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. 2017 Nov 1;23(21):6708-6720.
doi: 10.1158/1078-0432.CCR-17-0544. Epub 2017 Aug 1.

Diverse BRCA1 and BRCA2 Reversion Mutations in Circulating Cell-Free DNA of Therapy-Resistant Breast or Ovarian Cancer

Britta Weigelt  1 , Iñaki Comino-Méndez  2 , Ino de Bruijn  3 , Lei Tian  4 , Jane L Meisel  5   6 , Isaac García-Murillas  2 , Charlotte Fribbens  2   7 , Ros Cutts  2 , Luciano G Martelotto  3 , Charlotte K Y Ng  3   8   9 , Raymond S Lim  3 , Pier Selenica  3 , Salvatore Piscuoglio  3   8 , Carol Aghajanian  5 , Larry Norton  5 , Rajmohan Murali  3 , David M Hyman  5 , Laetitia Borsu  3 , Maria E Arcila  3 , Jason Konner  5 , Jorge S Reis-Filho  3 , Roger A Greenberg  4 , Mark E Robson  5 , Nicholas C Turner  10   7
Affiliations

Diverse BRCA1 and BRCA2 Reversion Mutations in Circulating Cell-Free DNA of Therapy-Resistant Breast or Ovarian Cancer

Britta Weigelt et al. Clin Cancer Res. .

Abstract

Purpose: Resistance to platinum-based chemotherapy or PARP inhibition in germline BRCA1 or BRCA2 mutation carriers may occur through somatic reversion mutations or intragenic deletions that restore BRCA1 or BRCA2 function. We assessed whether BRCA1/2 reversion mutations could be identified in circulating cell-free DNA (cfDNA) of patients with ovarian or breast cancer previously treated with platinum and/or PARP inhibitors.Experimental Design: cfDNA from 24 prospectively accrued patients with germline BRCA1 or BRCA2 mutations, including 19 patients with platinum-resistant/refractory ovarian cancer and five patients with platinum and/or PARP inhibitor pretreated metastatic breast cancer, was subjected to massively parallel sequencing targeting all exons of 141 genes and all exons and introns of BRCA1 and BRCA2 Functional studies were performed to assess the impact of the putative BRCA1/2 reversion mutations on BRCA1/2 function.Results: Diverse and often polyclonal putative BRCA1 or BRCA2 reversion mutations were identified in cfDNA from four patients with ovarian cancer (21%) and from two patients with breast cancer (40%). BRCA2 reversion mutations were detected in cfDNA prior to PARP inhibitor treatment in a patient with breast cancer who did not respond to treatment and were enriched in plasma samples after PARP inhibitor therapy. Foci formation and immunoprecipitation assays suggest that a subset of the putative reversion mutations restored BRCA1/2 function.Conclusions: Putative BRCA1/2 reversion mutations can be detected by cfDNA sequencing analysis in patients with ovarian and breast cancer. Our findings warrant further investigation of cfDNA sequencing to identify putative BRCA1/2 reversion mutations and to aid the selection of patients for PARP inhibition therapy. Clin Cancer Res; 23(21); 6708-20. ©2017 AACR.

