Circulating cell-free DNA (cfDNA) has been correlated with allograft rejection in organ transplant recipients and is proposed as a biomarker to assess graft injury that may be reflective of allograft rejection or immunosuppression toxicity. We have validated a method for quantifying donor-derived cfDNA (dd-cfDNA) based on targeted amplification of SNPs and next-generation sequencing (NGS) that does not require genotyping of the donor or recipient (AlloSure®). The test measures alleles for 266 SNPs distributed across the genome and quantifies the proportion of dd-cfDNA present in the recipient's plasma. The aim of this study was to identify relationships between levels of dd-cfDNA in kidney transplant patients and the degree of HLA mismatch with the donor, living or deceased donor type, and donor relationship to recipient. Testing was performed using cfDNA extracted from plasma collected in Streck Cell-Free DNA BCT blood collection tubes from kidney recipients enrolled in a multicenter observational study. Median values of dd-cfDNA were calculated from all visits for each recipient. Recipients were stratified by the total number of mismatches in HLA A, B and DR loci. Further stratifications were based on the donor type (living or deceased) as well as relationship to the donor (related or unrelated). Median dd-cfDNA levels were calculated from 208 recipients with 4 or more visits (median of 6 visits each). There were no statistically significant differences in median levels of dd-cfDNA associated with degree of donor HLA match. The between-patient variability trended higher for mismatched recipients compared to those with no mismatches to their donor and for recipients who received a kidney from a deceased donor compared to those recipients who received a kidney from a living donor, but the differences were not statistically significant. In samples collected on a surveillance schedule from study recipients, degree of HLA mismatch at HLA A, B, and DR were not predictive of dd-cfDNA overall nor when stratified by donor type and donor relationship. These results indicate that the level of dd-cfDNA in kidney recipients is independent of these variables and that these variables do not confound interpretation of the AlloSure results.

The use of circulating cell-free DNA (cfDNA) as a biomarker in transplant recipients offers advantages over invasive tissue biopsy as a quantitative measure for detection of transplant rejection and immunosuppression optimization. However, the fraction of donor-derived cfDNA (dd-cfDNA) in transplant recipient plasma is low and challenging to quantify. Previously reported methods to measure dd-cfDNA require donor and recipient genotyping, which is impractical in clinical settings and adds cost. We developed a targeted next-generation sequencing assay that uses 266 single-nucleotide polymorphisms to accurately quantify dd-cfDNA in transplant recipients without separate genotyping. Analytical performance of the assay was characterized and validated using 1117 samples comprising the National Institute for Standards and Technology Genome in a Bottle human reference genome, independently validated reference materials, and clinical samples. The assay quantifies the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs. The dd-cfDNA assay can reliably measure dd-cfDNA (limit of blank, 0.10%; limit of detection, 0.16%; limit of quantification, 0.20%) across the linear quantifiable range (0.2% to 16%) with across-run CVs of 6.8%. Precision was also evaluated for independently processed clinical sample replicates and is similar to across-run precision. Application of the assay to clinical samples from heart transplant recipients demonstrated increased levels of dd-cfDNA in patients with biopsy-confirmed rejection and decreased levels of dd-cfDNA after successful rejection treatment. This noninvasive clinical-grade sequencing assay can be completed within 3 days, providing the practical turnaround time preferred for transplanted organ surveillance.

