Citations

Abstract Throughout differentiation, mature B cells express distinct combinations of the BAFF and APRIL receptors, BAFF-R, TACI, and BCMA. The patterns of B lineage cell receptor expression reflect their stage of differentiation and impart the ability to respond to ligands, in some cases delivering a powerful anti-apoptotic signal. B cell malignancies arise from each stage of differentiation, typically exhibit the patterns of receptor expression that reflect their cell of origin, and have been shown to exploit the generally anti-apoptotic effects of BAFF and/or APRIL. For example, there is evidence for a role for BAFF in mature B cell cancers, including B cell chronic lymphocytic leukemia (B-CLL). As the majority of circulating CLL B cells are quiescent cells, prolonged survival is a significant hallmark, a trait that signals through BAFF-R could initiate or reinforce. Therapeutic strategies designed to interrupt this pro-survival pathway have thus far primarily focused on blocking ligand binding. Therapeutic modalities impacting receptor expression may be similarly effective. However, despite the apparent precise activation stage-dependent orchestration of B cell BAFF-R, TACI, and BCMA expression, the genetic mechanisms regulating expression of the three receptors remain undefined, and the question of whether each receptor governs expression of the other two remains unanswered. In agreement with previous studies in our own lab and others, analyses of the receptor profiles of CLL B cells continue to show BAFF-R surface expression, albeit at lower levels than seen on normal peripheral B cells, and the curious variable presence of BCMA and TACI. Similarly, multiple myeloma (MM) plasma cells (PCs), like normal PCs, uniformly lack BAFF-R expression, express BCMA, and variably express TACI. Our current study explores the mechanisms of receptor regulation in B cells, with an emphasis on BAFF-R, the receptor that is most consistently expressed on the CLL B cell population and that has the most clearly defined survival function. We began by analyzing the BAFF-R gene’s genomic context. We identified a possible regulatory element 2 kb upstream of the first exon with significant homology across seven mammalian species that overlapped a cluster of B cell lineage transcription factor binding sites, and, thus, we called the 2.5 kb directly upstream of the gene the putative BAFF-R promoter. We cloned the region and created promoter-reporter vectors in which the full-length promoter and 0.5 kb 5’ truncations thereof drive firefly luciferase production. While studies of primary B cells continue, studies with malignant B cell lines suggest that we have successfully cloned a powerful positive regulatory region. Specifically, B cell lines that express surface BAFF-R show positive inductions of firefly to control renilla luciferase activity in all of the promoter constructs over the empty construct with the greatest promoter activity in the longest constructs: 6-, 18-, and 3-fold inductions with the 2.5 kb promoter in RAJI, Loukes, and MEC-1 cells, respectively. To further test the promoter specificity, we transfected malignant PC lines, ALMC-1, ALMC-2, and KAS-6/1, which are negative by RT-PCR and surface staining for BAFF-R. These lines showed little promoter activity over baseline, with fold inductions between 0.5 and 2.5 for all of the promoter constructs. These results suggest that the MM lines no longer express the transcription factors required to drive BAFF-R expression and underscore our conclusion that we have identified the BAFF-R promoter. At the same time, investigations into epigenetic modification may reveal a crucial level of control. The transcriptional start site of the BAFF-R gene falls within a region of high CG content, and may be a possible CpG island. Upon treatment with the methyltransferase inhibitor, 5-azacytidine, primary blood B cells and MM cells showed no change in receptor expression. However, in CLL B cells, treatment of cultured cells caused a slight (9%) decrease in BAFF-R expression and prevented TACI upregulation in cells stimulated with CpG (a 76% increase fell to 36%). This evidence suggests that methylation indirectly suppresses expression of BAFF-R and TACI. It is essential to understand the regulation of survival receptors critical to normal B lineage cell survival, which may also be crucial for their malignant counterparts, in order to target those mechanisms as therapy.

