Human Tumor Primary Cultures Research Articles

Abstract 4910: Human tumor primary culture analyses for the prediction of response to chemotherapy, targeted agents, drug combinations and metabolic inhibitors: The role of ex vivo analysis of programmed cell death in cancer therapy

Abstract There is growing interest in the use of human tumor primary cultures for the prediction of response to drugs and drug combinations in cancer medicine. While the field has witnessed numerous attempts to apply human tissue for the prediction of response, fundamental errors in methodologic approaches severely limited the predictive validity and clinical applicability of these important technologies. The first error was a focus upon drug induced cell growth inhibition. This lead to the failure of the clonogenic (human tumor stem cell) and H3*Thymidine incorporation platforms in the 1970’s and 1980’s. As the concept of apoptosis arose following landmark observations in 1972, cell death endpoints became predominant including mitochondrial function (MTT, XTT), ATP content (luciferase) and delayed loss of membrane integrity (DISC, EVA/PCD) among others. These improvements led to more biologically relevant measures. However, a second hurdle arose as investigators chose to amplify subculture and propagate tumors into what are now referred to as human tumor organoids. While these 3-D cultures are more reflective of tumor cell-cell interactions found in cancers, they cannot recreate the cell-vasculature, cell-stroma, inflammatory environment nor cancer associated fibroblast interactions with human cancer cells required for accurate response prediction. To address these shortcomings we developed the ex vivo analysis of programmed cell death (EVA/PCD) platform that applies native state tumor explants directly from surgical specimens to select drugs and combinations. To date we have reported 11,680 individual analyses. Positive prospective results have been reported in breast cancer (TTP p < 0.03); ovarian cancer (TTP p = 0.05), lung cancer (2-fold improvement in ORR; TTP p =0.035) and recently ovarian and uterine cancer (DFS p = 0.038) with two meta-analyses confirming the predictive validity in hematologic and solid tumors. With over 20 years of CLIA-approved laboratory experience in the study of human tumor primary culture explants, the EVA/PCD platform provides improved response and time to progression and offers the opportunity to streamline drug development, curtail costs and avoid futile care in patients with advanced and refractory malignancies. Citation Format: Adam J. Nagourney, Steven S. Evans, Paulo D'Amora, Nise Yamaguchi, Paula Bernard, Federico Francisco, Joshua B. Gipoor, Robert Alan Nagourney. Human tumor primary culture analyses for the prediction of response to chemotherapy, targeted agents, drug combinations and metabolic inhibitors: The role of ex vivo analysis of programmed cell death in cancer therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4910.

Therapeutic Targeting of P53: A Comparative Analysis of APR-246 and COTI-2 in Human Tumor Primary Culture 3-D Explants

Background:TP53 is the most commonly mutated gene in human cancer with loss of function mutations largely concentrated in "hotspots" affecting DNA binding. APR-246 and COTI-2 are small molecules under investigation in P53 mutated cancers. APR binds to P53 cysteine residues, altering conformation, while COTI-2 showed activity in P53 mutant tumors by a computational platform. We compared APR-246 and COTI-2 activity in human tumor explants from 247 surgical specimens. Methods: Ex vivo analyses of programmed cell death measured drug-induced cell death by delayed-loss-of-membrane integrity and ATP content. The LC50s were compared by Z-Score. Synergy was conducted by the method of Chou and Talalay, and correlations were performed by Pearson moment. Results: APR-246 and COTI-2 activity favored hematologic neoplasms, but solid tumor activity varied by diagnosis. COTI-2 and APR-246 activity did not correlate (R = 0.1028) (NS). COTI-2 activity correlated with nitrogen mustard, cisplatin and gemcitabine, doxorubicin and selumetinib, with a trend for APR-246 with doxorubicin. For ovarian cancer, COTI-2 showed synergy with cisplatin at 25%. Conclusions: COTI-2 and APR-246 activity differ by diagnosis. A lack of correlation supports distinct modes of action. Cisplatin synergy is consistent with P53's role in DNA damage. Different mechanisms of action may underlie disease specificity and offer better disease targeting.