Abstract Background: While representing the largest fraction of women diagnosed with primary breast cancer, older postmenopausal women with ER+, HER2− tumors are less responsive to chemoendocrine therapy than younger women and have been underrepresented in molecular profiling of randomized trials. IBCSG Trial IX, a randomized controlled trial in postmenopausal women, median age 61y, with node negative disease, failed to demonstrate the benefit of preceding tamoxifen (T) by 3 cycles of CMF for ER+ tumors. We sought to determine if MS, a proliferation score, could identify a subset of women who differentially benefit from addition of chemotherapy to T in this trial. Methods: From 1988–1999, 1669 eligible patients (1040 with ER+, HER2− tumors) were randomized to CMF→T vs T. Disease-free survival (DFS) was the primary trial endpoint; breast cancer-free interval (BCFI) which excludes second (non-breast) malignancies and censors deaths without prior cancer event was also evaluated. Analysis was limited to the first 7 years of follow-up. From 671 (ER+, HER2−) available subjects, 568 were successfully profiled by RT-PCR. The mRNA expression levels of 14 equally-weighted proliferation genes and 3 normalization genes were used to generate MS; predetermined binary categorization of MS was used. Analysis of this post hoc, pre-specified study used results from centralized laboratory IHC and Cox models to assess the predictive value of MS on DFS and BCFI, adjusting for traditional risk factors of local treatment, age, ER, PR, Ki67, tumor size and grade. Results: Subgroups of MS (low, 169 samples (30%) and high, 399 samples (70%)) were identified. MS by treatment interaction was significant for DFS and BCFI (each p ≤ 0.004). Among patients with low MS, CMF→T improved DFS (HR 0.19, 95% CI 0.06–0.59) and BCFI (HR 0.19, 95% CI 0.05–0.72) vs T; 7y DFS was 95% vs 83% with CMF→T vs T. Among patients with high MS, CMF→T did not improve DFS (HR 1.27, 95% CI 0.79–2.05) or BCFI (HR 1.37, 95% CI 0.80–2.33) and 7y DFS of 81% for CMF→T and T. Continuous MS was moderately correlated with log Ki67 (r = 0.47) but not correlated with ER or PR. The MS by treatment interaction remained significant with Ki67 in the model. Conclusions: Low MS was associated with differential benefit favoring those women receiving CMF→T vs T alone for both DFS and BCFI in the first 7 years. The effect was independent of traditional risk factors including Ki67. Hence this study, which is unconfounded by chemotherapy-induced ovarian ablation in younger women, identifies a subset of postmenopausal women with ER+, HER2− tumors that benefit from CMF chemotherapy. This seemingly incongruous observation is consistent with a) the prior observation that only the low-proliferation subgroup by PAM50 11-gene signature benefits from the addition of weekly paclitaxel to adjuvant FEC (GEICAM/9906), b) the ability of MS to identify a subset of women with tumors with disseminated luminal progenitor cells activated through the agonistic activity of tamoxifen, and c) the repetitive dosing of cyclophosphamide and taxol being hypothesized to act via tumor stroma/anti-angiogenesis. The relative contribution of these factors is under investigation. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD10-03.

10586 Background: Teschendorff et al. (Breast Cancer Res 2008) reported a 7-gene, immune response-enriched gene expression classifier using microarrays that was prognostic in women with ER-negative breast cancer. To facilitate further evaluation of the prognostic value of a classifier in archival fixed tumor tissue, we developed two multiplex TaqMan assays to quantify the expression levels of these genes, then trained and validated a 7-gene immune response score (IRS). Methods: RNA was extracted from FFPE sections of 185 ER-negative breast cancer patient samples from Mayo Clinic (n=81), Guy’s Hospital (n=50) and California Pacific Medical Center (CPMC) (n=27). Patients (N+ and N-) did not receive adjuvant therapy and were followed for a minimum of 5 years. Amplification of i) LY9, TNFRSF17, HLA-F, and IGLC2 and ii) XCL2, SPP1, C1QA and two reference genes NUP214 and PPIG were carried out in two multiplex RT-PCR TaqMan reactions and relative expression levels were determined using the DDCT method. The training set, comprised of the Mayo Clinic samples, was used to develop an IRS by using a summation of expression levels of the 7 genes. Association of the IRS with distant metastasis was assessed using a Cox proportional hazards model. The score was subsequently tested in a validation set (Guy’s + CPMC). Results: The amplification efficiency of each gene within the multiplex reactions was comparable to singleplex reactions. Duplicate amplifications showed excellent assay reproducibility and sensitivity (R2=0.98). The hazard ratio for a single standard deviation decrease in the IRS was 1.45 (95% CI 0.99-2.12, p=0.056) in the training set and 2.18 (1.32-3.61, p=0.002) in the validation set. ROC curves of continuous IRS to predict distant metastasis within 5 years had AUC of 0.74, 95% CI = 0.60-0.88. Conclusions: Robust multiplex TaqMan assays for FFPE tissue extracted RNA were developed for a 7-gene IRS. Increases in risk are associated with decreases in the IRS. Immune related gene expression clearly plays an important role in the spectrum of risk of ER-negative tumors. Additional signatures are in development that further support this conclusion.

