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Single Mouse Testing Design: Conventional testing, where 10 mice per treatment group are used, is resource-intensive, and allows for only a few models to be explored. More recently, we have adopted Single Mouse Testing (SMT) to evaluate drugs in a large number of xenograft models, an approach that significantly increases the inclusion of genetic diversity for a given cancer type. The SMT design is validated by a retrospective analysis of > 2100 tumor-drug studies undertaken by the Pediatric Preclinical Testing Program, where the response of a tumor in one mouse, selected at random from the group, was compared to the median group response. This analysis showed that the SMT accurately predicted responses in 78% of studies. Allowing for a deviation of ± one response classification [e.g., stable disease (SD) vs. partial response (PR)], the concordance was 95%. Further, the SMT analysis was accurate in identifying the antitumor activity of 66 of 67 drugs in terms of the objective response rate determined for each drug over a range of tumor models. Prospective studies with up to 90 ALL models and up to 50 solid tumor models show that SMT has similar concordance with conventional testing. Importantly, using SMT, one can potentially incorporate up to 20-fold the number of models for evaluation of an agent, encompassing many diseases, or encompassing the genetic diversity of a given disease.

1. Single Mouse Trials: (adapted from Murphy B, Yin H, Maris JM, Kolb EA, Gorlick R, Reynolds CP, Kang MH, Keir ST, Kurmasheva RT, Dvorchik I, Wu J, Billups CA, Boateng N, Smith MA, Lock RB, Houghton PJ. Evaluation of Alternative In Vivo Drug Screening Methodology: A Single Mouse Analysis. Cancer Res. 2016;76(19):5798-809. Epub 2016/08/09. doi: 10.1158/0008-5472.CAN-16-0122. PubMed PMID: 27496711; PubMed Central PMCID: PMC5050128,
2. Ghilu S, Li Q, Fontaine SD, Santi DV, Kurmasheva RT, Zheng S, Houghton PJ. Prospective use of the single-mouse experimental design for the evaluation of PLX038A. Cancer Chemother Pharmacol. 2020;85(2):251-63. Epub 2020/01/14. doi: 10.1007/s00280-019-04017-8. PubMed PMID: 31927611; PubMed Central PMCID: PMC7039322).

Solid Tumor Models: For solid tumor experiments, an event is defined as the quadrupling of a mouse’s tumor volume from day 0 (or baseline measurement). The exact time-to-event (in days) is estimated by interpolating between the measurements directly preceding and following the event, assuming log-linear growth. Differences in event-free survival (EFS) between experimental groups (e.g., treated vs controls) are tested using the Peto and Peto modification of the Gehan-Wilcoxon test (α = 0.05, two-sided alternative).

Initial tumor volume (V0) for solid tumor experiments is measured at initiation of treatment. The mean and standard deviation of V0 is computed within each treatment group, and comparisons between treatment groups are performed using the Wilcoxon rank sum test. At subsequent tumor measurements, the relative tumor volume (RTV) is defined for each mouse as the ratio of its current tumor volume divided by V0. At the conclusion of the experiment, the minimum RTV (minRTV) for each mouse is computed across all measurements except the initial (baseline) one. The mean and standard deviation within each treatment group of minRTV is computed, and comparisons between treatments groups are performed using the Wilcoxon rank sum test.

At each tumor measurement, overall mouse bodyweight is also measured, either individually by mouse or by cage. The mean body weight within each treatment group is graphed by timepoint and visually inspected for patterns which might indicate toxicity or disease-related weight loss. Animals with weight loss 20% or greater are euthanized.

The objective response measure (ORM) categories are progressive disease (PD, which is subdivided into progressive disease without and with growth delay, PD1 and PD2, respectively, defined only for treated mice), stable disease (SD), partial response (PR), complete response (CR), and maintained complete response (MCR).