Abstract 4062: Activity of 3 Bromopyruvate in human tumor primary culture 3-dimensional explants using ex vivo analysis of programmed cell death

Abstract Altered metabolism is a hallmark of human malignancy. The “Warburg Effect “reflecting increased glycolytic activity in the presence of oxygen represents an opportunity to target specific features of cancer to induce selective toxicity to malignantly transformed cells. 3-bromopyruvate (3-BP) is a pyruvate analogue that inhibits hexokinase II at the mitochondrial level resulting in cancer cell death. Numerous studies have confirmed activity for 3-BP in human SW480 and HT 29 colon carcinoma, glioblastoma (U373MG), HTP-1 leukemic and other cell lines. Our interest in exploring the activity of 3-BP in human tumor primary culture explants led to the application of the ex vivo analysis of programmed cell death in 19 patient tumors. The intent was to develop concertation ranges for future studies and explore disease-specific activity. Methods: Fresh Sterile tumor samples were mechanically and enzymatically disaggregated and 3-dimensional organoids of desired size isolated by density centrifugation. Blood samples were isolated by density centrifugation. Cell suspensions in modified RPMI media were exposed to serial dilutions of 3-BP for 72 hours and then assessed for cell death by morphology, staining characteristics and ATP content by luciferase. Viability was scored against saline-exposed controls, normalized to 100%. Results: 3BP reveals activity in human tumor explants. Activity varied by cancer type. Hematologic tumors including NHL, CLL and AML were found sensitive as were several solid tumors including GI and lung cancers. Of interest lower degrees of activity were observed in breast and ovarian cancers that are generally considered chemo-sensitive . Activity in 2 fibrolamellar cancers could suggest a novel therapy for this drug refractory neoplasm. Studies are underway examining 3-BP correlations with other classes of drugs and metabolic inhibitors as well as 3-BP combinatorial potential. Supported by the Fibro fighters Foundation Citation Format: Adam Jeremiah Nagourney, Paulo D'Amora, Steven Scott Evans, Paula Bernard, Federico Francisco, Young Ko, Thomas Stockwell, Maxine Johnson, Robert Alan Nagourney. Activity of 3 Bromopyruvate in human tumor primary culture 3-dimensional explants using ex vivo analysis of programmed cell death [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 4062.

Drugging the undruggable-targeting P53: A comparative analysis of APR-246 and coti-2 in human tumor primary culture explants.

e15092 Background: The TP53 tumor suppressor is the most commonly mutated gene in human cancer. P53 protein comprised of 393 amino acids functions as a tetramer with the loss of function mutations concentrated in “hotspots” that often affect DNA binding domains. APR-246 and COTI-2 are novel small molecules that are under clinical investigation for the treatment of P53 mutated cancers. APR, a quinuclidine binds to P53 cysteine residues altering the functional conformation while COTI is a thio-semicarbazone, found active in P53 mutant tumors using a novel computational platform. As the mechanisms of action may differ we undertook a comparative analysis of these compounds in 3D human tumor explants derived from surgical specimens. Methods: Ex Vivo Analyses of Programmed Cell Death (EVA/PCD) (D’Amora, P et al Gyn. Oncol. 2021) was applied to 236 human tumor primary culture explants isolated from surgical specimens. After disaggregation, micro-spheroids, isolated by precise density centrifugation were exposed to APR, COTI and other agents for 72-96 hours. Drug-induced cell death was measured by delayed-loss-of-membrane integrity and ATP content (luciferase). Dose response curves were interpolated to LC50 values. Synergy analysis used the method of Chou & Talalay. Correlation coefficients used Pearson moment. Results: APR and COTI reveal the highest activity in hematologic neoplasms. Solid tumor activity patterns diverged with COTI active in gynecologic malignancies and NSCLC and moderately active in breast and colorectal while APR is active in colorectal but less active in breast and NSCLC. Pearson moments for COTI vs APR revealed an R value = 0.146 (NS). COTI activity correlated with Nitrogen Mustard and Alpelisib (PIK3CA) while APR activity correlated with Doxorubicin and Everolimus. COTI combinations focused upon ovarian cancers identified synergy in 25% and antagonism in 18%. Conclusions: P53’s function as the “guardian of the genome” is consistent with our findings that many cytotoxic and targeted agents that induce cell death reveal correlations with APR and COTI. While APR affects the structural conformation of P53, COTI may function downstream via oxidative, metabolic or other stress responses. The correlation coefficient for APR vs COTI at 0.146 (NS) suggests distinct mechanisms of action. This is supported by different disease-specific-profiles and correlations with other drugs. COTI synergy in ovarian cancers, the target of current clinical trials at 25% is below expectations. With differing disease specificities, selecting between these and related agents to optimize clinical outcomes may require a more granular examination of cellular mechanisms of response and resistance. Primary culture analyses offer insights into drug mechanisms of action, combinatorial potential and have the capacity to improve patient selection.

