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Purpose: Resistance to anti-HER2 therapies in HER2+ breast cancer can occur through activation of alternative survival pathways or reactivation of the HER signaling network. Here we employed BT474 parental and treatment-resistant cell line models to investigate a mechanism by which HER2+ breast cancer can reactivate the HER network under potent HER2-targeted therapies.Experimental Design: Resistant derivatives to lapatinib (L), trastuzumab (T), or the combination (LR/TR/LTR) were developed independently from two independent estrogen receptor ER+/HER2+ BT474 cell lines (AZ/ATCC). Two derivatives resistant to the lapatinib-containing regimens (BT474/AZ-LR and BT474/ATCC-LTR lines) that showed HER2 reactivation at the time of resistance were subjected to massive parallel sequencing and compared with parental lines. Ectopic expression and mutant-specific siRNA interference were applied to analyze the mutation functionally. In vitro and in vivo experiments were performed to test alternative therapies for mutant HER2 inhibition.Results: Genomic analyses revealed that the HER2L755S mutation was the only common somatic mutation gained in the BT474/AZ-LR and BT474/ATCC-LTR lines. Ectopic expression of HER2L755S induced acquired lapatinib resistance in the BT474/AZ, SK-BR-3, and AU565 parental cell lines. HER2L755S-specific siRNA knockdown reversed the resistance in BT474/AZ-LR and BT474/ATCC-LTR lines. The HER1/2–irreversible inhibitors afatinib and neratinib substantially inhibited both resistant cell growth and the HER2 and downstream AKT/MAPK signaling driven by HER2L755S in vitro and in vivo.Conclusions: HER2 reactivation through acquisition of the HER2L755S mutation was identified as a mechanism of acquired resistance to lapatinib-containing HER2-targeted therapy in preclinical HER2-amplified breast cancer models, which can be overcome by irreversible HER1/2 inhibitors. Clin Cancer Res; 23(17); 5123–34. ©2017 AACR.

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Supplementary tables S1-S4

Introduction: Neuroblastoma (NBL) is the most common type of extracranial pediatric solid tumor. NBL exhibits a wide spectrum of phenotypes, from low-risk cases that spontaneously regress to high-risk cases with 50% mortality, and it is responsible for 12% to 15% of deaths due to childhood cancer. While genome-wide association studies have identified a handful of risk loci, the germline genetic origins of sporadic NBL remain largely unknown. Notably, NBL is one of several childhood cancers that tend to co-occur with specific birth defects, which could point to novel genetic origins. For example, the risk of neuroblastoma is increased 3- to 7-fold among children with certain congenital heart defects (CHD). It is hypothesized that this co-occurrence arises from pleiotropic defects in the neural crest cells that are progenitors of both NBL and some heart structures. Based on these lines of evidence, we hypothesize that novel germline risk loci for NBL can be identified through pleiotropic analyses of CHD and NBL cohorts. Therefore, in this study we seek to identify germline risk loci for neuroblastoma by jointly analyzing CHD and NBL data sets with a primary focus on de novo variants. Methods: Using whole genome sequencing data from the Gabriella Miller Kids First Data Resource Center, we will identify NBL and CHD associated loci by testing for high rates of de novo variants (DNV) in each cohort separately and in the two cohorts combined. The testing strategy compares the rate of DNV to the expected locus-specific mutation rate. In our primary analyses, we will focus on genes known to be associated with CHD in order to reduce the multiple testing penalty for that family of loci. A broader analysis will explore coding and regulatory loci genome wide to identify loci associated jointly with NBL and CHD. Results: We acquired whole genome sequencing data of parent-offspring trios for NBL (n=609) and CHD (n=711) from the Gabriella Miller Kids First Data Resource Center. The probands in the NBL cohort was 54% male and 81% Non-Hispanic White, with a median age at diagnosis of 16 months. The probands in the CHD cohort was 59% male and 60% Non-Hispanic White. The most common CHD phenotypes were ventricular septal defect (46%), tetralogy of Fallot (32%), hypoplastic left heart syndrome (28%), atrial septal defect (25%), and right aortic arch (25%). Analyses to identify DNV are ongoing. Conclusion: The identification of germline risk loci associated with NBL will help to inform the developmental origins of this important childhood cancer and may inform novel risk stratification protocols and therapies. This study also aims to help explain the NBL-CHD co-occurrence, and that could shed light on the development of other neural crest cell derived cancers. Citation Format: Alexander Renwick, Yao Yu, Chad Huff, Philip J. Lupo. De novo variants associated with neuroblastoma and congenital heart defects: evidence of pleiotropic effect from 1311 WGS trios [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2506.

