Steven S. Evans

INTRAVENOUS vs INTRAPERITONEAL SENSITIZER: IMPLICATIONS FOR INTRAPERITONEAL PHOTODYNAMIC THERAPY

Photodynamic therapy (PDT) is a potential treatment for peritoneal carcinomatosis. However, little data is available regarding the relative distribution of sensitizer to tumor and intra‐abdominal organs, optimal route of sensitizer administration, and maximal tolerated light dose. Tumor and normal tissue sensitizer levels were measured by tissue extraction 3, 24, 48 and 72 h after 10 mg/kg of Photofrin II was given intraperitoneally (IP) or intravenously (IV) in a mouse peritoneal tumor model, and the maximal tolerated PDT light dose determined. Equivalent tumor sensitizer levels were obtained regardless of the route of sensitizer administration. Route of administration, however, did affect the kinetics of tumor sensitizer elimination, with the half‐time for elimination (T1/2) 113.6 h for IP drug and 60.6 h for IV drug. Route of administration also affected sensitizer levels in several intra‐abdominal organs, resulting in somewhat higher tumor to liver and kidney levels at 24 and 72 h after IP sensitizer administration. Despite these tissue distribution differences, route of sensitizer administration did not significantly affect PDT toxicity or mortality when mice were treated with 630 nm light. The maximum tolerated light dose was 1.04 J/cm2. These parameters will prove helpful in designing large scale animal trials assessing the efficacy and safety of intra‐abdominal PDT.

Metformin activity in advanced adrenocortical carcinoma: A comparative analysis in human tumors.

e14568Background: The biguanide metformin is a widely used oral hypoglycemic. One mode of action is the inhibition of mitochondrial complex I and activation of AMP kinase with down-regulation of mT...

Abstract TMEM-021: CORRELATION BETWEEN DRUG SENSITIVITY AND METABOLOMIC SIGNATURES WITH OBJECTIVE RESPONSE (OR), TIME TO PROGRESSION (TTP), AND OVERALL SURVIVAL (OS) IN ADVANCED EPITHELIAL OVARIAN CANCER (EOC): A PHASE II STUDY

INTRODUCTION: Personalized oncology has advanced through the application of genomic, transcriptomics & proteomic platforms. BCR-abl; EGFr & ALK have provided drug-able targets & companion diagnostics in several diseases, yet many human transforming events are polygenic & incompletely understood at a genetic level. The recognition that oncogenesis reflects changes in both the cell and its micro-environment has renewed interest in whole cell experimental models that capture native-state cell-cell, -stroma & -vascular signaling. Phenotypic platforms like the Ex vivo Analysis of Programmed Cell Death (EVA-PCD) and Metabolomic Analyses have been shown to correlate significantly with response, time to progression & survival in cancer patients. PURPOSE: To correlate drug response profiles measured by EVA-PCD with Metabolomic Profiles measured by Mass Spectrometry, in patients with newly diagnosed, advanced epithelial ovarian cancer (EOC). Procedures: After signing informed consent, patients with advanced EOC undergoing cytoreductive surgery submit tumor tissue for EVA/PCD and blood samples for Mass Spectrometry. EVA/PCD analyses using morphologic (delayed loss of membrane integrity) and metabolic (ATP-content, mitochondrial metabolism) endpoints are used to generate dose-response curves, interpolated to provide LC50 values. The LC50 values for Cisplatin and the principal platinum-based combinations are used to define drug sensitive/drug resistant profiles for correlation with plasma-sample lipidomic analytes measured by Mass Spectrometry. Metabolite profiles from 21 participants are under analysis for more than 180 different circulating components including: amino acids, acylcarnitines, phosphatidylcholines, sphingomyelins and hexoses simultaneously identified/quantified in a targeted approach by UPLC/Mass Spectrometry. Associations between metabolite concentrations and drug sensitivity/resistance as well as clinical response parameters in EOC patients are being analyzed using adjusted linear regression models. Pairwise relationships among metabolites are investigated and illustrated by Gaussian graphical models (GGMs). RESULTS: To date, 21EOC patients have been accrued and are under analysis with a target accrual of 50. CONCLUSIONS: The growing recognition that malignant transformation represents changes in cellular metabolism offers the opportunity to interrogate human tumor biology at the level of metabolomics. This study will be among the first to correlate EOC drug response, time to progression and survival with metabolomic profiles and may offer new insights into ovarian carcinogenesis and cellular mechanisms of drug resistance with the potential to develop new predictive & prognostic models for advanced EOC. Funding: Sao Paulo State Research Foundation (FAPESP): grants 2014/19171-2 / 2015/16921-3 (Sao Paulo, SP, Brazil) and grants from Memorial Medical Center Foundation (Long Beach, CA, USA). Citation Format: Paulo D9Amora MD, PhD; Ismael DCG Silva MD, PhD; 142 MD, PhD; Marcia Batista Salzgeber; Manoel JBC Girao MD, PhD; Robert Bristow MD; Steven Evans BS, MA; Philip J. DiSaia MD and Robert Nagourney MD. CORRELATION BETWEEN DRUG SENSITIVITY AND METABOLOMIC SIGNATURES WITH OBJECTIVE RESPONSE (OR), TIME TO PROGRESSION (TTP), AND OVERALL SURVIVAL (OS) IN ADVANCED EPITHELIAL OVARIAN CANCER (EOC): A PHASE II STUDY [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-021.