MON-466 Transcriptome Dynamics of in vitro Human Pituitary Tumor Primary Culture

Lack of an immortalized or long-term hormone-secreting human pituitary tumor in vitro cell line has significantly hampered studies of etiopathogenesis and development of targeted therapies for pituitary tumors. Various media, vessels, substrates and additives have been explored to optimize primary cultures. Some methods have prolonged cell survival time but maintaining hormone secretion is still a major challenge. We have recently developed methodology to maintain robust hormone secretion in human prolactinoma, somatotroph and corticotroph tumors in vitro for up to 9 months. To gain insight into the changes in gene expression profiles that occurred prior to/coincident with loss of hormone secretion during in vitro human pituitary tumor cultures, we compared the global transcriptiome landscape in serial passages from two human prolactinoma primary cultures. We obtained 27.1 million quality filtered reads per sample of which ~90% were exonic reads detecting an average of 16,000 genes. 3D principal component analysis identified 3 distinct clusters, with close similarity between cell passages 1-2, those beyond passage 4-5 and a stand-alone passage 3. Analysis of differential gene expression (DEG) over time using DAVID knowledgebase and hierarchical clustering demonstrated that most changes in gene expression occurred in the first 1-2 weeks in in vitro culture. During the first week of in vitro culture, although the cells maintained robust PRL secretion, reduced activation of angiogenesis and growth signal pathways (PI-3K, MAPK pathways, cell adhesion, GTPase activity, cell proliferation and cell-cell junction assembly) was observed. In the following 1-2 weeks, the cell population maintained PRL secretion but exhibited continued loss of survival signals, with increased expression of extracellular matrix (ECM) and surface adhesion molecules consistent with development of a fibroblastic phenotype that dominated later stage cultures. Based on our findings, we propose that the molecular events underpinning loss of hormone production in primary pituitary tumor cultures represent transition from endocrine cells to pituitary specific fibroblastic folliculo-stellate cell likely selected by the in vitro culture conditions. Our data further reveal that underexpression of several GPCRs and resultant downregulation of the PKA pathway may lead to loss of hormone production in parallel with loss of cell survival signals. In summary, our study defines a temporal progressive transcriptome transition signature to explain loss of hormone secretion and morphological alterations observed during human pituitary tumor in vitro culture and may provide molecular targets to further optimize our culture methodology to attain “immortalized” hormone-producing human pituitary tumor cell-lines.

