Stromal Cell Supported High-Throughput Drug Testing Of Primary Leukemia Cells For Comprehensive Assessment Of Sensitivity To Novel Therapies

Abstract Background Ex vivo drug sensitivity testing of cancer cells taken directly from patients would significantly facilitate optimization of clinical therapies. However, in the past, such testing has been performed in suboptimal conditions, where patient cells gradually stop proliferating and undergo apoptosis, with poor translation of Results. More reliable prediction of drug sensitivity is needed and recent focus has been directed towards Methods that take into account the supporting impact of the surrounding tumor microenvironment. Primary leukemia cell viability and long-term survival ex vivo can be promoted with co-culture Methods using stromal cells (McMillin et al. 2013). While high throughput (HT) drug testing enables rapid assessment of sensitivity to 100s of drugs or drug combinations, application of co-culture Methods is challenging considering the mixed readouts from multiple cell types. In this study we describe a HT platform based on stroma-conditioned medium for assessing the anti-leukemic activity of compounds against fresh and vital biobanked primary leukemia samples ex vivo. Methods Stroma-conditioned medium (CM) was collected from the HS-5 human bone marrow (BM) cell line and combined with RPMI medium for drug sensitivity testing. Mononuclear cell medium (Promocell) was used as the standard medium comparison. Sensitivity of primary leukemia or healthy cells to 306 approved and investigational drugs was measured at 5 different concentrations covering a 10,000-fold concentration range. Cell viability was measured after 72 h with the CellTiter-Glo assay and dose response curves generated for each tested drug. Drug sensitivity scores (DSS) were calculated based on the area under the dose response curve. Here, we compared comprehensive drug sensitivity ex vivo responses between stroma-conditioned medium and standard medium using mononuclear cells from 8 acute myeloid leukemia (AML) patients and 4 healthy donors. Results HS-5 CM supported fresh and biobanked primary AML cells, promoting proliferation and overall survival. Freshly isolated AML cells had a mean viability of 123% after 3 days in CM compared to 59% in the absence of CM. The viability of biobanked cells was 85% with CM vs. 20% in conventional medium. Improved ex vivo cell survival increased the therapeutic window of drug sensitivity testing and more drugs could be assessed with CM compared to conventional medium. Results from different healthy samples tested with the same type of medium were highly similar, but sensitivities differed significantly when comparing CM to standard medium Results. In contrast, drug sensitivity Results of AML cells from different patients were more diverse, reflecting the heterogeneity of the disease. However, comparison of CM and standard medium drug sensitivities of cells from individual AML patients showed modest differences that were primarily indicative of the increased proliferation of cells incubated with CM. Overall, both AML and healthy cells showed greater sensitivity to anti-mitotic drugs when incubated with CM. For example, the average DSS of vinblastine for healthy controls was 17 in CM vs. 9 in standard medium. In addition, AML cells often exhibited increased sensitivity to JAK inhibitors such as ruxolitinib when tested with CM compared to standard medium (DSS 14 vs. 9). In contrast, stress-related protein-targeting drugs (e.g. HSP90 inhibitors) and certain tyrosine kinase inhibitors (e.g. dasatinib, quizartinib) exhibited reduced efficacy when AML cells were incubated with CM compared to conventional media. This may be due to soluble factors present in CM that mimic the protection provided by the BM niche. Conclusions Our data support the concept that conditioned medium from stromal cells improves application of drug sensitivity testing to AML patient samples ex vivo. Stromal medium supports both fresh and biobanked AML cells, likely providing environmental cues present in the BM niche and necessary for AML cell growth and survival. This may lead to more reliable ex vivo assessment of the anti-leukemic activity of compounds for cells from leukemia patients. Importantly, stromal cell based conditions support the growth of vital biobanked leukemia samples and enable use of retrospective samples for a multitude of assays including HT drug testing. Disclosures: Porkka: Novartis: Consultancy, Research Funding, Speakers Bureau; BMS: Consultancy, Research Funding, Speakers Bureau. Kallioniemi:Medisapiens: Membership on an entity’s Board of Directors or advisory committees; Roche: Research Funding.

