Drug Sensitivity Testing for Cancer Therapy, Technique Analysis and Trend

: The techniques and qualities of drug sensitivity testing (DST) for anticancer treatment grew rapidly in the past two decades worldwide. Much of DST progress came from advanced systems of technical versatility (faster, high-throughput, high-sensitive and smaller in tumor quantity). As the earliest drug selective system, biomedical knowledge and technical advances for DST are mutually supported. More importantly, many pharmacological controversies are resolved by these technical advances. With this technical stride, clinical landscape of DST was entering into a new phase (>500 samples per testing and extremely low quantity of tumor cells). As a forerunner of drug selection system, DST awaits new version that can adapt to complicated therapeutic situations and diverse tumor categories in the clinic. By upholding this goal of pathogenic and therapeutic diversity, DST could eventually cure more cancer patients by establishing high-quality drug selection systems. To smooth DST develop, it needs to increasingly understanding of cancer biology, pathology and pharmacology (cancer heterogeneity, plasticity, metastasis and drug resistance) with well-informative parameters before chemotherapy. In this Article, medicinal and technical insights into DST are especially highlighted.

Successful Patient-Derived Organoid Culture of Gynecologic Cancers for Disease Modeling and Drug Sensitivity Testing

Developing reliable experimental models that can predict clinical response before treating the patient is a high priority in gynecologic cancer research, especially in advanced or recurrent endometrial and ovarian cancers. Patient-derived organoids (PDOs) represent such an opportunity. Herein, we describe our successful creation of 43 tumor organoid cultures and nine adjacent normal tissue organoid cultures derived from patients with endometrial or ovarian cancer. From an initial set of 45 tumor tissues and seven ascites fluid samples harvested at surgery, 83% grew as organoids. Drug sensitivity testing and organoid cell viability assays were performed in 19 PDOs, a process that was accomplished within seven days of obtaining the initial surgical tumor sample. Sufficient numbers of cells were obtained to facilitate testing of the most commonly used agents for ovarian and endometrial cancer. The models reflected a range of sensitivity to platinum-containing chemotherapy as well as other relevant agents. One PDO from a patient treated prior to surgery with neoadjuvant trastuzumab successfully predicted the patient’s postoperative chemotherapy and trastuzumab resistance. In addition, the PDO drug sensitivity assay identified alternative treatment options that are currently used in the second-line setting. Our findings suggest that PDOs could be used as a preclinical platform for personalized cancer therapy for gynecologic cancer patients.

FORECAST-1: Feasibility of organoid response assessment to define effective treatments for patients with colorectal cancer after failure of standard therapy.

e15571 Background: Many patients with metastatic colorectal cancer (mCRC) that have failed standard first- and second-line therapies remain fit and eager for further treatment. Therapeutic options include two agents with proven but modest survival benefit, a clinical trial of an investigational agent, rechallenge with agents successfully used in earlier lines of treatment, or agents that have shown some activity in non-randomised phase II studies. Patient derived tumouroid (PDT) are self-organizing three-dimensional in vitro models cultured from a fresh tumour biopsy. They potentially represent a robust personalized model for real-time drug sensitivity testing to determine optimal anti-cancer therapies for individual patients. Methods: FORECAST-1 is an observational, non-randomized study to determine the feasibility of PDT based drug sensitivity screening for patients with mCRC that have failed at least two lines of therapy. Once established, PDTs undergo drug sensitivity screening including agents regorafenib, trifluridine/tipiracil (TAS102), oxaliplatin, irinotecan, 5-fluorouracil (5FU), erlotinib (a surrogate EGFR-inhibitor), gemcitabine, pemetrexed and temozolomide. Patients are treated at clinician discretion, with clinical response to be correlated with PDT response. Results: Since September 2020 we have enrolled 18 of a planned 30 patients, median age 55 years (range 44 – 81). 17 patients were ECOG 0-1. Four patients had received prior TAS102 treatment. Biopsies were obtained from liver (n = 6), soft tissue/peritoneal metastases (5), lung (2), lymph node (2), primary (1), brain (1) and bone (1). Drug sensitivity testing has been performed on seven PDTs at a median of 62 days from biopsy (range 42 – 128), one PDT culture has failed. A standardised reporting template has been developed. Treatments received by patients to date include TAS102 (n = 5), regorafenib (2), FOLFOX (3), FOLFIRI (1) FOLFIRI plus EGFR-inhibitor (1), capecitabine plus bevacizumab (1) and an immunotherapy-based clinical trial (1). Four patients did not receive further anti-cancer therapy post enrolment, and one patient is yet to commence further systemic therapy. Conclusions: Enrolment to this feasibility study has been rapid demonstrating this to be an area of clinical need. PDTs have been successfully established from a high proportion of patients, but with variable growth rates. Methods to accelerate PDT culture and drug testing are being pursued to increase the feasibility of a planned prospective study of PDT sensitivity informing patient management. Clinical trial information: ACTRN12620001353987.

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

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.

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

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.

Mycobacteriophage-based test system for phenotypic drug sensitivity of clinical isolates of tuberculous mycobacteria

The issue of rapid phenotypic drug sensitivity testing of clinical isolates of tuberculous mycobacteria remains relevant.The objective of the study is to develop a new test system for rapid phenotypic drug sensitivity testing of MTB clinical isolates based on lytic mycobacteriophages, capable of testing resistance to first and second line drugs.Subjects and methods. Cultures of MTB (108 clinical isolates after primary culturing in Bactec MGIT) were incubated for 48 hours with addition of anti-tuberculosis drugs, then for 48 hours more after adding lytic mycobateriophage D29. The subsequent multiplex reaction of the polymerase chain reaction in real time allowed performing quantitative analysis of MTB DNA and mycobacteriophage DNA. The drug sensitivity of the tested sample was assessed by ranges of the differences in fluorescence threshold levels corresponding to the amount of mycobacteriophages between the control and test sample. At the same time, the drug sensitivity/resistance of all MTB clinical isolates after repeated culture was tested by Bactec MGIT which was adopted as a reference method.Results. Lytic mycobacteriophage-based drug sensitivity testing of 108 MTB isolated to four first line drugs and 90 isolates to six second line anti-tuberculosis drugs demonstrated a high level of concordance with the results of the Bactec MGIT system. It provided 99.5% sensitivity and 100% specificity of the method for 3 first-line drugs; it was slightly lower for ethambutol (86 and 96.9%, respectively), while for second line drugs, its sensitivity made 94.83% and specificity - 98.85%.