AHRR and SFRP2 in primary versus recurrent high-grade serous ovarian carcinoma and their prognostic implication.

BACKGROUND: The aim of this study was to analyse transcriptomic differences between primary and recurrent high-grade serous ovarian carcinoma (HGSOC) to identify prognostic biomarkers. METHODS: We analysed 19 paired primary and recurrent HGSOC samples using targeted RNA sequencing. We selected the best candidates using in silico survival and pathway analysis and validated the biomarkers using immunohistochemistry on a cohort of 44 paired samples, an additional cohort of 504 primary HGSOCs and explored their function. RESULTS: We identified 233 differential expressed genes. Twenty-three showed a significant prognostic value for PFS and OS in silico. Seven markers (AHRR, COL5A2, FABP4, HMGCS2, ITGA5, SFRP2 and WNT9B) were chosen for validation at the protein level. AHRR expression was higher in primary tumours (p < 0.0001) and correlated with better patient survival (p < 0.05). Stromal SFRP2 expression was higher in recurrent samples (p = 0.009) and protein expression in primary tumours was associated with worse patient survival (p = 0.022). In multivariate analysis, tumour AHRR and SFRP2 remained independent prognostic markers. In vitro studies supported the anti-tumorigenic role of AHRR and the oncogenic function of SFRP2. CONCLUSIONS: Our results underline the relevance of AHRR and SFRP2 proteins in aryl-hydrocarbon receptor and Wnt-signalling, respectively, and might lead to establishing them as biomarkers in HGSOC.

Clinical and molecular characteristics of HER2-low-positive breast cancer: pooled analysis of individual patient data from four prospective, neoadjuvant clinical trials.

BACKGROUND: The development of anti-HER2 antibody-drug conjugates opens new therapeutic options for patients with breast cancer, including patients with low expression of HER2. To characterise this new breast cancer subtype, we have compared the clinical and molecular characteristics of HER2-low-positive and HER2-zero breast cancer, including response to neoadjuvant chemotherapy and prognosis. METHODS: In this pooled analysis of individual patient data, we evaluated a cohort of 2310 patients with HER2-non-amplified primary breast cancer that were treated with neoadjuvant combination chemotherapy in four prospective neoadjuvant clinical trials (GeparSepto, NCT01583426; GeparOcto, NCT02125344; GeparX, NCT02682693; Gain-2 neoadjuvant, NCT01690702) between July 30, 2012, and March 20, 2019. Central HER2 testing was done prospectively before random assignment of participants in all trials. HER2-low-positive status was defined as immunohistochemistry (IHC) 1+ or IHC2+/in-situ hybridisation negative and HER2-zero was defined as IHC0, based on the American Society of Clinical Oncology/College of American Pathologists guidelines. Disease-free survival and overall survival data were available for 1694 patients (from all trials except GeparX) with a median follow-up of 46·6 months (IQR 35·0-52·3). Bivariable and multivariable logistic regression models and Cox-proportional hazards models were performed based on a predefined statistical analysis plan for analysis of the endpoints pathological complete response, disease-free survival, and overall survival. FINDINGS: A total of 1098 (47·5%) of 2310 tumours were HER2-low-positive and 1212 (52·5%) were HER2-zero. 703 (64·0%) of 1098 patients with HER2-low-positive tumours were hormone receptor positive, compared with 445 (36·7%) of 1212 patients with HER2-zero tumours (p<0.0001). HER2-low-positive tumours had a significantly lower pathological complete response rate than HER2-zero tumours (321 [29·2%] of 1098 vs 473 [39·0%] of 1212, p=0·0002). Pathological complete response was also significantly lower in HER2-low-positive tumours versus HER2-zero tumours in the hormone receptor-positive subgroup (123 [17·5%] of 703 vs 105 [23·6%] of 445, p=0·024), but not in the hormone receptor-negative subgroup (198 [50·1%] of 395 vs 368 [48·0%] of 767, p=0·21). Patients with HER2-low-positive tumours had significantly longer survival than did patients with HER2-zero tumours (3-year disease-free survival: 83·4% [95% CI 80·5-85·9] vs 76·1% [72·9-79·0]; stratified log-rank test p=0·0084; 3-year overall survival: 91·6% [84·9-93·4] vs 85·8% [83·0-88·1]; stratified log-rank test p=0·0016). Survival differences were also seen in patients with hormone receptor-negative tumours (3-year disease-free survival: 84·5% [95% CI 79·5-88·3] vs 74·4% [70·2-78.0]; stratified log-rank test p=0·0076; 3-year overall survival: 90·2% [86·0-93·2] vs 84·3% [80·7-87·3], stratified log-rank test p=0·016), but not in patients with hormone receptor-positive tumours (3-year disease-free survival 82·8% [79·1-85·9] vs 79·3% [73·9-83·7]; stratified log-rank test p=0·39; 3-year overall survival 92·3% [89·6-94·4] vs 88·4% [83·8-91·8]; stratified log-rank test p=0·13). INTERPRETATION: Our results show that HER2-low-positive tumours can be identified as new subgroup of breast cancer by standardised IHC, distinct from HER2-zero tumours. HER2-low-positive tumours have a specific biology and show differences in response to therapy and prognosis, which is particularly relevant in therapy-resistant, hormone receptor-negative tumours. Our results provide a basis for a better understanding of the biology of breast cancer subtypes and the refinement of future diagnostic and therapeutic strategies. FUNDING: German Cancer Aid (Deutsche Krebshilfe).

