Combination of laser microdissection, 2D-DIGE and MALDI-TOF MS to identify protein biomarkers to predict colorectal cancer spread

Chandra Kirana,Richard S Stubbs,Lifeng Peng,Hongjun Shi,John P Keating,Rose Miller,T William Jordan,Guy J Maddern,Corinne Glenn

doi:10.1186/s12014-019-9223-7

Abstract

Biomarkers are urgently required to support current histological staging to provide additional accuracy in stratifying colorectal cancer (CRC) patients according to risk of spread to properly assign adjuvant chemotherapy after surgery. Chemotherapy is given to patients with stage III to reduce the risk of recurrence but is controversial in stage II patients. Up to 25% of stage II patients will relapse within 5 years after tumor removal and when this occurs cure is seldom possible. The aim of this study was to identify protein biomarkers to stratify risk of spread of CRC patients. Laser micro-dissection was used to isolate cancer cells from primary colorectal tumors of stage II patients which did or did not metastasize within 5 years after surgical resection. Protein expression differences between two groups of tumors were profiled by 2D-DIGE with saturation CyDye labeling and identified using MALDI-TOF mass spectrometry. Evaluation of protein candidates was conducted using tissue micro array (TMA) immunohistochemistry on 125 colorectal tumor tissue samples of different stages. A total of 55 differentially expressed proteins were identified. Ten protein biomarkers were chosen based on p value and ratio between non metastasized and metastazised groups and evaluated on 125 tissues using TMA immunohistochemistry. Expression of HLAB, protein 14-3-3β, LTBP3, ADAMTS2, JAG2 and NME2 on tumour cells was significantly associated with clinical parameters related to tumour progression, invasion and metastasis. Kaplan–Meier survival curve showed strong expression of six proteins was associated with good CRC specific survival. Expression of HLAB, ADAMTS2, LTBP3, JAG2 and NME2 on tumour cells, was associated with tumour progression and invasion, metastasis and CRC specific survival may serve as potential biomarkers to stratify CRC patients into low and high risk of tumour metastasis. Combined methods of laser microdissection, 2D DIGE with saturation labelling and MALDI-TOF MS proved to be resourceful techniques capable of identifying protein biomarkers to predict risk of spread of CRC to liver.

Highlights

Colorectal cancer (CRC) remains the leading cause of cancer deaths in the Western countries
Combined methods of laser microdissection, 2D DIGE with saturation labelling and MALDI TOF/TOF proved to be resourceful methods capable to show proteins differences of stage II CRC patients whose tumours did not metastasis with those whose tumours metastasized to liver
HLAB, ADAMTS2, LTBP3, JAG2 and NME2 were significantly associated with tumor progression, vascular invasion and distant liver metastasis

Summary

Introduction

Colorectal cancer (CRC) remains the leading cause of cancer deaths in the Western countries. Metastatic dissemination from primary tumor accounts for over 90% of all colorectal cancer death [3]. Adjuvant chemotherapy for stage II CRC patients is still regarded as controversial [4,5,6]. About 25% of stage II CRC patients will develop metastasis after surgical removal of their primary tumor mainly to liver and 50–60% of stage III CRC patients will develop metastasis [7]. The overall survival rate for stage II CRC patients 5 years after surgery is approximately 70–80% and that for stage III patients is 30–60% [8]

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