In this study, we have developed a qRT-PCR grade index (PCR-GGI) composed of four genes associated with cell cycle progression and proliferation initially included in the GGI [2
]. This gene set includes MYBL2, KPNA2, CDC2
, which together cover all phases of the cell cycle. Indeed, MYBL2
encodes a nuclear protein involved in cell cycle progression; KPNA2
is involved in the import of proteins to the nuclear envelope and acts as a regulator of cell cycle checkpoint mediators; CDC2
encodes a catalytic subunit of the highly conserved protein kinase complex known as M-phase promoting factor (MPF) which is essential for G1/S and G2/M phase transitions; and, CDC20
encodes a protein acting as a regulatory protein interacting with several other proteins at multiple points in the cell cycle.
We demonstrated that a quantitative assessment of a very small number of genes is sufficient to recapitulate the performance of the original GGI, especially in ER-positive breast cancer. This could be explained by the fact that the expression levels of most genes of the GGI signature are highly correlated with one another. Two out of the four genes included in our PCR-GGI signature were also present in a number of previously reported prognostic signatures; MYBL2
was part of the Recurrence Score developed by Paik et al. [9
] as well as the microarray signatures defined by Naderi et al. [10
] and Miller et al. [11
], whereas the KPNA2
gene was present in the 76-gene signature of Wang et al. [7
], which has been applied successfully on a cohort of node-negative estrogen receptor positive breast cancer patients to predict benefit of tamoxifen [12
We further showed that the averaged expression of the four selected genes evaluated by qRT-PCR using FFPE samples accurately recapitulates the prognostic performance of the GGI. Moreover the PCR-GGI was conducted with success from 10 years old FFPE samples. Fourteen percent of the samples have been excluded from the study and this number is expected to be lower using recent FFPE samples.
Additionally, as illustrated in this manuscript, the PCR-GGI has several advantages which highlight its clinical relevance.
First, as illustrated previously for the GGI, the PCR-GGI is able to identify high and low-risk patients within the subgroup of patients with intermediate histological grade tumors, possibly improving treatment decision making for these patients.
Second, as the GGI, the PCR-GGI is able to identify a higher proportion of early breast cancer patients at low risk of recurrence than do the clinical guidelines. This means that the number of patients that would receive unnecessary treatment could be reduced by applying this molecular assay.
Third, the PCR-GGI is not subject to reproducibility and inter-observer variability problems such as the histological grading. Moreover, the PCR-GGI's procedure developed at Jules Bordet was easily transferred at the Josephine Nefkens Institute, Rotterdam following some inter-laboratory tests which gave great confidence in the reproducibility of the assessment of the PCR-GGI. Also, the PCR-GGI cut-off for positivity identified at the Jules Bordet Institute was easily applied on the Josephine Nefkens Institute assays.
Fourth, the PCR-GGI can classify ER-positive BC patients treated either with adjuvant tamoxifen only or first-line tamoxifen for advanced disease into clinically relevant subgroups. We first observed a statistically significant association between a high PCR-GGI score and a higher risk of recurrence in ER-positive BC samples from patients that received adjuvant tamoxifen for primary disease across two different BC populations. We also observed a statistically significant association between a high PCR-GGI score and a higher risk of progression in ER+ BC samples originated from patients that received first-line tamoxifen for advanced disease. However, the determination of ER positivity might also play a role in the assessment of the performance of the PCR-GGI since we report here a discordance of 25% between IHC and RT-PCR assessment of ER.
Finally, the PCR-GGI has two practical advantages: 1/ it requires only very small amounts of routinely available FFPE samples, whereas the original GGI requires fresh-frozen material, and 2/ it allows the use of a SYBR-based technology, instead of the specific Taqman probes (Applied Biosystems), providing the advantages of being easy to use and cheaper than specific probes. Therefore, we might envisage that this test could be carried out in local certified pathology laboratories.
Additionally, several studies have demonstrated that tumors with characteristics associated with poor histological grade and high proliferating index tended to respond better to chemotherapy [13
]. As our signature was derived from the histological grade we might then hypothesize that the PCR-GGI not only quantifies the likelihood of BC recurrence in women with node-negative ER+ BC, it might also predict the magnitude of chemotherapy benefit.