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Figures

Figure 1
Figure 1. BRCA1 open reading frame-restoring somatic mutations identified in cfDNA derived from ovarian cancer patients with BRCA1 germline mutations resistant/ refractory to platinum-based chemotherapy
Representation of the BRCA1 protein (top). Nucleotide and amino acid sequences for the affected genomic location shown are based on ENSEMBL transcript no. ENST00000357654.3. Representation of the predicted nucleotide and protein sequences for BRCA1 wild-type (WT), germline mutation and putative reversion mutations from ovarian cancer patient OCT5 (top) and OCT15 (bottom). These three putative BRCA1 reversion mutations were found to restore the BRCA1 open reading frame. Additional putative BRCA1 reversion mutations are shown in Supplementary Fig. S2. Predicted protein lengths are shown in bold. The base triplets affected by a mutation are marked in light blue, and the aberrant amino acids produced by a given mutation are marked in red. Green arrows indicate the restored open reading frames. AA, amino acid; ORF, open reading frame; WT, wild-type.
Figure 2
Figure 2. Validation of putative BRCA1 reversion mutation using dPCR and IR-induced BRCA1 foci formation
A, Validation of the putative BRCA1 c.85delG reversion mutation in cfDNA and tissue samples from patient OCT15 harboring a BRCA1 c.68–69delAG germline mutation using dPCR. Massively parallel sequencing libraries of germline DNA spiked in with 10, 100, and 1,000 BRCA1 c.85delG synthetic oligonucleotide molecules were used as controls and for BRCA1 c.85delG mutant gating (top). Massively parallel sequencing libraries from the plasma DNA (top right) and from three anatomically distinct ovarian tumor samples (i.e. ovary, peritoneum and fallopian tube; bottom) of case OCT15 were tested. The somatic BRCA1 c.85delG mutation was confirmed in the cfDNA but was not detected in the pretreatment ovarian cancer tissues. B, U2OS cells were transfected with pcDNA-BRCA1(Δ510–1283) and BRCA1 mutant plasmids (BRCA1 germline and/or respective putative BRCA1 reversion mutations of cases OCT1, OCT5, OCT10 and OCT15) or wild-type (WT) BRCA1 as control for 48 hrs (see Methods). Following 8Gy irradiation (IR), BRCA1 foci formation was assessed using immunofluorescence. Arrows indicate the BRCA1 reversion mutations partially restoring BRCA1 foci formation.
Figure 3
Figure 3. BRCA2 open reading frame-restoring somatic mutations identified in cfDNA derived from breast cancer patients with BRCA2 germline mutations after platinum-based chemotherapy
Representation of the BRCA2 protein (top). Nucleotide and amino acid sequences for the affected genomic location shown are based on ENSEMBL transcript no. ENST00000380152.7. Representation of the predicted nucleotide and protein sequences for the BRCA2 wild-type (WT), germline alteration and putative reversion mutations from patients A, 1109 and B, L031 are shown. The putative BRCA2 reversion mutations presented in this figure were validated independently using targeted amplicon re-sequencing. Predicted protein lengths are shown in bold. The base triplets affected by a mutation are marked in light blue, and the aberrant amino acids produced by a given mutation are marked in red. Gaps represent the germline and somatic BRCA2 reversion mutations identified. Four putative BRCA2 reversion mutations were found to co-localize with the germline alteration, which is underlined in red in the reversion mutation sequences on the left. Insertions are highlighted by green squares. Green arrows indicate the restored open reading frames. AA, amino acid; ORF, open reading frame; WT, wild-type.
Figure 4
Figure 4. Serial analysis of putative BRCA2 reversion mutations in cfDNA samples from breast cancer patient L031, and the interaction between reversion-mutant BRCA2, PALB2 and RAD51
A, CT images during the course of therapy of breast cancer patient L031 demonstrating the initial response and subsequent progression of the lesions. Plasma samples were obtained before and after treatment with the PARP inhibitor Talazoparib and after Capecitabine therapy (top). Mutant allele frequencies of two somatic BRCA2 reversion mutations identified by targeted massively parallel sequencing were assessed in two independent analyses in the plasma samples pre- and post PARP inhibitor treatment using targeted amplicon sequencing. B, 293T cells transfected with HA-BRCA2 wild-type (WT), HA-BRCA2 c.407delA germline mutant (GM) and HA-BRCA2 c.402_413delTCTAAATTCTTG somatic reversion-mutant plasmids Rev). Western blot performed using an anti-HA antibody revealed that the HA-BRCA2Rev was translated into mutant protein (predicted 3414AA) with a molecular weight similar to that of the wild-type protein (3418AA). The HA-BRCA2GM protein length is predicted to be 150AA. Immunoprecipitation of HA-BRCA2Rev and wild-type HA-BRCA2 revealed that HA-BRCA2Rev protein displays proficient interactions with PALB2 and RAD51 similar to that of the wild-type BRCA2 protein. AA, amino acid.
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