Abstract 4684: MYC inhibition in human malignancy by BET-Bromodomain inhibitor JQ1, using a 3-D human tumor primary culture micro-spheroid platform to explore clinically relevant observations

Abstract The transcription factor MYC is associated with the regulation of over 15% of human gene expression. Deregulation of MYC is found in up to 70% of human cancers. As MYC is responsible for the reprogramming of cell metabolism, growth and survival, it is an attractive therapeutic target, yet effective drug development has been slow. As the Bromodomain/BET proteins epigenetically regulate MYC, BET inhibitors like JQ1 have been developed as MYC therapeutics. We used the Ex Vivo Analysis of Programmed Cell Death (EVA/PCD) previously shown to correlate with response, time to progression and survival, to examine JQ1 activity in 46 human tumor specimens isolated from surgical biopsies. Methods: Surgical biopsies were mechanically and enzymatically disaggregated, with micro spheroids of desired size isolated by density centrifugation. Five-Point dose response curves were interpolated to provide LC50 values for comparison by Z-score. Synergy was conducted by median-effect. Results: JQ1 revealed activity in hematologic, breast, glial & gastrointestinal (GI) tumors. By rank order, JQ1 activity favored hematologic (AML & NHL) & breast tumors over Lung, Ovary and GI with inter-patient differences observed within tumor types. MYC association with histone acetylation and aurora kinase (AURK) activity, led to an exploration of JQ1 synergy with SAHA (HDACi), MLN 8054 & Tozasertib (both AURKi). JQ1 synergy analysis revealed SAHA > Tozasertib > MLN 8054. Conclusions: Based upon JQ1 activity, several tumor types appear to be candidates for agents that target MYC-mediated cell survival, offering potential treatment options for patients with advanced malignancies. JQ1 synergy with HDACi & AURKi suggests novel drug combinations for future development. As MYC signaling influences thousands of gene targets, phenotypic analyses like the EVA/PCD platform can offer insights into cellular cell death events that serve as surrogate markers for clinical response to MYC inhibition. Supported in part by the Vanguard Cancer Foundation, the Nagourney Institute and the Malcolm C. Todd Cancer Institute. Citation Format: Robert Alan Nagourney, Steven S. Evans, Alexander J. Nagourney, Paula J. Bernard, Federico R. Francisco, Milan Sheth, Nilesh Vora, Eknath Deo. MYC inhibition in human malignancy by BET-Bromodomain inhibitor JQ1, using a 3-D human tumor primary culture micro-spheroid platform to explore clinically relevant observations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4684. doi:10.1158/1538-7445.AM2017-4684

Targeting the ERK pathway for the treatment of Cushing's disease.

We recently demonstrated that the orphan nuclear receptor testicular receptor 4 (TR4) is a potent regulator of corticotroph tumor growth and hormone secretion. The Ras/Raf/MEK/ERK pathway is commonly overactivated in human tumors and we have demonstrated that corticotroph tumor TR4 is activated by ERK1/2-mediated phosphorylation. We evaluated effects of MEK-162, a selective, non-ATP-competitive allosteric inhibitor of MEK1/2, on murine and human in vitro and in vivo corticotroph tumor proliferation and adrenocorticotrophic hormone (ACTH) secretion. MEK-162 treatment dose-dependently inhibited corticotroph tumor proliferation, induced apoptosis, reduced pro-opiomelanocortin (POMC) mRNA levels and inhibited ACTH secretion in vitro. Similar findings were obtained in human corticotroph tumor primary cultures (n = 5). These actions of MEK-162 were augmented in the presence of TR4 overexpression, suggesting that TR4 levels may serve as a predictive biomarker of MEK-162 corticotroph tumor responsiveness. Additionally, MEK-162 treatment reduced TR4 protein expression and blocked recruitment of TR4 to bind its consensus site on the POMC promoter (−854bp to −637bp), elucidating multiple mechanisms to control TR4 corticotroph tumor actions. In a murine corticotroph tumor in vivo model of Cushing's disease, MEK-162 treatment inhibited tumor growth and reduced tumor-derived circulating plasma ACTH, and corticosterone levels. These results demonstrate the potent actions of MEK-162 to inhibit corticotroph tumor growth and hormone secretion in vitro and in vivo via TR4-dependent and independent mechanisms, and raise the possibility of MEK-162 as a novel therapy for Cushing's disease.

