ERBB2 Alterations Research Articles

ERBB2 gene as a potential therapeutic target in small bowel adenocarcinoma.

205 Background: Small bowel adenocarcinoma (SBA) is an aggressive tumor responsible for poor outcomes with an expected median OS at 5 years inferior to 30%. Because of its low incidence, few prospective studies have been performed leading to insufficient knowledge and absence of standard of care. Aiming to better understand the small bowel carcinogenesis process we screened for somatic mutations a large data set of patients in more than 740 mutational hotspots among 46 genes. Methods: In total, 83 SBA cases were selected from 2 European databases. The sequencing was performed using the Ion 316 Chip. We additionally looked into MSI status and ERBB2 expression (immunochemistry and FISH). Results: Tumors were mostly located in the duodenum (47%) and stage ≥ 3 (63%). Among the 46 genes investigated, 8 were mutated in more than 5% of the cases: KRAS, TP53, APC, SMAD4, PIK3CA, ERBB2, BRAF, and FBXW7. Compelling mutations (7 cases) and amplifications (3 cases) 12% of the patients had an ERBB2 alteration. In this group, ERBB2 alterations were positively associated with dMMR status (p=0.006) and APC mutations (p=0.02) but negatively associated with p53 mutations (p=0.038). Interestingly, ERBB2 mutations were mainly detected within tumors derived from the foregut while ERBB2 amplifications were limited those derived from the midgut. Conclusions: This study describes the first large screening of somatic mutations in SBA using next generation sequencing. ERBB2 mutation was revealed to be one of the most frequent alterations in SBA with a distribution dependent on tumor location. In most cases patients harbored the same p.L755S-ERBB2 mutation. In clinical practice, this study may suggest that more than 10% of the patients with SBA could be treated using anti-ERBB2-targeted agents.[Table: see text]

A High Frequency of Activating Extracellular Domain ERBB2 (HER2) Mutation in Micropapillary Urothelial Carcinoma

Micropapillary urothelial carcinoma (MPUC) is a rare and aggressive form of bladder cancer. We conducted genomic analyses [next-generation sequencing (NGS)] of MPUC and non-micropapillary urothelial bladder carcinomas (non-MPUC) to characterize the genomic landscape and identify targeted treatment options. DNA was extracted from 40 μm of formalin-fixed paraffin-embedded sections from 15 MPUC and 64 non-MPUC tumors. Sequencing (NGS) was performed on hybridization-captured, adaptor ligation-based libraries to high coverage for 3,230 exons of 182 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer. The results were evaluated for all classes of genomic alteration. Mutations in the extracellular domain of ERBB2 were identified in 6 of 15 (40%) of MPUC: S310F (four cases), S310Y (one case), and R157W (one case). All six cases of MPUC with ERBB2 mutation were negative for ERBB2 amplification and Erbb2 overexpression. In contrast, 6 of 64 (9.4%) non-MPUC harbored an ERBB2 alteration, including base substitution (three cases), amplification (two cases), and gene fusion (one case), which is higher than the 2 of 159 (1.3%) protein-changing ERBB2 mutations reported for urinary tract cancer in COSMIC. The enrichment of ERBB2 alterations in MPUC compared with non-MPUC is significant both between this series (P < 0.0084) and for all types of urinary tract cancer in COSMIC (P < 0.001). NGS of MPUC revealed a high incidence of mutation in the extracellular domain of ERBB2, a gene for which there are five approved targeted therapies. NGS can identify genomic alteration, which inform treatment options for the majority of MPUC patients.

Abstract B200: A new resource enabling the practical utility of targeted therapy in oncology clinics.