Abstract 2418: A comparison of NVP-AUY-922 (AUY) and Geldenamycin (G) in human tumor primary culture microspheroids

Introduction: Molecular chaperone heat shock protein 90 (HSP 90) maintains the stability and function of client proteins essential for malignant transformation. The ansamycin Geldenamycin (G) and its derivative 17-AAG are potent HSP90 inhibitors with activity observed in human tumors. The resorcinol-based HSP90 inhibitor AUY offers advantages with regard to solubility & DT-diaphorase independence. To evaluate and compare the activity of AUY & G we conducted ex vivo analysis of programmed cell death (EVA/PCD™) on microspheroids isolated from fresh human tumor specimens. Methods: The EVA/PCD™ method as previously reported (Nagourney, RA Anticancer Res. 2012) isolates microspheroids of desired size by mechanical & enzymatic disaggregation followed by precise density centrifugation. Drug activity is measured by delayed loss of membrane integrity & ATP content (luciferase). Lethal concentration (LC50) values are interpolated from 5-point dose response curves and compared by modified Z-Score. Concentrations ranges for AUY were 6.25-100 uM and for G 1.25-20 uM. Results: AUY and G activity compared in 22 parallel analyses by Pearson Moment revealed a correlation coefficient R = 0.59 (p Sarcoma >Melanoma >Ovary>NSCLC>Breast. ERBB2 (+) breast appeared more sensitive as did several EGFr mutation (+) NSCLC specimens. Metachronous assays conducted in 3 EGFr (+) NSCLC pts revealed activity for G following Erlotinib failure. Additional studies with AUY alone and in combination are underway. Conclusions: i) HSP-90 inhibitors are active in human tumor primary cultures ii) AUY and G activity correlate iii) HSP90 inhibitor activities reveal disease & patient-specific profiles iv) EVA/PCD™ analyses could facilitate the development of clinical strategies for the use of HSP90 inhibitors. Supported by The Vanguard Cancer Foundation & The Nagourney Institute. NVP-AUY-922 was the kind gift of Novartis AG. Citation Format: Robert Alan Nagourney, Paula J. Bernard, Federico Francisco, Meghan Cule, Ryan Wexler, Steven S. Evans. A comparison of NVP-AUY-922 (AUY) and Geldenamycin (G) in human tumor primary culture microspheroids. [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 2418. doi:10.1158/1538-7445.AM2015-2418

Abstract 4245: A comparative analysis of PI3K inhibitors in human tumor primary culture microspheroids

