Sample Preparation Approach Influences PAM50 Risk of Recurrence Score in Early Breast Cancer

The PAM50 gene expression subtypes and the associated risk of recurrence (ROR) score are used to predict the risk of recurrence and the benefits of adjuvant therapy in early-stage breast cancer. The Prosigna assay includes the PAM50 subtypes along with their clinicopathological features, and is approved for treatment recommendations for adjuvant hormonal therapy and chemotherapy in hormone-receptor-positive early breast cancer. The Prosigna test utilizes RNA extracted from macrodissected tumor cells obtained from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, RNA extracted from fresh-frozen (FF) bulk tissue without macrodissection is widely used for research purposes, and yields high-quality RNA for downstream analyses. To investigate the impact of the sample preparation approach on ROR scores, we analyzed 94 breast carcinomas included in an observational study that had available gene expression data from macrodissected FFPE tissue and FF bulk tumor tissue, along with the clinically approved Prosigna scores for the node-negative, hormone-receptor-positive, HER2-negative cases (n = 54). ROR scores were calculated in R; the resulting two sets of scores from FFPE and FF samples were compared, and treatment recommendations were evaluated. Overall, ROR scores calculated based on the macrodissected FFPE tissue were consistent with the Prosigna scores. However, analyses from bulk tissue yielded a higher proportion of cases classified as normal-like; these were samples with relatively low tumor cellularity, leading to lower ROR scores. When comparing ROR scores (low, intermediate, and high), discordant cases between the two preparation approaches were revealed among the luminal tumors; the recommended treatment would have changed in a minority of cases.

Micro-computed tomography: A novel diagnostic technique for the evaluation of gastrointestinal specimens

Micro-computed tomography (micro-CT) is a non-destructive modality that can be used to obtain high-resolution three-dimensional (3 D) images of the whole sample tissue; the usefulness of micro-CT has been reported for evaluation of breast cancer and lung cancer. However, this novel diagnostic technique has never been used for evaluating endoscopically resected gastrointestinal specimens. In the present study, we scanned 13 formalin-fixed paraffin-embedded (FFPE) tissue blocks of a normal human colon and gastric tissue samples using micro-CT. The evaluation comprised a comparison of the acquired whole block images with the images of the corresponding cross-sectional slice of the hematoxylin and eosin-stained slide. Micro-CT was able to produce images of the whole sample and clearly depict tissues such as glandular structures, muscularis mucosae, and blood vessels in the FFPE tissue blocks of normal gastrointestinal samples. Furthermore, the 3 D reconstructed could be used to create a cross-sectional image and reflected the surface structure of samples obtained from any site. Micro-CT has the potential to become a highly promising pathological diagnostic assistance tool for endoscopically resected gastrointestinal specimens in combination with conventional microscopic examination.

Interplay between copy number alterations and immune profiles in the early breast cancer Scandinavian Breast Group 2004-1 randomized phase II trial: results from a feasibility study

Emerging data indicate that genomic alterations can shape immune cell composition in early breast cancer. However, there is a need for complementary imaging and sequencing methods for the quantitative assessment of combined somatic copy number alteration (SCNA) and immune profiling in pathological samples. Here, we tested the feasibility of three approaches—CUTseq, for high-throughput low-input SCNA profiling, multiplexed fluorescent immunohistochemistry (mfIHC) and digital-image analysis (DIA) for quantitative immuno-profiling- in archival formalin-fixed paraffin-embedded (FFPE) tissue samples from patients enrolled in the randomized SBG-2004-1 phase II trial. CUTseq was able to reproducibly identify amplification and deletion events with a resolution of 100 kb using only 6 ng of DNA extracted from FFPE tissue and pooling together 77 samples into the same sequencing library. In the same samples, mfIHC revealed that CD4 + T-cells and CD68 + macrophages were the most abundant immune cells and they mostly expressed PD-L1 and PD-1. Combined analysis showed that the SCNA burden was inversely associated with lymphocytic infiltration. Our results set the basis for further applications of CUTseq, mfIHC and DIA to larger cohorts of early breast cancer patients.