Abstract Academic oncology now recognizes that a cancer's genetic signature may be as important in clinical decision-making as its tissue of origin. However, the degree to which this revolution in thinking has penetrated the clinic is unknown. Our commercial platform, which operates at the point of contact between patients and their physicians, is ideally positioned to address this question. After clinical samples provided by oncologists are analyzed by any molecular diagnostic platform, our internal experts interpret the results using our knowledge integration platform and provide treatment-strategy roadmaps to guide future interventions. Since 2008, approximately 4000 patients have been assessed using this methodology. Overall, the practical utility of the platform has been high: over 70% of cases across all cancer types have revealed actionable results, defined as a molecular profile that suggests either rational on- or off-label use of an FDA-approved agent or enrollment into a clinical trial. Among the 130 patients for whom follow-up is available, use of this platform has resulted in a known effect on therapy selection in 35% of cases and a potential future effect on therapy in an additional 61% of cases. The utility of the approach has also been demonstrated through acceptance by oncologists; 36% of patients who have engaged the service were referred by their oncologists. Although not rising to the rigor of prospective randomized trial data, information in our database may be used in hypothesis generation. For example, while ERBB2 amplification is common in breast cancer, ERBB2 mutations have been documented in a small percentage of non-small cell lung cancer (NSCLC), and were detected in 4% (19/450) of NSCLC cases in our dataset. Mutations or copy number alterations in ERBB2 have also been identified in our dataset in 4% (7/195) of colorectal cancers, 6% (4/68) of urothelial carcinomas, and 10% (4/39) of endometrial carcinomas. In one case, a 61-year-old woman with Stage IV NSCLC presented with a secondary metastasis on the pleura, undergoing treatment with carboplatin and pemetrexed chemotherapy, following earlier treatment of a primary lung tumor with surgery and radiation. Multiplex sequencing identified a somatic exon 20 insertion (AYVM 771-774) in ERBB2, and subsequent treatment with trastuzumab resulted in nearly two years of progression-free survival. These results are consistent with published case studies in this patient population and suggest that clinical trials examining trastuzumab in NSCLC patients with ERBB2 exon 20 mutations may be warranted. In conclusion, our data demonstrate the increasing importance of targeted therapy in clinical oncology. They also demonstrate how databases such as ours, which document uncommon targetable molecular alterations, may have significant impact on real-time treatment decision-making and have potential as hypothesis-generating resources for future clinical trials. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B200. Citation Format: Sheryl K. Elkin, Linda Call, Jo-Ellen Murphy, Kelly Sullivan, Jennifer Carter. A new resource enabling the practical utility of targeted therapy in oncology clinics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B200.

Abstract B122: A high frequency of activating extracellular domain ERBB2 (HER2) mutation in micropapillary urothelial carcinoma.

Abstract Background: Micropapillary urothelial carcinoma of the urinary bladder (MPUC) encompasses approximately 5% of all bladder cancers and comprises approximately 3,000 to 4,000 new cases diagnosed each year in the US. MPUUPC is a highly aggressive form of bladder cancer associated with distant metastases and shortened patient survival. Once MPUC recurs progresses from loco-regional and progresses to metastatic disease, there is no currently no recognized effective treatment. We conducted a genomic analysis of a series of patients with MPUC to characterize the genomic landscape of MUPUC and identify targeted treatment options for patients with this lethal form of urologic malignancy. Methods: DNA was extracted from 40 microns of formalin-fixed paraffin embedded (FFPE) sections from 15 MPUC and 64 non-MPUC. Sequencing to high, uniform coverage was performed on hybridization-captured, adaptor ligated, hybridization capturedion based libraries for for 3,230 exons of 182 cancer-related genes plus 37 introns offrom 14 genes frequently rearranged in cancer to high, uniform coverage and evaluated for all classes of genomic alteration. Results: Extracellular domain mutations of ERBB2 were identified in 6/15 (40%) MPUC including: S310F (4 cases), S310Y (1 case) and R157W (1 case). All 6 cases of MPUC with ERBB2 mutation were negative for ERBB2 amplification which was confirmed by immunohistochemistry (IHC) in the 3 cases where additional tissue was available. In contrast, 6/64 (9.4%) of non-MPUC harbored an ERBB2 alteration: base substitutions (3 cases), amplifications (2 cases) and gene fusion (1 case), which is higher than the 2/159 (1.3%) of protein changing ERBB2 mutations reported in urinary tract cancer in COSMIC. The enrichment of ERBB2 alterations in MPUC compared to non-MPUC is significant inbetween this series (p &amp;lt; 0.0084) and for all types of urinary tract cancer in COSMIC (p &amp;lt; 0.001). All 9 ERBB2 WT MPUC cases harbored at least 1 actionable alteration, including alterations in AKT1, AKT2, CCND1, EGFR, PIK3CA, PIK3R1 and RAF1. Conclusions: Comprehensive genomic profiling of MPUC revealed actionable genomic alterations in all 15 specimens including a high incidence of ERBB2 extracellular domain mutation. We conclude that genomic profiling of MUPUC samples can reveal actionable alterations that can inform potential targeted treatment decisions for the majority of patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B122. Citation Format: Kai Wang, Jeff S. Ross, Laurie M. Gay, Rami N. Al-Rohil, Tipu Nazeer, Christine E. Sheehan, Timothy A. Jennings, Geoff A. Otto, Amy Donahue, Jie He, Gary Palmer, Siraj Ali, Michelle Nahas, Geneva Young, Elaine LaBrecque, Garrett Frampton, Rachel Erlich, John A. Curran, Tina Brennan, Sean R. Downing, Roman Yelensky, Doron Lipson, Matthew J. Hawryluk, Vincent A. Miller, Philip J. Stephens. A high frequency of activating extracellular domain ERBB2 (HER2) mutation in micropapillary urothelial carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B122.

An analysis of ERBB2 alterations (amplifications and mutations) found by next-generation sequencing (NGS) in 2000+ consecutive solid tumor (ST) patients.