Abstract Background: This is an initial report of a correlative laboratory study of an ECOG clinical trial, E2903, to investigate an array of prognostic factors that are potentially associated with the clinical outcome in previously treated, high risk Chronic Lymphocytic Leukemia (CLL) patients. The ultimate goal of this study is to develop a prognostic model for this group of patients. Method: We have previously reported (ASH, 2007) on initial clinical and toxicity results using a combination of PCR followed by Campath. To date we have clinical response data on 37 patients and have conducted correlative prognostic factor analysis on these patients at entry to the study. The prognostic parameters assessed were fluorescence interphase hybridization (FISH) panel, CD38, ZAP-70 and IGVH (mutated vs. unmutated) status and plasma levels of pro- and anti-angiogenic cytokines. In addition, features such as clinical stage, age, sex and laboratory parameters including total white blood count, absolute lymphocyte count (ALC) and beta 2 microglobulin (b2m) were studied as predictors of response to PCR. Results: As of July 2008, the response data for the first 41 (the accrual goal is 110) patients who started the induction treatment are available. Of these 41, 3 patients are ineligible and one patient had no response data. The median age was 63 (range 51–76) with 29 males and 8 females, median b2m was 4 (range 2–28), 11 (31%) were Rai stage 0–2, and 26 (69%) were Rai 3–4. The median number of prior treatments was 2 (range 1–5); 15 of 23 (65%) patients tested for IGVH were unmutated, while on FISH analysis 4 had del 6q, 8 had del 11q, 4 had del 17p and 30 patients had complex FISH defects (>1 defect). Despite the prevalence of high risk features in this cohort of patients, 17 out of 37 responded (best response=partial response), 18 were stable and 2 had progressive disease with an overall response rate of 46%. Prognostic parameters most strongly associated with a clinical response included CD38 negative (p=0.008), ZAP-70 negative status (p=0.01) and mutated status (p=0.03). Responses were independent of age, sex, b2m, ALC, number of prior therapy or FISH defects. Responders showed a prolonged progression-free survival (PFS) compared to the PFS of non-responders (i.e., 2-yr PFS 29% vs. 0%, respectively, p<0.0001). Prognostic parameters most strongly associated with PFS included being both ZAP-70 negative (p=0.0009) and CD38 negative (p=0.004). Also, ZAP-70 negative and CD38 negative status was a strong predictor of favorable PFS (p=0.002). Mutated status was borderline significant (p=0.07) in univariate analysis and there was no obvious correlation of baseline angiogenic cytokine plasma levels with PFS. These results also hold in a multivariable Cox model. In the Cox model, mutated status was significantly associated with PFS (HR=0.2, p=0.01). Conclusion: We believe that the PCR regimen is highly effective at inducing responses in previously treated relapsed or refractory CLL patients who carry high risk prognostic markers. Thus, while no CRs were seen all but 2 patients had a PR or were stable after 6 cycles of PCR. The prognostic parameters most strongly associated with predictors of clinical outcome appear to be ZAP-70, CD38 and mutation status. The ability to attain a response also appears to be important for achieving a prolonged PFS. In summary, we believe that the strong clinical activity of PCR in a high risk group of CLL patients provides more evidence for pursuing the regimen as a platform for other combinations. In addition, we have generated a first level prognostic model that will continue to be refined as we proceed to complete this trial.

Abstract Background: Normal and tumor stem cells are characterized by high activity of multidrug resistance (MDR) transporters. One of these, ABCG2 (ATP-binding cassette, sub-family G member 2 protein), is an ATP dependent transporter and putative stem cell marker responsible for verapamil sensitive Hoechst efflux. While ABCG2 is known to be expressed in normal and leukemic stem cells, as well as a small population of normal lymphocytes and some B-cell malignancies, its expression in chronic lymphocytic leukemia (CLL) is unknown. It has been postulated that leukemic stem cells due to their quiescent nature and expression of MDR transporters represent a population resistant to therapy and that this residual population is critical for tumor persistence and recurrence. Hypothesis: We hypothesized that ABCG2 is expressed in a small percentage of primary CLL B cells; gene expression profiles of ABCG2 positive versus ABCG2 negative CLL B cells differ in respect to expression of self renewal and lymphoid development genes; the frequency of ABCG2+ CLL B cells increases after treatment in patients responding to therapy. Methods: We analyzed ABCG2 expression by primary CD5+, CD19+ CLL-B cells from untreated CLL patients of all Rai stages by flow cytometry. In a subset of patients we used fluorescence activated cell sorting (FACS) to sort CD19+, CD5+ ABCG2+ and CD19+, CD5+ ABCG2- cells. Gene expression profiling was then performed using the U133 plus 2.0 Affymetrix microarray platform. In a separate cohort of patients treated in a clinical trial of pentostatin, cyclophosphamide and rituximab (PCR), the percentage of ABCG2+, CD19+, CD5+, CD79b dim cells at baseline and then two months after completion of 6 cycles of PCR