Targeted RNAseq assay incorporating unique molecular identifiers for improved quantification of gene expression signatures and transcribed mutation fraction in fixed tumor samples.

BACKGROUND: Our objective was to assess whether modifications to a customized targeted RNA sequencing (RNAseq) assay to include unique molecular identifiers (UMIs) that collapse read counts to their source mRNA counts would improve quantification of transcripts from formalin-fixed paraffin-embedded (FFPE) tumor tissue samples. The assay (SET4) includes signatures that measure hormone receptor and PI3-kinase related transcriptional activity (SETER/PR and PI3Kges), and measures expression of selected activating point mutations and key breast cancer genes. METHODS: Modifications included steps to introduce eight nucleotides-long UMIs during reverse transcription (RT) in bulk solution, followed by polymerase chain reaction (PCR) of labeled cDNA in droplets, with optimization of the polymerase enzyme and reaction conditions. We used Lin's concordance correlation coefficient (CCC) to measure concordance, including precision (Rho) and accuracy (Bias), and nonparametric tests (Wilcoxon, Levene's) to compare the modified (NEW) SET4 assay to the original (OLD) SET4 assay and to whole transcriptome RNAseq using RNA from matched fresh frozen (FF) and FFPE samples from 12 primary breast cancers. RESULTS: The modified (NEW) SET4 assay measured single transcripts (p< 0.001) and SETER/PR (p=0.002) more reproducibly in technical replicates from FFPE samples. The modified SET4 assay was more precise for measuring single transcripts (Rho 0.966 vs 0.888, p< 0.01) but not multigene expression signatures SETER/PR (Rho 0.985 vs 0.968) or PI3Kges (Rho 0.985 vs 0.946) in FFPE, compared to FF samples. It was also more precise than wtRNAseq of FFPE for measuring transcripts (Rho 0.986 vs 0.934, p< 0.001) and SETER/PR (Rho 0.993 vs 0.915, p=0.004), but not PI3Kges (Rho 0.988 vs 0.945, p=0.051). Accuracy (Bias) was comparable between protocols. Two samples carried a PIK3CA mutation, and measurements of transcribed mutant allele fraction was similar in FF and FFPE samples and appeared more precise with the modified SET4 assay. Amplification efficiency (reads per UMI) was consistent in FF and FFPE samples, and close to the theoretically expected value, when the library size exceeded 400,000 aligned reads. CONCLUSIONS: Modifications to the targeted RNAseq protocol for SET4 assay significantly increased the precision of UMI-based and reads-based measurements of individual transcripts, multi-gene signatures, and mutant transcript fraction, particularly with FFPE samples.

Technical Validity of a Customized Assay of Sensitivity to Endocrine Therapy Using Sections from Fixed Breast Cancer Tissue.