11000 Background: Testing for ERBB2 amplification by FISH and IHC is routine in breast and gastro-esophageal cancer. There are 3 approved and multiple targeted therapies in clinical trials that rely on the results of these tests. ERBB2 amplification and activation by mutation/fusion has also been described in a wide variety of other ST. As these alterations are not routinely tested for but may predict response to anti-ERBB2 agents we sought to determine their frequency in an unselected cohort of specimens from advanced ST patients. Methods: We reviewed genomic profiles from the first 2,223 formalin-fixed, paraffin-embedded specimens received and analyzed by our CLIA-certified lab (Foundation Medicine) with our NGS platform. 3,230 exons in 182 cancer-related genes and 14 genes frequently rearranged were assayed for base pair substitutions, small insertions/deletions (indels), amplifications, and rearrangements. Results: 110/2,223 (4.9%) specimens had 116 ERBB2 alterations: 67 (58%) amplifications, 29 (25%) substitutions, 16 (14%) indels, 2 (2%) splice site variants and 2 (2%) translocations, including a potential fusion. Six samples (5%) had multiple alterations, and two had both ERBB2 substitution and amplification. 14 ST types had evidence of ERBB2 alterations including 29% of esophageal, 20% of uterine, 14% of breast, and 12% of stomach carcinomas. 6% of all lung cancer samples had ERBB2 alterations. Amplifications predominated, but lung specimens had predominantly indels. Durable responses exist to anti-ERBB2 agents in STs with activating ERBB2 mutations. Conclusions: Use of a broad NGS panel identifies an unprecedented number of actionable genomic changes including a significant rate of ERBB2 alterations across 14 different solid tumor types. The discovery of unanticipated ERBB2 amplifications and activating mutations in a wide variety of ST highlights the need to study a broad range of genes at a high level of sensitivity and specificity when searching for novel targets of therapy. Widespread use of this approach could provide more treatment options and enable more rapid accrual to ongoing and planned trials of agents targeting pathways under study.

Highlights of This Issue

Next-generation sequencing (NGS) can be applied to routine formalin-fixed paraffin-embedded clinical samples as a means to further classify the lobular phenotype of invasive breast cancer into tumors with and without E-cadherin (CDH1) mutations. When Ross and colleagues used this approach to evaluate invasive lobular carcinoma (ILC) with CDH1 mutation, they found a significant enrichment of genomic alterations in the targetable HER2 (ERBB2) gene. Of the 22 CDH1-mutated ILCs evaluated in this study, 6 (27%) featured ERBB2 alterations, only 1 of which was a classic gene amplification that was detectable by the routine slide-based ERBB2/HER2 tests in common practice (immunohistochemistry and FISH). The 5 gene amplification-negative ERBB2 alterations detected included 4 base substitutions (mutations) and 1 novel ERBB2-GRB7 gene fusion. Recent evidence suggests that ERBB2 mutations may be sensitive to standard anti-HER2 targeted therapies in breast and other cancers. Thus, given the relatively high incidence of treatable ERBB2 alterations in relapsed and metastatic CDH1-mutated ILC, the use of NGS to identify these alterations which cannot be detected by routine slide-based tests has the potential to change routine clinical practice.Treatment of patients with BRAFV600E colorectal cancer is a critical unmet medical need. To address this deficiency, Coffee and colleagues have developed a novel genetically engineered mouse model for BRAFV600E colorectal cancer. Their results suggest that BRAFV600E tumors are not sensitive to single-agent BRAF inhibition and implicate concurrent signaling through the parallel phosphoinositide 3-kinase (PI3K)/mTOR signaling axis. Using a combination of in vitro and in vivo approaches, the authors show that simultaneous targeting with both a BRAF inhibitor and a PI3K/mTOR inhibitor may be required for successful treatment in this population of colorectal cancer patients.Targeting “undruggable targets,” such as transcription factors, is a challenge. Varsano and colleagues address this task by focusing on altering the transcription factor's subcellular localization. Based on their findings that subcellular localization dictates ATF2 oncogenic (nuclear) or tumor-suppressive (cytosolic) function, they developed a high-content screen and used it to identify small molecules that promote ATF2 nuclear export. The hits identified were shown to sensitize melanoma to cell death along the protein kinase C signaling pathway, which drives ATF2 nuclear localization and oncogenic activity in these tumors. The proof-of-concept screen reported in this study provides the foundation for larger screening campaigns as well as further development and characterization of the small molecules that were identified.Jimeno and colleagues evaluated the Smoothened receptor inhibitor IPI-926 in advanced cancer patients with a focus on basal cell carcinoma of the skin. The uniqueness of this phase I trial is that IPI-926 is a direct derivative of cyclopamine, the plant alkaloid whose discovery enhanced our understanding of the hedgehog pathway (HhP). The HhP is dysregulated in a variety of solid tumors and is proposed to provide key growth and survival signals to tumor cells. IPI-926 was administered with a tolerable toxicity profile in patients with advanced solid tumors and has shown substantial efficacy in patients with basal cell carcinoma.