therapy where patients had minimal residual disease (MRD) was assessed and correlated with clinical response. Results: ABCG2+ CD19+, CD5+ detectable populations were seen in all 20 CLL assessed patients (median percentage 0.6%; range 0.08%–3.8%). There was no difference in percentage of ABCG2+ cells based on Rai stage, IGVH mutational status, Zap70 or CD38 expression. Preliminary analysis of the gene expression profiling of ABCG2 positive versus negative CLL B cells from four randomly selected patients revealed significantly higher expression of genes associated with self-renewal, cell cycle and early B-cell development including: cyclin-dependent kinase inhibitor 1C (CDKN1C, p=0.034), transcription factor 7-like 2 (TCF7L2, involved in WNT pathway regulation, p=0.016), beta-catenin (p=0.034) and pre-B-cell colony enhancing factor 1 (PBEF-1, p=0.037). Flow based assessment of the levels of ABCG2 positive populations at baseline and after therapy with PCR in patients with minimal residual disease showed a dramatic increase in frequency of ABCG2 positive CLL B cells. The percentage of ABCG2+ cells went from a median level of 0.19% (range 0.04%–0.19%) prior to therapy to a median level of 10.93% (range 0.15%–25.12%), p<0.001. In contrast two patients who did not reach MRD (partial responses by NCI-WG criteria) had no significant increase in percentage of ABCG2 positive cells (0.14%; 0.23% and 0.16%; 0.21% prior and after therapy, respectively, p=0.68). Conclusion: Our data indicate that ABCG2 positive CLL B-cells constitute 0.1–3.8% of circulating CLL B-cells in untreated patients. The frequency of ABCG2+ CLL B-cells appears to dramatically increase after therapy in the MRD state; this could be related to their relative resistance to therapy and/or a shift from extravascular compartments post therapy. Since ABCG2 positive CLL B-cells demonstrate expression of early B-cell development and self-renewal genes we believe that that this population could represent a putative self renewing CLL B-cell compartment. Further studies to characterize features of ABCG2 CLL-B –cells in relation to their capacity to be self renewing and resistance to therapy are warranted.

Abstract Within the past five years studies published from this laboratory and others have identified or confirmed CD38 as a marker for aggressive disease in B cell chronic lymphocytic leukemia (B-CLL). To learn more about the possible role of CD38 in this disease, we performed gene expression profiling (GEP; Affymetrix U133A platform) on primary leukemic B cells isolated from CD38 positive and negative patients. We and others have previously shown that the leukemic compartment within some patients consists of both CD38neg and CD38pos CLL B cells. To optimize identification of differentially expressed genes associated with CD38 expression or lack thereof, we restricted our gene profiling to patient cells that were unimodally negative for CD38 and those that expressed unimodally high levels of CD38. In both groups of patients, CD19 positive cells were purified using magnetic bead cell sorting on a Miltenyi AutoMacs prior to isolation of total RNA. To determine genes that appear to be differentially expressed in this set of experiments, we used a recently developed model-based algorithm that uses the normalized probe-level data as well as information about the underlying experimental design. Reassuringly, the gene expression profiles within the CD38pos versus the CD38neg cohort maintain congruency within each cohort when analyzing the gene expression data set as a whole, as well as within categories such as adhesion, signaling, and transcriptional molecules. Using this analytical approach, several genes emerged as interesting candidates for exploration into the nature of the CD38pos association with disease aggressiveness. Interestingly, although peripheral blood CLL B cells are overwhelmingly in the resting, G0, stage of the cell cycle, several cell cycle related genes were expressed at higher levels in CD38pos patient leukemic cells but not in CD38neg leukemic patients. Moreover, these genes encompass protein families that function at different stages throughout the cell cycle. These genes include: anaphase promoting complex subunit 5 (APC5); cell division cycle (cdc) family members; a checkpoint protein CHFR; the microtubule and actin associated protein, MACF1; the mitotic spindle checkpoint protein, MAD1L1; the DNA replication regulator, MCM7; STAG3, a meiotic regulator of chromosome segregation; and several activators of the ubiquitin pathway, including UBE2N, a known regulator of DNA repair enzymes (p-values of <0.01, <0.01, <0.01, 0.01, 0.02, <0.01, 0.01, and 0.01, respectively). We have begun to confirm these findings at the protein translation level. Through immunoblotting, preliminary data show that 3 out of 5 CD38pos and 0 out of 5 CD38neg patients’ leukemic cells express detectable levels of APC5, suggesting that CD38pos cells, or at least a subpopulation of cells, express APC5. Taken together, these gene and protein expression data suggest that at least a subpopulation of the leukemic cells from CD38pos CLL patients have more recently completed the cell cycle and maintain expression of specific cell cycle related genes. Alternatively, it is possible that this subpopulation is better prepared to re-enter the cell cycle if it encounters proliferative stimuli in other microenvironments.

