Clinical Response to a Lapatinib-Based Therapy for a Li-Fraumeni Syndrome Patient with a Novel HER2 V659E Mutation

Published OnlineFirst August 15, 2013; DOI: 10.1158/2159-8290.CD-13-0132

RESEARCH BRIEF

Violeta Serra 1 , Ana Vivancos 1 , Xose S. Puente 4 , Enriqueta Felip 1 , Daniel Silberschmidt 1 , Ginevra Caratù 1 , Josep-Lluís Parra 1 , Leticia De Mattos-Arruda1 , Judit Grueso 1 , Javier Hernández-Losa 2 , Joaquín Arribas 1 , 3, Ludmila Prudkin 1 , Paolo Nuciforo 1 , Maurizio Scaltriti 5 , Joan Seoane 1 , 3, and José Baselga 1 , 5

ABSTRACT Genomic characterization of recurrent breast and lung tumors developed over the course of 10 years in a 29-year-old patient with a germline TP53 mutation (Li- Fraumeni Syndrome) identifi ed oncogenic alterations in the HER2 and EGFR genes across all tumors, including HER2 amplifi cations, an EGFR -exon 20 insertion, and the fi rst-in-humans HER2 V659E mutation showing a phenotypic convergent evolution toward HER2 and EGFR alterations. Following the identifi cation of HER2-activating events in the most recent lung carcinoma and in circulating tumor cells, we treated the reminiscent metastatic lesions with a lapatinib-based therapy. A symptomatic and radiologic clinical response was achieved. HER2 V659E sensitivity to lapatinib was confi rmed in the laboratory.

SIGNIFICANCE: The precise knowledge of the genomic alterations present in tumors is critical to selecting the optimal treatment for each patient. Here, we report the molecular characterization and clinical response to a lapatinib-based therapy for the tumors of a Li-Fraumeni patient showing prevalence of HER2 and EGFR genomic alterations. Cancer Discov; 3(11); 1–7. ©2013 AACR.

INTRODUCTION was detected in a control chest X-ray and was conﬁ rmed by computed tomography (CT) scan. A CT-guided needle We describe the case of a 29-year-old nonsmoker female aspiration of the lesion showed positivity for CK7 and who, in 2003, underwent a bilateral mastectomy due to a TTF1, suggesting a primary lung adenocarcinoma. A left malignant phyllodes tumor (MPT) and to a bilateral ductal lower lobectomy and lymphadenectomy was subsequently carcinoma in situ (DCIS; Fig. 1A ). The patient was treated performed and the pathology analysis showed a moder- with adjuvant tamoxifen from 2003 until 2008 (Fig. 1B ). In ately differentiated invasive lung adenocarcinoma with a November 2007, a nodule in the lower lobe of the left lung bronchioloalveolar pattern [non–small cell lung carcinoma

Authors’ Afﬁ liations: 1 Vall d’Hebron Institut d’Oncologia; 2 Vall d’Hebron Insti- Box 20, New York, NY 10065. Phone: 212-639-8000; Fax: 212-794-3182; tut de Recerca; 3 Institució Catalana de Recerca i Estudis Avançats (ICREA), E-mail: [email protected] ; and Joan Seoane, Translational Research Barcelona; 4 Instituto Universitario de Oncología, Universidad de Oviedo, Program, Vall d’Hebron Institut d’Oncologia, PgValld’Hebron 119-129, Oviedo, Spain; and 5 Human Oncology & Pathogenesis Program, Memorial 08035 Barcelona, Spain. Phone: 34-93-4894167; E-mail: jseoane@vhio Sloan-Kettering Cancer Center, New York, New York .net Note: Supplementary data for this article are available at Cancer Discovery doi: 10.1158/2159-8290.CD-13-0132 Online (http://cancerdiscovery.aacrjournals.org/). ©2013 American Association for Cancer Research. Corresponding Authors: José Baselga, Human Oncology & Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue,

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Therapeutic Response to Novel HER2 Mutation RESEARCH BRIEF