Integration of Targeted Gene Expression Profiling and FDG-PET Radiomics Uncovers Radiometabolic Signatures Associated with Outcome in Diffuse Large B-Cell Lymphoma

Metabolic rewiring is a hallmark of cancer and a predominant feature of aggressive lymphoproliferative disorders such as diffuse large B-cell lymphomas (DLBCL), which need a reshaped metabolism in order to meet the increased demands related to rapid cell proliferation. Emerging evidence indicates that chemoresistance is closely related to altered metabolism in cancer. However, the relationship between metabolic rewiring and chemoresistance in lymphoma is yet to be elucidated. Radiomic analysis applied to functional imaging with fluoroedoxyglucose positron emission tomography (FDG-PET) provides a unique opportunity to explore DLBCL metabolism. In this study we hypothesized that distinct gene expression (GEP) signatures might be correlated with specific FDG-PET radiomics signatures, which in turn could be associated with resistance to standard chemoimmunotherapy and DLBCL outcome. First, we retrospectively analyzed a discovery cohort of 48 consecutive DLBCL patients (pts) treated at our center with standard first line R-CHOP/R-CHOP-like chemoimmunotherapy from 2010 to 2018, with available formalin-fixed paraffin embedded (FFPE) tissue from the initial diagnostic biopsy and FDG-PET radiomics data extracted from the same target lesion. Median follow-up was 55 months (range 18-110). We profiled this cohort with targeted-GEP (T-GEP) (NanoString platform), using a custom panel to define the cell of origin (COO) and MYC/BCL-2 levels, and a dedicated panel comprising 180 genes encompassing the most relevant cancer metabolism pathways. By applying the maxstat package we found that a 6-gene metabolic signature was strongly associated with outcome and outperformed the COO, the MYC/BCL-2 status and the International Prognostic Index (IPI) score for progression free survival (PFS) and overall survival (OS) in multivariate analysis. The 6-gene metabolic signature included genes regulating oxidative metabolism and fatty acid oxidation (SCL25A1, PDK4, PDPR) which were upregulated, and was inversely associated with genes involved in glycolytic pathways (MAP2K1, HIF1A, GBE1) which were downregulated. Notably 5-year PFS and OS were 100% and 95% in metabolic signature (met-Sig) low pts vs 24% and 45% in met-Sig high pts respectively (p<0.0001 for PFS and OS). There was no significant association between the COO, MYC/BCL-2 levels, standardized uptake value (SUV), and the 6-gene signature. The prognostic value of the 6-gene signature for OS was validated in 2 large publicly available cohorts of 469 (Sha et al. J Clin Oncol 2019) and 233 (Lenz et al. N Eng J Med 2005) pts. Next, we integrated PET radiomics and T-GEP data. Radiomics analysis (LifeX package) was performed by applying regions of interest semi-automatically, using a 25% SUV max threshold for segmentation. Fifty-five radiomic features (RFs) were extracted and 10 RFs significantly correlated either positively or negatively with the T-GEP metabolic signature (Spearman). After stability evaluation, applying a stepwise feature selection procedure, 4 RFs (Histo Curtosis, Histo Energy, Shape Sphericity, NGLDM Contrast) were used to generate a radiomic signature (hereafter called radiometabolic signature) characterized by the most significant correlation with both the metabolic T-GEP signature (r=0.43, p=0.0027) and PFS (p=0.004). These results (obtained analyzing the lesion of the initial diagnostic biopsy), were confirmed using different target lesions (i.e. the most FDG-avid and the largest lesion), and were validated in a second independent cohort of 64 patients (validation cohort) treated at our center in the same period of time (with no FFPE tissue available). A multivariate analysis performed in the whole cohort of 112 pts (discovery + validation) indicated that the radiometabolic signature retained independent prognostic value in relation to the IPI score and metabolic tumor volume. The robustness of the radiometabolic signature was further confirmed by using a second segmentation method (fixed 2.5 SUV max threshold). These data indicate that oxidative metabolic rewiring could be a powerful adverse prognostic predictor, suggesting the possibility of targeting oxidative metabolism to overcome chemorefractoriness in DLBCL. This study provides the proof of principle for the use of FDG-PET radiomics as a tool for non-invasive assessment of cancer metabolism, and for predicting metabolic vulnerabilities in DLBCL. Figure 1 Figure 1. Disclosures Tarella: ADC-THERAPEUTICS: Other: ADVISORY BOARD; Abbvie: Other: ADVISORY BOARD. Pileri: CELGENE: Other: ADVISORY BOARD; ROCHE: Other: ADVISORY-BOARD; NANOSTRING: Other: ADVISORY BOARD. Derenzini: TAKEDA: Research Funding; BEIGENE: Other: ADVISORY BOARD; ASTRA-ZENECA: Consultancy, Other: ADVISORY-BOARD; TG-THERAPEUTICS: Research Funding; ADC-THERAPEUTICS: Research Funding.