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

Abstract 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 <0.005). Activities for AUY & G by disease were generally similar favoring Heme >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

Abstract 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 (LC50's) 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< 0.01); BYL vs. LY (N=7, R=0.74, P=0.1); BEZ vs. LY (N=61, R=0.52, P<0.001). A comparison of activity for hematologic (Heme) vs. Solid tumors (solid) for LY: Heme > 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

Cyclin-dependent kinases as therapeutic targets: Examination of palbociclib (PD 0332991) and flavopiridol in human tumor primary culture microspheroids.

e13504 Background: The cyclin dependent kinases (CDK) are a family of enzymes that mediate cell cycle progression. CDK dysregulation is a common event in human carcinogenesis, making CDK inhibition...

An examination of crizotinib activity in human tumor primary culture micro-spheroids isolated from patients with advanced non-small cell lung cancer.

e13558 Background: The amino-pyridine, Crizotinib (Criz) (PF02341066, Xalkori), active against c-MET is an inhibitor of anaplastic lymphoma kinase (ALK). Identification of ALK-gene rearrangement in NSCLC led to clinical trials & FDA approval. Recognition of ROS-1 mutations as Criz targets provided additional therapy options. ALK mutations found in NSCLC also occur in lymphoma, neuro & myofibroblastic tumors but may participate in the oncogenesis of other tumors. Methods: We used ex vivo analysis of programmed cell death (EVA/PCD) (Nagourney, R. Curr Treat. Op Oncol, 2006) to examine Criz activity in human tumor 1°culture micro-spheroids, from 60 surgical specimens, with a focus on NSCLC. Using metabolic (ATP-content; mitochondrial) & morphologic (membrane integrity) endpoints, dose response curves were interpolated to LC50 values for comparison of activity by patient & tumor type. Patients were screened for ALK & ROS-1 by FISH. Results: ALK (+) tumors reveal lower LC50’s (3.4 uM) vs. ROS-1 (+) (11.5 uM), despite clinical responses in both groups. A Criz-responding patient, at 2nd biopsy for progression, reverted to Criz-resistance but developed collateral sensitivity to cytotoxics that provided durable response. Despite FISH (-), a 39 y/o nonsmoker male, revealed exquisite sensitivity to Criz by repeat EVA/PCD. At our insistence, FISH conducted at a 2nd reference lab correctly identified ALK(+) qualifying for Criz, to which he responded, now at year 2. Using low LC50 as a phenotypicmarker of Criz responsiveness, we identified activity in an extremely rare pediatric sarcoma patient. When, secondarily screened, patient found ALK (+), followed by rapid objective response to Criz. Conclusions: Primary culture analyses provide insights into Criz activity including ALK (+) vs. ROS-1(+), individual patient response profiles, and the identification of Criz candidates, who might otherwise not be screened for sensitizing mutations. By capturing human tumors in their “native” state EVA/PCD offers opportunities to study Criz for unrecognized targets and analyze novel strategies including synergy & sequence dependence, less readily examined with genomic platforms.

International collaboration in personalized medicine for the treatment of advanced and drug-refractory cancers: Clinical application of human tumor primary culture analyses.