BACKGROUND: We translated a multigene expression index to predict sensitivity to endocrine therapy for Stage II-III breast cancer (SET2,3) to hybridization-based expression assays of formalin-fixed paraffin-embedded (FFPE) tissue sections. Here we report the technical validity with FFPE samples, including preanalytical and analytical performance. METHODS: We calibrated SET2,3 from microarrays (Affymetrix U133A) of frozen samples to hybridization-based assays of FFPE tissue, using bead-based QuantiGene Plex (QGP) and slide-based NanoString (NS). The following preanalytical and analytical conditions were tested in controlled studies: replicates within and between frozen and fixed samples, age of paraffin blocks, homogenization of fixed sections versus extracted RNA, core biopsy versus surgically resected tumor, technical replicates, precision over 20 weeks, limiting dilution, linear range, and analytical sensitivity. Lin's concordance correlation coefficient (CCC) was used to measure concordance between measurements. RESULTS: SET2,3 index was calibrated to use with QGP (CCC 0.94) and NS (CCC 0.93) technical platforms, and was validated in two cohorts of older fixed samples using QGP (CCC 0.72, 0.85) and NS (CCC 0.78, 0.78). QGP assay was concordant using direct homogenization of fixed sections versus purified RNA (CCC 0.97) and between core and surgical sample types (CCC 0.90), with 100% accuracy in technical replicates, 1-9% coefficient of variation over 20 weekly tests, linear range 3.0-11.5 (log2 counts), and analytical sensitivity ≥2.0 (log2 counts). CONCLUSIONS: Measurement of the novel SET2,3 assay was technically valid from fixed tumor sections of biopsy or resection samples using simple, inexpensive, hybridization methods, without the need for RNA purification.

The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues.

BACKGROUND: Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. METHODS: Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. RESULTS: Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63-0.66) and between technical replicates (median expression difference 0.13-0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31-0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91-0.96). CONCLUSIONS: The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

Tumour buds determine prognosis in resected pancreatic ductal adenocarcinoma.

BACKGROUND: The prognostic effect of tumour budding was retrospectively analysed in a cohort of 173 patients with resected pancreatic ductal adenocarcinomas (PDACs) of the prospective clinical multicentre CONKO-001 trial. METHODS: Haematoxylin and eosin (H&E)-stained whole tissue slides were evaluated. In two independent approaches, the mean number of tumour buds was analysed according to the consensus criteria in colorectal cancer, in one 0.785 mm2 field of view and additionally in 10 high-power fields (HPF) (HPF = 0.238 mm2). RESULTS: Tumour budding was significantly associated with a higher tumour grade (p < 0.001) but not with distant or lymph node metastasis. Regardless of the quantification approach, an increased number of tumour buds was significantly associated with reduced disease-free survival (DFS) and overall survival (OS) (10 HPF approach DFS: HR = 1.056 (95% CI 1.022-1.092), p = 0.001; OS: HR = 1.052 (95% CI 1.018-1.087), p = 0.002; consensus method DFS: HR = 1.037 (95% CI 1.017-1.058), p < 0.001; OS: HR = 1.040 (95% CI 1.019-1.061), p < 0.001). Recently published cut-offs for tumour budding in colorectal cancer were prognostic in PDAC as well. CONCLUSIONS: Tumour budding is prognostic in the CONKO-001 clinical cohort of patients. Further standardisation and validation in additional clinical cohorts are necessary.

Clinical relevance and concordance of HER2 status in local and central testing-an analysis of 1581 HER2-positive breast carcinomas over 12 years.

Human epidermal growth factor receptor 2 (HER2) is a central predictive biomarker in breast cancer. Inaccurate HER2 results in different laboratories could be as high as 20%. However, this statement is based on data generated more than 13 years ago and may not reflect the standards of modern diagnostic pathology. We compared central and local HER2 testing in a total of 1581 HER2-positive tumors from five clinical trials. We evaluated the clinical relevance for pathological complete response (pCR) and disease-free survival in a subgroup of 677 tumors, which received an anti-HER2 therapy. Over the period of 12 years, the discordance rate for HER2 decreased from 52.4 (GeparTrio) to 8.4% (GeparSepto). Discordance rates were significantly higher in hormone receptor (HR)-positive tumors (26.6%), compared to HR-negative tumors (16.3%, P<0.0001), which could be explained by a different distribution of HER2 mRNA levels in HR-positive and HR-negative tumors. pCR rates were significantly lower in discordant tumors (13.7%) compared to concordant tumors (32.2%, GeparQuattro and GeparQuinto, P<0.001). In survival analysis, tumors with discordant HER2 testing had a reduced overall survival (OS) in the HR-negative group (P=0.019) and a trend for improved OS in the HR-positive group (P=0.125). The performance of local HER2 testing was considerably improved over time and has reached a 92% concordance, which shows that quality initiatives in diagnostic pathology are working. Tumors with discordant HER2 testing had a reduced therapy response and different survival rates.

Standardized evaluation of tumor-infiltrating lymphocytes in breast cancer: results of the ring studies of the international immuno-oncology biomarker working group.