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Myeloma Drug Sensitivity Testing to Optimize Retreatment with Anti-CD38 Monoclonal Antibodies in Daratumumab-Refractory Patients

Blood ◽

10.1182/blood-2020-137819 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 37-38

Author(s):

Olivia Perez De Acha ◽

Beau M Idler ◽

Zachary J Walker ◽

Peter A Forsberg ◽

Tomer M Mark ◽

...

Keyword(s):

Research Funding ◽

Drug Sensitivity ◽

Drug Class ◽

Ex Vivo ◽

Partner Choice ◽

Sensitivity Testing ◽

Drug Sensitivity Testing ◽

Cd38 Expression ◽

Cell Subpopulations ◽

20 Nm

Background: Daratumumab-refractory multiple myeloma (MM) patients have limited treatment options and a dire prognosis. Daratumumab (Dara) targets the overexpressed myeloma antigen CD38, and its mechanism of resistance has been partially associated with downregulation of CD38 expression (Nijhof et al. Blood, 2016). Dara is a key agent in the relapsed setting and is being integrated into upfront treatment. In addition, isatuximab (Isa) has become the second FDA-approved anti-CD38 monoclonal antibody (mAb). Thus, when and how patients can be retreated with this important drug-class has become a critical question. We developed a platform for measuring drug efficacy ex vivo, including mAbs, termed Myeloma Drug Sensitivity Testing (My-DST) (Walker et al. Blood Advances, 2020). Here, we addressed whether My-DST could determine if retreatment with the anti-CD38 mAbs Dara and Isa can be effective in patients that had previously developed Dara-resistance. Methods: Bone marrow aspirates were obtained from the hematologic tissue bank (HTB) from patients at the University of Colorado Blood Cancer Program after informed consent and IRB approval. Mononuclear cells (MNCs) were isolated by Ficoll density gradient centrifugation, cells incubated for 48 h with 20 nM Dara, Isa and untreated controls in triplicate wells. Flow cytometry was performed on a BD FACSCelesta. To identify the viable MM population, samples were stained with fluorophore-conjugated mAbs to CD38, CD138, CD45, CD19, CD56 and CD46. Stained samples were washed and resuspended in Live/Dead dye Near-IR. Results were analyzed through FlowJo and Graphpad Prism software. Results: My-DST for Dara (20 nM) cytotoxicity has been performed in 67 patients, 52 of which were Dara-naïve and 15 of which were clinically Dara-refractory. Of Dara-naïve patients, 38/52 (73%) showed >20% reduction in viable MM cells whereas only 3/15 (20%) of the Dara-refractory patients showed >20% decrease (Fig 1A). Provocatively, the patients that showed potential "re-sensitization" had been off Dara for >12 months, and there was a significant correlation between response to Dara and months off treatment (r= -0.5096, p= 0.0457, Fig 1B). Furthermore, we evaluated two timepoints in one of the responders (HTB1749), and the later sample (HTB1749.3) showed a deeper response, further supporting the correlation. Surprisingly, in Dara-refractory patients the median CD38 expression was not significantly different between the three ex vivo responders and those who did not respond (p = 0.439, Fig 1C). Isa was tested in seven of the Dara-refractory patient samples, showing >20% decrease in viable MM cells in five, of which three did not respond ex vivo to Dara (Fig 1D). Interestingly, the Isa responders were only off Dara treatment for an average of 7.5 months. We further investigated HTB1059.3 and HTB1749.2, which responded to Dara, and found that these patients had multiple distinct MM cell subpopulations with different levels of CD38 expression, with the subpopulation with higher CD38 MFI accounting for the decreased viability (Fig 1E-F). When we compared HTB1059.3 with a prior sample available, these differential populations were not present before Dara treatment (data not shown), indicating these subpopulations evolved after treatment. Likewise, the same phenomenon of subpopulations with differential CD38 expression was observed in the Dara-refractory patients who responded to Isa (Fig 1G-H). Conclusion: These data support the possibility of retreatment with anti-CD38 mAbs in patients who once became refractory to Dara. Although our findings need to be confirmed with additional samples, they suggest that Isa may have efficacy earlier in this setting, supporting an approach to switch agents when retreating with this drug class. Anti-CD38 mAb sensitivity in the Dara-refractory population appears to be heavily influenced by the different CD38 expression levels on the heterogeneous MM cell subpopulations that emerge when a patient is off Dara for a period of time. Furthermore, My-DST with anti-CD38 mAbs may be applied to help guide the treatment approach in this population. Still, the presence of CD38-low subpopulations in these patients may represent resistant cells that shorten the remission times on retreatment. Thus, the combination drug partner choice will likely be critical to successfully optimizing depth and duration of response in Dara-refractory patients. Figure 1 Disclosures Forsberg: Genentech, Inc., Sanofi, Karyopharm, Abbvie: Research Funding; Celgene: Speakers Bureau. Mark:Bristol-Myers Squibb: Research Funding; Janssen: Research Funding; Celgene: Consultancy; Amgen: Consultancy; Kayopharm: Consultancy; Janssen: Consultancy; Sanofi: Consultancy; Takeda: Consultancy. Sherbenou:Oncopeptides Inc.: Research Funding.

