FGFR1 amplification in breast carcinomas: a chromogenic in situhybridisation analysis

Amplification of 8p11.2-p12 is reported to be found in up to 10–15% of all breast cancers [1–5]. For a long time it was believed that the fibroblast growth factor receptor 1 (FGFR1) would be the target oncogene of 8p11.2-p12 amplifications [1, 2, 5, 6]. Recent studies, however, have called into question the role of FGFR1 as the 'amplicon driver', given that not all cell lines and breast cancers with 8p11.2-p12 amplification overexpressed the FGFR1 gene and that FGFR1 protein and mRNA expression was much more pervasive than gene amplification [7–9]. Other oncogene candidates for the amplicon on 8p11.2-p12 have been put forward, including zinc finger protein 703 (FLJ14299), SPFH domain family member 2 (SPFH2, also known as C8orf2), subunit of RNA polymerase III transcription initiation factor (BRF2) and RAB11 family interacting protein 1 (RAB11FIP1) [7].

Gelsi-Boyer and colleagues [3] have demonstrated more recently that the 8p11.2-p12 amplicon is much more complex than previously anticipated and that it comprises at least four independent cores, which can be amplified independently [3]. While FLJ14299, SPFH2, proline synthetase cotranscribed homologue (PROSC), BRF2, and RAB11FIP1 were associated with core A1, golgin subfamily a 7 (GOLGA7) was correlated with A3, and MYST histone acetyltransferase 3 (MYST3) and miR172-resistant version of AP2 (AP2M3) were associated with A4. The genes whose expression correlated with amplification of core A2 included LSM1 homologue, U6 small nuclear RNA associated (LSM1, also known as cancer-associated Sm-like (CASM)), DDHD domain containing 2 (DDHD2), phosphatidic acid phosphatase type 2 domain containing 1B (HTPAP), Wolf–Hirschhorn syndrome candidate 1-like 1 (WHSC1L1), TM2 and FGFR1 [3]. Interestingly, amplification of core 2, but not the other cores, was associated with shorter metastasis-free survival [3].

FGFR1 gene encodes a tyrosine kinase receptor that is part of the fibroblast growth factor and growth factor receptors family [10–12]. FGFR1 expression has been shown to play pivotal roles in mammary development and breast cancer tumourigenesis [12, 13]. Activation of FGFR1 in a transgenic mouse model resulted in increased luminal cell proliferation, activation of mitogen-activated protein kinase and Akt, lateral budding and, eventually, alveolar hyperplasia and invasive lesions [11, 12]. In addition, we have recently demonstrated that when core 2 of the 8p11.2-p12 is amplified, the FGFR1 gene shows increased levels of mRNA and protein expression [14]. Furthermore, we have also determined in vitro that FGFR1 signalling is paramount for the survival of a FGFR1 amplified breast cancer cell line [14].

The prognostic impact of FGFR1 amplification in breast cancer still remains unclear. In previous studies analysing FGFR1 amplification by means of Southern blot or fluorescent in situ hybridisation, conflicting results were observed: while FGFR1 amplification proved to be associated with positivity for oestrogen receptor (ER) in one study [1], Prentice and colleagues found no association between FGFR1 amplification and clinicopathological parameters or patients' survival [4]. More recently, using probes for both RAB11FIP1 and FGFR1, Letessier and colleagues [15] demonstrated that cases with 8p12 amplification have a significantly shorter metastasis-free survival [15].

Chromogenic in situ hybridisation (CISH) is a technique that allows for a concurrent analysis of the gene copy number and morphological features of the cells [16–21]. Although results obtained with CISH show an excellent concordance with those obtained with fluorescent in situ hybridisation [21], CISH has proven useful for high-throughput copy number assessment, given that it can be easily applied to tissue microarrays and the analysis can be performed with a conventional light microscope [16–21]. No studies analysing FGFR1 amplification, as defined by CISH, in a large cohort of breast cancer patients have so far been performed. Using a previously described method [18], inhouse probes specific for FGFR1 were generated and we set out to characterise the prevalence of FGFR1 amplification in a large community-based cohort of breast cancers and its correlations with traditional clinicopathological features, immunohistochemical markers, and disease-free and overall survival.

After excluding the uninformative tissue microarray cores, results on FGFR1 amplification were available for 496 tumours. Forty-three tumours (8.7%) showed either >5 signals or large gene clusters in >50% of neoplastic cells (Figure 1).

Complete clinical follow-up information was available for 478 patients for whom FGFR1 CISH results were optimal. The median follow-up period was 58 months (range 1–192 months). During this period, a total of 73 (14.7%) patients died from breast cancer. Of all cases, 116 (24.3%) cases were grade 1, 141 (29.5%) cases were grade 2, and 221 (46.2%) were grade 3. From the available data, 153 (32.2%) of the patients had lymph-node-positive disease, 130 (27.8%) had positive vascular invasion, and 311 (65.1%) had tumour size ≥1.5 cm. Recurrence occurred in 147 cases (30.8%), and distant metastases developed in 84 cases (17.6%). A total of 149/496 (30%) patients received tamoxifen, 5/496 (1%) received tamoxifen and Zoladex, and 309/496 (62.3%) received no endocrine treatment. There was no significant difference between the disease-free interval (P = 0.761) or overall survival (P = 0.225) between those patients that received hormone therapy compared with those patients that did not. A total of 48/496 (9.7%) patients received chemotherapy.