Background: The proliferation genes of metastatic expression scores for ER-positive tumors are thought to be critical. While the correlation between these scores and distant metastasis has clinical utility, further improvements of predicted risk and a better understanding of the underlying biological processes is desirable. Since cell survival, proliferation, and cellular metabolism are integrated, we asked if expressions of the genes of the glycolytic pathway are associated with predicted metastatic risk. Methods: RNA was extracted from 46 breast cancer tumor blocks collected at the Blumenthal Cancer Center with Institutional Review Board approval. A previously described metastasis score (MS) (Tutt et al) consisting of 14 proliferation genes was determined by RT-PCR and with categorization as either high or low risk. These tumors had been previously profiled using Oncotype Dx® and categorized using Oncotype Dx® and TAILORx cut points. Thirteen glycolysis pathway genes (GLUT1, HK1, HK2, PKM1, PKM2, ENO3, LDHA, LDHB, PGK1, PKFFB4, PFKL, PFKM, and KHK) were profiled. Data were analyzed using Eisen9s Cluster program. Results: In a preliminary analysis of 46 tumors, we identified 27 tumors that were predicted to be at high risk for distant metastasis based on MS. Clustal analysis of the glycolysis pathway genes divided the tumors into two major groups. Of the 20 tumors that displayed higher levels of glycolytic expression, 19 had a high MS and 1 had a low MS. Of the 26 tumors with lower glycolytic expression, 8 had a high MS and 18 had a low MS. Four of the 20 higher glycolytic expression group were categorized as high risk using Oncotype Dx® cutpoints while 5 were categorized as high risk using TAILORx cutpoints. Of the 26 lower glycolytic expression group, 1 tumor was classified as high risk by either Oncotype Dx® or TAILORx criteria. Seven tumors in each of the higher and lower glycolytic expression groups were classified as intermediate risk with Oncotype Dx® cutpoints;12 and 19 were classified as intermediate risk in the higher and lower glycolytic expression groups, respectively, with TAILORx cutpoints. A 2.5 to 12 fold range in glycolytic gene expression was observed between the tumors; the largest ranges were observed with LDHA, LDHB, PGK1 and PKM1. Discussion: Tumors that were highly proliferative based on MS showed higher expression of the glycolysis pathway genes. However, there was a subset of tumors that displayed discordant proliferative and glycolytic expression. Although the tumors with high MS cluster together, the expression level of the composite genes differ between tumors, suggestive that different pathways may be involved. The predominance of intermediate Oncotype Dx® categorized tumors made the interpretation of association difficult. Studies with clinical outcome data will be required to assess the utility of the inclusion of glycolytic gene expression in predicting metastatic risk. 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 5145. doi:1538-7445.AM2012-5145

Targeting tumour metabolism is becoming a major new area of pharmaceutical endeavour. Consequently, a systematic search to define whether there are specific energy source dependencies in tumours, and how these might be dictated by upstream driving genetic mutations, is required. The PI3K-AKT-mTOR signalling pathway has a seminal role in regulating diverse cellular processes including cell proliferation and survival, but has also been associated with metabolic dysregulation. In this study, we sought to define how mutations within PI3KCA may affect the metabolic dependency of a cancer cell, using precisely engineered isogenic cell lines. Studies revealed gene expression signatures in PIK3CA mutant cells indicative of a consistent up-regulation of glycolysis. Interestingly, the genes up- and down-regulated varied between isogenic models suggesting that the primary node of regulation is not the same between models. Additional gene expression changes were also observed, suggesting that metabolic pathways other than glycolysis, such as glutaminolysis, were also affected. Nutrient dependency studies revealed that growth of PIK3CA mutant cells is highly dependent on glucose, whereas glutamine dependency is independent of PIK3CA status. In addition, the glucose dependency exhibited by PIK3CA mutant cells could not be overridden by supplementation with other nutrients. This specific dependence on glucose for growth was further illustrated by studies evaluating the effects of targeted disruption of the glycolytic pathway using siRNA and was also found to be present across a wider panel of cancer cell lines harbouring endogenous PIK3CA mutations. In conclusion, we have found that PIK3CA mutations lead to a shift towards a highly glycolytic phenotype, and that despite suggestions that cancer cells are adept at utilising alternative nutrient sources, PIK3CA mutant cells are not able to compensate for glucose withdrawal. Understanding the metabolic dependencies of PIK3CA mutant cancers will provide critical information for the design of effective therapies and tumour visualisation strategies.

