A Phase II Trial of Guadecitabine in Children and Adults with SDH-Deficient GIST, Pheochromocytoma, Paraganglioma, and HLRCC-associated Renal Cell Carcinoma

Abstract

Purpose:

Succinate dehydrogenase deficient (dSDH) tumors, including pheochromocytoma/paraganglioma, hereditary leiomyomatosis and renal cell cancer-associated renal cell carcinoma (HLRCC-RCC), and gastrointestinal stromal tumors (GISTs) without KIT or platelet derived growth factor receptor alpha mutations are often resistant to cytotoxic chemotherapy, radiotherapy, and many targeted therapies. We evaluated guadecitabine, a dinucleotide containing the DNA methyltransferase inhibitor decitabine, in these patient populations.

Patients and Methods:

Phase II study of guadecitabine (subcutaneously, 45mg/m²/day for 5 consecutive days, planned 28-day cycle) to assess clinical activity (according to Response Evaluation Criteria in Solid Tumors v.1.1) across 3 strata of patients with dSDH GIST, pheochromocytoma/paraganglioma, or HLRCC-RCC. A Simon optimal two-stage design (target response rate 30% rule out 5%) was used. Biologic correlates (methylation and metabolites) from peripheral blood mononuclear cells (PBMCs), serum, and urine were analyzed.

Results:

Nine patients (7 with dSDH GIST, 1 each with paraganglioma and HLRCC-RCC, 6 females and 3 males, age range 18–57 years-old) were enrolled. Two patients developed treatment-limiting neutropenia. No partial or complete responses were observed (range 1–17 cycles of therapy). Biologic activity assessed as global demethylation in PBMCs was observed. No clear changes in metabolite concentrations were observed.

Conclusions:

Guadecitabine was tolerated in patients with dSDH tumors with manageable toxicity. While 4/9 patients had prolonged stable disease there were no objective responses. Thus, guadecitabine did not meet the target of 30% response rate across dSDH tumors at this dose, though signs of biologic activity were noted.

INTRODUCTION

Loss of the succinate dehydrogenase (SDH) complex or fumarate hydratase leads to the accumulation of the Krebs cycle metabolites succinate and fumarate, inhibiting α-ketoglutarate-dependent dioxygenases¹. SDH deficiency (dSDH) has now been identified in 88% of patients with pediatric gastrointestinal stromal tumors (GIST) and those tumors that do not present KIT or platelet derived growth factor receptor alpha (PDGFRA) mutations, which are often described as “wild-type” GIST². GISTs are the most common sarcomas of the gastrointestinal tract^3–5. These dSDH GIST are generally resistant to targeted therapy with tyrosine kinase inhibitors such as imatinib^6–8, as well as chemotherapy and radiotherapy⁹. Thus, novel agents are urgently needed for these deadly tumors. dSDH has also been identified in 10–30% of patients with pheochromocytoma and paraganglioma^10–12, and similarly, abnormalities in fumarate hydratase have been detected in 93% of patients with hereditary leiomyomatosis and renal cell cancer-associated renal cell carcinoma (HLRCC-RCC)^13–15.

The specific mechanism or mechanisms of tumorigenesis due to dSDH is not known, and until recently a lack of preclinical models has hindered development of new treatments for these tumors. Prior attempts to target the metabolic derangements present in dSDH GIST with the tyrosine kinase inhibitor vandetanib⁷ or the IGF-1R inhibitor linsitinib⁸ were successfully completed but did not demonstrate clinical activity. However, α-ketoglutarate-dependent dioxygenases are important regulators of DNA demethylation, and consequently dSDH-mediated increases in succinate and fumarate may lead to global DNA hypermethylation. The potential relevance of DNA hypermethylation in dSDH GIST is supported by global epigenetic dysregulation¹⁶ and decreased levels of the downstream Krebs cycle intermediate 5-hydroxymethylcytosine¹⁷ found in clinical samples. This suggests a potential therapeutic role for agents which can reverse DNA hypermethylation in dSDH tumors. Guadecitabine (SGI-110), a dinucleotide containing the small molecule inhibitor decitabine, functions as a DNA methyltransferase inhibitor. Guadecitabine has entered phase 1 and phase 2 clinical trials in acute myeloid leukemia/myelodysplastic syndrome^18,19, hepatocellular carcinoma²⁰, and ovarian cancer^21,22. We hypothesized that guadecitabine would have activity in dSDH tumors including GIST, pheochromocytoma/paraganglioma, and HLRCC-RCC, and tested this hypothesis in a small phase II study.

PATIENTS AND METHODS

Patient Population

Patients ≥12 years of age with measurable disease of histologically-confirmed recurrent or refractory/unresectable dSDH: (1) GIST; (2) pheochromocytoma/paraganglioma; or (3) HLRCC-RCC. For GIST and HLRCC-RCC, previously untreated patients with metastatic and/or unresectable disease were eligible. For pheochromocytoma/paraganglioma, progression following ¹³¹I-meta-iodobenzylguanidine (MIBG) in patients with MIBG avid tumors or cytotoxic chemotherapy (dacarbazine or temozolomide) was required prior to enrollment on this trial unless the patient refused or the investigator deemed being treated on this trial prior to cytotoxic chemotherapy was in the best interest of the patient. Other eligibility criteria included recovery from toxic effects of prior therapy to grade 1; Eastern Cooperative Oncology Group (ECOG) performance status ≤ 2, or Lansky/Karnofsky score ≥ 60%; interval from prior therapy ≥ 4 weeks from prior surgical procedures with complete healing of surgical sites; ≥ 28 days from a last dose of cytotoxic chemotherapy, monoclonal antibody, or any investigational agent; ≥ 28 days from prior biological therapy including biological response modifiers (e.g. cytokines) immunomodulatory agents, vaccines, and differentiating agent; and ≥ 4 weeks from external beam radiotherapy. Patients were required to have normal organ and marrow function including adequate renal function (age-adjusted normal serum creatinine, or a creatinine clearance ≥60 mL/min/1.73 m²) and adequate liver function [total bilirubin within normal institutional limits, alanine aminotransferase, and aspartate aminotransferase ≤2.5x institutional upper limit of normal]. Adequate bone marrow function was required and defined as an absolute neutrophil count ≥1,500/ μL and transfusion-independent platelet count of ≥100,000/ μL.

