Multiclass cancer classification in fresh frozen and formalin-fixed paraffin-embedded tissue by DigiWest multiplex protein analysis.

Histomorphology and immunohistochemistry are the most common ways of cancer classification in routine cancer diagnostics, but often reach their limits in determining the organ origin in metastasis. These cancers of unknown primary, which are mostly adenocarcinomas or squamous cell carcinomas, therefore require more sophisticated methodologies of classification. Here, we report a multiplex protein profiling-based approach for the classification of fresh frozen and formalin-fixed paraffin-embedded (FFPE) cancer tissue samples using the digital western blot technique DigiWest. A DigiWest-compatible FFPE extraction protocol was developed, and a total of 634 antibodies were tested in an initial set of 16 FFPE samples covering tumors from different origins. Of the 303 detected antibodies, 102 yielded significant correlation of signals in 25 pairs of fresh frozen and FFPE primary tumor samples, including head and neck squamous cell carcinomas (HNSC), lung squamous cell carcinomas (LUSC), lung adenocarcinomas (LUAD), colorectal adenocarcinomas (COAD), and pancreatic adenocarcinomas (PAAD). For this signature of 102 analytes (covering 88 total proteins and 14 phosphoproteins), a support vector machine (SVM) algorithm was developed. This allowed for the classification of the tissue of origin for all five tumor types studied here with high overall accuracies in both fresh frozen (90.4%) and FFPE (77.6%) samples. In addition, the SVM classifier reached an overall accuracy of 88% in an independent validation cohort of 25 FFPE tumor samples. Our results indicate that DigiWest-based protein profiling represents a valuable method for cancer classification, yielding conclusive and decisive data not only from fresh frozen specimens but also FFPE samples, thus making this approach attractive for routine clinical applications.

Distinct immune evasion in APOBEC-enriched, HPV-negative HNSCC.

Immune checkpoint inhibition leads to response in some patients with head and neck squamous cell carcinoma (HNSCC). Robust biomarkers are lacking to date. We analyzed viral status, gene expression signatures, mutational load and mutational signatures in whole exome and RNA-sequencing data of the HNSCC TCGA dataset (n = 496) and a validation set (DKTK MASTER cohort, n = 10). Public single-cell gene expression data from 17 HPV-negative HNSCC were separately reanalyzed. APOBEC3-associated TCW motif mutations but not total single nucleotide variant burden were significantly associated with inflammation. This association was restricted to HPV-negative HNSCC samples. An APOBEC-enriched, HPV-negative subgroup was identified, that showed higher T-cell inflammation and immune checkpoint expression, as well as expression of APOBEC3 genes. Mutations in immune-evasion pathways were also enriched in these tumors. Analysis of single-cell sequencing data identified expression of APOBEC3B and 3C genes in malignant cells. We identified an APOBEC-enriched subgroup of HPV-negative HNSCC with a distinct immunogenic phenotype, potentially mediating response to immunotherapy.

Proteogenomic systems analysis identifies targeted therapy resistance mechanisms in EGFR-mutated lung cancer.

Cancer precision medicine largely relies on knowledge about genetic aberrations in tumors and next-generation-sequencing studies have shown a high mutational complexity in many cancers. Although a large number of the observed mutations is believed to be not causally linked with cancer, the functional effects of many rare mutations but also of combinations of driver mutations are often unknown. Here, we perform a systems analysis of a model of EGFR-mutated nonsmall cell lung cancer resistant to targeted therapy that integrates whole exome sequencing, global time-course discovery phosphoproteomics and computational modeling to identify functionally relevant molecular alterations. Our approach allows for a complexity reduction from over 2,000 genetic events potentially involved in mediating resistance to only 44 phosphoproteins and 35 topologically close genetic alterations. We perform single- and combination-drug testing against the predicted phosphoproteins and discovered that targeting of HSPB1, DBNL and AKT1 showed potent antiproliferative effects overcoming resistance against EGFR-inhibitory therapy. Our approach may therefore be used to complement mutational profiling to identify functionally relevant molecular aberrations and propose combination therapies across cancers.

Sphingosine Kinase 1 Regulates Inflammation and Contributes to Acute Lung Injury in Pneumococcal Pneumonia via the Sphingosine-1-Phosphate Receptor 2.