Abstract Motexafin gadolinium (MGd, Xcytrin®) is a tumor-selective redox mediator that oxidizes intracellular reducing metabolites and protein thiols, thereby generating reactive oxygen species (ROS) through a process known as redox cycling. B-cell chronic lymphocytic leukemia (CLL) has elevated levels of the antioxidant ascorbate, and is known to be sensitive to redox stress. Since MGd can generate ROS through oxidation of ascorbate, we reasoned that MGd would be cytotoxic to CLL cells. Mononuclear cells were isolated from the blood of CLL patients (n=15) characterized by Rai Stage, CD38, FISH and immunoglobulin heavy chain variable (Ig VH) mutational status. CLL cells were incubated in vitro over 2–5 days with MGd at concentrations ranging from 0–50 uM. Cytotoxicity was assessed by flow cytometry using annexin V binding/propidium iodide uptake. Accumulation of MGd in CLL cells was demonstrated by flow cytometry using drug fluorescence (>650 nm). MGd-induced cytotoxicity was found to be dose-dependent over a range of 2.5 to 50 uM in cells from 11 of 15 CLL patients. CLL cells from 11/15 patients treated with 50 uM MGd and 4/15 patients treated with 10 uM MGd had cytotoxicity levels at least 2 fold above baseline. MGd uptake was similar in sensitive and resistant CLL cells, implying that mechanisms other than uptake are responsible for the differential responses. CLL cells that were sensitive to MGd treatment showed an increase in caspase-3 activity and cleavage of the caspase substrate PARP was found by immunoblot suggesting that MGd induced apoptosis. The induction of apoptosis was found to be exclusive of Rai stage, CLL FISH defects, CD38 expression or Ig VH mutational status. Cells from 5 CLL patients were studied for drug synergy using seven concentrations of MGd (7.5 – 50 uM) and the active moiety F-ara-A of fludarabine (0.25 –2.0 uM). The cells were treated with each drug individually or both drugs for 48 hours prior to analysis for cytotoxicity. MGd was found to exhibit additive activity (N=4) or synergistic (N=1) effects on CLL B cytotoxicity when combined with fludarabine. Thus, our pre-clinical results reveal that MGd is cytotoxic to CLL cells in vitro through induction of apoptosis. Furthermore, MGd shows additive or synergistic activity when combined with fludarabine. These results support the evaluation of MGd in ongoing CLL clinical trials utilizing this agent alone or in combination with fludarabine.

Abstract The protein tyrosine kinase ZAP-70, known to be critical for T cell development and T cell receptor signaling, has become a potential prognostic tool for B cell chronic lymphocytic leukemia (B-CLL). Recent studies have suggested that those patients with ZAP-70 positive leukemic cells have a poorer disease prognosis than do patients whose leukemic cells lack ZAP-70 expression. In addition, ZAP-70 expression occurs more frequently in CLL B cells that express unmutated immunoglobulin (Ig) VH region genes. However, several questions remain regarding ZAP-70 expression in B-CLL. First, is ZAP-70 expression in some patients with B-CLL the consequence of malignant transformation or do normal human B cells express ZAP-70 under certain conditions of stimulation? Secondly, does the expression of ZAP-70 reflect a more activated phenotype, thus resulting in the more aggressive phenotype of ZAP-70+ B-CLL? To begin to address these questions, we focused our studies on ZAP-70 expression in normal, human B cells. CD19+ B cells were isolated by magnetic bead separation from normal human peripheral blood (PB) or tonsil and spleen surgical waste tissue. We first used three-color flow cytometry to evaluate ZAP-70 expression in CD19+ versus CD3+ lymphocytes. While PB B cells were negative for ZAP-70 expression, we were able to detect a subset of CD19+/ZAP-70+ cells in both spleen and tonsil and this subset expressed ZAP-70 at levels comparable to those of CD3+ T cells. To verify the flow cytometry results, we evaluated total ZAP-70 expression by immunoblotting and observed readily detectable ZAP-70 in both splenic and tonsillar CD19+ cells. Based on the observation that ZAP-70 was found in B cells of tonsil and spleen, but not PB, we