47 Background: IHC is the present standard for measuring estrogen (ER) and progesterone receptor (PR) expression for breast cancer. However, the lack of concordance between testing laboratories and the critical patient treatment decisions made with results prompted ASCO/CAP to recommend guidelines and proficiency testing requirements to ensure accuracy. Quantitative RT-PCR (qRT-PCR) is an alternative method for ER and PR testing and while concordance with IHC has been reported, additional testing is merited. This study compares ER, PR and HER2 status determined by IHC and qRT-PCR. Methods: FFPE tissues of ER(+) tumors collected and tested at the Blumenthal Cancer Center were studied. Expression levels of ESR1, PGR, and ERBB2 were determined by a multiplex qRT-PCR TaqMan assay and by the Oncotype Dx assay. Pre-established cutpoints were used to determine positivity. Only samples with qRT-PCR and corresponding IHC data were used, resulting in 144 ER, 128 PR, and 107 HER2 comparisons. ESR1 and PGR expression levels determined by the two qRT-PCR assays were also compared. Results: Of the144 IHC ER(+) samples, 142 were positive and 2 were negative by both qRT-PCR assays. All 120 IHC PR(+) samples were positive by both qRT-PCR assays. Of the 8 IHC PR(-) samples, 5 were negative and 3 were positive by both qRT-PCR assays. Of the 107 IHC HER2(-) samples, all but 2 were negative by qRT-PCR. One sample was positive by both qRT-PCR assays; one was positive by Oncotype Dx only. The expression levels determined by the qRT-PCR assays showed good correlation for ESR1 (r=0.85) and PGR (r=0.9). Conclusions: Concordance between qRT-PCR and IHC was 98.6% for ER and 97.7% for PR. Except for a single HER2 determination, there was 100% concordance between the two qRT-PCR assays. The discordant sample was HER2 (+) by multiplex qRT-PCR and “equivocal” by Oncotype Dx. Excellent correlation was also observed in the mRNA expression levels. The absence of HER2(+) and ER(-) samples are limitations of this study. These results suggest that qRT-PCR is a promising alternative method to IHC for determining hormone receptor status. Additional testing of samples with endocrine therapy outcomes including ER(-) samples would be beneficial.

Abstract Background: During tumorigenesis, cells acquire mutations that confer selective advantages for growth. These mutations may reprogram cellular metabolism to meet the requirements of rapid proliferation and survival in an environment of fluctuating and scarce nutrients. Phosphoinositide-3-kinases (PI3Ks) play key roles in fundamental cellular processes and are frequently mutated in a broad range of cancers. The aim of this study was to investigate how a single endogenous activating mutation in PI3K, H1047R, affects the metabolic programming of human breast epithelial cells. Methods: We utilized X-MAN isogenic cells derived from MCF10a mammary epithelial cells, which harbour a single H1047R PIK3CA mutation introduced via AAV-mediated homologous recombination and compared them to matched parental cells with a wild type (WT) allele. This system allows use of endogenous promoters thereby more accurately recapitulating the genomic architecture in human tumours. Metabolic gene expression profiles were measured by RT-PCR. Results: We identified a multi-gene expression signature indicative of increased dependency on the glycolytic pathway. Hexokinase 2, which is involved at an early stage of the glycolytic pathway, was strongly up-regulated along with the lactate transporters MCT1 and 4. The fructose transporter GLUT5 was nearly undetectable in H1047R PIK3CA cells, suggesting a strong preference for glucose and elevated levels of aerobic glycolysis. PI3K mutant cells also down-regulated genes in the pentose phosphate pathway-shunt, tricarboxylic acid cycle and glutaminolysis pathways, indicating that aerobic glycolysis is heavily favoured. Treatment with the PI3K inhibitor GDC-0941 led to a general reversal of gene expression for these pathways, confirming the PI3K specificity of these metabolic alterations. We proceeded to investigate growth dependence of the H1047R PIK3CA and parental MCF10a cells on various nutrient sources (e.g. glucose, fructose, galactose and glutamine). While the growth rates of