A Tumor sites

N2(2R) N1(10) N2(4R) PLC N2(7) MPT

DCIS N1(12) DCIS

NSCLC NSCLC PE

B Treatment timelines Year 2003 2008 2012

Disease MPT, DCIS NSCLC left NSCLC right PE, PLC

Bilateral mastectomy Lobectomy Segmentectomy Lymphadenectomy Lymphadenectomy Treatment TAM CDDP/DOC CDDP/PM Lap/P Lap/T

Figure 1. Tumor sites and treatment timelines. A, the different tumor localization for the MPT of 2 cm, the right DCIS of 1 cm (pT1mic, grade 3) and the left DCISs of 0.8 and 1 cm (pTis, grade 3), the non–small cell lung carcinoma (NSCLC) of the left lung of 1.6 cm (pT2N1), and the NSCLC of the right lung of 1 cm (T1N2M1). The pulmonary lymphangitic carcinomatosis (PLC) and the pleural effusion (PE) were not measurable. The hillar lymph nodes N1(10) (n = 2, of 0.5 cm), peribronchial N1(12) (n = 1, of 1 cm), subcarinal N2(7) (n = 2, of 9 and 3 mm), and paratracheal N2(2R) (n = 2, of 1.6 and 1 cm), N2(4R) (n = 1, of 3 cm), and N1(10) (n = 1, of 2 cm by CT scan) are also shown. B, the treatment timelines received by the patient. From 2003 until 2008, the patient received adjuvant tamoxifen (TAM). In 2008, following the lobectomy she received six cycles of cisplatin/docetaxel (CDDP/DOC). After the segmentectomy in 2012, she received two cycles of cisplatin/pemetrexed (CDDP/PM). On the basis of the identiﬁ cation of HER2-V659E, she received Lap/P, (oral daily lapatinib 1,000 mg/intravenous weekly paclitaxel 80 mg/m2 ).

(NSCLC) left] with visceral pleural invasion and involve- activating mutation HER2 V659E was identifi ed (Fig. 2 ). This ment of three N1 lymph nodes without N2 involvement result led us to consider that the tyrosine kinase inhibitor (pT2N1). The patient received four cycles of postoperative lapatinib could be a benefi cial treatment option. Although adjuvant cisplatin/docetaxel chemotherapy. In December this clinical decision was hypothesis-driven, we confi rmed 2011, a follow-up CT scan detected a 1-cm nodule in the that HER2 V659E-expressing cells were sensitive to lapatinib right lower lobe of the lung. In parallel, the left lung images (Fig. 3 ). Lapatinib was given daily at a dose of 1,000 mg in could not rule out the presence of pulmonary lymphangitic combination with paclitaxel 80 mg/m2 . Shortly after start- carcinomatosis (PLC). A segmentectomy of the right lower ing therapy, the patient experienced pain relief at the lower lung lobe and lymph node sampling was performed, show- left thorax and a pronounced improvement in breathing. ing a 1-cm TTF1+ invasive adenocarcinoma (suggesting lung Two months after the beginning of the treatment, a CT carcinoma origin, NSCLC right) and extensive invasion scan revealed a reduction in the pleural effusion in addition of N2 lymph nodes in the 2R, 4R, and 7 regions [N2(2R), to shrinkage of the nodule in the left hilus (Fig. 4 ). Three N2(4R), and N2(7)]. Given the pattern of tumors developed months after the beginning of the lapatinib/paclitaxel treat- by the patient, genetic counseling was provided and revealed ment, the administration of paclitaxel was discontinued that the patient carried a TP53R248W germline mutation, because of toxicity. Treatment with lapatinib continued, and leading to the diagnosis of Li-Fraumeni Syndrome (LFS). trastuzumab, an anti-HER2 antibody, was added (initial A postoperative CT scan confi rmed the presence of PLC in dose of 8 mg/kg intravenously, followed by 6 mg/kg every the left lung, and the patient started cisplatin/pemetrexed 3 weeks). The observation that HER2 was amplifi ed in cir- chemotherapy. After two chemotherapy cycles, the patient culating tumor cells (CTC) at this point in time, in addition reported the appearance of pain in the lower left thorax, to reports describing the clinical benefi t of trastuzumab in and a CT scan showed the presence of a new pleural effu- the context of HER2-mutant NSCLC, supported this thera- sion in the left lung, a 2-cm nodule in the left hilus, and peutic approach ( 1, 2 ). The disease was further stabilized for the previously detected PLC. As a result of whole-exome an additional 6 months. In total, the clinical benefi t lasted sequencing of her most recent tumor, the NSCLC right, an over 9 months.

