Background. PI3K/Akt/mTOR is one of the most important pathways for the regulation of cell survival, proliferation and apoptosis. Mutational events occurring in this pathway could lead to malignant transformation and endocrine resistance in breast cancer. The mTOR inhibitor Everolimus (EVE) interferes with cellular proliferation by binding FKB12 protein. EVE has been definitely approved thanks to BOLERO-2 phase III study, which showed a significant prolongation of Progression Free Survival (PFS) due to the addiction of EVE to Exemestane therapy, compared to Exemestane alone, in Hormonal Receptors-positive (RO+) and HER2-negative (HER2-) metastatic breast cancer patients. Hortobagyi et al. performed Next Generation Sequencing on 227 BOLERO-2 samples of primary breast carcinoma, to study the potential correlation between genetic alterations and EVE efficacy. A greater incidence of mutations in PIK3CA, PTEN, CCND1 and FGFR1/2 genes was detected and it was observed that patients with no or only one genetic alteration in these genes derive the most benefit from EVE therapy. To our knowledge, no previous research has evaluated the mutational status both in primary and metastatic breast cancers. Materials and methods. Aim of this study was to evaluate the molecular profile in primitive breast cancers (21 ductal carcinomas, 3 lobular carcinomas and 1 colloid carcinoma) and visceral metastases (hepatic and pulmonary), in 25 patients with advanced breast cancer (RO+ HER2-) treated with EVE in combination with Exemestane. Thirty-three DNA samples from 25 patients were examined, 13 from primary0 breast cancers and 20 from metastatic lesions. In 8 patients, both the primary tumor and the corresponding metachronous metastasis were evaluated. Genomic DNA samples from FFPE tumoral tissue were analized by using OncoCarta v2.0 panel on Mass Array Sequenom platform. A preliminar Multiplex-PCR, followed by SAP-dephosphorylating reaction and iPLEX-primer specific extension, was performed to detect more than 150 single nucleotide variations in mutational hotspots from 18 implicated genes (AKT1, BRAF, CTNNB1, FBX4, FBXW7, FGFR2, FGFR3, GNAQ, KIT, KRAS, MAP2K1, MAP2K2, NRAS, PDGFRa, PIK3CA, PTPN11, SOS1, TP53). Differences were evaluated using Chi-Square and Fisher Tests. Survival analysis was conducted using Kaplan-Meyer curves. Results. Overall, 11 DNA samples, out of the 33 examined, were mutated (33%). Mutations were found in 10 ductal carcinomas and in the colloid carcinoma. Five mutations were detected in primary breast lesions and 6 in metastatic ones. All mutations consisted of a single-nucleotide variation resulting in aminoacidic substitution. Among primary lesions, mutations were detected in the following genes: PIK3CA (E545K), FBX4 (G30N), KIT (S709F), MAP2K1 (D67N), FBXW7 (R465C). They occurred with a frequency of 3%, respectively, namely in 1 out of 33 samples each. Only in the AKT1 gene the same mutation (E17K) was found in 2 DNA primary lesion samples. In metastatic lesions, BRAF (R444W), KIT (G565R), TP53 (R273H), FBXW7 (R479Q), CTNNB1 (S45F), PIK3CA (E545K), AKT (E17K) were mutated. Notably, mutations were found exclusively in primary lesions or in metastatic ones, while only in one patient both primary and secondary lesions were mutated; however, these mutations occurred in two different genes: MAP2K1 (D67N) in breast, FBXW7 (R479Q) in metastasis. Of notice, a reduction in PFS was observed in one patient which carried 3 different mutations (FBX4, PIK3CA, KIT) in the primary tumor (3.4 month versus an average of 5 month) whereas a significantly increased PFS (15.9 month) was detected in a case with 2 mutations (PIK3CA, AKT1) in metastatic lesion. Conclusion. Although the number of patients and samples is quite limited, our findings in mutational status support literature evidence, as genes most frequently mutated were PIK3CA, AKT1 and FBXW7, even if the percentage of PIK3CA and AKT1 mutations was less than expected. No correlations between primary and metastatic mutational status were detected.

Molecular Profile, as detected with Mass-Array Spectrometry (Sequenom platform), in primary and metastatic breast carcinoma treated with Exemestane + Everolimus / Manfredini, S.; Bettelli, S.; Filieri, M. E.; Caprera, C.; Ficarra, G.; Piacentini, F.; Maiorana, A.. - In: PATHOLOGICA. - ISSN 0031-2983. - 2:107(2015). ((Intervento presentato al convegno Congresso Annuale di Anatomia Patologica SIAPEC-IAP tenutosi a Milano nel 23-25 Settembre 2015.