MIBI staining v2

This protocol is the standard FFPE tissue staining procedure recommended for Multiplex Ion Beam Imaging Time of Flight instrument (MIBI_TOF) developed in the Sean C. Bendall and Michael R. Angelo labs. The protocol has been successfully used for MIBI and is the result of extensive optimization experiments. It is inspired from state-of-the art of immunohistochemistry staining procedures but differs in some very important steps, namely, glutaraldehyde fixation and final washes prior tissue dehydration. Failure to follow exactly all steps described in this procedure may result in inconsistencies in output data after MIBI_TOF acquisition.

Targeted Next-Generation Sequencing of Cancer-Related Genes in a Norwegian Patient Cohort With Head and Neck Squamous Cell Carcinoma Reveals Novel Actionable Mutations and Correlations With Pathological Parameters

BackgroundTargeted next-generation sequencing (NGS) is increasingly applied in clinical oncology to advance personalized treatment. Despite success in many other tumour types, use of targeted NGS panels for assisting diagnosis and treatment of head and neck squamous cell carcinomas (HNSCC) is still limited.AimThe focus of this study was to establish a robust NGS panel targeting most frequent cancer mutations in long-term preserved formalin-fixed paraffin-embedded (FFPE) tissue samples of HNSCC from routine diagnostics.Materials and MethodsTumour DNA obtained from archival FFPE tissue blocks of HNSCC patients treated at Haukeland University Hospital between 2003-2016 (n=111) was subjected to mutational analysis using a custom made AmpliSeq Library PLUS panel targeting 31 genes (Illumina). Associations between mutational burden and clinical and pathological parameters were investigated. Mutation and corresponding clinicopathological data from HNSCC were extracted for selected genes from the Cancer Genome Atlas (TCGA) and used for Chi-square and Kaplan-Meier analysis.ResultsThe threshold for sufficient number of reads was attained in 104 (93.7%) cases. Although the specific number of PCR amplified reads detected decreased, the number of NGS-annotated mutations did not significantly change with increased tissue preservation time. In HPV-negative carcinomas, mutations were detected mainly in TP53 (73.3%), FAT1 (26.7%) and FLG (16.7%) whereas in HPV-positive, the common mutations were in FLG (24.3%) FAT1 (17%) and FGFR3 (14.6%) genes. Other less common pathogenic mutations, including well reported SNPs were reproducibly identified. Presence of at least one cancer-specific mutations was found to be positively associated with an extensive desmoplastic stroma (p=0.019), and an aggressive type of invasive front (p=0.035), and negatively associated with the degree of differentiation (p=0.041). Analysis of TCGA data corroborated the association between cancer-specific mutations and tumour differentiation and survival analysis showed that tumours with at least one mutation had shorter disease-free and overall survival (p=0.005).ConclusionsA custom made targeted NGS panel could reliably detect several specific mutations in archival samples of HNSCCs preserved up to 17 years. Using this method novel associations between mutational burden and clinical and pathological parameters were detected and actionable mutations in HPV-positive HNSCC were discovered.