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A Molecular alterations

Tumor HER2 Tumor ER PR HER2 Chr 17 EGFR PTEN Year area FISH sample (%) (%) IHC polysomy (H score) (H score) (%) ratio

MPT 80 ND ND ND ND ND 290 40 (60-40-0-0)

280 2003 DCIS right 25 70 60 3+ 3.58 No ND (0-10-0-90)

200 DCIS left 23 5 2 3+ 4.15 No ND (0-0-100-0)

2008 200 NSCLC left 75 ND ND 0 1.11 Yes 130 (0-30-40-30)

NSCLC right 40 ND ND 0 2.38 Yes 120 0 (100-0-0-0) 2012

N2(4R) 35 ND ND 0 1.71 Yes 280 50 (50-50-0-0)

B Exome sequencing AGAP2 ARID1B B4GALT2 C15orf2 C1orf9 C9orf82 CC2D2A DST DYRK4 FLG2 GAD2 GNL3 GPR65 GTPBP2 HDAC9 HNRNPA2B1 INTS2 KIAA0907 KIAA1147 KIF6 KLHL30 LDHAL6A MESTP3 MUC3A NAALADL2 OTUD7B PAPD4 PHYH PLCB1 PRDM5 PRR5L RAD50 RASGRF1 RNF146 SEC63 SERAC1 SLC1A3 SLC45A4 SPTY2D1 SYNE2 TAS2R7 TLL1 TTBK2 VEZT VPS41 WFDC13 ZNF14 ZNF408 CAPN6 CLASP1 EIF4A3 FAM65A GLRB GYG1 MEPCE MYO1E NDUFC2 NUP214 PARD6B PPP1R15B SEL1L3 SGK2 TAGAP TCF12 TMPO TOMM70A TRPC5 TTC30B USP40 DYNC2H1 EMR1 FAM47A FAM83D HLCS IGDCC3 SETBP1 TIE1 TKTL1 TMPRSS3 UBR5 ZNF527 PJA1 C5 C9orf79 CA13 CEP68 DGKH DSC1 ERBB2 IPCEF1 MTFR1 NTRK1 PABPC1L PLK2 PLXNC1 PREX2 RBM22 RYR2 SEMA4F TEP1 TGS1 TKTL1 ZIM2 ZNF518B ZNF608 ZNF716 KHDC1 MYOZ3 UMODL1 ASB3 CHAD DYNC1H1 EGFR OS9 SEC16B ERBB2 year Tumor sample DCIS right 2003 DCIS left 2008 NSCLC left NSCLC right 2012 N2(4R)

Mutations Indels Amplifications

C HER2 and EGFR alterations

Year Tumor sample HER2 EGFR

DCIS right Amp — 2003 DCIS left Amp —

2008 NSCLC left — p.A767_S768insSVD

NSCLC right Amp, p.V659E — 2012 N2(4R) 20% cells Amp p.A767_S768insSVD

Figure 2. Molecular characterization of the tumors. A, the estrogen receptor (ER), progesterone receptor (PR), HER2, EGF receptor (EGFR), and PTEN levels were quantifi ed by immunohistochemistry (IHC) or FISH. ND, not determined. B, exome sequencing analysis in fi ve tumor samples. Blue represents mutations and yellow insertion–deletion alterations. In bold, Cancer Gene Census–annotated genes. H score = (0 × percentage of unstained cells + 1 × percentage of weakly stained cells + 2 × percentage of moderately stained cells + 3 × percentage of strongly stained cells). For PTEN, the respective percent- ages are provided in parentheses. The table in C summarizes the HER family molecular alterations in the fi ve tumors analyzed. Amp, amplifi cation.