OBJECTIVES: Severe pneumonia may evoke acute lung injury, and sphingosine-1-phosphate is involved in the regulation of vascular permeability and immune responses. However, the role of sphingosine-1-phosphate and the sphingosine-1-phosphate producing sphingosine kinase 1 in pneumonia remains elusive. We examined the role of the sphingosine-1-phosphate system in regulating pulmonary vascular barrier function in bacterial pneumonia. DESIGN: Controlled, in vitro, ex vivo, and in vivo laboratory study. SUBJECTS: Female wild-type and SphK1-deficient mice, 8-10 weeks old. Human postmortem lung tissue, human blood-derived macrophages, and pulmonary microvascular endothelial cells. INTERVENTIONS: Wild-type and SphK1-deficient mice were infected with Streptococcus pneumoniae. Pulmonary sphingosine-1-phosphate levels, messenger RNA expression, and permeability as well as lung morphology were analyzed. Human blood-derived macrophages and human pulmonary microvascular endothelial cells were infected with S. pneumoniae. Transcellular electrical resistance of human pulmonary microvascular endothelial cell monolayers was examined. Further, permeability of murine isolated perfused lungs was determined following exposition to sphingosine-1-phosphate and pneumolysin. MEASUREMENTS AND MAIN RESULTS: Following S. pneumoniae infection, murine pulmonary sphingosine-1-phosphate levels and sphingosine kinase 1 and sphingosine-1-phosphate receptor 2 expression were increased. Pneumonia-induced lung hyperpermeability was reduced in SphK1 mice compared with wild-type mice. Expression of sphingosine kinase 1 in macrophages recruited to inflamed lung areas in pneumonia was observed in murine and human lungs. S. pneumoniae induced the sphingosine kinase 1/sphingosine-1-phosphate system in blood-derived macrophages and enhanced sphingosine-1-phosphate receptor 2 expression in human pulmonary microvascular endothelial cell in vitro. In isolated mouse lungs, pneumolysin-induced hyperpermeability was dose dependently and synergistically increased by sphingosine-1-phosphate. This sphingosine-1-phosphate-induced increase was reduced by inhibition of sphingosine-1-phosphate receptor 2 or its downstream effector Rho-kinase. CONCLUSIONS: Our data suggest that targeting the sphingosine kinase 1-/sphingosine-1-phosphate-/sphingosine-1-phosphate receptor 2-signaling pathway in the lung may provide a novel therapeutic perspective in pneumococcal pneumonia for prevention of acute lung injury.

Semiconductor sequencing: how many flows do you need?

MOTIVATION: Semiconductor sequencing directly translates chemically encoded information (A, C, G or T) into voltage signals that are detected by a semiconductor device. Changes of pH value and thereby of the electric potential in the reaction well are detected during strand synthesis from nucleotides provided in cyclic repeated flows for each type of nucleotide. To minimize time requirement and costs, it is necessary to know the number of flows that are required for complete coverage of the templates. RESULTS: We calculate the number of required flows in a random sequence model and present exact expressions for cumulative distribution function, expected value and variance. Additionally, we provide an algorithm to calculate the number of required flows for a concrete list of amplicons using a BED file of genomic positions as input. We apply the algorithm to calculate the number of flows that are required to cover six amplicon panels that are used for targeted sequencing in cancer research. The upper bounds obtained for the number of flows allow to enhance the instrument throughput from two chips to three chips per day for four of these panels. AVAILABILITY AND IMPLEMENTATION: The algorithm for calculation of the flows was implemented in R and is available as package ionflows from the CRAN repository. CONTACT: jan.budczies@charite.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Comparison of targeted next-generation sequencing and Sanger sequencing for the detection of PIK3CA mutations in breast cancer.