hypothesized that ZAP-70 may be associated with activation status since tonsillar and splenic B cells will include a population of activated B cells. To examine this issue, we sorted CD19+/CD38+ from CD19+/CD38− tonsillar cells and again evaluated ZAP-70 expression. Whereas both populations expressed ZAP-70, CD38+ B cells expressed ZAP-70 at a higher level suggesting that ZAP-70 may be associated with a more activated phenotype. To address the issue of activation, we activated CD19+ PB B cells with a cocktail of CD40L, IL-10, IL-4 and IL-6 for 3 days and analyzed ZAP-70 expression. Indeed, we were able to induce ZAP-70 expression in a subpopulation of blood B cells. We also assessed ZAP-70 phosphorylation following tonsillar B cell receptor (BCR) ligation and we observed that phosphorylation of ZAP-70 (tyrosine 493) occurred within 10 min. of BCR stimulation. Finally, we assessed whether ZAP-70 expression in B-CLL could be associated with an activated cell phenotype. By stimulating Ig VH non-mutated and mutated B-CLL patient samples with the CD40L cocktail, we observed an increase in ZAP-70 expression in non-mutated CLL by immunoblotting. These results demonstrate expression of ZAP-70 in a subset of normal B cells that express an activated phenotype. Moreover, our results also suggest that ZAP-70 levels in CLL B cells may also be increased following cellular activation. Given that non-mutated type B-CLL expresses ZAP-70 more frequently than mutated type B-CLL, ZAP-70 expression may simply reflect a more activated population of leukemic cells that may correlate with progressive disease. Continuing investigation into the role of ZAP-70 in normal B cell development and B-CLL is clearly warranted.

Abstract Background: Endx is a Tam metabolite with promising antitumor activity in Tam and aromatase inhibitor (AI) resistant estrogen receptor (ER) positive (+) metastatic breast cancer (MBC). Methods: This randomized phase II study compared progression-free survival (PFS) and toxicity of Endx 80 mg/day to Tam 20 mg/day in patients (pts) with ER+ MBC. Eligibility included postmenopausal women, ECOG PS 0-2, prior progression on AI but not Tam (unlimited endocrine therapy [ET] lines allowed), and a preregistration biopsy confirming ER+ (>10% nuclear staining) and HER2-negative MBC. Stratified randomization was used balancing prior CDK 4/6 inhibitor (CDK 4/6i) use and/or everolimus (yes/no), measurable disease (yes/no) and endocrine resistance (primary/secondary) between arms. Pts randomized to Tam were allowed to cross over to Endx at progression. Due to short expected median PFS, differences in PFS were assessed using approaches for interval-censored data (ICD). 40 eligible pts were to be randomized to each arm so a one-sided alpha=0.10 generalized log-rank test (GLRT) would have a 90% chance of detecting a 50% decrease in hazard of disease progression with Endx (median: 6 months) relative to Tam (median: 3 months). Secondary endpoints include clinical benefit rate (stable or partial response > 6 cycles) (CBR) for measurable and non-measurable disease. Pharmacokinetic (PK) data were obtained Day (d) 1 (4 hour), end of cycle 2, and at time of progression. Results: From March 2015 to March 2017, 108 women with endocrine refractory recurrent or MBC were preregistered. 27 pts did not register due to: biopsy demonstrating ER-/HER2- (3 pts), ER+/HER2+ (5 pts), cancer other than breast (3 pts), no cancer in specimen (6 pts), brain metastases (2 pts), acute infection (1 pt), progression on or recent use of Tam (2 pts), or pt refusal (5 pts). 