RESULTS observed in NSCLC right in 65% of tumor cells, whereas no amplifi cation was found in NSCLC left. Interestingly, 23% of Molecular Characteristics of Tumors and CTCs tumor cells presented HER2 amplifi cation in N2(4R) (Sup- To identify potential actionable mutations, molecu- plementary Fig. S1). Similarly, CTCs exhibited HER2 gene lar pathologic analysis and exome sequencing of the MPT, amplifi cation. PTEN (and PIK3CA later on) were assessed DCISs, NSCLCs, and N2(4R) were performed ( Fig. 1A and B ). because of their potential negative predictive value toward Tumors were analyzed for the expression level of HER2, HER2/EGFR therapy (3–6 ). Although PIK3CA was wild-type EGF receptor (EGFR), estrogen receptor (ER), progesterone in all samples, PTEN expression displayed inter- and intratu- receptor (PR), and PTEN by immunohistochemistry (IHC), moral heterogeneity ( Fig. 2A ). and HER2 and EGFR gene amplifi cation by FISH (Fig. 2A ). Whole-exome sequencing was performed on DNA High expression of EGFR was found in the MPT and in the extracted from formalin-fi xed paraffi n-embedded (FFPE) N2(4R). HER2 overexpression was observed in both DCISs by tumor samples and from peripheral blood. We fi rst identifi ed IHC, and HER2 gene amplifi cation was confi rmed by FISH. the TP53-R248W mutation in the blood sample, confi rm- All three lung lesions showed Chr17 polysomy (the chro- ing the diagnosis of LFS. However, the analysis of somatic mosome that bears HER2 ). HER2 gene amplifi cation was variants presented two main diffi culties. The percentage

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A Biochemical activity of lapatinib B Antiproliferative activity of lapatinib Vector HER2 V659E

Lapatinib Vector 1.4 pHER2 Wild-type 1.2 V659E HER2 1.0 0.8 pEGFR 0.6 EGFR 0.4

Relative cell growth 0.2 pHER3 0.0 0.1 1 10 100 HER3 Lapatinib (µmol/L) pAKT

AKT

pERK

ERK

p4EBP1

Figure 3. HER2 V659E response to lapatinib in MCF10A cells. A, MCF10A cells overexpressing empty vector control, HER2 wild-type, or HER2 V659E were treated with increasing doses of lapatinib (0, 0.125, 0.5, and 2 μmol/L). Immunoblotting of the total and phosphorylated proteins was conducted. pERK, pAKT, and p4EBP1 are downstream biomarkers of HER2 activation. B, MCF10A cells overexpressing empty vector control, HER2 wild-type, or HER2V659E treated with increasing doses of lapatinib. Cell proliferation was superior in HER2 V659E-overexpressing cells. Error bars indicate SD of six independent experiments. Patient plasma concentration of 2.43 μg/mL = 2.58 μmol/L based on a 1,250 mg/daily dose.

of tumoral cells within the samples was diverse, and the insertions positioned at the loop following the C-helix of tumor samples were fi xed and conserved in paraffi n for up EGFR (for example, p.M766_A767insASV) have been shown to 10 years, generating DNA of variable quality. The analy- to be activating mutations, indicating that the insertion sis of MPT was not possible due to the low quality of harbored by our patient is likely to result in constitutive the DNA, and for the other samples, a high number of receptor activation. HER2 and EGFR somatic mutations were otherwise low-frequency C>T/G>A transitions was detected. confi rmed by capillary sequencing (Supplementary Fig. S2B). This likely represents cytosine deamination events caused by Interestingly, all tumors presented alterations in either HER2 the fi xation/storage of the samples and not tumor-specifi c or EGFR , both members of the HER family of tyrosine kinase somatic mutations, as suggested by the linear correlation receptors (Fig. 2C ). This indicates that HER-mediated signal- between number of C>T/G>A substitutions and the length ing confers a selective advantage to the tumors and exempli- of storage of the sample (data not shown). Both factors fi es a phenotypic convergent evolution toward a hyperactive prompted us to use stringent criteria for the identifi cation of HER pathway in all tumors of the LFS patient. genomic alterations (Fig. 2B ). Among the somatic mutations, A comparison of the somatic mutations and indels found in we identifi ed a transmembrane domain mutation of HER2 , the fi ve tumors analyzed showed that both DCISs were inde- HER2 -p.V659E (69% mutation allele frequency, MAF), in the pendent primary tumors, as they did not share any genomic NSCLC right. This mutation was a dinucleotide change [659 alteration between them or with the rest of the tumors V(GTT)-> E(GAA)] that has not been previously identifi ed (Fig. 2B and Supplementary Table S1). However, the analysis in human tumors and is orthologous to the well-described of the NSCLCs and the N2(4R) metastasis showed that these oncogenic mutation found in rat Her2 [664 V(GTG)->E(GAG) three samples were related. In support of this assessment, ( 7, 8 ); Supplementary Fig. S2A]. The high MAF of the HER2- the NSCLC left and the N2(4R) shared fi ve indels (including p.V659E mutation was in agreement with the HER2 amplifi - EGFR p.A767_S768insSVD) and two nonsynonymous muta- cation identifi ed by FISH. In addition, we identifi ed an indel tions, indicating that N2(4R) is a metastasis derived from in exon 20 of EGFR [p.A767_S768insSVD ( 9 )], in the 2008 the NSCLC left. On the other hand, N2(4R) and NSCLC tumor, NSCLC left (25% MAF), which was also present in the right contained cells with HER2 amplifi cation and, moreover, N2(4R) lymph metastases (11% MAF). EGFR exon 20 inser- both NSCLCs shared one indel as well as polysomy of chro- tions are infrequent, accounting for up to 4% of all EGFR mosomes 14, 16, and 17 (Supplementary Fig. S3A and S3B). mutations (9 ), and although no functional studies of our These results suggested a phylogenetic relationship between patient- specifi c EGFR indel have been reported, neighboring them.