BACKGROUND: Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha, PIK3CA, is one of the most frequently mutated genes in breast cancer, and the mutation status of PIK3CA has clinical relevance related to response to therapy. The aim of our study was to investigate the mutation status of PIK3CA gene and to evaluate the concordance between NGS and SGS for the most important hotspot regions in exon 9 and 20, to investigate additional hotspots outside of these exons using NGS, and to correlate the PIK3CA mutation status with the clinicopathological characteristics of the cohort. METHODS: In the current study, next-generation sequencing (NGS) and Sanger Sequencing (SGS) was used for the mutational analysis of PIK3CA in 186 breast carcinomas. RESULTS: Altogether, 64 tumors had PIK3CA mutations, 55 of these mutations occurred in exons 9 and 20. Out of these 55 mutations, 52 could also be detected by Sanger sequencing resulting in a concordance of 98.4 % between the two sequencing methods. The three mutations missed by SGS had low variant frequencies below 10 %. Additionally, 4.8 % of the tumors had mutations in exons 1, 4, 7, and 13 of PIK3CA that were not detected by SGS. PIK3CA mutation status was significantly associated with hormone receptor-positivity, HER2-negativity, tumor grade, and lymph node involvement. However, there was no statistically significant association between the PIK3CA mutation status and overall survival. CONCLUSIONS: Based on our study, NGS is recommended as follows: 1) for correctly assessing the mutation status of PIK3CA in breast cancer, especially for cases with low tumor content, 2) for the detection of subclonal mutations, and 3) for simultaneous mutation detection in multiple exons.

An analysis of PIK3CA hotspot mutations and response to neoadjuvant therapy in breast cancer patients from four prospective clinical trials.

PURPOSE: The PI3K signaling pathway is frequently dysregulated in breast cancer (BC), and mutations in PIK3CA, are relevant for therapy resistance in HER2pos BC. Mutations in exons 9 or exon 20 may have different impact on response to neoadjuvant chemotherapy-based treatment regimens. EXPERIMENTAL DESIGN: We investigated PIK3CA mutations in 1691 early BC patients, randomized in four neoadjuvant multicenter trials: GeparQuattro (NCT00288002), GeparQuinto (NCT00567554), GeparSixto (NCT01426880) and GeparSepto (NCT01583426). The role of different PIK3CA exons and hotspots for pathological complete response (pCR) after neoadjuvant chemotherapy (NACT) and patient survival was evaluated for distinct molecular subgroups and anti-HER2 treatment procedures. RESULTS: A total of 302 patients (17.9%) of the full cohort of 1691 patients had a tumor with a PIK3CA mutation, with a different prevalence in molecular subgroups: luminal/HER2neg 95 of 404 patients (23.5%), HER2pos 170 of 819 patients (20.8%) and TNBC 37 of 431 patients (7.9%). We identified mutations in PIK3CA exon 20 to be linked with worse response to anti-HER2 treatment (OR=0.507, 95%CI 0.320-0.802, p=0.004), especially in HR positive HER2 positive BC (OR=0.445, 95%CI 0.237-0.837, p=0.012). In contrast, exon 9 hotspot mutations p.E452K and p.E545K revealed no noteworthy differences in response therapy response. Luminal/HER2neg patients show a trend to have worse treatment response when PIK3CA was mutated. Interestingly, patients with residual disease after neoadjuvant treatment, have better survival when PIK3CA was mutated. CONCLUSIONS: PIK3CA hotspot mutation p.H1047R are associated with worse pCR rates after NACT in HER2pos BC, while hotspot mutations in exon 9 seems to have less impact.

Proteomic profiling reveals ACSS2 facilitating metabolic support in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by genomic aberrations in oncogenes, cytogenetic abnormalities, and an aberrant epigenetic landscape. Nearly 50% of AML cases will relapse with current treatment. A major source of therapy resistance is the interaction of mesenchymal stroma with leukemic cells resulting in therapeutic protection. We aimed to determine pro-survival/anti-apoptotic protein networks involved in the stroma protection of leukemic cells. Proteomic profiling of cultured primary AML (n = 14) with Hs5 stroma cell line uncovered an up-regulation of energy-favorable metabolic proteins. Next, we modulated stroma-induced drug resistance with an epigenetic drug library, resulting in reduced apoptosis with histone deacetylase inhibitor (HDACi) treatment versus other epigenetic modifying compounds. Quantitative phosphoproteomic probing of this effect further revealed a metabolic-enriched phosphoproteome including significant up-regulation of acetyl-coenzyme A synthetase (ACSS2, S30) in leukemia-stroma HDACi treated cocultures compared with untreated monocultures. Validating these findings, we show ACSS2 substrate, acetate, promotes leukemic proliferation, ACSS2 knockout in leukemia cells inhibits leukemic proliferation and ACSS2 knockout in the stroma impairs leukemic metabolic fitness. Finally, we identify ACSS1/ACSS2-high expression AML subtype correlating with poor overall survival. Collectively, this study uncovers the leukemia-stroma phosphoproteome emphasizing a role for ACSS2 in mediating AML growth and drug resistance.