Purpose: There is emerging evidence that children with multiple congenital anomalies have a higher risk of developing cancer compared to unaffected children. However, there have been few population-based estimates of cancer risk among children with well-described patterns of recurrent multiple congenital anomalies (MCAs). VACTERL association is one such pattern that is defined by the presence of ≥3 of the following in the absence of a genetic diagnosis: vertebral defects, anal atresia, cardiac defects, tracheoesophageal fistula, renal, and limb anomalies. VACTERL occurs in approximately 5% of children with Fanconi anemia (FA), a known cancer predisposition syndrome. In spite of this, there are no population-based estimates of cancer risk in children with VACTERL. Therefore, we investigated if VACTERL confers an elevated risk of pediatric cancer by examining a birth cohort of >7 million children from three U.S. states (Texas, North Carolina, and Oklahoma) for the period 1997-2013. Methods: Demographic and diagnostic data from birth certificates, birth defects registries, and cancer registries were linked in each state and pooled for analysis. For this analysis, children with chromosomal anomalies were excluded. We used Cox proportional hazards models to evaluate the risk of cancer before 19 years of age in three birth defect groups: (1) VACTERL; (2) ≥3 major anomalies but not VACTERL; and (3) 1 to 2 major anomalies. A hazard ratio (HR) and 95% confidence interval (CI) was calculated in each group for: (1) overall cancer risk and (2) risk of embryonal tumors (e.g., neuroblastoma, medulloblastoma, hepatoblastoma) based on reported associations between birth defects and these pediatric cancers. Results: In our cohort of 7,767,786 births, we identified: 2,090 children (0.02%) with VACTERL; 32,558 children with ≥3 non-VACTERL defects; and 201,871 children with 1 to 2 defects. Respectively, these three groups had 10, 167, and 684 cases of cancer. Compared to children without birth defects, children with VACTERL (HR=3.9, 95% CI: 2.1 - 7.2), ≥3 non-VACTERL defects (4.2, 3.6 - 4.9), and 1 to 2 defects (2.7, 2.5 - 2.9), were all more likely to develop cancer. Risk was higher for embryonal tumors among children with VACTERL (10.4, 4.7 - 23.1) than for those with ≥3 non-VACTERL defects (5.6, 4.2 - 7.4) or 1 to 2 defects (3.9, 3.4 - 4.5). Conclusion: In this population-based assessment, we demonstrated that children with VACTERL were more likely than other groups to develop cancer, particularly embryonal tumors, although the absolute cancer risk was Citation Format: Alexander Renwick, Jeremy M. Schraw, Tania A. Desrosiers, Amanda E. Janitz, Michael E. Scheurer, Mark A. Canfield, Peter H. Langlois, Angela E. Scheuerle, Sharon E. Plon, Philip J. Lupo. A population-based assessment of cancer risk in children with recurrent multiple congenital anomalies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1998.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer diagnosed in more than 200,000 women each year1 and is recalcitrant to targeted therapies(2,3). Although TNBCs harbor multiple hyperactive receptor tyrosine kinases (RTKs)(4–8), RTK inhibitors have been largely ineffective in TNBC patients thus far. We developed a broadly effective therapeutic strategy for TNBC that is based on combined inhibition of receptors that share the negative regulator PTPN12. Previously, we and others identified the tyrosine phosphatase PTPN12 as a tumor suppressor that is frequently inactivated in TNBC(9,10). PTPN12 restrains several RTKs(9,11–17), suggesting that PTPN12 deficiency leads to aberrant activation of multiple RTKs and a co-dependency on these receptors. This in turn leads to the therapeutic hypothesis that PTPN12-deficient TNBCs may be responsive to combined RTK inhibition. However, the repertoire of RTKs that are restrained by PTPN12 in human cells has not been systematically explored. By methodically identifying the suite of RTK substrates (MET, PDGFRb, EGFR, and others) inhibited by PTPN12, we rationalized a combination RTK-inhibitor therapy that induced potent tumor regression across heterogeneous models of TNBC. Orthogonal approaches revealed that PTPN12 was recruited to and inhibited these receptors after ligand stimulation, thereby serving as a feedback mechanism to limit receptor signaling. Cancer-associated mutation of PTPN12 or reduced PTPN12 protein levels diminished this feedback mechanism, leading to aberrant activity of these receptors. Restoring PTPN12 protein levels restrained signaling from RTKs, including PDGFRb and MET, and impaired TNBC survival. In contrast with single agents, combined inhibitors targeting the PDGFRb and MET receptors induced the apoptosis in TNBC cells in vitro and in vivo. This therapeutic strategy resulted in tumor regressions in chemo-refractory patient-derived TNBC models. Notably, response correlated with PTPN12 deficiency, suggesting that impaired receptor feedback may establish a combined addiction to these proto-oncogenic receptors. Taken together, our data provide a rationale for combining RTK inhibitors in TNBC and other malignancies that lack receptor-activating mutations.

