Pre-analytical effects on whole transcriptome and targeted RNA sequencing analysis in cytology: The effects of prolonged time in storage of effusion specimens prior to preservation.

OBJECTIVES: To investigate the pre-analytics of the molecular testing of cytology specimens, we studied the effects of time in refrigerator storage (4°C) of malignant effusions on RNA sequencing (RNAseq) results. METHODS: Ten effusion specimens were stored in a refrigerator (4°C) for different durations (day 0, 1, 4, and 7). All specimens were prepared as cytospins fixed in either Carnoy's solution or 95% ethanol (EtOH) and in an RNA preservative for a fresh frozen (FF) high-quality reference. Whole transcriptome (wt) and targeted (t)RNAseq of two multigene expression signatures were performed. We then compared transcript expression levels (including mutant allele fraction) according to pre-analytical variables using a concordance correlation coefficient (CCC) and a mixed effect model. RESULTS: Sequencing results were mostly stable over increasing time in storage. Cytospins fixed in Carnoy's solution were more concordant with FF samples than cytospins fixed in 95% EtOH at all timepoints. This finding was consistent for both wtRNAseq (averages: day 0 CCC = 0.98 vs 0.91; day 7 CCC = 0.88 vs 0.78) and tRNAseq methods (averages: day 0 CCC = 0.98 vs 0.81; day 7 CCC = 0.98 vs 0.90). Cytospins fixed in Carnoy's solution did not show significant changes in expression over timepoints or between expression signatures, whereas 95% EtOH did. CONCLUSION: RNAseq can be accurately performed on effusion specimens after prolonged refrigerator storage. RNA extracted from scraped cytospin slides fixed in Carnoy's solution was marginally superior to 95% EtOH fixation, but either method had comparable analytic performance to high-quality FF RNA 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.

Spatially resolved analyses link genomic and immune diversity and reveal unfavorable neutrophil activation in melanoma.

Complex tumor microenvironmental (TME) features influence the outcome of cancer immunotherapy (IO). Here we perform immunogenomic analyses on 67 intratumor sub-regions of a PD-1 inhibitor-resistant melanoma tumor and 2 additional metastases arising over 8 years, to characterize TME interactions. We identify spatially distinct evolution of copy number alterations influencing local immune composition. Sub-regions with chromosome 7 gain display a relative lack of leukocyte infiltrate but evidence of neutrophil activation, recapitulated in The Cancer Genome Atlas (TCGA) samples, and associated with lack of response to IO across three clinical cohorts. Whether neutrophil activation represents cause or consequence of local tumor necrosis requires further study. Analyses of T-cell clonotypes reveal the presence of recurrent priming events manifesting in a dominant T-cell clonotype over many years. Our findings highlight the links between marked levels of genomic and immune heterogeneity within the physical space of a tumor, with implications for biomarker evaluation and immunotherapy response.

Genomic and immune heterogeneity are associated with differential responses to therapy in melanoma.

Appreciation for genomic and immune heterogeneity in cancer has grown though the relationship of these factors to treatment response has not been thoroughly elucidated. To better understand this, we studied a large cohort of melanoma patients treated with targeted therapy or immune checkpoint blockade (n = 60). Heterogeneity in therapeutic responses via radiologic assessment was observed in the majority of patients. Synchronous melanoma metastases were analyzed via deep genomic and immune profiling, and revealed substantial genomic and immune heterogeneity in all patients studied, with considerable diversity in T cell frequency, and few shared T cell clones (<8% on average) across the cohort. Variables related to treatment response were identified via these approaches and through novel radiomic assessment. These data yield insight into differential therapeutic responses to targeted therapy and immune checkpoint blockade in melanoma, and have key translational implications in the age of precision medicine.

Integrated molecular analysis of tumor biopsies on sequential CTLA-4 and PD-1 blockade reveals markers of response and resistance.

Immune checkpoint blockade produces clinical benefit in many patients. However, better biomarkers of response are still needed, and mechanisms of resistance remain incompletely understood. To address this, we recently studied a cohort of melanoma patients treated with sequential checkpoint blockade against cytotoxic T lymphocyte antigen-4 (CTLA-4) followed by programmed death receptor-1 (PD-1) and identified immune markers of response and resistance. Building on these studies, we performed deep molecular profiling including T cell receptor sequencing and whole-exome sequencing within the same cohort and demonstrated that a more clonal T cell repertoire was predictive of response to PD-1 but not CTLA-4 blockade. Analysis of CNAs identified a higher burden of copy number loss in nonresponders to CTLA-4 and PD-1 blockade and found that it was associated with decreased expression of genes in immune-related pathways. The effect of mutational load and burden of copy number loss on response was nonredundant, suggesting the potential utility of a combinatorial biomarker to optimize patient care with checkpoint blockade therapy.