Kaplan–Meier survival analysis revealed an association between FGFR1 amplification and a shorter overall survival (P = 0.01, log-rank test) (Figure 2a). A trend for a shorter disease-free survival and FGFR1 amplification was found (P < 0.07, log-rank test) (Figure 2b). On multivariate Cox hazard analysis adjusted for tumour grade, size and lymph node status, for ER status, and for FGFR1 amplification, it was found that the FGFR1 amplification was a significant predictor of poor overall survival independent of the other known prognostic parameters (P < 0.04) (Table 3).

In recent years it has been demonstrated that CISH is a useful technique to determine gene copy numbers and gene amplification on formalin-fixed, paraffin-embedded tissue sections [16–21]. Unlike fluorescent in situ hybridisation, CISH allows a direct comparison between morphological features of neoplastic cells and the presence of gene amplification [16–21]. Furthermore, CISH analysis is relatively quick; in the present study, the whole analysis of FGFR1 amplification in a cohort of 880 patients took 2 weeks and, although only one tissue microarray core per tumour was analysed, 56% cases rendered optimal results.

We demonstrate in the present study that FGFR1 amplification is found in 8.7% of breast cancers, which is in agreement with previous studies [1–4, 29]. Unlike previous studies where FGFR1 amplification was determined by Southern blot analysis [1], our results and those obtained with other in situ methods [3, 4, 15] did not show any correlation between FGFR1 amplification and low histological grade or positivity for ER. On the other hand, our results demonstrate that FGFR1 amplification is an independent predictor of poor outcome, especially for patients with ER-positive breast cancers. Interestingly, the impact of FGFR1 amplification was stronger on overall survival than disease-free survival (that is, higher hazard ratios on multivariate analysis). This may stem from the fact that locoregional recurrences were included as events for disease-free survival analysis, and that overall survival considered only breast-cancer-related deaths as events. Alternatively, this may reflect the association between FGFR1 amplification and the development of distant metastasis (P = 0.05) or a shorter survival after the first distant recurrence event.

Our group [14] and others [3] have demonstrated that when FGFR1 is specifically amplified (that is, amplification of core A2 of the 8p11.2-p12 amplicon) it is also overexpressed, and that FGFR1 signalling is important for the survival of a cell line that harbours FGFR1 amplification and high-level gene expression [14, 30]. Taken together, these results suggest that, in a significant proportion of cases with core A2 amplification, FGFR1 may be the actual amplicon driver [6]. We could not correlate FGFR1 amplification with expression in this study, as it was not possible to optimise antibodies for FGFR1 immunohistochemical analysis on tissue microarrays due to the highly fixation-dependent nature of the commercially available antibodies (data not shown).

The FGFR1 gene encodes a tyrosine kinase receptor that has been shown to play an important role in mammary gland development [12, 13, 31]. Previous studies have shown in vitro and in vivo that FGFR1 overexpression has oncogenic properties [10, 12–14, 31]. Furthermore, FGFR1 has been implicated in the tumourigenesis of haematological malignancies, where it is frequently involved in balanced chromosomal translocations, including cases of chronic myeloid leukaemia (BCR-FGFR1 fusion) and the 8p11 myeloproliferative syndrome/stem cell leukaemia–lymphoma syndrome, which is characterised by myeloid hyperplasia and non-Hodgkin's lymphoma with chromosomal translocations fusing several genes, the most common being a fusion between ZNF198 and FGFR1 [32]. In preclinical models, the PKC412 tyrosine kinase inhibitor has been shown to successfully inhibit the growth of proliferation of ZNF198-FGFR1-transformed Ba/F3 cells and to prolong the survival of animals with a ZNF198-FGFR1-induced stem cell leukaemia–lymphoma syndrome [33]. Targeting FGFR1 signalling with RNA interference or with the SU5402 FGFR1 tyrosine kinase inhibitor has been shown to decrease cell survival in a breast cancer cell line with FGFR1 amplification [14].

Taken together, our results demonstrate that FGFR1 amplification is found in 8.7% of breast cancers and is an independent predictor of outcome. Although large studies correlating FGFR1 amplification with mRNA and protein expression are still needed, the functional data demonstrating that FGFR1 signalling is required for the survival of breast cancer cells harbouring FGFR1 amplification [14], the relatively high prevalence of FGFR1 amplification in breast cancer and the independent prognostic information provided by FGFR1 amplification status support the idea that this gene may be a useful therapeutic target for a subgroup of breast cancer patients with FGFR1 gene amplification [14].