Clinical relevance of H-RAS, K-RAS, and N-RAS mRNA expression in primary breast cancer patients

Abstract

Purpose

The RAS family comprises three proto-oncogenes (H-RAS, K-RAS, and N-RAS) and is among the most widely studied of oncogenes. The present study aimed at investigating the clinical relevance of mRNA levels of the three isoforms in a large group of breast cancer patients with a long-term follow-up.

Methods

198 previously untreated patients were enrolled in the study. mRNA levels of K-RAS, H-RAS, and N-RAS were measured using microarray (Affymetrix HG-U133A).

Results

Elevated H-RAS levels were found significantly more frequently in patients with larger (p = 0.021) and ER-positive tumors (p = 0.048), while elevated K-RAS levels were associated with nodal positivity (p = 0.001) and HER2-positivity (p = 0.010). Patients with high N-RAS mRNA levels were more likely to be diagnosed with triple-negativity (p < 0.001) and higher grading (p = 0.001). Patients with high K-RAS levels were more likely to show an elevated H-RAS (p = 0.003). After a median follow-up of 183 months, patients with high N-RAS expression had significantly reduced overall survival (OS) compared with patients with low N-RAS (mean: 146.9 vs. 211.0 months; median 169.3 vs. not reached; p = 0.009). In patients with non-metastatic disease at the time of tissue sampling, mean disease-free survival (DFS) was 150.1 months for patients with high N-RAS versus 227.7 months with low N-RAS; median DFS was not reached (p = 0.004). The expression of H-RAS and K-RAS was not associated with DFS/OS. In the multivariable analysis, distant metastasis, HER2 positivity, and elevated N-RAS mRNA levels independently predicted reduced OS, while nodal status, HER2 status, and N-RAS predicted reduced DFS.

Conclusions

Elevated N-RAS mRNA levels predict impaired clinical outcome; hypothetically, further exploration of the RAS signaling pathway might enable identifying potential targeted treatment strategies. The association between high N-RAS levels and the most aggressive among breast cancer subtypes, the triple-negative phenotype, for which targeted approaches are still lacking, underlines the need to further investigate the RAS family.

REMARK checklist

Item to be reported		Page no.
Introduction
1	State the marker examined, the study objectives, and any pre-specified hypotheses	3
Materials and methods
Patients
2	Describe the characteristics (e.g., disease stage or co-morbidities) of the study patients, including their source and inclusion and exclusion criteria	5
3	Describe treatments received and how chosen (e.g., randomized or rule-based)	5
Specimen characteristics
4	Describe type of biological material used (including control samples) and methods of preservation and storage	5
Assay methods
5	Specify the assay method used and provide (or reference) a detailed protocol, including specific reagents or kits used, quality control procedures, reproducibility assessments, quantitation methods, and scoring and reporting protocols. Specify whether and how assays were performed blinded to the study endpoint	5
Study design
6	State the method of case selection, including whether prospective or retrospective and whether stratification or matching (e.g., by stage of disease or age) was used. Specify the time period from which cases were taken, the end of the follow-up period, and the median follow-up time	5
7	Precisely define all clinical endpoints examined	5–6
8	List all candidate variables initially examined or considered for inclusion in models	6
9	Give rationale for sample size; if the study was designed to detect a specified effect size, give the target power and effect size.	5–6
Statistical analysis methods
10	Specify all statistical methods, including details of any variable selection procedures and other model-building issues, how model assumptions were verified, and how missing data were handled	6
11	Clarify how marker values were handled in the analyses; if relevant, describe methods used for cut point determination	6 Table 2
Results
Data
12	Describe the flow of patients through the study, including the number of patients included in each stage of the analysis (a diagram may be helpful) and reasons for dropout. Specifically, both overall and for each subgroup extensively examined report the numbers of patients and the number of events	7–8
13	Report distributions of basic demographic characteristics (at least age and sex), standard (disease-specific) prognostic variables, and tumor marker, including numbers of missing values	7–8, Table 1
Analysis and presentation
14	Show the relation of the marker to standard prognostic variables	7–8 Table 1
15	Present univariable analyses showing the relation between the marker and outcome, with the estimated effect (e.g., hazard ratio and survival probability). Preferably provide similar analyses for all other variables being analyzed. For the effect of a tumor marker on a time-to-event outcome, a Kaplan–Meier plot is recommended	7–8 Figures 4, 5, and 6
16	For key multivariable analyses, report estimated effects (e.g., hazard ratio) with confidence intervals for the marker and, at least for the final model, all other variables in the model	8 Table 3
17	Among reported results, provide estimated effects with confidence intervals from an analysis in which the marker and standard prognostic variables are included, regardless of their statistical significance	7–8
18	If done, report results of further investigations, such as checking assumptions, sensitivity analyses, and internal validation
Discussion
19	Interpret the results in the context of the pre-specified hypotheses and other relevant studies; include a discussion of limitations of the study	9–11
20	Discuss implications for future research and clinical value	9–11