Multiple independent studies have shown that tumor-infiltrating lymphocytes (TIL) are prognostic in breast cancer with potential relevance for response to immune-checkpoint inhibitor therapy. Although many groups are currently evaluating TIL, there is no standardized system for diagnostic applications. This study reports the results of two ring studies investigating TIL conducted by the International Working Group on Immuno-oncology Biomarkers. The study aim was to determine the intraclass correlation coefficient (ICC) for evaluation of TIL by different pathologists. A total of 120 slides were evaluated by a large group of pathologists with a web-based system in ring study 1 and a more advanced software-system in ring study 2 that included an integrated feedback with standardized reference images. The predefined aim for successful ring studies 1 and 2 was an ICC above 0.7 (lower limit of 95% confidence interval (CI)). In ring study 1 the prespecified endpoint was not reached (ICC: 0.70; 95% CI: 0.62-0.78). On the basis of an analysis of sources of variation, we developed a more advanced digital image evaluation system for ring study 2, which improved the ICC to 0.89 (95% CI: 0.85-0.92). The Fleiss' kappa value for <60 vs ≥60% TIL improved from 0.45 (ring study 1) to 0.63 in RS2 and the mean concordance improved from 88 to 92%. This large international standardization project shows that reproducible evaluation of TIL is feasible in breast cancer. This opens the way for standardized reporting of tumor immunological parameters in clinical studies and diagnostic practice. The software-guided image evaluation approach used in ring study 2 may be of value as a tool for evaluation of TIL in clinical trials and diagnostic practice. The experience gained from this approach might be applicable to the standardization of other diagnostic parameters in histopathology.

Neoadjuvant carboplatin in patients with triple-negative and HER2-positive early breast cancer (GeparSixto; GBG 66): a randomised phase 2 trial.

BACKGROUND: Preclinical data suggest that triple-negative breast cancers are sensitive to interstrand crosslinking agents, and that synergy may exist for the combination of a taxane, trastuzumab, and a platinum salt for HER2-positive breast cancer. We therefore aimed to assess the efficacy of the addition of carboplatin to neoadjuvant therapy for triple-negative and HER2-positive breast cancer. METHODS: Patients with previously untreated, non-metastatic, stage II-III, triple-negative breast cancer and HER2-positive breast cancer were enrolled. Patients were treated for 18 weeks with paclitaxel (80 mg/m(2) once a week) and non-pegylated liposomal doxorubicin (20 mg/m(2) once a week). Patients with triple-negative breast cancer received simultaneous bevacizumab (15 mg/kg intravenously every 3 weeks). Patients with HER2-positive disease received simultaneous trastuzumab (8 mg/kg initial dose with subsequent doses of 6 mg/kg intravenously every 3 weeks) and lapatinib (750 mg daily). Patients were randomly assigned in a 1:1 ratio with dynamic allocation and minimisation, stratified by biological subtype and Ki-67 level to receive, at the same time as the backbone regimens, either carboplatin (AUC 1·5 [2·0 for the first 329 patients] once a week) or no carboplatin. The primary endpoint the proportion of patients who achieved a pathological complete response (defined as ypT0 ypN0), analysed for all patients who started treatment; a p value of less than 0·2 was deemed significant for the primary endpoint. This trial is registered with ClinicalTrials.gov, number NCT01426880. FINDINGS: 296 patients were randomly assigned to receive carboplatin and 299 to no additional carboplatin, of whom 295 and 293 started treatment, respectively. In this final analysis, 129 patients (43·7%, 95% CI 38·1-49·4) in the carboplatin group achieved a pathological complete response, compared with 108 patients (36·9%, 31·3-42·4) without carboplatin (odds ratio 1·33, 95% CI 0·96-1·85; p=0·107). Of the patients with triple-negative breast cancer, 84 (53·2%, 54·4-60·9) of 158 patients achieved a pathological complete response with carboplatin, compared with 58 (36·9%, 29·4-44·5) of 157 without (p=0·005). Of the patients with HER2-positive tumours, 45 (32·8%, 25·0-40·7) of 137 patients achieved a pathological complete response with carboplatin compared with 50 (36·8%, 28·7-44·9) of 136 without (p=0·581; test for interaction p=0·015). Haematological and non-haematological toxic effects that were significantly more common in the carboplatin group than in the no-carboplatin group included grade 3 or 4 neutropenia (192 [65%] vs 79 [27%]), grade 3 or 4 anaemia (45 [15%] vs one [<1%]), grade 3 or 4 thrombocytopenia (42 [14%] vs one [<1%]), and grade 3 or 4 diarrhoea (51 [17%] vs 32 [11%]); carboplatin was more often associated with dose discontinuations (141 [48%] with carboplatin and 114 [39%] without carboplatin; p=0·031). The frequency of grade 3 or 4 haematological events decreased from 82% (n=135) to 70% (n=92) and grade 3 or 4 non-haematological events from 78% (n=128) to 59% (n=77) in the carboplatin arm when the dose of carboplatin was reduced from AUC 2·0 to 1·5. INTERPRETATION: The addition of neoadjuvant carboplatin to a regimen of a taxane, an anthracycline, and targeted therapy significantly increases the proportion of patients achieving a pathological complete response. This regimen seems to increase responses in patients with triple-negative breast cancer, but not in those with HER2-positive breast cancer. FUNDING: GlaxoSmithKline, Roche, and Teva.