Relapsed Classic E-Cadherin (CDH1)–Mutated Invasive Lobular Breast Cancer Shows a High Frequency ofHER2(ERBB2) Gene Mutations

We queried whether comprehensive genomic profiling using a next-generation sequencing-based assay could identify novel and unanticipated targets of therapy for patients with relapsed invasive lobular carcinoma (ILC). DNA sequencing (Illumina HiSeq 2000) was conducted for 3,320 exons of 182 cancer-related genes and 37 introns of 14 genes frequently rearranged in cancer on indexed, adaptor-ligated, hybridization-captured libraries using DNA isolated from formalin-fixed paraffin-embedded sections from 22 histologically verified ILC. A total of 75 genomic alterations were identified with an average of 3.4 alterations per tumor (range, 1-6), of which 35 were actionable for an average of 1.59 actionable alterations per patient (range, 0-3). Nineteen of 22 (86%) of the ILC samples harbored at least one actionable alteration. Six (27%) cases featured alterations in ERRB2 including 4 (18%) with ERBB2 mutation, 1 (5%) with an ERBB2 gene fusion, and 1 (5%) with an ERBB2 copy number gain (amplification). The enrichment of ERBB2 mutations/fusion in CDH1-mutated ILC (5 of 22, 23%) compared with the 5 ERBB2 mutations in a series of 286 non-CDH1-mutated breast cancers from which the ILC cases were obtained (5 of 286, 2%) was significant (P = 0.0006). Comprehensive genomic profiling of relapsed CDH1-mutated ILC revealed actionable genomic alterations in 86% of cases, featured a high incidence of ERBB2 alterations, and can reveal actionable alterations that can inform treatment decisions for patients with ILC.

929 NEXT-GENERATION DEEP SEQUENCING IDENTIFIES RECURRENT AND TARGETABLE GENETIC ALTERATIONS IN HIGH GRADE BLADDER UROTHELIAL CARCINOMA

INTRODUCTION AND OBJECTIVES: High grade urothelial carcinoma (UC) is an aggressive malignancy with limited therapeutic options in the advanced, metastatic, or recurrent disease setting. Targeted genetic analysis of high grade tumors was performed to determine the prevalence of known cancer genes and to identify potential targets for therapy. METHODS: Frozen high grade bladder tumors and matched germline blood DNA were obtained from 60 patients undergoing transurethral resection (TUR) or cystectomy. Specimens were analyzed using a targeted, deep-sequencing assay designed to identify point mutations, indels, and copy number alterations in 275 cancer-associated genes. RESULTS: The cohort was predominantly male (N 49, 81%) with a median age of 71 (IQR 61, 76). Frozen tumor was obtained from radical cystectomy(RC) in 53 patients (88%), TUR in 6 (10%), and partial cystectomy in 1 (2%). 11 patients (18%) had a history of intravesical therapy. Tumor pathologic stage was Ta in 5 (8%), T1 in 5 (8 %), T2 in 11 (18%), T3 in 30 (50%), and T4 in 9 (15%). Of the 53 patients undergoing RC and lymph node dissection, 17 received neoadjuvant chemotherapy (32%) and 24 (45%) had lymph node metastases. Mean target coverage for all sequenced exons was 512X. The most common genomic event was somatic mutation of TP53, identified in 33 patients (55%). RB mutations were identified in 11 (18%). Alterations in the PI3K/AKT/mTOR signaling pathway were also common; 12 patients had mutations in PIK3CA (20%), and non-overlapping mutations were identified in other genes within the pathway including AKT1, PTEN, and MTOR. Frequent mutations in chromatin remodeling genes were also identified. KDM6A mutations were identified in 21 tumors (35%), MLL2 in 18 (30%), and ARID1A in 17 (28%). Overall, 60% of all analyzed tumors harbored potentially targetable genomic alterations, including those with alterations in ERBB2, BRAF, and FGFR3 (Figure). CONCLUSIONS: Genetic alterations are frequent in high grade bladder UC. Over half of the tumors analyzed harbor alterations in genes that have been targeted for therapeutic benefit in other solid tumors, thus providing a compelling case for clinical trials using novel agents. Analysis for correlations with clinical outcomes is ongoing to assess for the prognostic significance of these genetic events. Source of Funding: Sidney Kimmel Center for Prostate and Urologic Cancers, Michael and Zena Wiener Research and Therapeutics Program in Bladder Cancer