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Ex Vivo Analysis of Primary Tumor Specimens for Evaluation of Cancer Therapeutics

Annual Review of Cancer Biology ◽

10.1146/annurev-cancerbio-043020-125955 ◽

2020 ◽

Vol 5 (1) ◽

Author(s):

Cristina E. Tognon ◽

Rosalie C. Sears ◽

Gordon B. Mills ◽

Joe W. Gray ◽

Jeffrey W. Tyner

Keyword(s):

Primary Tumor ◽

Cancer Biology ◽

Drug Sensitivity ◽

Ex Vivo ◽

Publication Date ◽

Sensitivity Testing ◽

Drug Activity ◽

Drug Sensitivity Testing ◽

Cancer Pathogenesis ◽

Tumor Types

The use of ex vivo drug sensitivity testing to predict drug activity in individual patients has been actively explored for almost 50 years without delivering a generally useful predictive capability. However, extended failure should not be an indicator of futility. This is especially true in cancer research, where ultimate success is often preceded by less successful attempts. For example, both immune- and genetic-based targeted therapies for cancer underwent numerous failed attempts before biological understanding, improved targets, and optimized drug development matured to facilitate an arsenal of transformational drugs. Similarly, directly assessing drug sensitivity of primary tumor biopsies—and the use of this information to help direct therapeutic approaches—has a long history with a definitive learning curve. In this review, we survey the history of ex vivo testing and the current state of the art for this field. We present an update on methodologies and approaches, describe the use of these technologies to test cutting-edge drug classes, and describe an increasingly nuanced understanding of tumor types and models for which this strategy is most likely to succeed. We consider the relative strengths and weaknesses of predicting drug activity across the broad biological context of cancer patients and tumor types. This includes an analysis of the potential for ex vivo drug sensitivity testing to accurately predict drug activity within each of the biological hallmarks of cancer pathogenesis. Expected final online publication date for the Annual Review of Cancer Biology, Volume 5 is March 4, 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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Enhanced Ex vivo drug sensitivity testing of chronic lymphocytic leukaemia using refined DiSC assay methodology

Leukemia Research ◽

10.1016/0145-2126(95)00127-1 ◽

1996 ◽

Vol 20 (2) ◽

pp. 143-153 ◽

Cited By ~ 29

Author(s):

Andrew G. Bosanquet ◽

Philip B. Bell

Keyword(s):

Chronic Lymphocytic Leukaemia ◽

Drug Sensitivity ◽

Lymphocytic Leukaemia ◽

Ex Vivo ◽

Sensitivity Testing ◽

Drug Sensitivity Testing ◽

Disc Assay

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Abstract 2153: Ex vivo drug sensitivity testing of primary cells for precision cancer medicine

10.1158/1538-7445.sabcs18-2153 ◽

2019 ◽

Author(s):

Sergey G. Kuznetsov ◽

Alexander Ianevski ◽

Evgeny Kulessky ◽

Karoliina Laamanen ◽

Elina Lehtinen ◽

...