Interferon-stimulated gene, 15 kDa (ISG15) in ovarian high-grade serous carcinoma: prognostic impact and link to NF-κB pathway.

The ubiquitin-like protein interferon-stimulated gene, 15 kDa (ISG15) plays an ambiguous role in the progression and response to chemotherapy of solid cancers. We aimed to investigate the prognostic impact of ISG15 and its link to the nuclear factor κB pathway in ovarian high-grade serous carcinoma. Immunohistochemistry was performed in a cohort of 128 primary ovarian high-grade serous carcinomas treated with standard surgery and adjuvant chemotherapy using tissue microarrays. In addition, 28 matched relapsed carcinomas were investigated. ISG15 protein expression was significantly increased in relapsed carcinomas as compared to primary tumors (P=0.027). In primary carcinoma, ISG15 was positively associated with total inhibitor of κB α (IκBα) (P=0.001) as well as nuclear and cytoplasmic phospho-IκBα (p-IκBα) expression (P=0.039 and P=0.002, respectively). Patients with ISG15-positive carcinomas had a significantly longer overall survival in univariate analysis (P=0.002), and in multivariate analysis [hazard ratio=0.35 (95% confidence interval, 0.14-0.84, P=0.019)]. ISG15 is a potential prognostic marker in high-grade serous carcinoma of the ovary. Its impact on survival might be explained by its tight link to the nuclear factor κB pathway, and the further evaluation of the interplay between ISGylation machinery and nuclear factor κB, particularly with regard to response to chemotherapy, would be desirable.

HER2 and ESR1 mRNA expression levels and response to neoadjuvant trastuzumab plus chemotherapy in patients with primary breast cancer.

INTRODUCTION: Recent data suggest that benefit from trastuzumab and chemotherapy might be related to expression of HER2 and estrogen receptor (ESR1). Therefore, we investigated HER2 and ESR1 mRNA levels in core biopsies of HER2-positive breast carcinomas from patients treated within the neoadjuvant GeparQuattro trial. METHODS: HER2 levels were centrally analyzed by immunohistochemistry (IHC), silver in situ hybridization (SISH) and qRT-PCR in 217 pretherapeutic formalin-fixed, paraffin-embedded (FFPE) core biopsies. All tumors had been HER2-positive by local pathology and had been treated with neoadjuvant trastuzumab/ chemotherapy in GeparQuattro. RESULTS: Only 73% of the tumors (158 of 217) were centrally HER2-positive (cHER2-positive) by IHC/SISH, with cHER2-positive tumors showing a significantly higher pCR rate (46.8% vs. 20.3%, P <0.0005). HER2 status by qRT-PCR showed a concordance of 88.5% with the central IHC/SISH status, with a low pCR rate in those tumors that were HER2-negative by mRNA analysis (21.1% vs. 49.6%, P <0.0005). The level of HER2 mRNA expression was linked to response rate in ESR1-positive tumors, but not in ESR1-negative tumors. HER2 mRNA expression was significantly associated with pCR in the HER2-positive/ESR1-positive tumors (P = 0.004), but not in HER2-positive/ESR1-negative tumors. CONCLUSIONS: Only patients with cHER2-positive tumors - irrespective of the method used - have an increased pCR rate with trastuzumab plus chemotherapy. In patients with cHER2-negative tumors the pCR rate is comparable to the pCR rate in the non-trastuzumab treated HER-negative population. Response to trastuzumab is correlated to HER2 mRNA levels only in ESR1-positive tumors. This study adds further evidence to the different biology of both subsets within the HER2-positive group.