Wilms tumor is the most common childhood renal cancer. To identify mutations that predispose to Wilms tumor, we are conducting exome sequencing studies. Here we describe 11 different inactivating mutations in the REST gene (encoding RE1-silencing transcription factor) in four familial Wilms tumor pedigrees and nine non-familial cases. Notably, no similar mutations were identified in the ICR1000 control series (13/558 versus 0/993; P < 0.0001) or in the ExAC series (13/558 versus 0/61,312; P < 0.0001). We identified a second mutational event in two tumors, suggesting that REST may act as a tumor-suppressor gene in Wilms tumor pathogenesis. REST is a zinc-finger transcription factor that functions in cellular differentiation and embryonic development. Notably, ten of 11 mutations clustered within the portion of REST encoding the DNA-binding domain, and functional analyses showed that these mutations compromise REST transcriptional repression. These data establish REST as a Wilms tumor predisposition gene accounting for ∼2% of Wilms tumor.

Purpose: Resistance to anti-HER2 therapies in HER2+ breast cancer can occur through activation of alternative survival pathways or reactivation of the HER signaling network. Here we employed BT474 parental and treatment-resistant cell line models to investigate a mechanism by which HER2+ breast cancer can reactivate the HER network under potent HER2-targeted therapies.Experimental Design: Resistant derivatives to lapatinib (L), trastuzumab (T), or the combination (LR/TR/LTR) were developed independently from two independent estrogen receptor ER+/HER2+ BT474 cell lines (AZ/ATCC). Two derivatives resistant to the lapatinib-containing regimens (BT474/AZ-LR and BT474/ATCC-LTR lines) that showed HER2 reactivation at the time of resistance were subjected to massive parallel sequencing and compared with parental lines. Ectopic expression and mutant-specific siRNA interference were applied to analyze the mutation functionally. In vitro and in vivo experiments were performed to test alternative therapies for mutant HER2 inhibition.Results: Genomic analyses revealed that the HER2L755S mutation was the only common somatic mutation gained in the BT474/AZ-LR and BT474/ATCC-LTR lines. Ectopic expression of HER2L755S induced acquired lapatinib resistance in the BT474/AZ, SK-BR-3, and AU565 parental cell lines. HER2L755S-specific siRNA knockdown reversed the resistance in BT474/AZ-LR and BT474/ATCC-LTR lines. The HER1/2–irreversible inhibitors afatinib and neratinib substantially inhibited both resistant cell growth and the HER2 and downstream AKT/MAPK signaling driven by HER2L755S in vitro and in vivo.Conclusions: HER2 reactivation through acquisition of the HER2L755S mutation was identified as a mechanism of acquired resistance to lapatinib-containing HER2-targeted therapy in preclinical HER2-amplified breast cancer models, which can be overcome by irreversible HER1/2 inhibitors. Clin Cancer Res; 23(17); 5123–34. ©2017 AACR.

