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. 2017 Apr;10(2):241-254.
doi: 10.1016/j.tranon.2017.01.008. Epub 2017 Feb 12.

Analysis of Chemopredictive Assay for Targeting Cancer Stem Cells in Glioblastoma Patients

Candace M Howard  1 , Jagan Valluri  2 , Anthony Alberico  3 , Terrence Julien  3 , Rida Mazagri  3 , Robert Marsh  3 , Hoyt Alastair  3 , Antonio Cortese  4 , Michael Griswold  5 , Wanmei Wang  5 , Krista Denning  6 , Linda Brown  6 , Pier Paolo Claudio  7
Affiliations

Analysis of Chemopredictive Assay for Targeting Cancer Stem Cells in Glioblastoma Patients

Candace M Howard et al. Transl Oncol. 2017 Apr.

Abstract

Introduction: The prognosis of glioblastoma (GBM) treated with standard-of-care maximal surgical resection and concurrent adjuvant temozolomide (TMZ)/radiotherapy remains very poor (less than 15 months). GBMs have been found to contain a small population of cancer stem cells (CSCs) that contribute to tumor propagation, maintenance, and treatment resistance. The highly invasive nature of high-grade gliomas and their inherent resistance to therapy lead to very high rates of recurrence. For these reasons, not all patients with similar diagnoses respond to the same chemotherapy, schedule, or dose. Administration of ineffective anticancer therapy is not only costly but more importantly burdens the patient with unnecessary toxicity and selects for the development of resistant cancer cell clones. We have developed a drug response assay (ChemoID) that identifies the most effective chemotherapy against CSCs and bulk of tumor cells from of a panel of potential treatments, offering great promise for individualized cancer management. Providing the treating physician with drug response information on a panel of approved drugs will aid in personalized therapy selections of the most effective chemotherapy for individual patients, thereby improving outcomes. A prospective study was conducted evaluating the use of the ChemoID drug response assay in GBM patients treated with standard of care.

Methods: Forty-one GBM patients (mean age 54 years, 59% male), all eligible for a surgical biopsy, were enrolled in an Institutional Review Board-approved protocol, and fresh tissue samples were collected for drug sensitivity testing. Patients were all treated with standard-of-care TMZ plus radiation with or without maximal surgery, depending on the status of the disease. Patients were prospectively monitored for tumor response, time to recurrence, progression-free survival (PFS), and overall survival (OS). Odds ratio (OR) associations of 12-month recurrence, PFS, and OS outcomes were estimated for CSC, bulk tumor, and combined assay responses for the standard-of-care TMZ treatment; sensitivities/specificities, areas under the curve (AUCs), and risk reclassification components were examined.

Results: Median follow-up was 8 months (range 3-49 months). For every 5% increase in in vitro CSC cell kill by TMZ, 12-month patient response (nonrecurrence of cancer) increased two-fold, OR=2.2 (P=.016). Similar but somewhat less supported associations with the bulk tumor test were seen, OR=2.75 (P=.07) for each 5% bulk tumor cell kill by TMZ. Combining CSC and bulk tumor assay results in a single model yielded a statistically supported CSC association, OR=2.36 (P=.036), but a much attenuated remaining bulk tumor association, OR=1.46 (P=.472). AUCs and [sensitivity/specificity] at optimal outpoints (>40% CSC cell kill and >55% bulk tumor cell kill) were AUC=0.989 [sensitivity=100/specificity=97], 0.972 [100/89], and 0.989 [100/97] for the CSC only, bulk tumor only, and combined models, respectively. Risk categorization of patients was improved by 11% when using the CSC test in conjunction with the bulk test (risk reclassification nonevent net reclassification improvement [NRI] and overall NRI=0.111, P=.030). Median recurrence time was 20 months for patients with a positive (>40% cell kill) CSC test versus only 3 months for those with a negative CSC test, whereas median recurrence time was 13 months versus 4 months for patients with a positive (>55% cell kill) bulk test versus negative. Similar favorable results for the CSC test were observed for PFS and OS outcomes. Panel results across 14 potential other treatments indicated that 34/41 (83%) potentially more optimal alternative therapies may have been chosen using CSC results, whereas 27/41 (66%) alternative therapies may have been chosen using bulk tumor results.

Conclusions: The ChemoID CSC drug response assay has the potential to increase the accuracy of bulk tumor assays to help guide individualized chemotherapy choices. GBM cancer recurrence may occur quickly if the CSC test has a low in vitro cell kill rate even if the bulk tumor test cell kill rate is high.