cells WT for PIK3CA were not significantly affected by glucose withdrawal and nutrient replacement, growth of mutant PIK3CA cells was dramatically compromised in the absence of glucose, revealing a specific dependency for this nutrient as an energy substrate. Consistent with PI3K activation mediating glucose dependency, treatment of H1047R PIK3CA cells with GDC-0941 promoted growth of the cells under low or no glucose conditions. Conclusion: Isogenic cells WT for PIK3CA are capable of utilizing a range of nutrient sources, while cells harboring H1047R PIK3CA appear hard-wired to exclusively utilize glucose. These data suggest that targeting glucose transporters or glycolytic enzymes may be a powerful therapeutic strategy for mutant PI3K-cancers. The overt glucose dependence of cells with this mutation may also offer routes to non-invasively image PI3K-pathway activated tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1274. doi:10.1158/1538-7445.AM2011-1274

Abstract Background: We previously reported that a published “Metastasis Score” (MS) could be used to evaluate the effects of a PI3K inhibitor (GDC-0941) on mutated human isogenic breast cancer cell lines. MS, based on the expression of14 genes, has been shown to predict distant metastasis in ER(+), node (−), breast cancer. We were interested to determine the impact of gefitinib (EGFR inhibitor) and PLX-4720 (selective inhibitor of BRAF V600E) on MS to assess the applicability of this score to a broader class of targeted agents. In addition, given the cross-talk between metabolism and proliferation, we also profiled genes involved in glycolysis, fatty acid metabolism and oxidative phosphorylation. Methods: Parental MCF10A (WT for all genes) and isogenic lines of MCF10A harboring PI3K (H1047R), p53 null or KRAS(G12V) mutations were cultured overnight in DMEM:F12 media under identical conditions. Cells were then treated with gefitinib, PLX4720, or DMSO (vehicle control) and further incubated for 24hr. Expression analysis was performed by RT-PCR. Results: The MS of the PI3K (H1047R) and p53 null lines was higher than the parental line and lower for the KRAS (G12V) line. Treatment of parental, PI3K and KRAS cell lines with gefitinib resulted in dose-dependent decreases in MS, as reported for GDC-0941, with a higher dose required to inhibit growth of PI3K (H1047R) cells. Treatment of p53 null cells with gefitinib, however, had only a modest effect on MS. In contrast to gefitinib, MS increased with PLX-4720 treatment in all 4 lines; the greatest increase was observed in KRAS (G12V) cells. The expression of metabolic genes differed significantly depending on the oncogenic mutation harbored by the cell line. ACTA2, ACLY, RPIA, KHK, GLS2 were most highly expressed in p53 null but lowest in KRAS (G12V), while GLUT5, PFKFB4, ENO3, SLC27A1, PGK2, GLUT1, TKT were expressed most highly in KRAS (G12V) but lowest in PI3K(H1047R). Upon treatment with gefitinib or PLX-4720, genes that were downregulated in the mutated lines generally showed a dose-dependent increase; those that were upregulated relative to the parental, showed a dose-dependent decrease with treatment. Discussion: The decrease in MS of the parental, PI3K (H1047R) and KRAS (G12V) cell lines treated with gefitinib supports the impact of this EGFR inhibitor on cell proliferation. The modest effect on MS in p53 null cells supports the findings that sensitivity to gefitinib requires active p53 in order to induce apoptosis through a p53-dependent pathway. Increases in MS were observed in all 4 cell lines treated with PLX-4720, with the largest increase observed in KRAS (G12V). The enhanced proliferation of a BRAF WT cell line with a KRAS mutation is consistent with the Ras-dependent nature of this pathway. The metabolic genes showed diverse expression patterns that differed with different oncogenic mutations and likely reflect the multiple mechanisms controlling metabolism in cancer. An improved understanding of the expression of metabolic genes relative to proliferation in cell lines with various oncogenic mutations may provide additional insights into the dysregulation of these cellular processes and the possible role of anti-metabolite intervention. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-04.