YB-1 dependent virotherapy in combination with temozolomide as a multimodal therapy approach to eradicate malignant glioma.

The human Y-box binding protein 1 (YB-1) is known to be a promising target for cancer therapy. We have demonstrated that YB-1 plays an important role in the adenoviral life cycle by regulating the adenoviral E2-gene expression. Thus, we studied the oncolytic effect of the recombinant adenovirus Ad-Delo3-RGD, in which the transactivation domain CR3 of the E1A protein is ablated to enable viral replication only in YB-1 positive cancer cells. In vitro Southern Blot analysis and cytopathic effect assays demonstrate high anti-glioma potency, which was significantly increased in combination with temozolomide (TMZ), daunorubicin and cisplatin. Since vascular endothelial growth factor (VEGF) is thought to promote the hypervascular phenotype of primary, malignant brain tumors, we also tested Ad-Delo3-RGD in regard to the inhibition of VEGF expression. Indeed, we found that Ad-Delo3-RGD induced VEGF down regulation, which was even amplified under hypoxic conditions. Tumor-bearing nudemice treated with the YB-1 dependent oncolytic adenovirus showed significantly smaller tumors than untreated controls. Furthermore, combination therapy with TMZ led to a regression in all treated animals with complete tumor regression in 33 % of analyzed mice, which was verified by bioluminescence imaging and histological studies. In addition, histopathological evaluation revealed enhanced apoptosis and a reduction in tumor vessel formation, indicating that Ad-Delo3-RGD has an anti-angiogenic effect in addition to its oncolytic capacity in vivo. Hence, our results demonstrate that the combination therapy of YB-1 dependent virotherapy and TMZ is effective in a xenograft glioma mouse model and might be useful in a YB-1 based clinical setting.

TFE3 activation in a TSC1-altered malignant PEComa: challenging the dichotomy of the underlying pathogenic mechanisms.

Perivascular epithelioid cell tumors (PEComas) form a family of rare mesenchymal neoplasms that typically display myomelanocytic differentiation. Upregulation of mTOR signaling due the inactivation of TSC1/2 (Tuberous Sclerosis 1 and 2) is believed to be a key oncogenic driver in this disease. Recently, a subgroup of PEComas harboring TFE3 (Transcription Factor E3) rearrangements and presenting with a distinctive morphology has been identified. TSC1/2 and TFE3 aberrations are deemed to be mutually exclusive in PEComa, with two different pathogenic mechanisms assumed to lead to tumorigenesis. Here, we challenge this dichotomy by presenting a case of a clinically aggressive TCS1-mutated PEComa displaying a TFE3-altered phenotype. FISH analysis was suggestive of a TFE3 inversion; however, RNA and whole genome sequencing was ultimately unable to identify a fusion involving the gene. However, a copy number increase of the chromosomal region encompassing TFE3 was detected and transcriptome analysis confirmed upregulation of TFE3, which was also seen at the protein level. Therefore, we believe that the TSC1/2-mTOR pathway and TFE3 overexpression can simultaneously contribute to tumorigenesis in PEComa. Our comprehensive genetic analyses add to the understanding of the complex pathogenic mechanisms underlying PEComa and harbor insights for clinical treatment options.

Immune-related Gene Expression Predicts Response to Neoadjuvant Chemotherapy but not Additional Benefit from PD-L1 Inhibition in Women with Early Triple-negative Breast Cancer.

