Comparison of the Ability of Different Clinical Treatment Scores to Estimate Prognosis in High-Risk Early Breast Cancer Patients: A Hellenic Cooperative Oncology Group Study

Abstract

Background-AimEarly breast cancer is a heterogeneous disease, and, therefore, prognostic tools have been developed to evaluate the risk for distant recurrence. In the present study, we sought to develop a risk for recurrence score (RRS) based on mRNA expression of three proliferation markers in high-risk early breast cancer patients and evaluate its ability to predict risk for relapse and death. In addition the Adjuvant! Online score (AOS) was also determined for each patient, providing a 10-year estimate of relapse and mortality risk. We then evaluated whether RRS or AOS might possibly improve the prognostic information of the clinical treatment score (CTS), a model derived from clinicopathological variables.MethodsA total of 1,681 patients, enrolled in two prospective phase III trials, were treated with anthracycline-based adjuvant chemotherapy. Sufficient RNA was extracted from 875 samples followed by multiplex quantitative reverse transcription-polymerase chain reaction for assessing RACGAP1, TOP2A and Ki67 mRNA expression. The CTS, slightly modified to fit our cohort, integrated the prognostic information from age, nodal status, tumor size, histological grade and treatment. Patients were also classified to breast cancer subtypes defined by immunohistochemistry. Likelihood ratio (LR) tests and concordance indices were used to estimate the relative increase in the amount of information provided when either RRS or AOS is added to CTS.ResultsThe optimal RRS, in terms of disease-free survival (DFS) and overall survival (OS), was based on the co-expression of two of the three evaluated genes (RACGAP1 and TOP2A). CTS was prognostic for DFS (p<0.001), while CTS, AOS and RRS were all prognostic for OS (p<0.001, p<0.001 and p = 0.036, respectively). The use of AOS in addition to CTS added prognostic information regarding DFS (LR-Δχ2 8.7, p = 0.003), however the use of RRS in addition to CTS did not. For estimating OS, the use of either AOS or RRS in addition to CTS added significant prognostic information. Specifically, the use of both CTS and AOS had significantly better prognostic value vs. CTS alone (LR-Δχ2 20.8, p<0.001), as well as the use of CTS and RRS vs. CTS alone (LR-Δχ2 4.8, p = 0.028). Additionally, more patients were scored as high-risk by AOS than CTS. According to immunohistochemical subtypes, prognosis was improved in the Luminal A (LR-Δχ2 7.2, p = 0.007) and Luminal B (LR-Δχ2 8.3, p = 0.004) subtypes, in HER2-negative patients (LR-Δχ2 23.4, p<0.001) and in patients with >3 positive nodes (LR-Δχ2 23.9, p<0.001) when AOS was added to CTS.ConclusionsThe current study has shown a clear benefit in predicting overall survival of high-risk early breast cancer patients when combining CTS with either AOS or RRS. The combination of CTS and AOS adds significant prognostic information compared to CTS alone for DFS, while the combination of CTS with either AOS or RRS has better prognostic value than CTS alone for OS. These findings could possibly add on the information needed for the best risk prediction strategy ...

Evaluation of the prognostic value of CD3, CD8, and FOXP3 mRNA expression in early‐stage breast cancer patients treated with anthracycline‐based adjuvant chemotherapy

“…To assure optimal reactivity, immunostaining was applied 7 to 10 days after sectioning at the Laboratory of Molecular Oncology of the Hellenic Foundation for Cancer Research, Aristotle University of Thessaloniki School of Medicine. The staining procedures for HER2 (A0485 polyclonal antibody, dilution 1:200, Dako, Glostrup, Denmark), estrogen receptor (ER, clone 6F11, dilution 1:70, NovocastraTM, Leica Biosystems, Newcastle, UK), progesterone receptor (PgR, clone 1A6, dilution 1:70, NovocastraTM, Leica Biosystems), and Ki67 (clone MIB‐1, dilution 1:70, Dako) were performed using a Bond MaxTM autostainer (Leica Microsystems, Wetzlar, Germany), as previously described in detail …”

“…More than one FFPE section (2‐8 sections, 10 μm thick) was used for RNA extraction when the tumor surface of a given sample was less than 0.25 cm 2 . From each FFPE section or macrodissected tissue fragments, RNA was extracted using a standardized fully automated isolation method for total RNA from FFPE tissue, based on germanium‐coated magnetic beads (XTRAKT kit, STRATIFYER Molecular Pathology GmbH, Cologne, Germany) in combination with a liquid handling robot (XTRAKT XL, STRATIFYER Molecular Pathology GmbH), as previously described in detail . The method involves extraction‐integrated deparaffinization and DNase I digestion steps.…”

“…For each primer/probe set, the amplification efficiency was tested, aiming to reach comparable efficiency of >90% (efficiency range from 91% to 108%). Primers and hydrolysis probes were diluted to 100 μmol/L, using a stock solution with nuclease‐free water (Life Technologies GmbH, Darmstadt, Germany) . qRT‐PCR was applied for the relative quantification (RQ) of RANK, OPG, and RANKL.…”

“…When the difference between the duplicate CT values for a given sample was >0.50, the sample was reassessed in triplicates (repeats). Relative expression levels of the target transcripts were calculated as 40‐DCT values (DCT = mean CT target gene − mean CT housekeeping gene) to yield positively correlated numbers and to facilitate comparisons . CD3, CD8, and FOXP3 results were available for all 826 eligible samples, after the above‐mentioned repeats were completed.…”

“…The staining procedures for HER2 (A0485 polyclonal antibody, dilution 1:200, Dako, Glostrup, Denmark), estrogen receptor (ER, clone 6F11, dilution 1:70, NovocastraTM, Leica Biosystems, Newcastle, UK), progesterone receptor (PgR, clone 1A6, dilution 1:70, NovocastraTM, Leica Biosystems), and Ki67 (clone MIB-1, dilution 1:70, Dako) were performed using a Bond MaxTM autostainer (Leica Microsystems, Wetzlar, Germany), as previously described in detail. [31][32][33][34][35]…”