Abstract Targeting tumor metabolism is becoming a major new area of pharmaceutical endeavor. Consequently, a systematic search to define whether there are specific energy source dependencies in tumors, and how these might be dictated by upstream driving genetic mutations, is now required. This is particularly relevant given the potential ability of cancer cells to switch between metabolic pathways. The PI3K-Akt-mTOR signaling pathway has a seminal role in regulating diverse cellular processes including cell proliferation and survival. This pathway has also been associated with metabolic dysregulation, particularly gluconeogenesis, largely through effects on Akt and mTOR. We have used isogenic cell models, wherein homologous recombination is used to specifically delete endogenous mutant PI3KCA alleles or introduce PIK3CA activating mutations in human cell lines. By performing rtPCR on candidate metabolic genes, we have found gene expression signatures indicative of a consistent up-regulation of glycolysis in PIK3CA mutant cells. Interestingly, the genes up- and down-regulated varied between isogenic models suggesting that the primary node of regulation is not the same between models. In MCF10A breast epithelial ‘knock-in’ lines, hexokinase 2 (HK2) is the primary target of up-regulation. However, in HCT116 colon cancer cells that are heterozygous for the H1047R PIK3CA mutation, knocking-out mutant PIK3CA resulted in a marked reduction of Glut1. Altered expression of asparagine synthetase (ASNS) and glutaminase (GLS2) was also observed in both models, suggestive of a potential increased dependency on glutaminolysis, while up-regulation of the fatty acid transporter SLC27A2 was observed in HCT116 PIK3CA mutant cells. To determine whether any of these metabolic expression changes impart hard-wired nutrient dependencies, we next performed nutrient switching experiments. These demonstrated that growth of PIK3CA mutant cells is highly dependent on glucose, whereas glutamine dependency is independent of PIK3CA status. In addition, the glucose dependency exhibited by PIK3CA mutant cells could not be overridden by supplementation with other nutrients such as alternative monosaccharides, fatty acids or aspartic acid. The extent of glucose dependency of the PIK3CA mutant cells suggested that targeted disruption of the glycolytic pathway should inhibit growth, even in the presence of glucose. The use of hexokinase 2 siRNA to disrupt glycolysis did indeed result in anti-proliferative effects that were selective for the PIK3CA mutant cells. In conclusion, we have demonstrated that glycolysis-associated genes are up-regulated in PIK3CA mutant cells and that the shift to a glycolytic phenotype does not appear to be controlled by a single rate limiting molecule. Despite suggestions that cancer cells are adept at utilizing alternative nutrient sources, this study has demonstrated that PIK3CA mutant cell are not able to compensate for glucose withdrawal. This specific dependence on glucose for growth, identified using precise isogenic models, was also present across a wider panel of cancer cell lines harboring endogenous PIK3CA mutations. Understanding the metabolic dependencies of PIK3CA mutant cancers will provide critical information for the design of effective therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B140.

Abstract Background: Activation of signaling pathways, loss of tumor suppressors and oncogenic mutations often lead to dysregulation of glycolysis and tumorigenesis. We performed expression analysis on genetically-defined human isogenic breast cancer cell lines and with PI3K inhibitor exposure to determine impact on a patient-relevant Metastasis Score (MS) (proliferation index) and an assembled “glycolytic index”. A better understanding of the links between cellular metabolism and proliferation may yield insights into tumor cell biology and shed light on potential companion diagnostic and therapeutic opportunities. Methods: A parental MCF10A and three isogenic cell lines harboring K-ras (G12V), PI3Kα (H1047R), and p53 null were grown overnight in the presence of high levels of growth factors in tissue culture flasks. The parental and PI3Kα cells were treated with 0.3 µM, 1 µM,3 µM GDC-0941 or DMSO. All cell lines were incubated for an additional 24 hr. RNA was extracted with RNeasy (Qiagen) and expression analysis performed by RT-PCR. A total of 28 genes were profiled that included 14 genes in a metastasis score (MS) (Tutt et al), 11 glycolysis-associated genes and 3 reference genes. Expression of each gene was calculated using the ΔΔCT method and “summed” with equal weighting to yield an MS and a “glycolytic index”. Results: The MS and phenotypes for the four untreated cell lines were similar under the monolayer culture conditions used. The parental and PI3Kα cells had a similar “glycolytic index” while p53 null and K-ras cells had