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. 2019 Nov 5;116(45):22730-22736.
doi: 10.1073/pnas.1911385116. Epub 2019 Oct 17.

Whole-exome sequencing of cervical carcinomas identifies activating ERBB2 and PIK3CA mutations as targets for combination therapy

Luca Zammataro  1 , Salvatore Lopez  1   2 , Stefania Bellone  1 , Francesca Pettinella  1 , Elena Bonazzoli  1 , Emanuele Perrone  1   3 , Siming Zhao  4 , Gulden Menderes  1 , Gary Altwerger  1 , Chanhee Han  1 , Burak Zeybek  1 , Anna Bianchi  1 , Aranzazu Manzano  1 , Paola Manara  1 , Emiliano Cocco  5 , Natalia Buza  6 , Pei Hui  6 , Serena Wong  6 , Antonella Ravaggi  7 , Eliana Bignotti  7 , Chiara Romani  7 , Paola Todeschini  7 , Laura Zanotti  7 , Franco Odicino  7 , Sergio Pecorelli  7 , Carla Donzelli  8 , Laura Ardighieri  8 , Roberto Angioli  9 , Francesco Raspagliesi  10 , Giovanni Scambia  3   11 , Jungmin Choi  12   13 , Weilai Dong  12 , Kaya Bilguvar  14 , Ludmil B Alexandrov  15 , Dan-Arin Silasi  1 , Gloria S Huang  1 , Elena Ratner  1 , Masoud Azodi  1 , Peter E Schwartz  1 , Valentina Pirazzoli  16 , Amy L Stiegler  17 , Titus J Boggon  17 , Richard P Lifton  12 , Joseph Schlessinger  18 , Alessandro D Santin  1
Affiliations

Whole-exome sequencing of cervical carcinomas identifies activating ERBB2 and PIK3CA mutations as targets for combination therapy

Luca Zammataro et al. Proc Natl Acad Sci U S A. .

Abstract

The prognosis of advanced/recurrent cervical cancer patients remains poor. We analyzed 54 fresh-frozen and 15 primary cervical cancer cell lines, along with matched-normal DNA, by whole-exome sequencing (WES), most of which harboring Human-Papillomavirus-type-16/18. We found recurrent somatic missense mutations in 22 genes (including PIK3CA, ERBB2, and GNAS) and a widespread APOBEC cytidine deaminase mutagenesis pattern (TCW motif) in both adenocarcinoma (ACC) and squamous cell carcinomas (SCCs). Somatic copy number variants (CNVs) identified 12 copy number gains and 40 losses, occurring more often than expected by chance, with the most frequent events in pathways similar to those found from analysis of single nucleotide variants (SNVs), including the ERBB2/PI3K/AKT/mTOR, apoptosis, chromatin remodeling, and cell cycle. To validate specific SNVs as targets, we took advantage of primary cervical tumor cell lines and xenografts to preclinically evaluate the activity of pan-HER (afatinib and neratinib) and PIK3CA (copanlisib) inhibitors, alone and in combination, against tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway (71%). Tumors harboring ERBB2 (5.8%) domain mutations were significantly more sensitive to single agents afatinib or neratinib when compared to wild-type tumors in preclinical in vitro and in vivo models (P = 0.001). In contrast, pan-HER and PIK3CA inhibitors demonstrated limited in vitro activity and were only transiently effective in controlling in vivo growth of PIK3CA-mutated cervical cancer xenografts. Importantly, combinations of copanlisib and neratinib were highly synergistic, inducing long-lasting regression of tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway. These findings define the genetic landscape of cervical cancer, suggesting that a large subset of cervical tumors might benefit from existing ERBB2/PIK3CA/AKT/mTOR-targeted drugs.

Keywords: HER2/neu; PIK3CA; cervical cancer; copanlisib; neratinib.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Somatic variation pattern underlying cervical cancer. Distribution of number of protein-altering somatic mutations and copy number variations in 69 cervical cancers. Representative significantly mutated genes are listed vertically.
Fig. 2.
Fig. 2.
Schematic representation of ERBB2, PIK3CA, and HUWE1 functional domains and mutation conservation analysis. Somatic mutations observed are shown. Green dots represent each case of the indicated mutation, and purple and black dots represent truncating alterations.
Fig. 3.
Fig. 3.
ERBB2 extracellular domain mutant cervical cancers are sensitive to ERBB2 inhibition by afatinib or neratinib in vitro and in vivo. (A and B) Mean IC50 values of ERBB2-mutated cervical carcinoma (CC) compared to ERBB2 wild-type CC cell lines (P < 0.0001) in response to afatinib (A) and to neratinib (B). (C) Tumor growth inhibition and (D) overall survival (OS) in response to afatinib and neratinib in a representative Her2/neu-mutated xenograft (CVX-4). The control group received vehicle per os (PO), the afatinib group 25 mg/kg afatinib, and the neratinib group 40 mg/kg neratinib (tumor volume [TV] control vs. afatinib P = 0.001 and control vs. neratinib P = 0.0002, OS control vs. afatinib P = 0.0001 and OS control vs. neratinib P = 0.0001).
Fig. 4.
Fig. 4.
Combinations of pan-HER and PIK3CA inhibitors induce synergistic in vitro and in vivo responses in tumors with derangements in the ERBB2/PI3K/AKT/mTOR pathway. (A) Cell viability of the PIK3CA-mutated and amplified CVX-5 primary cell line treated with neratinib, copanlisib, and their combination for 72 h. Cell viability was analyzed by flow cytometry and was normalized to the mean of the untreated control (Materials and Methods) and expressed as mean ± SEM; 3 independent experiments were performed (*P < 0.05 when compared to the control, to neratinib, and to copanlisib). (B and C) In vivo treatment of CVX-5 xenografts. Mice were treated with vehicle, neratinib (20 mg/kg), copanlisib (5 mg/kg), or the combination of the two for up to 30 d. Tumor volumes are reported as mean ± SEM. Tumor growth is significantly different between control (CTRL) and the treated groups (CTRL vs. copanlisib, neratinib, and combination, P = 0.001, P = 0.0002, and P < 0.0001, respectively). Similarly, the OS shows significant difference (P = 0.0001, P = 0.0001, and P < 0.0001, respectively). DMSO, dimethyl sulfoxide; IV, intravenous.
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