SummaryDefining the molecular networks that drive breast cancer has led to therapeutic interventions and improved patient survival. However, the aggressive triple-negative breast cancer subtype (TNBC) remains recalcitrant to targeted therapies because its molecular etiology is poorly defined. In this study, we used a forward genetic screen to discover an oncogenic network driving human TNBC. SCYL1, TEX14, and PLK1 (“STP axis”) cooperatively trigger degradation of the REST tumor suppressor protein, a frequent event in human TNBC. The STP axis induces REST degradation by phosphorylating a conserved REST phospho-degron and bridging REST interaction with the ubiquitin-ligase βTRCP. Inhibition of the STP axis leads to increased REST protein levels and impairs TNBC transformation, tumor progression, and metastasis. Expression of the STP axis correlates with low REST protein levels in human TNBCs and poor clinical outcome for TNBC patients. Our findings demonstrate that the STP-REST axis is a molecular driver of human TNBC.

Significant variation exists for maternal nurturing ability in inbred mice. Although classical mapping approaches have identified quantitative trait loci (QTL) that may account for this variation, the underlying genes are unknown. In this study, lactation performance data among the mouse diversity panel were used to map genomic regions associated with this variation. Females from each of 32 inbred strains ( n = 8–19 dams/strain) were studied during the first 8 days of lactation by allowing them to raise weight- and size-normalized cross-foster litters (10 pups/litter). Average daily weight gain (ADG) of litters served as the primary indicator of milk production. The number of pups successfully reared to 8 days (PNUM8) also served as a related indicator of maternal performance. Initial haplotype association analysis using a Bonferroni-corrected, genome-wide threshold revealed 10 and 15 associations encompassing 11 and 13 genes for ADG and PNUM8, respectively. The most significant of these associated haplotype blocks were found on MMU 8, 11, and 19 and contained the genes Nr3c2, Egfr, Sec61g, and Gnaq. Lastly, two haplotype blocks on MMU9 were detected in association with PNUM8. These overlapped with the previously described maternal performance QTL, Neogq1. These results suggest that the application of in silico QTL mapping is a useful tool in discovering the presence of novel candidate genes involved in determining lactation capacity in mice.

MYC (also known as c-MYC) overexpression or hyperactivation is one of the most common drivers of human cancer. Despite intensive study, the MYC oncogene remains recalcitrant to therapeutic inhibition. MYC is a transcription factor, and many of its pro-tumorigenic functions have been attributed to its ability to regulate gene expression programs. Notably, oncogenic MYC activation has also been shown to increase total RNA and protein production in many tissue and disease contexts. While such increases in RNA and protein production may endow cancer cells with pro-tumour hallmarks, this increase in synthesis may also generate new or heightened burden on MYC-driven cancer cells to process these macromolecules properly. Here we discover that the spliceosome is a new target of oncogenic stress in MYC-driven cancers. We identify BUD31 as a MYC-synthetic lethal gene in human mammary epithelial cells, and demonstrate that BUD31 is a component of the core spliceosome required for its assembly and catalytic activity. Core spliceosomal factors (such as SF3B1 and U2AF1) associated with BUD31 are also required to tolerate oncogenic MYC. Notably, MYC hyperactivation induces an increase in total precursor messenger RNA synthesis, suggesting an increased burden on the core spliceosome to process pre-mRNA. In contrast to normal cells, partial inhibition of the spliceosome in MYC-hyperactivated cells leads to global intron retention, widespread defects in pre-mRNA maturation, and deregulation of many essential cell processes. Notably, genetic or pharmacological inhibition of the spliceosome in vivo impairs survival, tumorigenicity and metastatic proclivity of MYC-dependent breast cancers. Collectively, these data suggest that oncogenic MYC confers a collateral stress on splicing, and that components of the spliceosome may be therapeutic entry points for aggressive MYC-driven cancers.