Does long-term survival in patients with pancreatic cancer really exist? Results from the CONKO-001 study.

BACKGROUND: Long-term survival (LTS) in patients (pts) with pancreatic cancer is still uncommon, little data is available to identify long-term survivors. The CONKO-001 study, which established gemcitabine after resection as adjuvant therapy, may provide data to answer this question. METHODS: CONKO-001 pts with an overall survival ≥5 years were compared to those who survived <5 years. Central re-evaluation of primary histology was performed. Univariate analysis with the χ(2) -test identified qualifying factors. Logistic regression was used to investigate the influence of these covariates on LTS. RESULTS: Of the evaluable 354 CONKO-001 pts, 54 (15%) with an overall survival ≥5 years were identified. It was possible to obtain tumor specimens of 39 pts (72%). Histological re-evaluation confirmed adenocarcinoma in 38 pts, 1 showed a high-grade neuroendocrine tumor. Univariate analysis for all 53 LTS pts with adenocarcinoma compared to the remaining 300 non-LTS pts revealed as relevant active treatment, tumor grading, tumor size, lymph nodes. No significance could be demonstrated for resection margin, sex, age, Karnofsky performance status, CA 19-9 at study entry. In multivariate analysis, tumor grading, active treatment, tumor size, lymph node involvement were independent prognostic factors for LTS. CONCLUSION: Long-term survival can be achieved in adenocarcinoma of the pancreas.

Assessment of stained direct cytology smears of breast cancer for whole transcriptome and targeted messenger RNA sequencing.

BACKGROUND: Rather than surgical resection, cytologic specimens are often used as first-line clinical diagnostic procedures due to higher safety, speed, and cost-effectiveness. Archival diagnostic cytology slides containing cancer can be equivalent to tissue biopsies for DNA mutation testing, but the accuracy of transcriptomic profiling by RNA sequencing (RNA-seq) is less understood. METHODS: This study compares the results from whole transcriptome RNA-seq and a targeted RNA-seq assay of stained cytology smears (CS) versus matched tumor tissue samples preserved fresh-frozen (FF) and processed as formalin-fixed paraffin-embedded (FFPE) sections. Cellular cytology scrapes from all 11 breast cancers were fixed and stained using three common protocols: Carnoy's (CS_C) or 95% ethanol (CS_E) fixation and then Papanicolaou stain or air-dried then methanol fixation and DiffQuik stain (CS_DQ). Agreement between samples was assessed using Lin's concordance correlation coefficient. RESULTS: Library yield for CS_DQ was too low, therefore it was not sequenced. The distributions of concordance correlation coefficient of gene expression levels in comparison to FF were comparable between CS_C and CS_E, but expression of genes enriched in stroma was lower in cytosmear samples than in FF or FFPE. Six signatures showed similar concordance to FF for all methods and two were slightly worse in CS_C and CS_E. Genomic signatures were highly concordant using targeted RNA-seq. The allele fraction of selected mutations calculated on cytosmear specimens was highly correlated with FF tissues using both RNA-seq methods. CONCLUSION: RNA can be reliably extracted from cytology smears and is suitable for transcriptome profiling or mutation detection, except for signatures of tumor stroma.

RANK Expression as an Independent Predictor for Response to Neoadjuvant Chemotherapy in Luminal-Like Breast Cancer: A Translational Insight from the GeparX Trial.