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Radiologic response to lapatinib/paclitaxel

March 19, 2012 June 11, 2012

PLC

Figure 4. Radiologic response to lapatinib/paclitaxel. The ﬁ gure shows images from a CT scan of the pleural effusion [PE; and N2(10) lymph node adenopathy; green arrows] and the PLC (diffused image) previous to, and their reduction under, lapatinib/paclitaxel treatment.

Functional Analysis of HER2 V659E that lapatinib at clinically achievable concentrations could be The identifi cation of the HER2V659E in the most recent benefi cial for the patient. tumor prompted us to consider a therapeutic approach based on lapatinib. Studies using the MMTV-c-neu mouse model (a mouse model for breast cancer generated by the overexpres- DISCUSSION sion of the rat ortholog of HER2 V659E ) have shown evidence of The identifi cation of tumor driver genetic alterations, like the antitumor activity of lapatinib ( 10, 11 ). the ones observed in the HER family of tyrosine kinase We decided to functionally validate the sensitivity of receptors, has led to novel therapeutic approaches that have HER2V659E to lapatinib in a preclinical in vitro model. HER2V659E dramatically improved patient outcomes. The paradigmatic was overexpressed in MCF10A cells and, as expected, high levels example is the discovery of HER2 gene amplifi cation in breast of HER2 V659E led to hyperphosphorylation of the receptor and cancer and the clinical response of HER2 -amplifi ed tumors to activation of the downstream signaling ( Fig. 3A ). Lapatinib the anti-HER2 monoclonal antibody trastuzumab (13, 14 ). inhibited HER2 V659E and EGFR signaling in a dose-dependent In addition to trastuzumab, the tyrosine kinase inhibitor manner. At low concentrations of lapatinib the HER2 V659E - lapatinib has also been approved as the therapy for HER2- mutant cells were less sensitive to treatment than control cells, amplifi ed tumors, thanks to its ability to reversibly inhibit suggesting that the mutation could confer resistance to the the intracellular tyrosine kinase activity of HER2 and down- treatment under these conditions, an observation that was stream pathways, such as phosphoinositide 3-kinase (PI3K)/ extended to other EGFR/HER2 –targeting agents (Supplemen- AKT and extracellular signal—regulated kinase (ERK; refs. tary Fig. S4). Importantly, when the inhibitor was used at a 15, 16). clinically achievable concentration of 2 μmol/L ( 12 ), MCF10A- In addition to HER2 gene amplifi cation, recent studies expressing HER2 V659E exhibited the same sensitivity to lapat- have shown that the HER2 gene can also be altered by somatic inib as control cells (Fig. 3B ). This reinforced our hypothesis gene point mutations and insertions and deletions (indels;

