Molecularly determined total tumour load in lymph nodes of stage I-II colon cancer patients correlates with high-risk factors. A multicentre prospective study

Abstract

Stage I-II (pN0) colorectal cancer patients are surgically treated although up to 25 % will eventually die from disease recurrence. Lymph node (LN) status is an independent prognostic factor in colorectal cancer (CRC), and molecular tumour detection in LN of early-stage CRC patients is associated with an increased risk of disease recurrence and poor survival. This prospective multicentre study aimed to determine the relationship between LN molecular tumour burden and conventional high-risk factors in stage I-II colon cancer patients. A total of 1940 LN from 149 pathologically assessed pN0 colon cancer patients were analysed for the amount of tumour cytokeratin 19 (CK19) messenger RNA (mRNA) with the quantitative reverse transcription loop-mediated isothermal amplification molecular assay One-Step Nucleic Acid Amplification. Patient's total tumour load (TTL) resulted from the sum of all CK19 mRNA tumour copies/μL of each positive LN from the colectomy specimen. A median of 15 LN were procured per case (IQR 12;20). Molecular positivity correlated with high-grade (p < 0.01), mucinous/signet ring type (p = 0.017), male gender (p = 0.02), number of collected LN (p = 0.012) and total LN weight per case (p < 0.01). The TTL was related to pT stage (p = 0.01) and tumour size (p < 0.01) in low-grade tumours. Multivariate logistic regression showed independent correlation of molecular positivity with gender, tumour grade and number of fresh LN [AUC = 0.71 (95 % CI = 0.62-0.79)]. Our results show that lymph node CK19 mRNA detection correlates with classical high-risk factors in stage I-II colon cancer patients. Total tumour load is a quantitative and objective measure that may help to better stage early colon cancer patients.

Keywords: Colorectal neoplasms; Cytokeratin 19; Lymph nodes; Molecular pathology; Neoplasm staging.

Fig. 1

a A primary high-grade mucinous carcinoma with signet ring cells. b Positive CK19 immunohistochemistry from the same case. c Amplification curve of CK19 mRNA from a positive LN, assessed through turbidity variation (Y-axis) over time (X-axis, minutes). (Fig. 1a, b, scale bar 250 μm)

Fig. 2

Study flow diagram

Fig. 3

OSNA results and correlation with LN retrieval and pT stage, regarding number of LN retrieved (a), time spent on fresh LN search in minutes (b) and total weight of fresh LN per case (gr) (c). OSNA positive cases held significantly more LN due to a higher fresh LN yields. d Shows that most cases in every pT stage held up to 3 OSNA positive LN. e Although not significant, a trend was observed between pT stage and TTL

Fig. 4

ROC curve of the multivariate logistic regression model for OSNA results including gender, grade and the number of fresh lymph nodes as variables predicting molecular positivity. Continuous line: All data (0.71 (95 % CI = 0.62–0.79); Dashed line: 10-fold CV (AUC = 0.67 [95 % CI = 0.59–0.76])