Keyword(s):

Drug Sensitivity ◽

Ex Vivo ◽

Primary Cells ◽

Sensitivity Testing ◽

Drug Sensitivity Testing ◽

Cancer Medicine

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Stroma-free long-term growth of primary acute myeloid leukemia (AML) cells.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.e18535 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. e18535-e18535

Author(s):

Jing Chen ◽

Glorymar Ibanez Sanchez ◽

Paul Calder ◽

Mark G. Frattini

Keyword(s):

Growth Factors ◽

Conditioned Medium ◽

Stromal Cells ◽

Drug Sensitivity ◽

Growth Conditions ◽

Leukemic Cells ◽

Sensitivity Testing ◽

Drug Sensitivity Testing

e18535 Background: To individualize therapy for relapsed/refractory AML patients, optimal in-vitro culture conditions to support primary leukemic cells are essential for drug sensitivity testing. Our lab has validated a high throughput chemosensitivity assay with primary AML cells maintained by growth factors (cytokines); however, growth factors have not been shown to support long-term assays of primary AML cells. Stromal cells of the tumor microenvironment are crucial to maintain normal hematopoiesis and leukemic cells and have been shown to support long term in-vitro expansion of primary AML cells. However, there is little information characterizing these growth conditions. The aim of this study was to compare long-term proliferation and phenotypes of primary AML cells with growth factors or stromal support to best determine their utility as a platform for drug sensitivity testing in functional assays. Methods: Patient-derived AML cells were cultured in 96-well plates in: 1) cell culture medium only 2) Human HS5 or HS27 stromal cells 3) HS5 or HS27 stroma-conditioned media or 4) cytokine cocktail. Viability readout by Guava ViaCount and leukemic cell surface phenotypes by fluorescently-conjugated antibodies were performed weekly over 3 weeks. Results: Primary AML cells cultured with only cytokines maintained proliferation at 3 weeks. In comparison, AML cells cultured in HS5 stroma-conditioned medium also maintained proliferation at a similar rate at 3 weeks, while co-culture with HS5 stromal cells demonstrated significantly higher proliferation. Leukemic immunophenotypes were maintained for all growth conditions over 3 weeks. Conclusions: Contrary to known data, primary AML cells with cytokines continued to expand at 3 weeks, at a similar rate to HS5 stroma-conditioned medium, a finding that has not been reported. Consistent with previous studies, we confirmed that stromal cells such as HS5 can provide long-term in-vitro expansion of primary AML cells, which cannot be substituted by stroma-conditioned medium. The ability to maintain long-term expansion of primary AML cells by both cytokines and stromal cells sets up a platform for a direct comparison of high throughput drug sensitivity testing under these growth conditions.

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Discovery of Novel Drug Sensitivities in T-Prolymphocytic Leukemia (T-PLL) By High-Throughput Ex Vivo Drug Testing and Genetic Profiling

Blood ◽

10.1182/blood.v124.21.917.917 ◽

2014 ◽

Vol 124 (21) ◽

pp. 917-917

Author(s):

Emma I Andersson ◽

Leopold Sellner ◽

Malgorzata Oles ◽

Tea Pemovska ◽

Paavo Pietarinen ◽

...