4/81 pts who registered were excluded due to ineligibility (3 pts) or refusal to start protocol treatment (1 pt). The study cohort consisted of 40 pts randomized to Endx and 37 pts to Tam. The median (m) number of ETs in the metastatic setting was 2 (range 1-4) for each arm including prior CDK 4/6i (Endx: 42.5%, Tam: 29.7%) and everolimus (Endx: 35.0%, Tam: 40.5%). The m cycle number was 6 for Endx (range: 1-35) and 3 for Tam (range: 1-42). PFS for Endx was not significantly different compared to Tam (HR= 0.77; 95% CI: 0.49-1.22, GLRT p=0.309; mPFS: Endx 130 days [95% CI: 76-138 days] and Tam 42 days [95%CI: 24-129 days]). However, PFS was significantly longer in pts with no prior CDK 4/6i in the Endx arm (GLRT p=0.002; HR(no/yes)=0.31; 95%CI: 0.15-0.65) but not in the Tam arm (GLRT p=0.708) (unplanned analysis). Severe (grade (G) 3+) toxicities included: Endx G3 hypertriglyceridemia (3 pts); Tam: G3 hypertension with G2 stroke (1 pt), G3 thromboembolic event (1 pt), and G3 abdominal, bone and liver pain (1 pt). In Endx arm, d1 m Endx plasma concentration (conc) was 216 ng/ml (n=17; range: 144-400). For Tam arm, d1 m Tam conc was 17 ng/ml (n=17; range:11-23) (Endx not detectable). For the 25 pts that crossed over to Endx, CBR was 28.0% (90% CI: 14.0-46.2%) and 14 pts had pk data at progression. A lower median Endx conc (6 ng/ml range: 3.3-16.3) was observed in Tam patients at progression who then had Endx clinical benefit compared to Tam pts without clinical benefit after Endx crossover (median Endx 12 ng/ml; range 4.4-36.6).Conclusions: In endocrine-resistant breast cancer, Z-Endx was not significantly superior to Tam, but clinical benefit was observed in 28% that crossed over to Endx after Tam progression. In pts with no prior CDK 4/6i, the observation of significantly longer PFS in the Endx arm is hypothesis generating. Support: U10CA180821, U10CA180882, U24CA196171, U10CA180820 (ECOG-ACRIN), https://acknowledgments.alliancefound.org; Clinical Trials.gov Identifier:NCT02311933 Citation Format: Matthew P. Goetz, Vera J Suman, Joel M Reid, Mary Kuffel, Sarah A Buhrow, Renee M McGovern, John Black, Travis Dockter, William F Symmans, Minetta C Liu, John R Hawse, James Doroshow, Anna M Storniolo, Jerry M Collins, Howard Streicher, Matthew M Ames, James N Ingle, Ann Partridge, Lisa Carey. A randomized phase II trial of tamoxifen versus Z-endoxifen HCL in postmenopausal women with metastatic estrogen receptor positive, HER2 negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD7-06.

Abstract Background The Circulating Cell-free Genome Atlas (CCGA; NCT02889978) study is a prospective, multi-center, observational, case-control study with longitudinal follow-up to support the development of a plasma cfDNA-based multi-cancer early detection assay. We previously reported that the fraction of tumor-derived cfDNA fragments in plasma (estimated tumor fraction [TF]) is associated with detection of multiple cancer types using a prototype whole-genome bisulfite sequencing (WGBS) assay in a pre-specified substudy. In addition, detectability of breast cancer varied by hormone-receptor (HR) status: HR negative (HR-) breast cancer had a higher detection rate than the overall breast cancer cohort. Here, we performed a planned in-depth analysis of the breast cancer cohort to understand the main clinical and biological determinants of detectability using our WGBS assay. Methods Blood samples were prospectively collected from participants (pts) with newly-diagnosed, untreated breast cancer. In a pre-specified CCGA substudy, 511 (31.4%) of 1,628 pts had a clinicopathologic diagnosis of breast cancer (any stage, excluding stage 0) and a WGBS result. Pre-specified biological and clinical factors were assessed for correlation with TF: clinical stage, pathologic N stage, lesion size (by imaging), T stage, histologic grade, hormone receptor (HR) status, and proliferative rate (Ki-67 by IHC [MIB-1 clone]) via univariate and multivariate analyses. Ki-67 was obtained from pathology reports provided by enrollment sites and also assessed centrally (Mayo Clinic [Rochester, MN]). Stage-specific differences in detection rates between HR positive (HR+) and HR- breast cancers due to biological differences such as histologic grade and/or proliferative rate was also assessed. Results Of 511 pts, 94 (18.4%; 48.9% HR- and 12.3% in HR+) had WGBS-detected cancer. Higher TF was significantly associated with cancer detection (p<0.001). In univariate analyses, the variables significantly associated with TF were clinical stage, pathological N stage, and histologic grade (p<0.001 each). Primary lesion size was modestly correlated with TF (all stages combined: Spearman's correlation coefficient rs=0.4, p<0.001), and when grouped by early and late stages (stages I/II: rs=0.27, p<0.01; stages