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Surgical Tumor Resection Deregulates Hallmarks of Cancer in Resected Tissue and the Surrounding Microenvironment.

Author(s): Chaubal R, Gardi N, Joshi S, Pantvaidya G, Kadam R, Vanmali V, Hawaldar R, Talker E, Chitra J, Gera P, Bhatia D, Kalkar P, Gurav M, Shetty O, Desai S, Krishnan NM, Nair N, Parmar V, Dutt A, Panda B, Gupta S, Badwe R

Publication: Mol Cancer Res, 2024, Vol. 22, Page 572-584

PubMed ID: 38394149 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared RNA expression profiles of tumor biopsies that were collected at the beginning (fully vascularized), during (partially devascularized) and following (fully devascularized) surgical resection for breast or head and neck cancer.  Multiple methods of analysis were used.  The following specimens were collected from 81 patients with treatment-naive breast cancer: 1.) a core needle biopsy of still vascularized tumor and normal adjacent tissue was collected at the start of surgery (immediately after anesthesia); 2.) a core needle biopsy of the tumor was obtained mid-way through surgery, after the tumor was partially devascularized; 3.) a tissue specimen was obtained at the end of surgery (specimen was fully devascularized).  The following specimens were collected from 10 patients with treatment-naive squamous carcinoma of head and neck : 1.) still vascularized tumor and normal adjacent tissue at the start of surgery; 2.) tumor specimen obtained mid-way through surgery, after the tumor was partially devascularized; 3.) a tumor specimen collected just after devascularization but before removal from body; 4.) a tumor specimen collected after devascularization and removal from body. After collection, each specimen was placed in RNAlater. RNA was extracted using Trizol, followed by the Pure-Link RNA Mini Kit. RNA was analyzed using the Agilent RNA 6000 Nano Kit and by NanoDrop spectrophotometer. Whole-transcriptome sequencing libraries were constructed from the RNA of 46 breast cancer patients, including 32 specimens with RNA available from 2 or 3 timepoints that was prepared with the TruSeq RNA Sample Prep Kit v2 and sequenced on an Illumina HiSeq 2000. Gene expression was compared among surgical timepoints from each patient. Validation was carried out using RNA from 43 breast cancer patients (including 13 samples that were analyzed using HiSeq) that was analyzed by NanoString nCounter gene expression profiling assays and RNA from 8 breast cancer patients (3 were also analyzed by NanoString) using the RT2 profiler qPCR assay. Expression of 20 differentially expressed genes between surgical timepoints and 23 hypoxia-related mRNAs were assayed by real-time PCR using an independent set of specimens collected at the same timepoints from 8 patients with treatment-naive breast cancer.  RNA from patients with head/neck cancer with a RIN <7 were labelled using the Illumina WGDASL assay, while specimens with RIN ≥7 were labeled using the Illumina Total Prep RNA Amplification Kit. Expression was analyzed in specimens with head/neck cancer using Illumina BeadArrays on a HiScan instrument. Gene expression was validated against data from the TCGA dataset.

   Summary of Findings:

   A total of 4,947 genes were deregulated in at least one of the 32 patients (multiple specimens per patient were collected) when surgical resection timepoints were compared. Of these, 737 genes were found to be deregulated in specimens from ≥3 patients. Of the 737 deregulated genes, 523 genes differed between fully vascularized and partially devascularized specimens, 423 genes differed between partially devascularized and fully devascularized specimens, and 250 genes differed between fully vascularized and fully devascularized specimens, with 141 genes found to be deregulated among all timepoints. Pathways analysis found that the deregulated genes between fully vascularized and partially devascularized specimens were enriched for biological pathways involved with stress responses, inflammation, epithelial markers, cellular invasion and migration and lipid and fat metabolism. The most enriched hallmarks included genes involved in TNFα signaling via NF-kB (inflammatory), epithelial-to-mesenchymal transition, KRAS signaling, late estrogen response and hypoxic stress response pathways. Canonical pathways that showed deregulation included the activator protein 1 (AP-1) transcription factor-network, extracellular matrix (ECM) protein-related genes and genes related to external stimuli, receptor binding, and hormone responses. Of the 737 deregulated genes, 85 were also deregulated during validation (NanoString and RT2 profiler) after correction for multiple testing. These validated genes showed enrichment for the p38MAPK cascade, cellular proliferation, cellular differentiation, and response to hormones.

   In tumor specimens from head/neck cancer patients, 30 genes were found to be differentially expressed between all 4 timepoints (beginning, partially devascularized, fully devascularized in body and following removal) that included many AP-1-related genes. A comparison of specimens from head/neck cancer patients found that AP-1–related genes were upregulated in tumor specimens collected after devascularization had begun relative to normal adjacent specimens collected prior to devascularization but not between the tumor specimen and normal adjacent specimen collected prior to devascularization. Analysis of normal adjacent breast specimens by NanoString also showed changes in AP-1 related genes as surgery progressed. The authors conclude that AP-1 signaling increases in both tumor and normal specimens with surgical progression. Interestingly, analysis of TCGA data also identified AP-1-related genes as the top differentially expressed genes in five of seven cancer types. Finally, the authors identified 164 genes that were deregulated over the course of surgery in all cohorts examined (in breast cancer specimens analyzed by RNAseq and NanoString and head/neck cancer specimens analyzed by microarray).

    

   Biospecimens

   • Tissue - Head and Neck
   • Tissue - Breast

   Preservative Types

   • RNAlater

   Diagnoses:

   • Neoplastic - Normal Adjacent
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   RNA	Next generation sequencing
   RNA	DNA microarray

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Biospecimen location	Head/neck Breast
   Biospecimen Acquisition	Time of biospecimen collection	At the start of surgery (immediately after anesthesia Mid-way through surgery after tumor was partially devascularized At the end of surgery
   Preaquisition	Warm ischemia time	Multiple collections during course of surgery

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