Activating Mutations in ERBB2 and Their Impact on Diagnostics and Treatment

Despite the ongoing “war on cancer,” cancer remains one of the major causes of human morbidity and mortality. A new paradigm of targeted therapies holds the most promise for the future, making identification of tumor-specific therapeutic targets of prime importance. ERBB2/HER2, best known for its role in breast cancer tumorigenesis, can be targeted by two types of pharmacological manipulation: antibody therapy against the extracellular receptor domain and small molecule compounds against the intracellular tyrosine kinase domain. Aberrant activation of ERBB2 by gene amplification has been shown to participate in the pathophysiology of breast, ovarian, gastric, colorectal, lung, brain, and head and neck tumors. However, the advent of next-generation sequencing technologies has enabled efficient identification of activating molecular alterations of ERBB2. In this review, we will focus on the functional role of these somatic mutations that cause ERBB2 receptor activation. We will additionally discuss the current preclinical and clinical therapeutic strategies for targeting mutationally activated ERBB2.

Use of next-generation sequencing (NGS) to detect high frequency of targetable alterations in primary and metastatic breast cancer (MBC).

10559 Background: The aim of this study was to assess the frequency of genomic alterations in breast cancer potentially treatable with approved targeted agents or investigational drugs in clinical trials. NGS was performed in a CLIA setting (Foundation Medicine). Methods: DNA was extracted from needle biopsies of 33 pre-therapy primary and 17 MBCs (mean age 52 yrs; 58% ER+, 20% HER2+, 30% triple negative) obtained prospectively for biomarker discovery and preserved in RNAlater. Patients with MBC received an average of 7 drugs (range 5-17) including adjuvant therapy before biopsy for this research; 13 biopsies were from soft tissues, 3 from liver and 1 from bone. Sequencing was targeted to 3230 exons in 182 cancer-related genes and 37 introns in 14 genes often rearranged in cancer. Average median depth was &gt;1200x. Results: All biopsies yielded sufficient DNA. NGS revealed a total of 117 known driver mutations across 36 genes (per-tumor average=2.5, range 1-6), including 37 base substitutions (32%), 28 indels (24%), 42 amplifications (36%) and 10 homozygous deletions (9%). NGS identified HER2 gene amplification in 6/7 cases scored HER2+ by FISH. The average number of functionally important alterations was surprisingly similar, 2.3 in primaries vs 2.8 in heavily treated MBCs (p=0.32). Remarkably, 25/33 (76%) of primary and 14/17 (82%) of MBCs had at least 1 genomic alteration targetable with an FDA approved drug or novel agent in clinical trials. These included: ERBB2 alterations (n=9), PIK3CA mutations (n=8), NF1 mutations (n=4, candidate for PI3K/MEK inhibitors), AKT1-3 mutations (n=5, PI3K inhibitors), BRCA1/2, (n=6, PARP inhibitors), and CCND2 (n=3)/CDKN2A (n=3) mutations (CDK inhibitors). Numerous other alterations with less apparent therapeutic implications were also observed. Conclusions: Comprehensive NGS profiling in breast cancer needle biopsies showed high frequency of genomic alterations linked to a clinical treatment option or clinical trials of targeted therapies. These results demonstrate it is feasible to use NGS to guide targeted therapy. Prospective testing of the diagnostic/predictive value of this patient selection approach is currently under way.

Frequency of actionable genomic alterations in early-stage lung adenocarcinoma (LA) detected by next-generation sequencing (NGS).

e17541 Background: Early stage LA (≤T1N0) has a variable natural history. Although many patients are cured (~2/3) with surgery, many tumors recur and better methods are needed to distinguish these subsets and tailor therapy accordingly. We undertook a pilot study of NGS in such tumors to characterize the frequency and diversity of actionable genomic alterations in this setting. Methods: Formalin-fixed paraffin embedded (FFPE) tissues from 21 archival samples collected from 2007 to 2011 were obtained commercially with accompanying clinical and pathologic data. Clinical characteristics: female (17)/male (4); T1N0 (18), AIS (2), MIA (1). Pathologic LA subtype: acinar (n=8), mucinous (n=4), lepidic (n=4), and mixed (n=2) in addition to AIS (n=2) and MIA (n=1). Targeted NGS performed in a CLIA laboratory (Foundation Medicine) gave evaluable results in all cases. Genomic libraries were captured for 3230 exons in 182 cancer-related genes plus 37 introns from 14 genes often rearranged in cancer and sequenced to average median depth of 640X with 99% of bases covered &gt;100X. Results: Forty-one driver mutations were identified in 17 genes among the 21 patients (avg= 1.9) including 28 base substitutions, 5 indels, 4 amplifications, 3 homozygous deletions and 1 rearrangement. Notably, 90% of cases (19/21) harbored at least one alteration that can be classified as actionable-linked to an approved therapy in LA or another tumor type or a clinical trial. Examples include alterations in EGFR (n=6), NF1 (n=2, PI3K/mTOR inhibitors), CDNK2A (n=1, CDK inhibitors), EML4-ALK (fusion, n=1, ALK inhibitor), ERBB2 (n=1, TKI inhibitors), STK11 (n=1, mTOR inhibitors), and MDM2 (n=1, nutlins). Conclusions: NGSidentified drivergenomic alterations in T1N0 LA, MIA and AIS. On average, two known driver mutations were identified per tumor, many of which are associated with existing or emerging targeted therapies. This provides rationale for future investigation of larger series of T1N0 LA, AIS and MIA, using NGS to evaluate the prognostic value of these changes and could presage adjuvant trials in higher risk patients.