PURPOSE: The GeparX study investigated whether denosumab as add-on treatment to nab-paclitaxel-based neoadjuvant chemotherapy (NACT) with two different schedules (125 mg/m² weekly vs. day 1, 8 every 22 days) may increase pathologic complete response (pCR) rate. The addition of denosumab to NACT did not improve pCR rates as recently published. In this study, we investigated whether receptor activator of nuclear factor-kappa B (RANK) expression, as part of the denosumab target pathway: (i) may retrospectively identify a subgroup of patients with additional clinical benefit of denosumab or (ii) may predict response to nab-paclitaxel NACT. EXPERIMENTAL DESIGN: RANK protein was IHC-stained on pre-therapeutic core biopsies from patients of the GeparX study (n = 667) with the antibody RANK/Envision System HRP (DAB) and was analyzed for the percentage of membranous RANK tumor cell staining (>5% RANKhigh vs. ≤5% RANKlow). RESULTS: We could not identify any patient subgroup with differential response under denosumab add-on treatment in patients with RANKhigh expression [139/667, 20.8%; OR, 0.86; 95% confidence interval (CI), 0.44-1.68; P = 0.667] or RANKlow expression (528/667 (79.2%) OR, 1.10; 95% CI, 0.78-1.56; P = 0.589; Pinteraction = 0.528). However, the pCR rate was higher in the RANKhigh subgroup compared with RANKlow (50% vs. 39%; OR, 1.52; 95% CI, 1.04-2.21; P = 0.037). RANK expression constituted an independent predictor of response to NACT frequently in patients with luminal-like subtype (HR+/HER2-; OR, 2.98; 95% CI, 1.30-6.79; P = 0.010). No predictive value of RANK expression among the different nab-paclitaxel regimens was observed. CONCLUSION: We report RANK expression to be an independent predictive biomarker for response to NACT in patients with luminal-like breast cancer.

Increased expression of IDO1 is associated with improved survival and increased number of TILs in patients with high-grade serous ovarian cancer.

BACKGROUND: The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) plays a crucial role in regulating the immune system's response to tumors, but its exact role in cancer, especially in high-grade serous ovarian cancer (HGSOC), remains controversial. We aimed to investigate the prognostic impact of IDO1 expression and its correlation with tumor-infiltrating lymphocytes (TILs) in HGSOC. METHODS: Immunohistochemical (IHC) staining and bioimage analysis using the QuPath software were employed to assess IDO1 protein expression in a well-characterized cohort of 507 patients with primary HGSOC. Statistical evaluation was performed using SPSS, and in silico validation considering IDO1 mRNA expression in bulk and single-cell gene expression datasets was conducted. Additionally, IDO1 expression in interferon-gamma (IFNG) stimulated HGSOC cell lines was analyzed. RESULTS: Our findings revealed that IDO1 protein and mRNA expression serve as positive prognostic markers for overall survival (OS) and progression-free survival (PFS) in HGSOC. High IDO1 expression was associated with a significant improvement in OS by 21 months (p < 0.001) and PFS by 6 months (p = 0.016). Notably, elevated IDO1 expression correlated with an increased number of CD3+ (p < 0.001), CD4+ (p < 0.001), and CD8+ TILs (p < 0.001). Furthermore, high IDO1 mRNA expression and protein level were found to be associated with enhanced responsiveness to pro-inflammatory cytokines, particularly IFNG. CONCLUSIONS: Our study provides evidence that IDO1 expression serves as a positive prognostic marker in HGSOC and is associated with an increased number of CD3+, CD4+ and CD8+ TILs. Understanding the intricate relationship between IDO1, TILs, and the tumor microenvironment may hold the key to improving outcomes in HGSOC.

LRP1B-a prognostic marker in tubo-ovarian high-grade serous carcinoma.

Low-density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a member of the LDL receptor family and has often been discussed as a tumor suppressor gene, as its down-regulation is correlated with a poor prognosis in multiple carcinoma entities. Due to the high metastasis rate into the fatty peritoneal cavity and current research findings showing a dysregulation of lipid metabolism in tubo-ovarian high-grade serous carcinoma (HGSC), we questioned the prognostic impact of the LRP1B protein expression. We examined a well-characterized large cohort of 571 patients with primary HGSC and analyzed the LRP1B protein expression via immunohistochemical staining (both in tumor and stroma cells separately), performed precise bioimage analysis with QuPath, and calculated the prognostic impact using SPSS. Our results demonstrate that LRP1B functions as a significant prognostic marker for overall survival (OS) and progression-free survival (PFS) in HGSC on the protein level. High cytoplasmic expression of LRP1B in tumor, stroma, and combined tumor and stroma cells has a significantly positive association with a mean prolongation of the OS by 42 months (P = .005), 29 months (P = .005), and 25 months (P = .001), respectively. Additionally, the mean PFS was 18 months longer in tumor (P = .002), 19 months in stroma (P = .004), and 19 months in both cell types combined (P = .01). Our results remained significant in multivariate analysis. We envision LRP1B as a potential prognostic tool that could help us understand the functional role of lipid metabolism in advanced HGSC, especially regarding liposomal medications.

Integration of metabolomics and expression of glycerol-3-phosphate acyltransferase (GPAM) in breast cancer-link to patient survival, hormone receptor status, and metabolic profiling.