PURPOSE: We evaluated mRNA signatures to predict response to neoadjuvant PD-L1 inhibition in combination with chemotherapy in early triple-negative breast cancer. EXPERIMENTAL DESIGN: Targeted mRNA sequencing of 2,559 transcripts was performed in formalin-fixed, paraffin-embedded samples from 162 patients of the GeparNuevo trial. We focused on validation of four predefined gene signatures and differential gene expression analyses for new predictive markers. RESULTS: Two signatures [GeparSixto signature (G6-Sig) and IFN signature (IFN-Sig)] were predictive for treatment response in a multivariate model including treatment arm [G6-Sig: OR, 1.558; 95% confidence interval (CI), 1.130-2.182; P = 0.008 and IFN-Sig: OR, 1.695; 95% CI, 1.234-2.376; P = 0.002), while the CYT metric predicted pathologic complete response (pCR) in the durvalumab arm, and the proliferation-associated gene signature in the placebo arm. Expression of PD-L1 mRNA was associated with better response in both arms, indicating that increased levels of PD-L1 are a general predictor of neoadjuvant therapy response. In an exploratory analysis, we identified seven genes that were higher expressed in responders in the durvalumab arm, but not the placebo arm: HLA-A, HLA-B, TAP1, GBP1, CXCL10, STAT1, and CD38. These genes were associated with cellular antigen processing and presentation and IFN signaling. CONCLUSIONS: Immune-associated signatures are associated with pCR after chemotherapy, but might be of limited use for the prediction of response to additional immune checkpoint blockade. Gene expressions related to antigen presentation and IFN signaling might be interesting candidates for further evaluation.

TP53 Mutations Predict Sensitivity to Adjuvant Gemcitabine in Patients with Pancreatic Ductal Adenocarcinoma: Next-Generation Sequencing Results from the CONKO-001 Trial.

PURPOSE: We performed next-generation sequencing (NGS) in the CONKO-001 phase III trial to identify clinically relevant prognostic and predictive mutations and conducted a functional validation in The Cancer Genome Atlas (TCGA) sequencing data. EXPERIMENTAL DESIGN: Patients of the CONKO-001 trial received curatively intended surgery for pancreatic adenocarcinoma (PDAC) followed by adjuvant chemotherapy with gemcitabine (Gem) or observation only (Obs). Tissue samples of 101 patients were evaluated by NGS of 37 genes. Cox proportional hazard models were applied for survival analysis. In addition, functional genomic analyses were performed in an NGS and RNA-sequencing dataset of 146 pancreatic tumors from TCGA. RESULTS: The most common mutations in the CONKO cohort were KRAS (75%), TP53 (60%), SMAD4 (10%), CDKNA2 (9%), as well as SWI/SNF (12%) complex alterations. In untreated patients, TP53 mutations were a negative prognostic factor for disease-free survival (DFS; HR mut vs. WT 2.434, P = 0.005). With respect to gemcitabine treatment, TP53 mutations were a positive predictive factor for gemcitabine efficacy [TP53mut: HR for DFS Gem vs. Obs, 0.235 (0.130 - 0.423; P < 0.001); TP53wt: HR for DFS Gem vs. Obs, 0.794 (0.417 - 1.513; P = 0.483)] with a significant test for interaction (P = 0.003). In the TCGA dataset, TP53 mutations were associated with shortened DFS. CONCLUSIONS: In CONKO-001, the benefit from adjuvant gemcitabine was confined to the TP53mut patient group. This potentially clinical relevant observation needs to be confirmed in independent prospective studies. The sensitivity of TP53mut PDAC to gemcitabine in CONKO-001 provides a lead for further mechanistic investigations.

Next generation sequencing of lung adenocarcinoma subtypes with intestinal differentiation reveals distinct molecular signatures associated with histomorphology and therapeutic options.