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Therapeutic Response to Novel HER2 Mutation RESEARCH BRIEF

refs. 17–23 ). Functional analysis in preclinical models has based treatment for our patient. Notably, this therapeutic shown that some of these HER2 gene mutations can induce regimen led to clinical benefi ts with an objective decrease in oncogenesis and sensitize tumors to anti-HER2 treatments tumor size and pleural effusion, relief of thoracic pain, and ( 23 ). However, little is known about the clinical response of breathing improvement. The potential role of HER tyrosine HER2 -mutant tumors to anti-HER2 therapies ( 1, 2 ), and novel kinase inhibitors in the therapy for HER2 -mutant tumors irreversible EGFR/HER2 inhibitors are currently under clini- deserves a systematic evaluation; therefore, a clinical trial cal evaluation for the treatment of HER2 -mutant metastatic with the irreversible HER2 inhibitor neratinib in metastatic breast cancer (NCT01111825) and NSCLC (NCT01827267). breast cancer is currently under way (NCT01111825), and Here, we report the molecular characterization and clinical a “basket” clinical trial in patients with HER2 mutations response to a lapatinib-based therapy for the tumors of a from any other histologic origin is also in an advanced Li-Fraumeni patient showing prevalence of HER2 and EGFR planning stage. oncogenic driver alterations. In this study, we have performed whole-exome sequencing of the breast and lung tumors of a patient with LFS METHODS and identifi ed driver genomic alterations that are actionable. Please see Supplementary Data for additional methods. The observation of a phenotypic convergent evolution of the MCF10A cells were cultured in complete growth media as recom- tumors of the patient toward a hyperactive HER2 or EGFR mended by the vendor (American Type Culture Collection), supple- pathways and the discovery of the HER2 V659E mutation (to mented with selection antibiotic (hygromycin). MCF10A cells were not authenticated as they had undergone few passages after their our knowledge yet unreported in human samples) in her purchase. most recent tumors, as well as the identifi cation of HER2 gene amplifi cation in the patient CTCs, has allowed us to select a Exome Sequencing benefi cial treatment for our patient. This work is an example The patient provided written informed consent for somatic and of how personalized characterization of tumors can lead to germline DNA analysis. FFPE tumor samples from six primary therapeutic success. Despite the diffi culty of using FFPE- tumors, including a MPT, two DCISs (right and one left), two NSCLCs archived material, we have observed that the patient gener- (left and right), and one metastatic lymph node N2(4R), along with ated different independent primary tumors and that the three a blood sample from the patient were subjected to whole-exome most recent tumors were interrelated. Our data suggest that sequencing. Samples were initially assessed for tumor content based the two lung tumors and the lymph node metastasis located on a hematoxylin and eosin staining (Fig. 2A ). Genomic DNA was at N2(4R) have common origins and share tumor clones, most extracted from FFPE tumor samples using the DNA RecoverAll likely due to cross-seeding of metastasis. Despite the complex- Total Nucleic Acid Isolation Kit for FFPE (Ambion) according to ity and diversity of genomic alterations as well as the intricate the manufacturer’s instructions. Germline DNA was extracted from whole blood using the QIAamp DNA Blood Midi Kit (Qiagen). relationship between the tumors, we