Abstract LB-88: Identification of recurrent oncogenic KIF5B-RET rearrangements in non-small cell lung cancer

Abstract Background: A large number of non-small cell lung cancer (NSCLC) cases from never/former light smokers can be explained by driving oncogenic alterations in EGFR, KRAS, ERBB2, BRAF, ALK and ROS1. EGFR and ALK targeted tyrosine kinase inhibitors are effective clinical therapies for EGFR mutant and ALK rearranged NSCLC, respectively. We wished to study cancers that do not harbor a known genomic alteration in order to identify novel therapeutic targets that may increase treatment options for NSCLC patients. Methods and Results: We initially sequenced a NSCLC specimen from a 44 year old Caucasian never smoker in a CLIA certified lab (Foundation Medicine) using a next generation sequencing assay that captures and sequences 2574 coding exons representing 145 cancer relevant genes plus 37 introns from 14 genes frequently rearranged in cancer. We identified an 11,294,741 bp pericentric inversion on chromosome 10 generating a novel gene fusion joining exons 1-15 of KIF5B to exons 12-20 of RET (K15:R12). This fusion gene contains the kinesin motor and coiled-coil domains of KIF5B and the entire tyrosine kinase domain of RET. Expression of RET was confirmed by immunohistochemistry (IHC) and cDNA sequencing detected a 7.3-fold RET expression increase beginning at exon 12 relative to exons 1-11. Screening of additional 117 NSCLC patients using RET IHC identified 22 positive cases; sequencing of 15/22 of these identified 1 additional patient with a KIF5B-RET fusion. We also evaluated 526 tumors from never/former limited smokers (121 Caucasian and 405 Asian) and identified 10 additional (1/121 Caucasian (0.8%) and 9/405 Asian (2%)) positive patients. All fusion positive tumors were wild type for known oncogenes (frequency of 6.3 % (10/159) in WT patients). Four unique KIF5B-RET variants were identified: 8 K15:R12 (variant 1), 3 K16:R12 (variant 2), 1 K22:R12 (variant 3) and 1 K15:R11 (variant 4). We introduced K15:R12 (variant 1) into Ba/F3 cells and observed IL-3 independent growth consistent with oncogenic transformation. These KIF5B-RET Ba/F3 cells were sensitive to sunitinib, sorafenib and vandetinib, multi-targeted kinase inhibitors that inhibit RET, but not gefitinib, an EGFR kinase inhibitor. Sunitinib, but not gefitinib, inhibited RET phosphorylation in the KIF5B-RET Ba/F3 cells. Conclusions: We identify a novel oncogenic KIF5B-RET fusion gene in a subset of NSCLC patients lacking other known driver mutations. Our findings suggest that RET kinase inhibitors should be tested in prospective clinical trials for therapeutic benefit in NSCLC patients bearing KIF5B-RET rearrangements. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-88. doi:1538-7445.AM2012-LB-88

Gene amplification and protein overexpression of EGFR and ERBB2 in sinonasal squamous cell carcinoma

Sinonasal squamous cell carcinomas (SNSCCs) are rare tumors with no etiologic link to tobacco or alcohol, as opposed to other squamous cell carcinomas of the head and neck. Despite improvements in the field of surgery and radiotherapy, patients with these tumors still face a very unfavorable prognosis, partly because of their localization in a complex anatomic area, which has special relevance for surgery and postoperative treatment. Therefore, there is a need for new therapeutic possibilities for patients with these tumors. Gene copy numbers of epidermal growth factor receptor (EGFR) and v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2) were analyzed by fluorescence in situ hybridization and multiplex ligand-dependent probe amplification, and protein expression was evaluated by immunohistochemistry in 54 SNSCC specimens. The results were correlated with clinicopathologic and follow-up data. EGFR gene copy number increases were observed in 20 of 45 tumors (44%), and 21 of 54 tumors (39%) had EGFR protein overexpression. Eight of 38 tumors (21%) had ERBB2 copy number increases, and 4 of 54 tumors (7%) exhibited elevated protein expression levels. Both copy number increases and protein overexpression of EGFR and ERBB2 were mutually exclusive. v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations were absent in 37 tumors that were analyzed. A substantial proportion of SNSCCs carried alterations in EGFR or ERBB2. Together with the absence of KRAS mutations, these findings indicate that therapies targeting these molecules may be promising additions to the therapeutic options for patients with SNSCC.