We examine length distributions of ~6000 human dinucleotide microsatellite loci, representing chromosomes 1–22, from the GDB database. Under the stepwise mutation model, results from theory and simulation are compared with the empirical data. In both constant and expanding population scenarios, a simple single-step model with parameters chosen to account for the observed variance of microsatellite lengths produces results inconsistent with the observed heterozygosity and the dispersion of length skewness. Complicating the model by allowing a variable mutation rate accounts for the homozygosity, and introducing a small probability of a large mutation step accounts for the dispersion in skewnesses. We discuss these results in light of the long-term evolution of microsatellites.

Identification of the host genetic factors that contribute to variation in vaccine responsiveness may uncover important mechanisms affecting vaccine efficacy. We carried out an integrative, longitudinal study combining genetic, transcriptional, and immunologic data in humans given seasonal influenza vaccine. We identified 20 genes exhibiting a transcriptional response to vaccination, significant genotype effects on gene expression, and correlation between the transcriptional and antibody responses. The results show that variation at the level of genes involved in membrane trafficking and antigen processing significantly influences the human response to influenza vaccination. More broadly, we demonstrate that an integrative study design is an efficient alternative to existing methods for the identification of genes involved in complex traits. DOI: http://dx.doi.org/10.7554/eLife.00299.001, eLife digest Vaccines increase resistance to disease by priming the immune system to respond to specific viruses or microorganisms. By presenting a weakened (or dead) form of a pathogen, or its toxins or surface proteins, to the immune system, vaccines trigger the production of antibodies against the virus or microorganism. If a vaccinated individual then encounters the pathogen, their immune system should be able to recognize and destroy it. Many vaccines also include a secondary agent, known as an adjuvant, to further stimulate the immune response. Influenza, an RNA virus commonly referred to as the ‘flu’, is an infectious disease that affects both birds and mammals. Seasonal epidemics occur each year affecting 2–7% of the population. According to the World Health Organization, influenza leads to nearly 5 million hospitalizations each year and causes up to half a million deaths. Vaccination is a primary strategy for the prevention of seasonal influenza, but responses to the vaccine vary markedly, partly because of variation in the genetic makeup or genotype of individuals. However, the details of how genes influence response to vaccination, and indeed susceptibility to influenza, remain unclear. To investigate the genetic basis of variation in the immune response of healthy adults to the seasonal influenza vaccine, Franco et al. combined information about the genotypes of individuals with measurements of their gene transcription and antibody response to vaccination. They identified 20 genes that contributed to differential immune responses to the vaccine. Almost half of these encode proteins that are not specifically associated with the immune system, but have more general roles in processes such as membrane trafficking and intracellular transport. Focusing on these genes may enable researchers to spot those individuals who are less likely to respond to a vaccine. It could also open up new avenues of research for vaccine development: rather than designing adjuvants that target known immune mechanisms, researchers should develop adjuvants that target the proteins encoded by these 20 genes. DOI: http://dx.doi.org/10.7554/eLife.00299.002

Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication. Using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focused on protein-truncating variants (PTVs) and a large-scale sequencing case-control replication experiment in 13,642 individuals, here we show that rare PTVs in the p53-inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and ovarian cancer. PPM1D PTV mutations were present in 25 out of 7,781 cases versus 1 out of 5,861 controls (P = 1.12 × 10(-5)), including 18 mutations in 6,912 individuals with breast cancer (P = 2.42 × 10(-4)) and 12 mutations in 1,121 individuals with ovarian cancer (P = 3.10 × 10(-9)). Notably, all of the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370-base-pair region in the final exon of the gene, carboxy-terminal to the phosphatase catalytic domain. Functional studies demonstrate that the mutations result in enhanced suppression of p53 in response to ionizing radiation exposure, suggesting that the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function effect typically associated with this class of variant, but instead probably have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the use of sequencing in their identification.

Nat. Genet. 47, 1471–1474 (2015); published online 9 November 2015; corrected after print 8 February 2016 In the version of this article initially published, the authors failed to acknowledge funding from the NIHR Biomedical Research Centre at Great Ormond Street Hospital for Children NHS FoundationTrust and University College London to Neil Sebire.

Background: Targeting HER2 with lapatinib (L), trastuzumab (T), or the LT combination, is effective in HER2+ breast cancer (BC), but acquired resistance commonly occurs. In our 12-week neoadjuvant trial (TBCRC006) of LT without chemotherapy in HER2+ BC, the overall pathologic complete response (pCR) rate was 27%. To investigate resistance mechanisms, we developed 10 HER2+ BC cell line models resistant (R) to one or both drugs (LR/TR/LTR). To discover potential predictive markers/therapeutic targets to circumvent resistance, we completed genomic profiling of the cell lines and a subset of pre-treatment specimens from TBCRC006. Methods: Parental (P) and LR/TR/LTR lines of 10 cell line models were profiled with whole exome/RNA sequencing. Mutations detected in R lines but not in P lines of the same model were identified. Mutation-specific Q-PCR was designed for sensitive quantification. Resistant cell and xenograft tumor growth were measured in response to drugs. Whole exome sequencing (>100X) and Ampliseq of 17 baseline tumor/normal pairs from TBCRC006 were performed. Results: We found and validated the HER2 L755S mutation in the BT474/ATCC-LTR line and BT474/AZ-LR line (in ∼30% of DNA/RNA), in which the HER pathway was reactivated for resistance. Overexpression of this mutation was previously shown to induce LR in HER2-negative BC cell lines, and resistant growth of BT474/AZ-LR line is significantly inhibited by HER2-L755S-specific siRNA knock-down, suggesting its role as an acquired L/LT resistance driver in HER2+ BC. Sequencing of BT474/AZ-LR single cell clones found the mutation in ∼30% of HER2 copies in every cell. Using mutation-specific Q-PCR, we found statistically higher HER2 L755S levels in two BT474 parentals compared to P lines of SKBR3, AU565, and UACC812. These data suggest that HER2 L755S resistant subclones preexist in the BT474 parentals and were selected by L treatment to become the major clone in the two R lines. The HER1/2 irreversible tyrosine kinase inhibitor (TKI) afatinib (Afa) robustly inhibited growth of BT474/AZ-LR and BT474/ATCC-LTR cells (IC50: Afa 0.02μM vs. L 3 μM) and BT474/AZ-LR xenografts. Whole exome sequencing/Ampliseq of TBCRC006 found the HER2 L755S mutation in 1/17 primaries. This patient did not achieve pCR. The variant was present in 2% of DNA on both platforms, indicating a subclonal event of the resistance mutation. Conclusion: Acquired L/LT resistance in the two BT474 R lines is due to selection of HER2 L755S subclones present in parental cells. The higher HER2 L755S levels in BT474 parentals compared with other parentals, and detection of its subclonal presence in a pre-treatment HER2+ BC patient, suggest that sensitive mutation detection methods will be needed to identify patients with potentially actionable HER family mutations in primary tumor. Treating this patient group with an irreversible TKI like Afa may prevent resistance and improve clinical outcome of this subset of HER2+ BC. Citation Format: Xiaowei Xu, Agostina Nardone, Huizhong Hu, Lanfang Qin, Sarmistha Nanda, Laura Heiser, Nicholas Wang, Kyle Covington, Edward Chen, Alexander Renwick, Tamika Mitchell, Marty Shea, Tao Wang, Carmine De Angelis, Alejandro Contreras, Carolina Gutierrez, Suzanne Fuqua, Gary Chamness, Chad Shaw, Marilyn Li, David Wheeler, Susan Hilsenbeck, Mothaffar Fahed Rimawi, Joe Gray, C.Kent Osborne, Rachel Schiff. Clonal evolution of the HER2 L755S mutation as a mechanism of acquired HER-targeted therapy resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 737. doi:10.1158/1538-7445.AM2015-737