observed alterations in Library preparation was performed following the standard Illumina HER2 and EGFR across all tumors. These results suggest a protocol (Genomic Sample Prep) with slight modifi cations for FFPE- predisposition toward genomic alterations in HER family derived DNA samples. One microgram of DNA was decross-linked receptor tyrosine kinases in the context of the p53 germline by heat incubation, fragmented, ends repaired, and an adenine was mutation of the LFS. Whether this constitutes a general mech- ligated to each of the 3′ ends, where sample-specifi c adaptors were anism in patients with LFS and the exact mechanism involved linked. Libraries were amplifi ed using eight to 12 cycles of PCR, and in this phenomenon is something that needs to be evaluated. exome enrichment was performed using specifi c biotinylated probes The HER2 V659E mutation identifi ed in our patient is ortholo- (SureSelect XT Human All Exon 50Mb; Agilent). After enrichment, gous to the HER2 / neuT (HER2 V664E) allele that was originally the exome libraries were PCR-amplifi ed, quantifi ed, and loaded in identifi ed in rat tumors induced by chemical mutagenesis (7, 8). a HiSeq2000 sequencer (Illumina). Paired-end 100 base sequences were obtained and aligned to the reference genome using Burrows- Preclinical models that allowed the successful development of Wheeler Aligner. This yielded a median depth of coverage in targeted anti-HER2 therapies were based on this mutant form of HER2 regions between 13× and 80×, and with more than 83% of the exome ( 24, 25 ). Nevertheless, mutations affecting the V659 residue having at least 10 reads. Tumor samples were compared with normal within the transmembrane domain of HER2 have not been DNA as previously described ( 27 ). To remove artifacts likely caused identifi ed in human tumors until now. Of note, the human by cytosine deamination due to fi xation/long-term storage of FFPE HER2V659E mutation necessarily implies a tandem dinucleotide samples, and due to the presence of a low tumor cell content of some change (TT>AA), a type of mutation that may arise from samples, the following fi ltering criteria were applied: somatic vari- induced pyrimidine adducts in response to radiotherapy ( 26 ). ants were called when supported by at least three reads, representing × The patient did not receive radiotherapy; however, the dinucle- at least 10% of the total reads and with a minimum coverage of 35 . otide genomic alteration might have been caused by another Putative somatic mutations were manually inspected to remove false positives in homopolymer stretches and close to indels. type of irradiation, including the one from the frequent X-ray/ CT scans conducted on the patient. Disclosure of Potential Confl icts of Interest In summary, the thorough molecular characterization of J. Baselga is employed as a consultant at Novartis and Genentech. tumors from a patient with LFS has led to the identifi cation No potential confl icts of interest were disclosed by the other authors. of alterations in HER2 and EGFR, among those the fi rst-in- V659E humans HER2 mutation. The genomic characterization The Editor-in-Chief of Cancer Discovery (J. Baselga) is an author of of the patient, together with the preclinical analysis showing this article. In keeping with the AACR’s Editorial Policy, the paper that cells bearing the HER2 V659E mutation remain sensitive to was peer reviewed and a member of the AACR’s Publications Com- clinically achievable doses of lapatinib, supported a lapatinib- mittee rendered the decision concerning acceptability.