Correlation between clinical characteristics, survival and genetic alterations in patients with hepatocellular carcinoma from Saudi Arabia

Amplification of the two oncogenes ERBB2 and MYC and deletion of the tumor suppressor gene TP53 are frequently encountered in cancerous tissues. The purpose of this study was to use the fluorescence in situ hybridization (FISH) technique for the assessment of ERBB2 and MYC amplification and TP53 deletion, and to relate these molecular markers to clinical and pathologic factors in Saudi patients with hepatocellular carcinoma. The study was conducted on 40 paraffin-embedded tissue samples originally taken from either hepatitis C virus (HCV)- or HBV-infected patients using the FISH technique. The level of ERBB2, MYC, and TP53 in the malignant group was significantly increased as compared to the control group. Of the 40 patients, 3 (7.5%) had amplification of ERBB2 gene, 4 (10%) different patients had amplification of MYC, and 26 patients (65%) had evidence of deletion of at least one allele on chromosome 17 for the TP53 gene in a high proportion of cells. There was a significant correlation between amplification of MYC oncogene and the number of tumor masses. Moreover, significant correlation was observed between poorly differentiated tumors when compared with moderate or well-differentiated tumors when MYC was analyzed. On the other hand, MYC failed to reveal any significant association between oncogene amplification and other clinicopathologic variables examined. Univariate analysis revealed a strong association between deletion of TP53 and multiple tumor mass ( P< 0.001). No statistical correlation could be detected between deletion of TP53 and tumor size, grade, stage, and tumor differentiation. No significant difference could be detected in the mean survival time of patients positive for the alteration of the genes compared to the patients who showed no alterations for the same genes. However, when the stage of the tumor was analyzed, there was a significant difference in the mean survival time between patients who showed gene alterations compared to patients with no changes in the studied genes. When overall survival was analyzed, only patients with MYC amplification had a lower median survival (20.75 months) than patients without MYC amplification (35.82, P=0.009). Genetic alterations of ERBB2 and TP53 genes had no effect on survival 2 (see Results). The combination of ERBB2, MYC, and TP53 could be useful markers to stratify patients into different risk groups.

Identification of a CpG Island Methylator Phenotype that Defines a Distinct Subgroup of Glioma

We have profiled promoter DNA methylation alterations in 272 glioblastoma tumors in the context of The Cancer Genome Atlas (TCGA). We found that a distinct subset of samples displays concerted hypermethylation at a large number of loci, indicating the existence of a glioma-CpG island methylator phenotype (G-CIMP). We validated G-CIMP in a set of non-TCGA glioblastomas and low-grade gliomas. G-CIMP tumors belong to the proneural subgroup, are more prevalent among lower-grade gliomas, display distinct copy-number alterations, and are tightly associated with IDH1 somatic mutations. Patients with G-CIMP tumors are younger at the time of diagnosis and experience significantly improved outcome. These findings identify G-CIMP as a distinct subset of human gliomas on molecular and clinical grounds.

A gene-alteration profile of human lung cancer cell lines

Aberrant proteins encoded from genes altered in tumors drive cancer development and may also be therapeutic targets. Here we derived a comprehensive gene-alteration profile of lung cancer cell lines. We tested 17 genes in a panel of 88 lung cancer cell lines and found the rates of alteration to be higher than previously thought. Nearly all cells feature inactivation at TP53 and CDKN2A or RB1, whereas BRAF, MET, ERBB2, and NRAS alterations were infrequent. A preferential accumulation of alterations among histopathological types and a mutually exclusive occurrence of alterations of CDKN2A and RB1 as well as of KRAS, epidermal growth factor receptor (EGFR), NRAS, and ERBB2 were seen. Moreover, in non-small-cell lung cancer (NSCLC), concomitant activation of signal transduction pathways known to converge in mammalian target of rapamycin (mTOR) was common. Cells with single activation of ERBB2, PTEN, or MET signaling showed greater sensitivity to cell-growth inhibition induced by erlotinib, LY294002, and PHA665752, respectively, than did cells featuring simultaneous activation of these pathways, underlining the need for combined therapeutic strategies in targeted cancer treatments. In conclusion, our gene-alteration landscape of lung cancer cell lines provides insights into how gene alterations accumulate and biological pathways interact in cancer.

