Prognostic significance of tumor genotypes and CD8+ infiltrates in stage I-III colorectal cancer

Abstract

Background: We explored the clinical significance of tumor genotypes and immunophenotypes in non-metastatic colorectal cancer (CRC).

Methods: In primary tumors (paraffin blocks) from 412 CRC patients treated with adjuvant chemotherapy, we examined pathogenic mutations (panel NGS; 347 informative); mismatch repair (MMR) immunophenotype (360 informative); and CD8+ lymphocyte density (high - low; 412 informative). The primary outcome measure was disease-free survival (DFS).

Results: We evaluated 1713 pathogenic mutations (median: 3 per tumor; range 0-49); 118/412 (28.6%) tumors exhibited high CD8+ density; and, 40/360 (11.1%) were MMR-deficient. Compared to MMR-proficient, MMR-deficient tumors exhibited higher CD8+ density (chi-square, p<0.001) and higher pathogenic mutation numbers (p=0.003). High CD8+ density was an independent favorable prognosticator (HR=0.49, 95%CI 0.29-0.84, Wald's p=0.010). Pathogenic BRCA1 and ARID1A mutations were inversely associated with each other (p<0.001), were not associated with MMR-deficiency or CD8+ density, but both independently predicted for unfavorable DFS (HR=1.98, 95%CI 1.12-3.48, p=0.018 and HR=1.99, 95%CI 1.11-3.54, p=0.020, respectively).

Conclusion: In non-metastatic CRC, high CD8+ lymphocyte density confers a favorable prognosis and may be developed as a single marker in routine diagnostics. The unfavorable prognostic effect of pathogenic BRCA1 and ARID1A mutations is a novel observation that, if further validated, may improve treatment selection.

Keywords: ARID1A; BRCA1; CD8; MMR; targeted NGS.

Figure 1. REMARK diagram

NGS: next generation sequencing; MMR: mismatch repair; MMR-P and MMR-D: proficient and deficient, respectively.

Figure 2. Map of pathogenic mutations in 332 CRC

(A) Out of 1713 pathogenic mutations, 32% were nonsense or frameshifts in tumor suppressors, while missense mutations were dominant in known oncogenes. We did not apply the classification of hypermutated and non-hypermutated tumors because we used a 59-gene panel only. However, it is apparent that most tumors (75%) carried more than 1 pathogenic mutation, the most frequent combination being APC & TP53 in 1/3 of tumors, co-mutated with KRAS in ¼ of the cases, while 10% of tumors carried more than 10 pathogenic mutations. Despite that the applied reading depth was very high in our cases (>1000X, compared to <50X in whole genome sequencing), the 4 most frequently mutated genes are in line with previous publications. The high incidence of BRCA1, PTEN, CDH1 and BRCA2 mutations is most probably a result of high reading depth and over-representation of these genes in the custom panel. Red dots: genes with site-specific differences in the distribution of pathogenic mutations. (B) Demonstrates the actual number of tumors with pathogenic mutations in the presented genes. The number of tumors with pathogenic mutations is shown for the 15 most frequently affected genes. Blue bars correspond to the number of pathogenic mutations per gene; tumor suppressor genes, e.g., APC, TP53, occasionally carried multiple mutations per tumor, which was not observed for oncogenes, e.g., KRAS, BRAF. (C) Comparison of mutation numbers in MMR-D and MMR-P tumors. Although MMR-D were in general richer in mutations compared to MMR-P tumors, MMR-P tumors with mutations in MMR genes (red dots) exhibited higher mutation numbers compared to MMR-D, probably because of co-mutated pathways. Green dots: four MMR-D tumors with concordant MMR gene mutation status. Blue lines: mean values.

Figure 3

Prognostic significance of (A) high CD8+ density; (B and C) BRCA1 and ARID1A pathogenic mutations.

Figure 4. Associations between DFS and relevant clinicopathological, mutational and immunophenotypic parameters