III/IV: rs=0.50, p<0.01); clinical T stage was significantly associated with TF only when all stages were combined (p<0.001). Proliferative rate (Ki-67) was positively correlated with TF at stages I/II (rs=0.29, p<0.05) but not III/IV (rs=-0.24, p=0.34); Ki-67 results obtained through Mayo were similar to that from enrollment sites (Pearson’s correlation coefficient rp=0.68, p<0.001). HR- breast cancers had significantly higher TF versus HR+ breast cancers only at stage II (p<0.001). In addition, compared to HR+ breast cancers, HR- breast cancers had a significantly higher proportion of high histologic grade (p<0.001 at stages I and II) and proliferative rate at earlier stages (p<0.05 at stages I, II, and III). In the multivariate analysis, clinical stage (stage III vs I and IV vs I) and HR status remained the most significantly associated with TF (p<0.001 each). Conclusions Using a prototype methylation-based plasma cfDNA cancer detection assay, TF was found to be associated with detection, and with clinical and biological features of breast cancer. Features routinely used to assess clinical aggressiveness (eg, HR- status and histologic grade) also demonstrated stage-specific associations with TF. Given the known relationship between TF and detection, these data suggest that clinical and biological cancer features may also provide insight into the variability of cancer detection using plasma-based cfDNA tests. Citation Format: Minetta C. Liu, Jodi M. Carter, Daniel W. Visscher, Karla Kopp, Rita Shaknovich, Xiaoji Chen, Kathryn N. Kurtzman, Shilpen Patel, Jacqueline D. Brooks, Carlo R. Cosenza, Jafi A. Lipson, Donald A. Richards, Fergus J. Couch, Zhao Dong, Hai Liu, Oliver Venn, Joerg Bredno, Eric T. Fung, Anne-Renee Hartman. Blood-based cancer detection in plasma cell-free DNA (cfDNA): Evaluating clinical and pathologic tumor characteristics in participants with breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-01.

9581 Background: AKT, a serine threonine protein kinase, is activated downstream of phosphatidylinositol 3-kinase (PI3K) which transmits signals from cytokines, growth factors and oncoproteins to multiple targets. Activated AKT regulates survival, proliferation, and growth. The PI3K/AKT pathway is downstream of most of the common growth factor tyrosine kinase receptors in cancer, e.g., EGFR, HER2, IGFR, etc., and is a driver of tumor progression in many cancers. AKT protein kinase is activated in many pediatric solid tumors, including glioblastoma, malignant rhabdoid tumors, neuroblastoma, synovial sarcoma, rhabdomyosarcoma and medulloblastoma. MK2206, an oral allosteric AKT1, 2,3 inhibitor, has demonstrated antitumor activity in in vitro and in vivo cancer models.A phase I trial evaluating MK2206 was conducted in children with refractory solid tumors. Methods: Using a rolling-6 design, MK2206 was administered either once every 7 days (schedule 1), or once every other day (schedule 2) in a 28-day cycle. Serial PK studies were obtained on day 1, cycle 1 and trough samples were obtained on days 7, 14, 21 and 28. Biological studies included analysis of PI3K/PTEN/AKT-cell signaling pathway in pre and post-therapy in PBMC and in tumors at diagnosis or recurrence. Results: Forty-five patients [23 males, median age 13.6 years (range 3.1-21.9)] with malignant glioma (14), ependymoma (4), hepatocellular carcinoma (3), gliomatosis cereberi (2) or other tumors (22) were enrolled; 34 were fully evaluable for toxicity (schedule 1 n=17; schedule 2 n=17). Schedule 1 DLTs included: grade 3 dehydration in 1/6 patients at 28 mg/m2; grade 4 hyperglycemia and neutropenia in 1/6 patients at 45 mg/m2. There were no DLTs at 35 mg/m2 and dose level 4 (58 mg/m2) is currently open for patient accrual with one enrollment. Schedule 2 DLTs included: grade 3 alkaline phosphatase in 1/6 patients at 90 mg/m2; grade 3 rash in 1/6 patients at 120 mg/m2), and grade 3 rash in 2/6 patients at 155 mg/m2. Conclusions: The recommended pediatric phase II dose of weekly MK2206 is 120 mg/m2 and the last cohort of patients to the every other day dosing schedule of MMK206 is enrolling. PK and PD data are currently being analyzed and will be presented.