OBJECTIVES: We aim to provide a better understanding of the molecular landscape of primary lung adenocarcinomas with intestinal differentiation. MATERIAL AND METHODS: Five invasive mucinous adenocarcinomas (IMA) and seven pulmonary enteric adenocarcinomas (PEAD) were included in this study. Furthermore, we analyzed six pulmonary colloid adenocarcinomas (CAD), including one primary tumor, one metastasis, and two sample pairs consisting of the primary colloid lung tumor and a matching metastasis and an acinar component, respectively. All samples were characterized using immunohistochemistry (TTF-1, CK7, CK20, CDX2, Ki-67, ALK and PD-L1) and a next generation sequencing panel covering 404 cancer-related genes (FoundationOne® gene panel). RESULTS AND CONCLUSION: While Ki-67 expression was comparably low in IMA (range: 8-15%) and in primary CAD (range: 5-8%), we observed considerably higher proliferation rates in the non-colloid tumor compartment (16%) and metastases (72%) from CAD, as well as in the PEAD-group (36-71%). The overall tumor mutational burden was lowest in IMA (2.5 mutations per megabase), intermediate in CAD (5.8 mutations per megabase) and highest in PEAD (16.8 mutations per megabase). KRAS mutations were frequent in all three tumor subtypes, but TP53 mutations were mostly limited to PEAD. While chromosomal alterations were rare in IMA, we discovered MYC amplifications in three of four CAD. Comparing primary and metastatic CAD, we observed the acquisition of multiple mutations and chromosomal alterations. PEAD had a variety of chromosomal alterations, including two cases with RICTOR amplification. PD-L1 expression (20%, 50% and 80% of tumor cells) was limited to three PEAD samples, only. In conclusion, we provide a detailed insight into the molecular alterations across and within the different subtypes of pulmonary adenocarcinomas with intestinal differentiation. From a clinical perspective, we provide data on potential treatment strategies for patients with PEAD, including immunotherapy.

Machine learning analysis of DNA methylation profiles distinguishes primary lung squamous cell carcinomas from head and neck metastases.

Head and neck squamous cell carcinoma (HNSC) patients are at risk of suffering from both pulmonary metastases or a second squamous cell carcinoma of the lung (LUSC). Differentiating pulmonary metastases from primary lung cancers is of high clinical importance, but not possible in most cases with current diagnostics. To address this, we performed DNA methylation profiling of primary tumors and trained three different machine learning methods to distinguish metastatic HNSC from primary LUSC. We developed an artificial neural network that correctly classified 96.4% of the cases in a validation cohort of 279 patients with HNSC and LUSC as well as normal lung controls, outperforming support vector machines (95.7%) and random forests (87.8%). Prediction accuracies of more than 99% were achieved for 92.1% (neural network), 90% (support vector machine), and 43% (random forest) of these cases by applying thresholds to the resulting probability scores and excluding samples with low confidence. As independent clinical validation of the approach, we analyzed a series of 51 patients with a history of HNSC and a second lung tumor, demonstrating the correct classifications based on clinicopathological properties. In summary, our approach may facilitate the reliable diagnostic differentiation of pulmonary metastases of HNSC from primary LUSC to guide therapeutic decisions.

Mutational Diversity and Therapy Response in Breast Cancer: A Sequencing Analysis in the Neoadjuvant GeparSepto Trial.

PURPOSE: Next-generation sequencing (NGS) can be used for comprehensive investigation of molecular events in breast cancer. We evaluated the relevance of genomic alterations for response to neoadjuvant chemotherapy (NACT) in the GeparSepto trial. EXPERIMENTAL DESIGN: Eight hundred fifty-one pretherapeutic formalin-fixed paraffin-embedded (FFPE) core biopsies from GeparSepto study were sequenced. The panel included 16 genes for mutational (AKT1, BRAF, CDH1, EGFR, ERBB2, ESR1, FBXW7, FGFR2, HRAS, KRAS, NRAS, SF3B1, TP53, HNF1A, PIK3CA, and PTEN) and 8 genes for copy-number alteration analysis (CCND1, ERBB2, FGFR1, PAK1, PIK3CA, TOP2A, TP53, and ZNF703). RESULTS: The most common genomic alterations were mutations of TP53 (38.4%) and PIK3CA (21.5%), and 8 different amplifications (TOP2A 34.9%; ERBB2 30.6%; ZNF703 30.1%; TP53 21.9%; PIK3CA 24.1%; CCND1 17.7%; PAK1 14.9%; FGFR 12.6%). All other alterations had a prevalence of less than 5%. The genetic heterogeneity in different breast cancer subtypes [lum/HER2neg vs. HER2pos vs. triple-negative breast cancer (TNBC)] was significantly linked to differences in NACT response. A significantly reduced pathologic complete response rate was observed in PIK3CA-mutated breast cancer [PIK3CAmut: 23.0% vs. wild-type (wt) 38.8%, P < 0.0001] in particular in the HER2pos subcohort [multivariate OR = 0.43 (95% CI, 0.24-0.79), P = 0.006]. An increased response to nab-paclitaxel was observed only in PIK3CAwt breast cancer, with univariate significance for the complete cohort (P = 0.009) and the TNBC (P = 0.013) and multivariate significance in the HER2pos subcohort (test for interaction P = 0.0074). CONCLUSIONS: High genetic heterogeneity was observed in different breast cancer subtypes. Our study shows that FFPE-based NGS can be used to identify markers of therapy resistance in clinical study cohorts. PIK3CA mutations could be a major mediator of therapy resistance in breast cancer.

