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Background and Aims: Hematologic malignancies account for approximately 10% of all cancers in the US. In order to improve the treatment and outcome of patients with hematologic malignancies, integration of accurate noninvasive molecular biomarkers to diagnose, monitor therapeutic response and identify disease subtype are critical. Recent advances in next generation sequencing (NGS) have accelerated the utilization of circulating nucleic acids for the development of non-invasive disease biomarkers. We have developed an RNA-Seq platform that allows robust quantification of the circulating cell-free messenger RNA (cf-mRNA) whole transcriptome. Here, we conducted cf-mRNA profiling on plasma samples collected from multiple myeloma and acute myeloid leukemia patients to investigate the feasibility of cell-free transcriptomic profiling as a monitoring tool for hematologic malignancies following bone marrow transplantation and therapeutic treatment. Methods: We performed cf-mRNA RNA-Sequencing on 96 longitudinally collected plasma samples from 3 multiple myeloma and 3 acute myeloid leukemia patients. Cf-mRNA profiles of patients were evaluated using GTEx (tissue type) and Blueprint (cell type), and dysregulation of gene-expression signatures were assessed following bone marrow transplantation and therapeutic treatment. In addition, we sequenced corresponding whole blood cells and buffy coat for comparison. Results: Plasma cf-mRNA profiling showed substantially higher proportion of transcripts from megakaryocyte and non-hematopoietic cells relative to whole blood cellular transcripts, suggesting that the molecular profile of cf-mRNA is more suitable for the assessment of gene-expression dysregulation of bone marrow than whole blood. We further report that non-invasive cf-mRNA profiling captures gene-expression alterations of cancer patients following chemotherapy and bone marrow transplantation. And we observed dysregulation of hematopoietic lineage-specific transcripts in circulation during bone marrow ablation and reconstitution. Lastly, we used cf-mRNA profiling to identify a malignant Ig clone for multiple myeloma patients and were able to monitor bone marrow reconstitution. Conclusions: We demonstrated that cf-mRNA profiling is an effective means to non-invasively assess molecular characteristics that are associated with multiple clinical parameters of hematological malignancies. Our study highlights the potential of cf-mRNA Seq as a platform to develop patient monitoring biomarkers for hematologic malignancies. Citation Format: Shusuke Toden, David A. Ross, Stephen R. Quake, John J. Sninsky. Utilization of Cell free messenger RNA Sequencing for non-invasive molecular profiling of multiple myeloma and Acute Myeloid Leukemia [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 5174.

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.

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.

Background: Different gene expression sets have been reported to reproducibly assess the metastatic potential of early stage operable breast tumors. Knowledge of the biology-based components of amalgamated expression scores may provide insight into the relative contributions of the cellular processes involved. We sought to compare a composite metastasis score (cMS) consisting of a previously reported Metastasis Score (MS), that serves essentially as a proliferation index, and the progesterone receptor (PR) with the Oncotype DX® Recurrence Score (RS) in a regional cancer center setting. Methods: We examined sections from 152 blocks collected in the Blumenthal Cancer Center that had Oncotype DX® scores (a commercially available service performed by Genomic Health Inc. (GHI)). The study protocol was approved by the Institutional Review Board of the Blumenthal Cancer Center. ER and PR were initially scored by IHC; HER2 was determined by FISH. RT-PCR multiplex TaqMan assays of a 14-gene Metastasis Score (Tutt et al. (2008)) and a multiplex assay for ER, PR and HER2 (Iverson et al. (2009)) were carried out. A cMS was generated with MS and PR using previously constructed weighting. Risk classification of cMS was based on a single pre-established cut point; GHI recommended cut points were used for categorized Oncotype DX® risk. Correlations were estimated by Pearson's product-moment correlation coefficient; a goodness-of-fit test was used to determine differences in the categorical proportions. Results: Eighty-nine (59%), 51 (34%), and 12 (8%) of the 152 tumors tested had low, intermediate and high Oncotype DX® Recurrence Scores, respectively. The continuous scores for cMS and RS were correlated (r=0.71). Of the 89 tumors categorized as low risk by RS, 78 (88%) and 11 (12%) were at low and high risk by cMS, respectively; all (12) of the tumors categorized as high risk by RS were high risk by cMS; and of the 51 tumors with intermediate RS, 27 (53%) were categorized as low risk and 24 (47%) as high risk by cMS. Correlations of RT-PCR PR levels with overall RS and cMS scores were 0.70 and 0.71, respectively. The MS had correlations of 0.50 and 0.80 with RS and cMS, respectively. The categorized risk proportions of this patient population differed from those reported by Paik et al. (2004) (p=1.47x10-7). Tumors with HER2 gene amplification by FISH [3/141 (2%)] were under-represented (p=0.016) relative to Paik et al. (2004) [55/715 (8%)]; the proportion of PR negative tumors for Paik et al. [126/668 (19%)] and this study [14/149 (9%)], were also dissimilar (p=0.006). Conclusions: Despite sharing few genes, the continuous scores for cMS and RS show good correlation. The correlation of PR with these two scores suggests the level of this hormone receptor makes a similar significant contribution to both scores in this patient population. MS was more highly correlated to cMS than RS. Distinguishable features of cMS relative to the amalgamated RS include binary categorization of a continuous score and availability and transparency of discrete and combined scores for the endocrine and proliferation components. Under-representation of HER2- amplified tumors suggests exclusion of this patient group from testing. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-07-05.

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 Eisen's 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

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.

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