunit PSMB4 was suggested to be a survival gene in an animal model of hepatocellular carcinoma and in glioblastoma cell lines. In pulmonary adenocarcinoma, a high expression of these genes was found to be associated with poor differentiation and survival. This study investigates the gene expression levels of 26S proteasome subunits in human pulmonary neuroendocrine tumours including typical (TC) and atypical (AC) carcinoid tumours as well as small cell (SCLC) and large cell (LCNEC) neuroendocrine carcinomas. MATERIAL AND METHODS: Gene expression levels of proteasomal subunits (PSMA1, PSMA5, PSMB4, PSMB5 and PSMD1) were investigated in 80 neuroendocrine pulmonary tumours (each 20 TC, AC, LCNLC and SCLC) and compared to controls. mRNA levels were determined by using TaqMan assays. Immunohistochemistry on tissue microarrays (TMA) was performed to determine the expression of ki67, cleaved caspase 3 and PSMB4. RESULTS: All proteasomal subunit gene expressions were significantly upregulated in TC, AC, SCLC and LCNEC compared to controls. PSMB4 mRNA is differently expressed between all neuroendocrine tumour subtypes demonstrating the highest expression and greatest range in LCNEC (p=0.043), and is significantly associated with proliferative activity (p=0.039). CONCLUSION: In line with other 26S proteasomal subunits PSMB4 is significantly increased, but differently expressed between pulmonary neuroendocrine tumours and is associated with the proliferative activity. Unlike in pulmonary adenocarcinomas, no association with biological behaviour was observed, suggesting that increased proteasomal subunit gene expression is a common and probably early event in the tumorigenesis of pulmonary neuroendocrine tumours regardless of their differentiation.

Different micro-RNA expression profiles distinguish subtypes of neuroendocrine tumors of the lung: results of a profiling study.

MicroRNAs (miRNAs) are a class of small (∼22 nucleotides), non-coding, highly conserved single-stranded RNAs with posttranscriptional regulatory features, including the regulation of cell proliferation, differentiation, survival, and apoptosis. They are deregulated in a broad variety of tumors showing characteristic expression patterns and can, thus, be used as a diagnostic tool. In contrast to non-small cell carcinoma of the lung neuroendocrine lung tumors, encompassing typical and atypical carcinoids, small cell lung cancer and large cell neuroendocrine lung cancer, no data about deregulation of tumor entity-specific miRNAs are available to date. miRNA expression differences might give useful information about the biological characteristics of these tumors, as well as serve as helpful markers.In 12 pulmonary neuroendocrine tumors classified as either typical carcinoid, atypical, large cell neuroendocrine or small cell lung cancer, screening for 763 miRNAs known to be involved in pulmonary cancerogenesis was conducted by performing 384-well TaqMan low-density array real-time qPCR. In the entire cohort, 44 miRNAs were identified, which showed a significantly different miRNA expression. For 12 miRNAs, the difference was highly significant (P<0.01). Eight miRNAs showed a negative (miR-22, miR-29a, miR-29b, miR-29c, miR-367*; miR-504, miR-513C, miR-1200) and four miRNAs a positive (miR-18a, miR-15b*, miR-335*, miR-1201) correlation to the grade of tumor biology. The miRNAs let-7d; miR-19; miR-576-5p; miR-340*; miR-1286 are significantly associated with survival. Members of the miR-29 family seem to be extremely important in this group of tumors. We found a number of miRNAs, which showed a highly significant deregulation in pulmonary neuroendocrine tumors. Moreover, some of these deregulated miRNAs seem to allow discrimination of the various subtypes of pulmonary neuroendocrine tumors. Thus, the analysis of specific sets of miRNAs can be proposed as diagnostic and/or predictive markers in this group of neoplasias.

FFPE tissue as a feasible source for gene expression analysis--a comparison of three reference genes and one tumor marker.

BACKGROUND: Formalin-fixation, paraffin-embedding is the standard processing technique for tumor tissue in modern pathology. New techniques such as cryo-conservation allow rapid fixation and long-time storage but come along with increased costs and enlarged storage complexity. However, formalin-fixed, paraffin-embedded (FFPE) tissue is available in a large quantity, making it the ideal material for retrospective studies. The following study was designed to investigate the influence of formalin-fixation on the quality of mRNA and applicability of FFPE-derived mRNA for gene expression analysis. Three potential reference genes for pulmonary tumors with neuroendocrine differentiation were included and tested for their robust expression. MATERIALS AND METHODS: Eighty specimens collected from 2005 to 2012 at the Institute of Pathology and Neuropathology at the University Hospital Essen were analyzed for their gene expression by using TaqMan(®) gene expression assays on demand (AoD). Three distinct potential reference genes (ACTB, GAPDH, HPRT1) were evaluated for their expression, and a proteasome subunit (PSMA1) was included in the analysis as tumor marker and functioned as an internal technical control. CONCLUSION: For GAPDH and ACTB, a highly significant correlation and consistent expression between the investigated entities was found, making them reliable reference genes for further research. Additionally, the feasibility for a FFPE tissue-based gene expression analysis was verified by showing that the mRNA quality is sufficient. When standardized FFPE preparation is performed carefully, sufficient mRNA can be isolated for reliable and successful gene expression analysis. That provides the basis the door for large, retrospective studies that correlate molecular and clinical follow-up data.