Multilayered Omics-Based Analysis of a Head and Neck Cancer Model of Cisplatin Resistance Reveals Intratumoral Heterogeneity and Treatment-Induced Clonal Selection.

Purpose: Platinum-based drugs, in particular cisplatin (cis-diamminedichloridoplatinum(II), CDDP), are used for treatment of squamous cell carcinoma of the head and neck (SCCHN). Despite initial responses, CDDP treatment often results in chemoresistance, leading to therapeutic failure. The role of primary resistance at subclonal level and treatment-induced clonal selection in the development of CDDP resistance remains unknown.Experimental Design: By applying targeted next-generation sequencing, fluorescence in situ hybridization, microarray-based transcriptome, and mass spectrometry-based phosphoproteome analysis to the CDDP-sensitive SCCHN cell line FaDu, a CDDP-resistant subline, and single-cell derived subclones, the molecular basis of CDDP resistance was elucidated. The causal relationship between molecular features and resistant phenotypes was determined by siRNA-based gene silencing. The clinical relevance of molecular findings was validated in patients with SCCHN with recurrence after CDDP-based chemoradiation and the TCGA SCCHN dataset.Results: Evidence of primary resistance at clonal level and clonal selection by long-term CDDP treatment was established in the FaDu model. Resistance was associated with aneuploidy of chromosome 17, increased TP53 copy-numbers and overexpression of the gain-of-function (GOF) mutant variant p53R248L siRNA-mediated knockdown established a causal relationship between mutant p53R248L and CDDP resistance. Resistant clones were also characterized by increased activity of the PI3K-AKT-mTOR pathway. The poor prognostic value of GOF TP53 variants and mTOR pathway upregulation was confirmed in the TCGA SCCHN cohort.Conclusions: Our study demonstrates a link of intratumoral heterogeneity and clonal evolution as important mechanisms of drug resistance in SCCHN and establishes mutant GOF TP53 variants and the PI3K/mTOR pathway as molecular targets for treatment optimization. Clin Cancer Res; 24(1); 158-68. ©2017 AACR.

Ioncopy: a novel method for calling copy number alterations in amplicon sequencing data including significance assessment.

Recently, it has been demonstrated that calling of copy number alterations (CNAs) from amplicon sequencing (AS) data is feasible. Most approaches, however, require non-tumor (germline) DNA for data normalization. Here, we present the method Ioncopy for CNA detection which requires no normal controls and includes a significance assessment for each detected alteration.Ioncopy was evaluated in a cohort of 184 clinically annotated breast carcinomas. A total number of 252 amplifications were detected, of which 183 (72.6%) could be validated by a call of an additional amplicon interrogating the same gene. Moreover, a total number of 33 deletions were found, whereof 27 (81.8%) could be validated. Analyzing the 16 most frequently amplified genes, validation rates of over 89% could be achieved for 11 of these genes. 11 of the top 16 genes showed significant overexpression in the amplified tumors. 89.5% of the HER2-amplified tumors were GRB7 and STARD3 co-amplified, whereas 68.4% of the HER2-amplified tumors had additional MED1 amplifications. Correlations between CNAs measured by amplicons in HER2 exons 19, 20 and 21 were strong (all R > 0.93). AS based detection of HER2 amplifications had a sensitivity of 90.0% and a specificity of 98.8% compared to the gold standard of HER2 immunohistochemistry combined with in situ hybridization.In summary, we developed and validated a novel method for detection and significance assessment of CNAs in amplicon sequencing data. Using Ioncopy, AS offers a straightforward and efficient approach to simultaneously analyze gene amplifications and gene deletions together with simple somatic mutations in a single assay.