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Altered phosphoinosotide metabolism is a hallmark of many human malignances. The phosphoinsotide kinases (PI3K) of which there are several sub-classes, have been explored as targets for cancer therapy. Beyond the first generation pan-PI3K inhibitors Wortmannin & LY294002, new, clinically active agents with subclass specificity and/or dual inhibitory functions (PI3K & mTOR) have been developed, including BEZ235 (PI3K/mTOR), GDC0941(αβδ), BYL719(α) & Idelalisib (δ). To examine the activity of these agents we applied Ex Vivo Analysis of Programmed Cell Death (EVA/PCD) (Nagourney, R et al Anticancer Res., 2012)to human tumor microspheroids, isolated directly from surgical specimens. Lethal concentrations (LC50s) interpolated from 5-point dose response curves were compared for activity and for correlation by Pearson Moment. Results: Micro-molar activity favored BEZ > BYL > LY. Correlation coefficients reveal BYL vs. BEZ (N=15, R =0.69, P Solid; BEZ: Solid > Heme; BYL: Heme > Solid with preliminary evidence for Idelalisib showing Heme > Solid. Conclusions: The PI3K inhibitors are active in both Heme and Solid tumors, with relative concordance between agents, but some evidence of disease specificity. Additional comparisons with GDC 0941 and drug combination studies are being conducted to further characterize the clinical activity and combinatorial potential of this class of agents. EVA/PCD analyses, conducted in human tumor primary cultures, have the capacity to evaluate drug activity, synergy and sequence dependence offering clinically relevant insights into novel drug applications. Citation Format: Robert Alan Nagourney, Paula J. Bernard, Federico Francisco, Steven S. Evans. A comparative analysis of PI3K inhibitors in human tumor primary culture microspheroids. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4245. doi:10.1158/1538-7445.AM2014-4245

Abstract 3525: Functional analysis of epidermal growth factor receptor tyrosine kinase inhibitors: A comparison of Afatanib, Lapatinib, and Gefitinib in human tumor primary cultures.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: Targeting epidermal growth factor receptor (EGFr) tyrosine kinases has provided effective therapies for NSCLC, breast & GI cancers. However, secondary mutations can confer resistance. New multi-targeted and/or irreversible inhibitors (TKIs) may overcome these mechanisms. We used the ex vivo analysis of programmed death (EVA/PCD) to compare the activity of the irreversible EGFr TKI, BIBW 2992 (Afatanib) with Gefitinib and Lapatinib and examined downstream signals with inhibitors of MEK/ERK & PI3K/mTOR. Methods: Human tumor 1° culture microspheroids isolated from 54 individual-patient specimens obtained at surgery or aspiration were examined for EGFr-TKI induced programmed cell death, using morphologic & metabolic endpoints as previously described (Nagourney R, Curr. Treat Opt in Oncol., 2006). Lethal concentration 50% (LC50’s) interpolated from 5-point dose response curves were compared and drug correlations were conducted by Pearson-Moment. Results: BIBW activity favored upper GI, NSCLC and ovary over breast and colorectal cancer. In NSCLC, EGFr mutation (+) tumors were significantly more sensitive than wild type to BIBW (P < .05). Patients relapsing after Erlotinib who carried T790M mutations remained BIBW sensitive despite Gefitinib resistance. BIBW activity correlated with Gefitinib (r = 0.35, P= 0.05) and the MEK/ERK inhibitor AZD 6244 (r = 0.43, P < 0.05) and trended to correlation with Lapatinib, but did correlate with mTOR or mTOR/PI3K inhibitors, Everolimus & BEZ235. Of 3 patients treated with Afatanib, 2 of 3 showed benefit, 1 durable SD & 1 durable PR. Conclusions: BIBW reveals activity in several tumor types including NSCLC, GI and ovary. In NSCLC LC50’s significantly favor EGFr mutation (+) ‘s with favorable results in T790M(+) patients. Correlations with AZD 6244 but not Everolimus or BEZ235, suggest MEK/ERK pathway to be the operative survival signal. EVA/PCD analysis in human tumor 1°cultures offers a platform to study novel strategies for EGFr-targeted therapies. Supported by the Vanguard Cancer Foundation & the Nagourney Institute. Citation Format: Robert Alan Nagourney, Paula J. Bernard, Eric Federico, Sophie Nguyen, Steven S. Evans. Functional analysis of epidermal growth factor receptor tyrosine kinase inhibitors: A comparison of Afatanib, Lapatinib, and Gefitinib in human tumor primary cultures. [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 3525. doi:10.1158/1538-7445.AM2013-3525