For ST experiments, ORM categories are defined as:

• PD when < 50% tumor regression throughout study and > 25% tumor growth at end of study
• PD1 when PD and the mouse’s time-to-event ≤ 200% the median time-to-event in control group
• PD2 when PD and the mouse’s time-to-event is > 200% the median time-to-event in control group
• SD when < 50% tumor regression throughout study and ≤ 25% tumor growth at end of study,
• PR when ≥ 50% tumor regression at any point during study, but measurable tumor throughout study period
• CR when disappearance of measurable tumor mass during the study period occurs up to two times consecutively or intermittently any number of times
• MCR when no measurable tumor mass for at least three consecutive readings at any time after treatment has been completed

Overall group response is determined by the median response among evaluable mice as follows: Each individual mouse is assigned a score from 0 to 10 based on their ORM: PD1 = 0, PD2 = 2, SD = 4, PR = 6, CR = 8, and MCR = 10, and the median for the group determines the overall response. If the median score is half-way between an ORM number category, the objective response is assigned to the lower response category (e.g., an objective response score of 9 is scored CR). Studies in which toxic deaths are greater than 25% or in which the control group is not SD or worse are considered unevaluable and are excluded from analysis. Treatment groups with PR, CR, or MCR are considered to have had an objective response. Agents inducing objective responses are considered highly active against the tested line, while agents inducing SD or PD2 are considered to have intermediate activity, and agents producing PD1 are considered to have a low level of activity against the tested line.

The average minimum relative tumor volume (minRTV) is also utilized as a tumor volume response measure. A value of 0 indicates that the tumor is no longer detectable, while values < 1.0 indicate some level of tumor regression. Analyses of minRTV are visualized using waterfall plots using the “waterfall statistic”, calculated as minRTV – 1 and expressed as a percentage, with -100% indicating complete resolution of disease and with negative values indicating some degree of tumor regression.

For combination testing projects, the primary objective is usually to demonstrate that the combination is significantly more effective than either agent utilized at their optimal single agent dose/schedule. This condition is termed therapeutic enhancement, which represents a therapeutic effect for which a tolerated regimen of a combination treatment exceeds the optimal effect achieved at any tolerated dose of monotherapy associated with the same drugs used in the combination. This definition is operationalized as follows: therapeutic enhancement is considered present when the tumor growth delay (T-C) for a combination is greater than the tumor growth delay for each of the single agents tested at their maximum tolerated dose (MTD) and when the EFS distribution for the combination treatment is significantly better than the EFS distributions for both of the single agents tested at their MTD. In order to control experiment-wise Type I error at 5%, statistical tests are evaluated at the Bonferroni-corrected significance level α = 0.01 due to the five comparisons being made (combination vs. agent 1 alone, combination vs. agent 2 alone, agent 1 vs. control, agent 2 vs. control, and combination vs. control). Testing is considered not evaluable for therapeutic enhancement if either single agent used alone produces a median EFS beyond the observation period. If a treatment group exhibits excessive toxicity (> 25% toxic deaths), therapeutic enhancement is not evaluated.

Liquid Tumor Models: For Acute Lymphoblastic Leukemia (ALL) experiments, an event is defined as %huCD45+ cells (in peripheral blood) exceeding 25% or an animal exhibiting signs of morbidity associated with >50% leukemia infiltration of at least two major organs. The exact time-to-event is estimated by interpolating between the measurements directly preceding and following the event, assuming log-linear growth. Differences in event-free survival (EFS) between experimental groups (e.g., treated vs controls) are tested using the Peto and Peto modification of the Gehan-Wilcoxon test (α = 0.05, two-sided alternative).

For ALL experiments, the initial %huCD45+ is measured at initiation of treatment, the minimum %huCD45+ achieved by each mouse is computed across all subsequent measurements. For both the initial %huCD45+ and minimum %huCD45+, the mean and standard deviation are computed within each treatment group, and comparisons between treatment groups are performed using the Wilcoxon rank sum test.

At each tumor measurement, overall mouse bodyweight is also measured, either individually by mouse or by cage. The mean body weight within each treatment group is graphed by timepoint and visually inspected for patterns which might indicate toxicity or disease-related weight loss. Animals with weight loss 20% or greater are euthanized.