Modulation of dendritic cell immunobiology via inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase.

The maturation status of dendritic cells determines whether interacting T cells are activated or if they become tolerant. Previously we could induce T cell tolerance by applying a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor (HMGCRI) atorvastatin, which also modulates MHC class II expression and has therapeutic potential in autoimmune disease. Here, we aimed at elucidating the impact of this therapeutic strategy on T cell differentiation as a consequence of alterations in dendritic cell function. We investigated the effect of HMGCRI during differentiation of peripheral human monocytes and murine bone marrow precursors to immature DC in vitro and assessed their phenotype. To examine the stimulatory and tolerogenic capacity of these modulated immature dendritic cells, we measured proliferation and suppressive function of CD4+ T cells after stimulation with the modulated immature dendritic cells. We found that an HMGCRI, atorvastatin, prevents dendrite formation during the generation of immature dendritic cells. The modulated immature dendritic cells had a diminished capacity to take up and present antigen as well as to induce an immune response. Of note, the consequence was an increased capacity to differentiate naïve T cells towards a suppressor phenotype that is less sensitive to proinflammatory stimuli and can effectively inhibit the proliferation of T effector cells in vitro. Thus, manipulation of antigen-presenting cells by HMGCRI contributes to an attenuated immune response as shown by promotion of T cells with suppressive capacities.

Effect of YB-1 on the regulation of micro RNA expression in drug-sensitive and drug-resistant gastric carcinoma cells.

The multifunctional Y-Box protein 1 (YB-1) exerts positive and negative regulatory effects on gene expression by different mechanisms. Since transcription can be controlled by micro RNAs (miRNAs), YB-1 could also cause effects on gene expression by regulation of cellular miRNAs. To test this hypothesis, a previously established and well-characterized cell model derived from drug-sensitive (EPG85-257P/tetR/YB-1) and multidrug-resistant (EPG85-257RDB/tetR/YB-1) gastric carcinoma cells, in which the expression of YB-1 can be inhibited by tetracycline-dependent triggering of the RNA interference (RNAi) pathway, was investigated concerning their miRNA expression profiles in the presence and absence of YB-1. Microarray hybridizations demonstrated that six miRNAs (miR-96*, miR-210, miR-503, miR-623, miR-1275, miR-1290) were up-regulated more than 1.5-fold in drug-sensitive cells following YB-1 inhibition, but no differences in miRNA expression could be detected in multidrug-resistant cells. Independent validation of these findings by quantitative real-time reverse transcriptase polymerase chain reaction did not confirm these effects. Likewise, an in silico analysis of potential regulatory effects of the miRNAs on their target genes did not support the potential miRNA regulatory effects of YB-1. In conclusion, the data provide evidence that YB-1 has no direct influence on global miRNA expression pattern in different variants of gastric carcinoma cells and, therewith, does not control gene expression by regulation of miRNAs.

Regulation of mRNA expression in drug-sensitive and drug-resistant gastric carcinoma cells is independent of YB-1 expression.

Y-Box protein 1 (YB-1) is a multifunctional cellular protein expressed in a range of mammalian cells, including human cancer cells. It is involved in the regulation of various genes including cancer-associated genes, but the full range of target genes and regulatory mechanisms have not been fully elucidated. To identify global mRNA expression patterns that are potentially regulated by YB-1, a previously established and well-characterized cell model derived from drug-sensitive (EPG85-257P/tetR/YB-1) and multidrug-resistant (EPG85-257RDB/tetR/YB-1) gastric carcinoma cells in which the expression of YB-1 can be inhibited by tetracycline-dependent activation of the RNA interference (RNAi) pathway, was analyzed by microarray technology. By this approach, various potentially regulated genes encoding members of important cellular pathways such as the Jak/STAT, VEGF and the MAP-kinase signaling pathways were identified. Independent validation of these findings by quantitative real-time reverse transcriptase polymerase chain reaction and Western blot did not confirm these regulatory effects. In conclusion, the findings suggest that YB-1 is not directly involved in the regulation of mRNA expression in drug-sensitive or drug-resistant gastric carcinoma cells.