Abstract 2856: Functional profile of BEZ-235 activity in human tumor primary culture microspheroids by ex vivo analysis of programmed cell death (EVA/PCD)

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Aberrant signaling through the phospho-inositol pathway via the gain of PI3K function or loss of PTEN is a hallmark of many malignancies. Drugs that inhibit this pathway offer novel therapeutic opportunities. The imidazoquinolone derivative, BEZ-235, reversibly inhibits signaling through PI3K & mTOR by competition at ATP binding sites. We used ex vivo analysis of programmed cell death (EVA/PCD) to examine the activity and clinical potential of BEZ 235 in human tumors isolated from 88 surgical tumor specimens. By interrogating drug effects in primary culture micro-spheroids, replete with vascular, stromal and inflammatory elements, the EVA/PCD platform can provide insights into cellular responses under native-state conditions. Lethal concentration 50% values (LC50s), interpolated from dose response curves, were used to compare the activity of BEZ-235 by diagnosis using modified Z-scores. Comparisons between BEZ activity and related inhibitors of PI3K (LY294002), mTOR (Everolimus) & AKT (Phen B15-kindly provided by Dr. Peter Houghton) were conducted by Pearson-Moment. By rank order, BEZ-235 activity revealed upper gastrointestinal, breast, NSCLC and hematological malignancies to have the most favorable profiles, while renal, ovarian, colon and sarcoma specimens fell in the more resistant range. Pearson moments revealed high correlation coefficients (R values) for LY294002=0.56 (P =0.001); Everolimus=0.51 (P= 0.005) & Phen B15=0.89(P= 0.005) all consistent with BEZ-235s known modes of action. As PI3K signaling is associated with inhibition of apoptosis, its up-regulation may confer collateral resistance to other stressors like growth factor withdrawal or cytotoxic drugs. Relationships between BEZ-235 and other classes of drugs are being examined to explore additional correlations and potential combination that could provide future therapeutic opportunities, as will be reported. Supported in part by the Vanguard Cancer Foundation. 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 2856. doi:1538-7445.AM2012-2856

Abstract 650: Ex vivo analysis of the mTOR/PI3K & MEK/ERK inhibitors, BEZ235 & AZD6244, alone and in combination in human tumor primary culture micro-spheroids: Exploration of horizontal pathway targeting

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Therapeutic targeting of signal transduction pathways offers fertile ground for the discovery of small molecule kinase inhibitors and novel combinations. The success of Imatinib in CML, the first clinically approved oral TKI, reflects in part, the linear association between the malignant phenotype and c-Abl activation. However, most solid tumors reveal complex interactions between signal pathways that cross talk at points of commonality, the subject of prior report (Nagourney, RA Proc. AACR, 2010). To examine the clinical potential of BEZ235 and AZD6244, inhibitors of the PI3K & MEK/ERK pathways, we applied Ex Vivo Analysis of Programmed Cell Death (EVA/PCD™) (Nagourney RA, Curr. Treat Options in Oncology, 2006) to tumor micro-spheroids isolated from 24 patients. Drugs were tested alone and in combination. Five point dose response curves were interpolated to provide LC50 values. Synergy was assessed by median effect. Activity comparisons were conducted by modified Z-Score. LC50 values by Z-score for AZD revealed Melanoma > Breast > NSCLC > Myeloma >Ovarian > Uterine >Sarcoma > Renal, while BEZ revealed Breast > Renal > NSCLC > Ovarian > Myeloma > Sarcoma > Uterine. By combining BEZ with AZD, we then explored the simultaneous inhibition of the MEK/ERK and PI3K/mTOR pathways. Results in 5 of the first 6 analyses revealed activity & synergy for the dual pathway inhibitor combination. These results support the clinical relevance of targeting the MEK/ERK and PI3K/mTOR pathways and more importantly suggest dual pathway inhibition (horizontal) to be a productive strategy for further clinical developmental. Disease specific profiles and sequence dependence are being explored and will be reported. Supported in part by the Vanguard Cancer Foundation and The Nagourney Institute. 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 650. doi:10.1158/1538-7445.AM2011-650