Molecular Profile, as detected with Mass-Array Spectrometry (Sequenom platform), in primary and metastatic breast carcinoma treated with Exemestane + Everolimus

S. Manfredini;S. Bettelli;M. E. Filieri;F. Piacentini;A. Maiorana
2015

Abstract

Background. PI3K/Akt/mTOR is one of the most important pathways for the regulation of cell survival, proliferation and apoptosis. Mutational events occurring in this pathway could lead to malignant transformation and endocrine resistance in breast cancer. The mTOR inhibitor Everolimus (EVE) interferes with cellular proliferation by binding FKB12 protein. EVE has been definitely approved thanks to BOLERO-2 phase III study, which showed a significant prolongation of Progression Free Survival (PFS) due to the addiction of EVE to Exemestane therapy, compared to Exemestane alone, in Hormonal Receptors-positive (RO+) and HER2-negative (HER2-) metastatic breast cancer patients. Hortobagyi et al. performed Next Generation Sequencing on 227 BOLERO-2 samples of primary breast carcinoma, to study the potential correlation between genetic alterations and EVE efficacy. A greater incidence of mutations in PIK3CA, PTEN, CCND1 and FGFR1/2 genes was detected and it was observed that patients with no or only one genetic alteration in these genes derive the most benefit from EVE therapy. To our knowledge, no previous research has evaluated the mutational status both in primary and metastatic breast cancers. Materials and methods. Aim of this study was to evaluate the molecular profile in primitive breast cancers (21 ductal carcinomas, 3 lobular carcinomas and 1 colloid carcinoma) and visceral metastases (hepatic and pulmonary), in 25 patients with advanced breast cancer (RO+ HER2-) treated with EVE in combination with Exemestane. Thirty-three DNA samples from 25 patients were examined, 13 from primary0 breast cancers and 20 from metastatic lesions. In 8 patients, both the primary tumor and the corresponding metachronous metastasis were evaluated. Genomic DNA samples from FFPE tumoral tissue were analized by using OncoCarta v2.0 panel on Mass Array Sequenom platform. A preliminar Multiplex-PCR, followed by SAP-dephosphorylating reaction and iPLEX-primer specific extension, was performed to detect more than 150 single nucleotide variations in mutational hotspots from 18 implicated genes (AKT1, BRAF, CTNNB1, FBX4, FBXW7, FGFR2, FGFR3, GNAQ, KIT, KRAS, MAP2K1, MAP2K2, NRAS, PDGFRa, PIK3CA, PTPN11, SOS1, TP53). Differences were evaluated using Chi-Square and Fisher Tests. Survival analysis was conducted using Kaplan-Meyer curves. Results. Overall, 11 DNA samples, out of the 33 examined, were mutated (33%). Mutations were found in 10 ductal carcinomas and in the colloid carcinoma. Five mutations were detected in primary breast lesions and 6 in metastatic ones. All mutations consisted of a single-nucleotide variation resulting in aminoacidic substitution. Among primary lesions, mutations were detected in the following genes: PIK3CA (E545K), FBX4 (G30N), KIT (S709F), MAP2K1 (D67N), FBXW7 (R465C). They occurred with a frequency of 3%, respectively, namely in 1 out of 33 samples each. Only in the AKT1 gene the same mutation (E17K) was found in 2 DNA primary lesion samples. In metastatic lesions, BRAF (R444W), KIT (G565R), TP53 (R273H), FBXW7 (R479Q), CTNNB1 (S45F), PIK3CA (E545K), AKT (E17K) were mutated. Notably, mutations were found exclusively in primary lesions or in metastatic ones, while only in one patient both primary and secondary lesions were mutated; however, these mutations occurred in two different genes: MAP2K1 (D67N) in breast, FBXW7 (R479Q) in metastasis. Of notice, a reduction in PFS was observed in one patient which carried 3 different mutations (FBX4, PIK3CA, KIT) in the primary tumor (3.4 month versus an average of 5 month) whereas a significantly increased PFS (15.9 month) was detected in a case with 2 mutations (PIK3CA, AKT1) in metastatic lesion. Conclusion. Although the number of patients and samples is quite limited, our findings in mutational status support literature evidence, as genes most frequently mutated were PIK3CA, AKT1 and FBXW7, even if the percentage of PIK3CA and AKT1 mutations was less than expected. No correlations between primary and metastatic mutational status were detected.
Congresso Annuale di Anatomia Patologica SIAPEC-IAP
Milano
23-25 Settembre 2015
Manfredini, S.; Bettelli, S.; Filieri, M. E.; Caprera, C.; Ficarra, G.; Piacentini, F.; Maiorana, A.
Molecular Profile, as detected with Mass-Array Spectrometry (Sequenom platform), in primary and metastatic breast carcinoma treated with Exemestane + Everolimus / Manfredini, S.; Bettelli, S.; Filieri, M. E.; Caprera, C.; Ficarra, G.; Piacentini, F.; Maiorana, A.. - In: PATHOLOGICA. - ISSN 0031-2983. - 2:107(2015). ((Intervento presentato al convegno Congresso Annuale di Anatomia Patologica SIAPEC-IAP tenutosi a Milano nel 23-25 Settembre 2015.
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