Keyword(s):

Drug Screening ◽

Research Funding ◽

Drug Testing ◽

Drug Response ◽

Drug Sensitivity ◽

Response Pattern ◽

Ex Vivo ◽

Drug Responses ◽

Ic50 Values ◽

Stat Pathway

Abstract Introduction T-PLL is a mature post-thymic T-cell neoplasm with an aggressive clinical course (5-year overall survival 21%). Almost 75% of T-PLL cases harbor chromosome 14 translocations resulting in aberrant activation of the proto-oncogene TCL1A. Furthermore, in the majority of T-PLL cases the ATM gene is mutated or deleted, and recently it was reported that mutations in genes involved in the JAK-STAT pathway were found in 76% of T-PLL cases. Due to the rareness and aggressive nature of the disease, clinical trials are difficult to execute. By using a high-throughput ex vivo drug sensitivity and resistance testing (DSRT) platform covering 306 approved and investigational oncology drugs we systematically investigated the heterogeneity of drug responses in PLL-patients. As the impact of mutations on drug sensitivity is not well understood we aimed to identify relevant associations between the drug responses and genetic lesions in T-PLL patients. Methods Primary cells (MNCs) from seven T-PLL patients were obtained for drug screening. Samples were seeded in 384-well plates and 306 active substances were tested using a 10,000-fold concentration range resulting in a dose-response curve for each compound. Cell viability was measured after 72 h incubation and differential drug sensitivity scores (sDSS), representing leukemia-specific responses, were calculated by comparing patient samples to healthy donors. Hierarchical clustering of the drug responses was performed with Cluster 3.0 and Java Tree View. To assess the performance of the drug screening platform we also exchanged six samples with the German Cancer Research Center in Heidelberg for a comparison of results between two independent drug screening systems. To understand heterogeneous pathway dependencies, drug sensitivities were correlated with somatic genetic variants and recurrent chromosomal aberrations. Genetic characterization was performed by exome sequencing of tumor and matched healthy cells to profile known recurrent genetic variants (ATM, STAT5b, IL2RG, JAK1, JAK3) as well as CNVs (TCL1A translocations, ATM deletions, recurrent chromosomal aberrations). Results Four out of seven patient samples showed high sensitivity to small molecule BCL2 inhibitors navitoclax (IC50: 10-68nM) and ABT-199 (IC50: 14-45nM) and to HDAC inhibitors panobinostat and belinostat (IC50: 2-65nM). Intriguingly, the CDK inhibitor SNS-032 was effective in 6/7 patient samples (IC50: 7-95nM). SNS-032 inhibits Cdk2, Cdk7 and Cdk9, which control transcription of anti-apoptotic proteins including MCL1 and XIAP. As the AKT1/MTOR pathway is activated in many T-PLL patients due to expression of the TCL1A oncoprotein, it was interesting to observe that patient samples did not show any response to AKT inhibitors (MK-2206 and GDC-0068 IC50 values >1000 nM) nor to MTOR inhibitors (rapalogs temsirolimus and everolimus). Similarly, T-PLL cells were insensitive to JAK-inhibitors. Clustering of drug responses from T-PLL patients with primary AML and ALL patient samples revealed the drug response profiles to be specific for T-PLL patients (Figure). 6/7 patients clustered together while the only patient (PLL4) in our cohort with confirmed mutations in the JAK-STAT pathway genes STAT5b (P702S) and IL2RG (K315E) exhibited a non-sensitive response pattern when compared to other samples (Figure). Interestingly, exome sequencing did not reveal any JAK mutations in our PLL-cohort (n=5) nor additional STAT5b or IL2RG mutations in other patients except in this unresponsive patient. In the comparison between the platforms the correlation of the censored IC50 values from the 60 overlapping drugs was r=0.75. Similar fits of dose-response curves were seen for most drugs, although there were notable exceptions, which may be due to divergent culture conditions and day of read-out. Conclusions Ex vivo drug testing of primary patient cells has the potential to provide novel personalized drug candidates (such as BCL2, HDAC and CDK inhibitors) for T-PLL. The drug response pattern was T-PLL specific warranting further clinical testing. Drug screening, mutation analysis and RNA sequencing of additional patients is currently ongoing (n=20) to validate whether drug responses can be predicted based on the mutation profile or aberrant gene expression. Figure Clustering of T-PLL, AML and ALL patient samples based on DSRT results. Figure. Clustering of T-PLL, AML and ALL patient samples based on DSRT results. Disclosures Kallioniemi: Medisapiens: Consultancy, Membership on an entity's Board of Directors or advisory committees. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

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