e13562 Background: Personalized oncology has advanced through genomic and proteomic platforms. BCR-abl; EGFr and ALK have provided drug-able targets and companion diagnostics in several diseases, yet many transforming events in humans are polygenic, complex and incompletely understood at a genomic level. Recognition that oncogenesis reflects changes in the cell and its micro environment has renewed interest in whole cell experimental models that capture native-state cell-cell, -stroma and -vascular signaling. Ex vivo analysis of programmed cell death (EVA/PCD) has been shown to correlate significantly with response, time to progression and survival (Nagourney, R. Curr. Treat Op Oncol, 2006).To explore EVA/PCD functional profiling in Brazil, hospital-based investigators, Centro de Genomas and Rational Therapeutics coordinated the transport of 67 surgical specimens for analyses. Methods: Dose-response curves using metabolic (ATP-content, mitochondrial) and morphologic endpoints, interpolated to LC50's and synergy by median-effect, were compared with databases to identify patient-specific profiles for cytotoxics, targeted agents and combinations. Reports provided day 7. Results: 62/67 (92%) provided adequate tumor for analysis; 39 male (58%); 28 (42%) female; 6 chemo-naive and 61 previously treated. Results provided for < 8 drugs in 14/62 (22%); 8-16 drugs in 34/62 (54%) and > 16 drugs in 14/62 (22%). Of 22 tumor types, breast (8); Melanoma (8); NSCLC (8); ovary (7); pancreas (7) and sarcoma (4) predominated. EVA/PCD was used to select the most active combinations. Agents selected, response rates and durations are being tabulated and will be reported. Conclusions: The transport, processing and reporting of human tumor primary culture analyses is feasible, providing results in 92% of specimens and median of 12 drugs evaluated (range 4-32). Preliminary outcomes in these drug-refractory patients reveal that novel, often unexpected drug combinations (Everolimus and Lapatinib in triple negative breast) were identified and provided objective tumor responses, supporting EVA/PCD in therapy selection (personalized therapy) and drug development.

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

Abstract 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 (TKI's) 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

Optimal combination therapy for advanced colorectal cancer: An examination of synergy and antagonism with EGFR inhibition.

e14075 Background: While FOLFOX +/- Bevacizumab is the most widely used combination in colorectal cancer (CRC), the EGFr antagonists Cetuximab & Panitumumab have not further enhanced activity. On the contrary, the addition of EGFR inhibitors to Irinotecan-based regimens at 1st & 2nd line has improved response and both progression freel & overall survival (Van Cutsem. 2010; Peeters. 2010.) Methods: We applied the ex vivo analysis of programmed cell death (EVA-PCD) (Nagourney, Curr Treat Opt Onc 2006) to examine interactions between EGFr antagonists and Irinotecan or FOLFOX in 534 human tumor primary culture microspheroids isolated from CRC surgical specimens or cytologically (+) fluids. Gefitinib was used as a surrogate for Cetuxima/Panitumumab activity. Tissue samples exposed to Oxaliplatin, 5FU, Irinotecan, and Oxaliplatin & 5FU (FOLFOX) provided dose response curves with and without Gefitinib for formal synergy analysis by median effect (Chou & Talalay). Results: Shown in table below. Conclusions: Results indicate superiority for Irinotecan combination with the EGFr antagonist Gefitinib. The percentage of synergistic interactions for Irinotecan & Gefitinib (41%) is demonstrably higher than that associated with FOLFOX & Gefitinib (4%). This suggests distinct modes of interaction between these classes of agents favoring Irinotecan and could have an impact upon future clinical trial designs. Recognizing the importance of Kras mutation, additional studies are underway to examine the impact of Kras mutation upon synergy. However, as wild-type and mutant samples would be expected to be equally distributed, subset analyses may reveal quantitative (higher % synergy) rather than qualitative differences (relative activity of Irinotecan vs FOLFOX). Results of these analyses will be reported. Supported in part by the Vanguard Cancer Foundation. [Table: see text]

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

Abstract 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 (LC50's), 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-235's 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

Functional analysis of PARP inhibitors AZD 2281 and BSI-201 in human tumor primary cultures: A comparison of activity and examination of synergy with cytotoxic drugs.

e13599 Background: Poly(ADP-ribose) polymerases (PARP) are activated in response to cellular injury. DNA damage from radiation and cytotoxic drugs results in the up-regulation of PARP 1/2, leading ...

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

Abstract 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

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

Abstract 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<0.02); B15 vs. Rapa (P<0.0025); Rapa vs. Gefitinib (P <0.001). Synergy analyses for Rapa & Gefitinib revealed synergy in 29%, additivity in 41%, sub-additivity in 13% and antagonism in 17%. Exploratory synergy analyses that combined Gefitinib & LY and Gefitinib & B-15, favored the Gefitinib& B-15 combination with 100% synergy in this small series. Conclusions: EVA/PCD analyses provide insights into cellular response to targeted therapies, alone and in combination. Correlative analyses identify points of commonality and/or disparity in downstream signaling that can be exploited in drug development and therapy. Synergy analyses reveal potentially important drug combinations. The EVA/PCD human tumor micro-spheroid platform provides a functional tool capable of streamlining drug development and cancer therapy. Supported by The Vanguard Cancer Foundation and the Nagourney Institute. 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.