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Authors’ Contributions inib resistance that is reversed by the mTOR/phosphatidylinositol 3-kinase inhibitor NVP-BEZ235. Cancer Res 2008 ; 68 : 9221 – 30 . Conception and design: V. Serra, J. Arribas, M. Scaltriti, J. Seoane, 7. Bargmann CI , Hung MC , Weinberg RA . Multiple independent activa- J. Baselga tions of the neu oncogene by a point mutation altering the trans- Development of methodology: V. Serra, A. Vivancos, L. De Mattos-Arruda, membrane domain of p185. Cell 1986 ; 45 : 649 – 57 . J. Hernández-Losa, J. Baselga 8. Bargmann CI , Hung MC , Weinberg RA . The neu oncogene encodes an epi- Acquisition of data (provided animals, acquired and managed dermal growth factor receptor-related protein . Nature 1986 ; 319 : 226 – 30 . patients, provided facilities, etc.): V. Serra, A. Vivancos, E. Felip, 9. Yasuda H , Kobayashi S , Costa DB . EGFR exon 20 insertion mutations G. Caratù, L. De Mattos-Arruda, J. Grueso, J. Hernández-Losa, in non–small-cell lung cancer: preclinical data and clinical implica- J. Arribas, L. Prudkin, P. Nuciforo, J. Baselga tions. Lancet Oncol 2012 ; 13 : e23 – 31 . Analysis and interpretation of data (e.g., statistical analysis, 10. Burke CL , Stern DF . Activation of Neu (ErbB-2) mediated by disulfi de biostatistics, computational analysis): V. Serra, A. Vivancos, X.S. bond-induced dimerization reveals a receptor tyrosine kinase dimer Puente, D. Silberschmidt, L. De Mattos-Arruda, J. Arribas, P. Nuciforo, interface. Mol Cell Biol 1998 ; 18 : 5371 – 9 . J. Seoane, J. Baselga 11. Roberts PJ , Usary JE , Darr DB , Dillon PM , Pfefferle AD , Whittle MC , Writing, review, and/or revision of the manuscript: V. Serra, et al. Combined PI3K/mTOR and MEK inhibition provides broad A. Vivancos, E. Felip, L. De Mattos-Arruda, L. Prudkin, P. Nuciforo, antitumor activity in faithful murine cancer models . Clin Cancer Res M. Scaltriti, J. Seoane, J. Baselga 2012 ; 18 : 5290 – 303 . Administrative, technical, or material support (i.e., reporting 12. Chu QS , Schwartz G , de Bono J , Smith DA , Koch KM , Versola MJ, et al. Phase I and pharmacokinetic study of lapatinib in combination or organizing data, constructing databases): V. Serra, J.-L. Parra, with capecitabine in patients with advanced solid malignancies. J Clin L. De Mattos-Arruda Oncol 2007 ; 25 : 3753 – 8 . Study supervision: V. Serra, J. Seoane, J. Baselga 13. Slamon DJ , Clark GM , Wong SG , Levin WJ , Ullrich A , McGuire WL . Human breast cancer: correlation of relapse and survival with ampli- Acknowledgments fi cation of the HER-2/neu oncogene. Science 1987 ; 235 : 177 – 82 . The authors thank the patient for her willingness to participate in 14. Slamon DJ , Leyland-Jones B , Shak S , Fuchs H , Paton V , Bajamon d e A , this study and her family for always supporting her positive attitude. et al. Use of chemotherapy plus a monoclonal antibody against HER2 The authors also thank Orland Díez and Judith Balmaña (for genetic for metastatic breast cancer that overexpresses HER2. N Engl J Med counseling), and Alicia Garcia and Cristina Saura (breast oncologists) 2001 ; 344 : 783 – 92 . for technical contribution and valuable discussions of the clinical case. 15. Wood ER , Truesdale AT , McDonald OB , Yuan D , Hassell A , Dicker - son SH , et al. A unique structure for epidermal growth factor recep- Grant Support tor bound to GW572016 (Lapatinib): relationships among protein conformation, inhibitor off-rate, and receptor activity in tumor cells. This work has been supported by the European Research Coun- Cancer Res 2004 ; 64 : 6652 – 9 . cil grants (ERC AdG09 250244 to J. Baselga and ERC 205819 to J. 16. Geyer CE , Forster J , Lindquist D , Chan S , Romieu CG , Pienkows k i T , Seoane), Instituto de Salud Carlos III (ISCIII) grants (PSO9/00623 to et al. Lapatinib plus capecitabine for HER2-positive advanced breast J. Baselga, PI070648 to J. Seoane, and PI1202536 to J. Arribas), and cancer. N Engl J Med 2006 ; 355 : 2733 – 43 . Redes Temáticas de Investigación Cooperativa en Salud (RETIC)-ISCIII 17. Banerji S , Cibulskis K , Rangel-Escareno C , Brown KK , Carter SL , (RD06/06/0020/0075 to J. Baselga, RD12/0036/0043 to J. Seoane, and Frederick AM , et al. Sequence analysis of mutations and transloca- RD12/0036/0042 to J. Arribas), Asociación Española contra el Cancer tions across breast cancer subtypes. Nature 2012 ; 486 : 405 – 9 . AECC grants (to J. Seoane and J. Arribas), a Rafael del Pino Founda- 18. Lee JW , Soung YH , Seo SH , Kim SY , Park CH , Wang YP , et al. Somatic tion grant (to J. Baselga), a private donation from the Orozco family mutations of ERBB2 kinase domain in gastric, colorectal, and breast through the Oncology Research Foundation, FERO (to J. Baselga), and carcinomas . 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OF7 | CANCER DISCOVERYNOVEMBER 2013 www.aacrjournals.org

Clinical Response to a Lapatinib-Based Therapy for a Li-Fraumeni Syndrome Patient with a Novel HER2V659E Mutation

Violeta Serra, Ana Vivancos, Xose S. Puente, et al.

Cancer Discovery Published OnlineFirst August 15, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/2159-8290.CD-13-0132

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