For ALL experiments, ORM categories are defined as:

• • PD when huCD45+ never < 1% during study period and mouse reaches event (huCD45+ > 25%) at some point during the study period
  • PD1 when PD and the mouse’s time-to-event ≤ 200% the median time-to-event in control group
  • PD2 when PD and the mouse’s time-to-event > 200% the median time-to-event in control group
  • SD when huCD45+ never < 1% and mouse never reaches event during the study period
  • PR when huCD45+ < 1% at least once during the study period, but not CR
  • CR when huCD45+ < 1% for only 2 consecutive weekly readings (once or more) during the study period, regardless of whether event is reached at a later timepoint
  • MCR when huCD45+ < 1% for at least 3 consecutive weekly readings at any time after treatment has been completed

When tested in conventional format, overall group response is determined by the median response among evaluable mice as follows: Each individual mouse is assigned a score from 0 to 10 based on their ORM: PD1 = 0, PD2 = 2, SD = 4, PR = 6, CR = 8, and MCR = 10, and the median for the group determines the overall response. If the median score is half-way between an ORM number category, the objective response is assigned to the lower response category (e.g., an objective response score of 9 is scored CR). Studies in which toxic deaths are greater than 25% or in which the control group is not SD or worse are considered unevaluable and are excluded from analysis. Treatment groups with PR, CR, or MCR are considered to have had an objective response. Agents inducing objective responses are considered highly active against the tested line, while agents inducing SD or PD2 are considered to have intermediate activity, and agents producing PD1 are considered to have a low level of activity against the tested line.

The average minimum %huCD45+ in the peripheral blood is also used as a response measure. A value of 0 indicates that the tumor is no longer detectable, while values < 1.0 indicate some level of tumor regression. Analyses of minimum huCD45+ are visualized using waterfall plots using a “waterfall statistic”, calculated as the difference between the average minimum huCD45+ and average baseline huCD45+ divided by the average baseline huCD45+ expressed as a percentage, with -100% indicating complete resolution of disease and with negative values indicating some degree of tumor regression.

For combination testing projects, the primary objective is usually to demonstrate that the combination is significantly more effective than either agent utilized at their optimal single agent dose/schedule. This condition is termed therapeutic enhancement, which represents a therapeutic effect for which a tolerated regimen of a combination treatment exceeds the optimal effect achieved at any tolerated dose of monotherapy associated with the same drugs used in the combination. This definition is operationalized as follows: therapeutic enhancement is considered present when the tumor growth delay (T-C) for a combination is greater than the tumor growth delay for each of the single agents tested at their maximum tolerated dose (MTD) and when the EFS distribution for the combination treatment is significantly better than the EFS distributions for both of the single agents tested at their MTD. In order to control experiment-wise Type I error at 5%, statistical tests are evaluated at the Bonferroni-corrected significance level α = 0.01 due to the five comparisons being made (combination vs. agent 1 alone, combination vs. agent 2 alone, agent 1 vs. control, agent 2 vs. control, and combination vs. control). Testing is considered not evaluable for therapeutic enhancement if either single agent used alone produces a median EFS beyond the observation period. If a treatment group exhibits excessive toxicity (> 25% toxic deaths), therapeutic enhancement is not evaluated.

CNS Models: For central nervous system (CNS) tumors, an event is defined as the animal becoming moribund or developing severe neurologic deficit, and the time-to-event is based on the day on which the event is noted. Differences in event-free survival (EFS) between experimental groups (e.g., treated vs controls) are tested using the Peto and Peto modification of the Gehan-Wilcoxon test (α = 0.05, two-sided alternative). For brain tumor experiments, MRI imaging is time consuming and involves a relatively high rate of animal death after scanning, probably due to anesthesia overdose. Therefore, statistical analysis for these experiments will most often be limited to comparisons of EFS.

To request drug testing services, please complete the “Request for in vivo testing” form and return as directed. Once received a meeting will be scheduled to discuss the project.

In addition to drug evaluation services, the TPC-DTC offers the following services:

1. 1. 1. 1. Genomics guidance for model selection, and analysis of model sensitivity data
         2. Tissue microarrays (TMAs) for IHC that encompass pediatric cancers including sarcomas (Ewing sarcoma, rhabdomyosarcoma, osteosarcoma), malignant rhabdoid tumor, Wilms tumor, neuroblastoma, brain tumors (medulloblastoma, ependymoma, glioma, PXA).
         3. Most tumor models listed in Table 1.
         4. Viable cryopreserved PDX tissue for establishing PDX models: Table 1. Fee per service ($100 per PDX model).
            For UTHSA faculty: Internal MTA form as a PDF, complete and email as directed.
            For requests from non-UTHSA users: Outgoing MTA form. Export as a PDF, complete and email as directed.

1. Conventional testing
2. Single mouse testing
3. TMAs
4. Snap-frozen samples (non-viable)
5. Cryopreserved samples (viable)
6. Model expansion (NSG mice)