Casting-type calcification breast cancer: Assessment of response to cytotoxic insult in human tumor primary culture spheroids

680 Mammographic casting-type calcifications (CTC) represent Grade 3 DCIS that are often associated with T1a/b invasive tumors. CTC’s have an unexpectedly poor prognosis, at odds with the expected outcome for this size category (Tabar L, Cancer, 2004; Zunzunegui RG, Arch Surg, 2003). CTC is associated with (+) lymph nodes, higher grade and early death. It has been suggested that these tumors are chemo- and radio-resistant with viable CTC tumors observed at surgery following neo-adjuvant therapies. To test this hypothesis, we applied an ex vivo analysis (EVA) of drug sensitivity, shown to correlate with response & survival (Nagourney R, J Clin Oncol 2000; Gyn Oncol 2003), to surgical biopsies of CTC. Two the first 3 specimens submitted provided adequate cells for analysis, 1 from a previously untreated patient and 1 from a patient after neo-adjuvant Doxorubicin/Cytoxan/Taxol. The histological appearance of the isolated micro-spheroids is one of large cells with abundant mucinous cytoplasm. Drug response profiles reveal striking degrees of resistance, with the ex vivo LC50 values for most agents tested exceeding the highest concentration ranges. Noteworthy was the high activity for Gefitinib, an agent that has generally shown modest activity in breast cancer specimens, as we have reported (Nagourney R, Proc AACR 2003). Compared to the overall breast tumor database, the CTC tumors are significantly more Gefitinib sensitive. These preliminary results suggest CTC tumors manifest resistance to conventional chemotherapy yet sensitivity to agents that target cellular survival capacity associated with EGFR pathway activation. If confirmed in ongoing analyses (to be reported), the poor prognosis of CTC cancers may reflect altered intracellular response and survival cascades that could provide novel avenues for the management of these high-risk patients. Supported by a research grant from the Vanguard Cancer Foundation. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Central Hospital, Malcolm Todd Cancer Institute, Rational Therapeutics Rational Therapeutics

Fludarabine as a modulator of cisplatin activity in human tumour primary cultures and established cell lines.

The potential of the purine analogue fludarabine (9-beta-D-arabinofuranosyl-2-fluoroadenine-5' monophosphate) as a modulator of cisplatin cytotoxicity was investigated in four established cell lines and 20 primary cultures of human melanoma and ovarian cancer. Tumour cells were exposed to fludarabine and cisplatin, alone or in combination, for 4 h. Fludarabine did not affect the growth of ovarian cancer cell lines, whereas it induced a marked and dose-dependent inhibition of proliferation in melanoma cell lines. In primary cultures of both histotypes, the purine analogue did not induce appreciable antiproliferative effects. Combined cisplatin-fludarabine treatment caused additive effects in all established cell lines. Conversely, a synergistic effect of the combination was seen in 5 of 10 melanoma and 4 of 10 ovarian cancer primary cultures, with a dose-modifying factor ranging from 2.1 to 3.9 for melanomas and from 4.0 to 7.5 for ovarian cancers, respectively. In the remaining cultures, the interaction between fludarabine and cisplatin was additive. The alkaline filter elution analysis performed on primary cultures showed that the synergistic interaction between the two drugs was paralleled by an increase in the extent and persistence of the cisplatin-induced DNA interstrand crosslinks. Our results indicate that fludarabine can enhance cisplatin cytotoxic activity in human tumour primary cultures from ovarian cancer and malignant melanoma. Such an effect may be partially due to an interference by fludarabine on cisplatin-induced DNA adduct metabolism and repair.