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Figures

Figure 1
Figure 1
Quadrant diagram of the relationship between TMZ CSC assay results (%-cell kill on the y-axis) and TMZ bulk tumor assay results (%-cell kill on the x-axis) characterized by 12-month recurrence outcomes. Solid circles represent treatment responders (patients who did not manifest a recurrence at 12 months), and open circles represent patients manifesting recurrence within 12 months from treatment. Optimal threshold referent lines from the logistic regression models (40% for CSC, 55% for bulk tumor) are illustrated.
Figure 2
Figure 2
Kaplan-Meier plots of tumor recurrence across the study period. Survival (tumor nonrecurrence) is shown stratified by dichotomized test results (TMZ optimal thresholds of CSCs> 40% and bulk test >55%); P for both <.001 in Cox proportional hazard models.
Figure 3
Figure 3
Comparison of most sensitive drug from a panel of various chemotherapies versus Temodar. (A) Pyramid plot of percent cell kill for the most cytotoxic drug and TMZ comparing CSC and bulk tests for each patient. Optimal therapies with the highest cell kill are shown in light colors and TMZ cell kill is shown in dark colors, with each row of the pyramid corresponding to results for a single patient. When the light bar is longer than the dark bar, a potentially more optimal therapy than TMZ is identified. CSC results outlined in red show patients whose CSC test identified an optimal therapy that was different than the optimal therapy identified by the bulk test, 17/41 patients, 42% (95% CI 26%-57%),P < .001. *Represents patient in Figure 4; **Represents patient in Figure 5. (B) Cell kill diagram for the panel of 15 tested therapies across all 41 patients with patient numbers on the x-axis and cell kill on the y-axis. TMZ response (numbered as drug 1) and the therapy with the optimal response (highest cell kill) are shown in bold for every patient; other therapies are shown with their numbers faded. 1: Temodar; 2: Vincristine; 3: Carboplatin; 4: Cisplatin; 5: Etoposide; 6: Methotrexate; 7: Arabinocide-C; 8: Oxaliplatin; 9: Irinotecan; 10: Avastin; 11: BCNU; 12: CCNU; 13: Procarbazine; 14: Irinotecan + Avastin; 15: Procarbazine + Vincristine + CCNU.
Figure 4
Figure 4
MRIs of partial response in a GBM of the frontal lobe in a patient treated with standard of care comprising maximal surgical resection and concurrent adjuvant TMZ/radiation therapy, and mean tumor volume of PDXs treated with i.p. injection of anticancer drugs. Top shows the percent of cell kill determined by ChemoID assay on bulk of tumor and on CSC and the patient outcome summary at 12 months. (A) Preoperative MRI shows an intraaxial enhancing mass centered in the left frontal lobe crossing the midline with a component of the mass within the left frontal lobe as well consistent with a butterfly lesion measuring 59.8 × 44.2 mm. There is a large amount of adjacent vasogenic edema and mass affect in both frontal lobes and mass affect upon both frontal lobes of the lateral ventricles with left to right midline shift with subfalcine herniation causing ventricular entrapment and ballooning/hydrocephalus of the right lateral ventricle. (B) MRI at 6 months. There is regressing of the mass post–surgical resection with chemotherapy and radiation therapy with mild foci of rim enhancement in the tumor resection bed 6 months posttherapy measuring 5.4 mm anteriorly and 7.3 mm posteriorly, which could represent residual disease versus enhancement related to postradiation necrosis. (C-D) MRI at 12 months. Clear recurrence in the tumor resection bed by 12 months evidenced by nodular enhancing foci in the posterior margin of the resection bed measuring 11.3 mm and 16.3 mm each. Linear dural enhancement is attributed to postsurgical enhancement from prior craniotomy. (E-F) MRI at 18 months. Progression of recurrent disease at 18 months post–initial therapy with large enhancing irregular infiltrative mass measuring 43.9 mm × 43.0 mm at the level of the corpus callosum and more superiorly in the left frontal lobe measuring 51.4 × 30.0 mm in the tumor resection cavity crossing the midline involving both frontal lobes, left more so than right, and invading the anterior genu of the corpus callosum, again with mass affect on the frontal horns of both lateral ventricles. (G) Line diagram of the mean volumes in mm3 (±SD) from weeks 2 to 8 of the PDX tumors in 10 NOD-Scid mice following 4 weeks of treatment with various anticancer drugs. The mean tumor volumes are indicated on the ordinate. Asterisks indicate weeks in which treatment was performed. On the right are indicated the different treatment arms. PBS: saline solution, negative control. TMZ (Temodar); Methotrexate (MTX). Irinotecan (CPT-11). Bevacizumab (Avastin). Irinotecan (CPT-11) + Bevacizumab (Avastin).
Figure 5
Figure 5
MRIs of response in a multifocal GBM in a patient treated with standard of care comprising of diagnostic biopsy and concurrent adjuvant TMZ/radiation therapy, and mean tumor volume of PDXs treated with i.p. injection of anticancer drugs. Top shows the percent of cell kill determined by ChemoID assay on bulk of tumor and on CSC and the patient outcome summary at 12 months. (A-B) Preoperative MRI. Multifocal GBM multiforme with multiple infiltrative irregular enhancing masses in the left temporal lobe. Two target lesions are detailed in the left temporal lobe with the (target 1) more anterior of which measuring 19.8 mm in long axis and the (target 2) more posterior of which measuring 37.6 mm in long axis. Two adjacent target lesions in the left occipital lobe are referenced, one of which has a more cystic component (target 3) with thin rim enhancement measuring 23.5 × 16.9 mm with more solid enhancing nodular component (target 4) along the left anterior aspect of the lesion measuring 15.7 mm in long axis. Other smaller nontarget enhancing lesions are present in the left temporal lobe. (C-D) MRI at 12 months. There is regression of the target and nontarget lesion post–surgical resection with adjunct chemotherapy and radiation 12 months later with target 1 measuring 12.4 mm in long axis, target 2 measuring 5.0 mm, target 3 measuring 17.9 mm in long axis, and target 4 measuring 8.2 mm. (E) MRI at 36 months. Complete response at 3-yearfollow-up of therapy without residual enhancing tumor in all target and nontarget lesions. (F) Line diagram of the mean volumes in mm3 (±SD) from weeks 2 to 8 of the PDX tumors in 10 NOD-Scid mice following 4 weeks of treatment with various anticancer drugs. The mean tumor volumes are indicated on the ordinate. Asterisks indicate weeks in which treatment was performed. On the right are indicated the different treatment arms. PBS: saline solution, negative control; TMZ (Temodar); Methotrexate (MTX); Irinotecan (CPT-11); Bevacizumab (Avastin); Irinotecan (CPT-11) + Bevacizumab (Avastin).
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