Changes in lipid metabolism are an important but not well-characterized hallmark of cancer. On the basis of our recent findings of lipidomic changes in breast cancer, we investigated glycerol-3-phosphate acyltransferase (GPAM), a key enzyme in the lipid biosynthesis of triacylglycerols and phospholipids. GPAM protein expression was evaluated and linked to metabolomic and lipidomic profiles in a cohort of human breast carcinomas. In addition, GPAM mRNA expression was analyzed using the GeneSapiens in silico transcriptiomics database. High cytoplasmic GPAM expression was associated with hormone receptor negative status (p = 0.013). On the protein (p = 0.048) and mRNA (p = 0.001) levels, increased GPAM expression was associated with a better overall survival. Metabolomic analysis by GC-MS showed that sn-glycerol-3-phosphate, the substrate of GPAM, was elevated in breast cancer compared to normal breast tissue. LC-MS based lipidomic analysis identified significantly higher levels of phospholipids, especially phosphatidylcholines in GPAM protein positive tumors. In conclusion, our results suggest that GPAM is expressed in human breast cancer with associated changes in the cellular metabolism, in particular an increased synthesis of phospholipids, the major structural component of cellular membranes.

High nuclear poly-(ADP-ribose)-polymerase expression is prognostic of improved survival in pancreatic cancer.

AIMS: Poly-(ADP-ribose)-polymerases (PARPs) act as post-translational modifiers of proteins that are mainly involved in the DNA repair machinery, and have recently been shown to be predictive of pathologically complete remission after chemotherapy in breast cancer. In the pancreas, PARP expression has so far only been studied in inflammatory conditions. Therefore, in this study, we investigated the relevance of PARP in pancreatic cancer. METHODS AND RESULTS: Cytoplasmic and nuclear PARP expression was assessed by immunohistochemistry in a population-based cohort of 178 adenocarcinomas of the pancreas and correlated with clinicopathological parameters. We found that low-level nuclear expression of PARP is associated with a poor prognosis (median survival 9.6 versus 14.5 months, P = 0.004). CONCLUSIONS: Our analysis shows that nuclear PARP is an independent prognostic marker with respect to standard clinicopathological parameters. These results suggest that PARP should be further explored as a predictive factor with respect to conventional chemotherapy and concepts of PARP inhibitor therapy in pancreatic cancer.

Decentral gene expression analysis: analytical validation of the Endopredict genomic multianalyte breast cancer prognosis test.

BACKGROUND: EndoPredict (EP) is a clinically validated multianalyte gene expression test to predict distant metastasis in ER-positive, HER2-negative breast cancer treated with endocrine therapy alone. The test is based on the combined analysis of 12 genes in formalin-fixed, paraffin-embedded (FFPE) tissue by reverse transcription-quantitative real-time PCR (RT-qPCR). Recently, it was shown that EP is feasible for reliable decentralized assessment of gene expression. The aim of this study was the analytical validation of the performance characteristics of the assay and its verification in a molecular-pathological routine laboratory. METHODS: Gene expression values to calculate the EP score were assayed by one-step RT-qPCR using RNA from FFPE tumor tissue. Limit of blank, limit of detection, linear range, and PCR efficiency were assessed for each of the 12 PCR assays using serial samples dilutions. Different breast cancer samples were used to evaluate RNA input range, precision and inter-laboratory variability. RESULTS: PCR assays were linear up to Cq values between 35.1 and 37.2. Amplification efficiencies ranged from 75% to 101%. The RNA input range without considerable change of the EP score was between 0.16 and 18.5 ng/µl. Analysis of precision (variation of day, day time, instrument, operator, reagent lots) resulted in a total noise (standard deviation) of 0.16 EP score units on a scale from 0 to 15. The major part of the total noise (SD 0.14) was caused by the replicate-to-replicate noise of the PCR assays (repeatability) and was not associated with different operating conditions (reproducibility). Performance characteristics established in the manufacturer's laboratory were verified in a routine molecular pathology laboratory. Comparison of 10 tumor samples analyzed in two different laboratories showed a Pearson coefficient of 0.995 and a mean deviation of 0.15 score units. CONCLUSIONS: The EP test showed reproducible performance characteristics with good precision and negligible laboratory-to-laboratory variation. This study provides further evidence that the EP test is suitable for decentralized testing in specialized molecular pathological laboratories instead of a reference laboratory. This is a unique feature and a technical advance in comparison with existing RNA-based prognostic multigene expression tests.