Identificación de alteraciones genéticas en oligodendrogliomas mediante amplificación dependiente de ligasa de múltiples sondas (MLPA) (multiple ligationdependent probe amplification)

Concurrent deletion at 1p/19q is a common signature of oligodendrogliomas, and it may be identified in low-grade tumours (grade II) suggesting it represents an early event in the development of these brain neoplasms. Additional non-random changes primarily involve CDKN2A, PTEN and EGFR. Identification of all of these genetic changes has become an additional parameter in the evaluation of the clinical patients' prognosis, including good response to conventional chemotherapy. Multiple ligation-dependent probe amplification (MLPA) analysis is a new methodology that allows an easy identification of the oligodendrogliomas' abnormalities in a single step. No need of the respective constitutional DNA from each patient is another advantage of this method. We used MLPA kits P088 and P105 to determine the molecular characteristics of a series of 40 oligodendrogliomas. Deletions at l p and 19q were identified in 45% and 65% of cases, respectively. Alterations of EGFR, CDKN2A, ERBB2, PTEN and TP53 were also identified in variable frequencies among 7% to 35% of tumours. These findings demonstrate that MLPA is a reliable technique to the detection of molecular genetic changes in oligodendrogliomas.

An integrative hypothesis about the origin and development of sporadic and familial breast cancer subtypes

Do breast cancer tumours have a common cell origin? Do different breast cancer molecular phenotypes arise from distinct cell types? The studies we have performed during the last few years in familial breast tumours (BRCA1, BRCA2 and non-BRCA1/2) widen questions about the development of sporadic breast cancer to hereditary breast cancer. Array-comparative genomic hybridisation (CGH) studies show universal genomic aberrations in both familial and sporadic breast cancer subtypes that may be selected in the breast tumour development. The inactivation of BRCA1 seems to play a critical role in oestrogen receptor (ER)-negative cancer stem cells (CSCs), driving the tumour development mostly towards a basal-like or, in some cases, to a luminal B phenotype, but other carcinogenetic events are proposed to explain the remaining tumour subtypes. The existence of common genomic alterations in basal-like, ERBB2 and luminal B breast tumours may suggest a common cell origin or clonal selection of these tumour subtypes, arising from an ER-negative CSC or from a progenitor cell (PC). Finally, specific genomic aberrations in ER-positive tumours could provide cellular proliferation advantages when the cells are exposed to oestrogen. We propose a combination of the CSC hypothesis (for the carcinogenesis processes) and the clonal selection model (in terms of tumour development). We uphold that the basal-like-, ERBB2- and luminal B-sporadic and familial tumour subtypes have an ER-negative breast stem/PC origin, whereas luminal A tumours arise from an ER-positive PC, supporting a hierarchical breast carcinogenesis model, whereas crucial genomic imbalances are clonally selected during the tumour development.

Type of TP53 mutation and ERBB2 amplification affects survival in node-negative breast cancer

Alterations of TP53 and ERBB2 have been shown to play important roles in the prognosis of breast cancer. The primary objective of this study is to characterize TP53 mutation types in node negative breast cancer and investigate their prognostic value, alone and in combination with ERBB2 amplification status. TP53 mutational status (exons 2-10) and ERBB2 amplification status were determined in tumor specimens from a prospective cohort of 543 women with node-negative breast cancer. During a median follow-up of 120 months, there were 111 disease recurrences, and 81 disease-related deaths (3 with cancer; 78 from cancer). Of 543 women, 133 (24.5%) carried mutations in exons 4-9 of the TP53 gene. Seventy-one (53.4%) of these mutations were missense; whereas 62 (46.6%) were protein-truncating mutations. Women whose tumors had missense TP53 mutations were found to be at significantly higher risk of recurrence and death compared to those with wild type TP53, and they also tended to have worse prognosis compared to those with truncating mutations. Those with short truncated proteins tended to have good prognosis compared to those with long truncated proteins, but the risk of recurrence and death did not differ between those whose tumors exhibited conserved versus non-conserved mutations. Missense mutations, in combination with ERBB2 amplification, were observed in 4.6% of the tumors and dramatically affected the disease-specific survival (DSS) and disease-free survival (DFS) of the breast cancer patients. Our study suggests that the type of TP53 mutation, especially missense mutation, is a strong prognostic indicator for DFS and DSS in node-negative breast cancer, particularly in combination with ERBB2 amplification.

MP-08.17: Mutations of epidermal growth factor receptor in hormone sensitive and refractory prostate cancers

Hormone refractory prostate cancer (HRPC) is heterogeneous and a significant cause of morbidity and mortality. Androgen receptor mutations and amplifications may explain relapse in some patients, but in approximately 70% of cases, alternative mechanisms must be invoked and type I receptor tyrosine kinases may play a role in mediating HRPC. In this study, EGFR and ERBB2 gene amplification and alteration were analyzed in HRPC.