elevated indices. Treatment of the parental and PI3Kα with GDC-0941, a PI3K inhibitor, showed a dose-dependent decrease in the expression of constituent MS genes and the glycolytic genes as well as the combined scores. The correlation coefficient between MS and the glycolytic index was 0.77. The four genes in MS with the greatest dose-dependent decrease in expression (BUB1, CCNB1, MYBL2 and UBE2S) are associated with CDK1, a seminal participant in cell cycle regulation. The two genes from the “glycolytic index” with the greatest decrease, HK and PKM, are directly involved in glycolysis. The effect of GDC-0941, as reflected by MS and the “glycolytic index” was more pronounced in PI3Kα than the parental cells (p-value=0.0005). Conclusion: The good correlation observed between the MS and “glycolytic index “in the treated cell lines supports an association between proliferation and metabolism upon restriction of cell signal transduction. The dose-dependent expression decreases in the two indices and their constituent genes, suggest that this PI3K inhibitor successfully perturbs both fundamental processes of the cell. Given the ultimate targeting of proliferation suppression, these scores may serve as useful tools in evaluating therapeutic agents, independent of the specific intended pathways. The similar proliferation levels observed in untreated MCF10A and three mutated cell lines suggest either that single oncogenic mutations are insufficient to affect MS or that the in vitro conditions are masking the metastatic/invasive phenotype. Studies are underway to evaluate cells grown in 3D Matrigel that more accurately mimic the tumor microenvironment. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-19.

Abstract Background: Human derived cell lines represent an important model system for fundamental research and translational medicine. We sought to gather patient-relevant information by examining a previously published metastasis score (MS) (proliferation index) for operable breast cancer in well-characterized isogenic cell lines. We chose to examine the impact on MS of specific genetic alterations in known oncogenes and consequence of treatment with a class I PI3K inhibitor. Investigation of the proliferation index of cells without the complexity of cell mixtures found in tumor microenvironment may shed light on tumor cell-specific transcriptional dysregulation. Methods: Nontumorigenic MCF10A and isogenic cell lines harboring (G12V), PI3Kα (H1047R) and p53 (null) mutations were cultured in T75cm tissue culture flasks overnight. MCF10A and isogenic PI3Kα (H1047R) cell lines were seeded and allowed to adhere overnight before addition of 0.3 µM, 1 µM and 3 µM GDC-0941 or DMSO control and incubated for 24 hours. Total RNA was prepared using RNeasy Kit (Qiagen) from cells. Expression profiling was carried out using RT-PCR for the previously described metastasis score (Tutt et al, 2009) that includes 14 genes and 3 reference genes. Results: MCF10A and the isogenic mutant cell lines had equivalent levels of proliferation as measured by MS and similar phenotypes under these monolayer culture conditions with high levels of growth factors. Modest dose-dependent decreases in the expression level of the constituent MS genes (1.5-3 fold) were observed with GDC-0191 exposure that translated into a marked decrease in overall MS (14 to 4) due to their equally weighted additive impact. The four genes that demonstrated the greatest decrease were CCNB1, BUB1, MYBL2, and UBE2S. These genes are highly associated with cyclin-dependent kinase (CDK1), a key player in cell cycle regulation. The MS score for these isogenic lines are also being defined under in vitro assay conditions that more accurately mimic the tumor microenvironment; in particular a 3-dimentional Matrigel assay that unleashes a marked differential metastatic/invasive phenotype in mutant vs. WT cells. Conclusion: MCF10A and isogenic cell lines harboring KRAS (G12V), PI3Kα (H1047R) and p53 (null) mutations had equivalent levels of proliferation as measured by MS. These results suggest that a combination rather than a single oncogenic mutant are required to alter the MS, although in vitro conditions likely play an important role in fully unmasking genotype-specific phenotypes in these clean gain-of-function models. Exposure of parental and PI3K (H1047R) cell lines to PI3K inhibitor GDC-0941 led to a dose-dependent decrease in constituent genes and composite MS. These results suggest that even though genes in the PI3K/PTEN pathway are not included in the molecular score, perturbation of this signaling pathway leads to alteration of the MS, by affecting cell cycle progression. Future studies are required to determine if alteration of MS in cell lines treated with different targeted oncogenic pathway inhibitors will recapitulate patient tumor response. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A33.