Background: Targeting HER2 with lapatinib (L), trastuzumab (T), or the LT combination, is effective in HER2+ breast cancer (BC), but acquired resistance commonly occurs. In our 12-week neoadjuvant trial (TBCRC006) of LT without chemotherapy in HER2+ BC, the overall pathologic complete response rate (pCR) was 27%. To investigate resistance mechanisms our lab developed 10 HER2+ BC cell lines resistant (R) to these drugs (LR/TR/LTR). To discover potential predictive markers/therapeutic targets to circumvent resistance, we completed genomic profiling of the cell line panel and a subset of pre-treatment baseline specimens from TBCRC006. Methods: Parental (P) lines and LR/TR/LTR derivatives of 9 HER2+ BC cell line models were profiled with whole exome and RNA sequencing. Mutations detected in R lines but not in same-model P lines were identified. cDNAs were assessed by targeted Sanger sequencing. Single cells of the BT474AZ-LR line were cloned and their cDNAs were sequenced. Mutant-specific Q-PCR was designed to sensitively quantify mutations. Whole exome sequencing (minimum depth 100X) of 17 baseline tumor/normal pairs from TBCRC006 were performed on Illumina HiSeq. Results: We found and validated the HER2 L755S mutation in the BT474ATCC-LTR line and the BT474AZ-LR line (∼30% of DNA/RNA/cDNA in BT474AZ-LR), in which the HER pathway was reactivated to cause resistance. Overexpression of this mutation was previously shown to induce L resistance in HER2-negative BC cell lines, suggesting a role as an acquired L/LT resistance driver in HER2+ BC. Sanger sequencing of BT474AZ-LR single cell clones found the HER2 L755S mutation in every clone but only in ∼30% of the HER2 copies. Using sensitive mutant-specific Q-PCR, we found statistically higher levels of HER2 L755S expression in BT474ATCC-P and BT474AZ-P compared to parentals of other HER2+ BC cell lines (UACC812/AU565/SKBR3/SUM190). These data suggest that this mutation exists subclonally within BT474 parental lines and was selected to become the more dominant population in the two resistant lines. The HER1/2 irreversible tyrosine kinase inhibitor (TKI) afatinib (Afa) robustly inhibited growth of both BT474ATCC-LTR/AZ-LR cells (IC50: Afa 0.02µM vs. L 3 µM). Western blots confirmed inhibition of the HER and downstream Akt and MAPK signaling in the LR cells by Afa. Sequencing of TBCRC006 baseline samples found the HER2 L755S mutation in 1/17 subjects. This patient did not achieve pCR after neoadjuvant LT treatment. The variant was present in 2% of the reads, indicating it as a subclonal event in this patient’s baseline tumor. Conclusion: Acquired resistance in two of our BT474 LR/LTR lines is due to selection of HER2 L755S subclones present in the parental cell population. The higher HER2 L755S levels detected in BT474 parentals compared with other HER2+ BC parental lines, and detection of its subclonal presence in a pre-treatment HER2+ BC patient, suggest that sensitive mutation detection methods will be needed to identify patients with potentially actionable HER family mutations in primary tumor. Treating this patient group with an irreversible TKI like Afa may prevent resistance and improve clinical outcome of this subset of HER2+ BC. Citation Format: Xiaowei Xu, Agostina Nardone, Huizhong Hu, Lanfang Qin, Sarmistha Nanda, Laura M Heiser, Nicholas Wang, Kyle R Covington, Edward S Chen, Alexander Renwick, Tao Wang, Carmine De Angelis, Alejandro Contreras, Carolina Gutierrez, Suzanne AW Fuqua, Gary C Chamness, Chad Shaw, David A Wheeler, Joe W Gray, Susan G Hilsenbeck, Mothaffar F Rimawi, C Kent Osborne, Rachel Schiff. Clonal evolution of the HER2 L755S mutation leads to acquired HER-targeted therapy resistance that can be reversed by the irreversible HER1/2 inhibitor afatinib [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-03.