Functional analysis of human tumor response to signal transduction inhibitors: Ex vivo analysis of programmed cell death (EVA-PCD) in 1° culture microspheroids

Advances in cancer therapy increasingly reflect the application of genomics and proteomics to target tumor-specific phenomena. Several insights have accelerated this process including the primacy cell survival signals in carcinogenesis and drug resistance and the growing appreciation of tumor biology as contextual. Ex vivo analysis of programmed cell death (EVA/PCD™) addresses both issues by measuring metabolic and morphologic features of drug induced cell death (apoptotic and non-apoptotic) using native state 1° cultures isolated from surgical specimens and cytologically (+) fluids. The predictive validity of the EVA/PCD™ established for cytotoxics, e.g,. 2CDA in hairy cell leukemia; purine-alkylator synergy in NHL (Nagourney et al, B.J. Cancer, ‘93) and platin-gemcitabine activity/synergy in breast and gynecologic cancers (Nagourney et al, Deoxynucleoside analogs in cancer therapy, Humana Press, 2006) has led to this platforms current focus on signal transduction with a growing emphasis on NSCLC. A recent report in NSCLC provided an ORR of 62%, med TTP of 9.5 mos and med OS of 22.3 mos, including the identification of durable responders to EGFr-TKIs (Nagourney RA Proc ASCO, 2010). An EVA/PCD™ exploration of “vertical” and “horizontal” signal inhibition (Nagourney et al Proc AACR, 2010) favored “horizontal” strategies in NSCLC providing very durable clinical responses with dual EGFr/VEGFr and clinical response for dual EGFr/mTOR salvage following single agent EGFr-TKI failure. By expanding beyond current FDA-approved drugs into the new agents that target PI3K, AKT, TORC1&2, MEK/ERK and c-MET, the EVA/PCD™ can facilitate developmental therapeutics. Analyses are now examining the activity and synergy of EGFr-TKIs with VEGF, mTOR, MEK/ERK, PI3K, AKT and c-MET inhibitors in NSCLC and other diseases as will be reported. Supported in part by a research grant from Biophysica, Ltd, the Vanguard Cancer Foundation and The Nagourney Institute.

Abstract 1764: Horizontal and vertical signal pathway inhibition in human tumor primary culture micro-spheroids

Introduction: Signal transduction pathways are targets for small molecule tyrosine and serine-threonine kinase inhibitors. Redundancy and cross talk between pathways can complicate the application of genomic signatures but proteomic and functional platforms may more closely approximate tumor phenotypes. We used EVA/PCD (ex vivo analysis of programmed cell death), a functional platform measuring both apoptotic & non-apoptotic events to examine the PI3K and EGFr pathways in human 1° culture micro-spheroids. Methods: 337 tumor specimens provided from the OR were mechanically and enzymatically disaggregated and exposed to EGFr (Gefitinib), AKT (B-15), PI3K (LY294002) & mTOR (Rapamycin) inhibitors with LC50 values interpolated from 5-point dose response curves. Synergy was assessed by median effect and correlation coefficients by Pearson Moment. Results: Parallel Pearson Moment analyses in 51 specimens by 2-tailed T, revealed LY vs. B15 (NS); LY vs. Rapa (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1764.