Modern microscopy- and flow cytometry-based analyses indicate tumor cell populations can be markedly heterogeneous. Yet, the vast majority of approaches used to investigate tumorigenic mechanisms and therapeutic responses are based upon average single-point analysis of bulk populations of cells, where subpopulation responses are undetectable and therefore unexplored. For example, breast cancers are generally classified by IHC as either estrogen receptor-alpha positive (ER+) or negative (ER-); however, in ER+ tumors, there is a broad range of ER levels in individual cells. Despite a growing awareness of the importance of tumor cell heterogeneity and outlier biology in cancer therapeutics, there is a paucity of data on the potential interplay between multiple nuclear receptors and coregulators, their heterogeneity of expression and functions, and their combinatorial role in determining therapeutic response, or in sensing environmental stressors (e.g., endocrine disruptors). To specifically quantify biological responses on an individual cell basis rather than averaged responses from bulk populations, we performed single cell-based high content analysis (HCA) to examine MCF-7 breast cancer cells in terms of simultaneous expression of several type-1 nuclear receptors (e.g., Estrogen, Androgen and Glucocorticoid receptors - ER, AR and GR) and coregulators (e.g., SRC-2 and SRC-3). Quantitative immunofluorescence reveals a wide range of heterogeneity in the expression and ratios of ER, AR, and GR. Multi-parametric analyses describing NR expression and spatial distribution were used to evaluate ligand dose responses and time pattern of the three nuclear receptors, simultaneously. Next, we are expanding our analysis to endogenous target gene expression via mRNA fluorescence in situ hybridization (RNA FISH) and to cell cycle marker analysis to link heterogeneity of NRs/coregulators expression with transcriptional output and cell cycle phases. We are also implementing robust analysis pipelines to describe and quantify NRs and coregulators patterns of phenotypic heterogeneity to create “biological response fingerprints,” which we are using to classify the effects of individual or mixed ligand treatments with particular focus on chemotherapeutic drugs and endocrine disruptors. We are also starting to expand our HCA approach to other breast cancer cell lines moving towards primary tumors microarrays analysis to evaluate the prognostic/diagnostic significance of NR heterogeneity. Collectively, these high throughput-amenable approaches are poised to greatly aid in the understanding the role of heterogeneity in breast cancer, and serve as a robust model for next-generation drug screening and endocrine disruptor assays moving toward personalized medicine. Citation Format: Fabio Stossi, Alexander Renwick, Radhika D. Dandekar, Justin Y. Newberg, Arvind Rao, Michael A. Mancini. Single cell-based image analysis of nuclear receptor levels and activity in breast cancer cells reveals heterogeneous subpopulations: Development of biological response fingerprints for ligands and endocrine disruptors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3558. doi:10.1158/1538-7445.AM2013-3558