This trial conformed to the Declaration of Helsinki and Good Clinical Practice guidelines and was approved by the National Cancer Institute’s Institutional Review Board. Investigators obtained written informed consent from all patients or their legal guardians, indicating their understanding of the investigational nature and risks of this study. Assent was obtained according to institutional guidelines.

Drug administration and study design

The study (NCT03165721) was conducted as a single-site Simon optimal two-stage phase II trial in 3 strata of patients with one of the three eligible diagnoses with the primary objective to assess clinical activity (complete response or partial response) of guadecitabine to rule out an unacceptably low overall response rate of 5% (p0=0.05), in favor of a response rate of 30% (p1=0.30). With α=0.10 and β=0.10, if ≥1 of 7 patients in stage 1 responded in a given stratum, enrollment would be expanded in that stratum to 21 patients, and if ≥3 of 21 responded, guadecitabine would be considered active in that stratum. Guadecitabine was supplied to the National Cancer Institute (sponsor: Cancer Therapy Evaluation Program) by Astex Pharmaceuticals, Inc. (Pleasanton, CA, USA) and was administered under an investigator-held Investigational New Drug application. Guadecitabine was administered subcutaneously at a dose of 45mg/m²/day for 5 consecutive days on a planned 28-day cycle.

Toxicity assessment and disease evaluation

Monitoring for guadecitabine-related toxicity included physical examination with vital sign measurements (including observation in clinic following initial injection of guadecitabine) as well as complete blood counts with differential, serum chemistries including electrolytes, calcium, phosphate, magnesium, creatinine, glucose, blood urea nitrogen, albumin, aspartate aminotransferase, alanine aminotransferase, total bilirubin, total protein, creatine phosphokinase, and urinalysis at baseline and prior to each subsequent cycle. Pregnancy testing was done at baseline and prior to each cycle in female patients of childbearing potential. Adverse events were graded according to the Common Terminology Criteria for Adverse Events Version 5.0.

Response was evaluated using Response Evaluation Criteria in Solid Tumors guideline version 1.1²³ at baseline and prior to cycle 2 and then prior to every other cycle. Assessment of disease was performed using radiologic evaluation which could include computerized tomography and/or magnetic resonance imaging, and/or 2[18F]fluoro-2-deoxy-D-glucose -positron emission tomography as clinically indicated. A consistent method of disease evaluation was used for each patient throughout the study.

Definition of treatment-limiting toxicity

Any toxicity of grade 3 or higher that could not be resolved/reversed within 72 hours with supportive care was considered a treatment-limiting toxicity (TLT), as was any persistent grade 2 toxicity that was intolerable to the patient. Guadecitabine was held for TLT until resolution to grade 1 or baseline, and then could be resumed with up to two dose reductions (approximately 30% each, 30mg/m²/dose at dose level −1 and 20mg/m²/dose at dose level −2).

Quality of life assessment

Health-related quality of life was evaluated by patient and parent report (for pediatric patients) using the Distress Thermometer (DT) and Patient-Reported Outcomes Measurement Information System (PROMIS) short-form measures (anxiety, depression, fatigue, pain interference, and physical function). The measures were administered to all consenting English- and Spanish-speaking patients with parallel Parent Proxy instruments administered to parents of patients ages 12 to 17. These instruments were administered at baseline, after cycle 4 and after each subsequent 4^th cycle, as well as at the time a patient was taken off treatment.

Pharmacokinetics

Decitabine was extracted from heparinized plasma samples using solid-phase extraction, then analyzed using an LC-MS/MS assay with a calibrated range of 5–1000 ng/mL. Non-compartmental analysis was performed using Phoenix WinNonlin, v8.2 (Certara, Princeton, NJ)(RRID:SCR_021370), validated per FDA 21 CFR Part 11 regulations, to estimate pharmacokinetic parameters. Specifically, the maximum plasma concentration (C_MAX) was recorded as observed values, as was the time to C_MAX (T_MAX). The area under the plasma concentration vs time curve (AUC) to the last time point was calculated using the linear up/log down trapezoidal rule. If possible, with sufficient terminal data, an elimination rate was calculated, from which half-life (t_1/2), AUC to time infinity, apparent clearance (CL/F) and volume of distribution (V_z/F) were calculated.

Correlative studies of guadecitabine activity

Peripheral blood mononuclear cell (PBMC) global methylation was assessed by whole genome bisulfate sequencing²⁴ at baseline and on day 14 of cycle 1. Briefly, peripheral blood was collected in ethylenediaminetetraacetic acid tubes, centrifuged, and then the buffy coat was resuspended in phosphate-buffered saline and stored at −20°C until further processing. Subsequently, the buffy coat was resuspended in phosphate-buffered saline for germline DNA extraction using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany). 100 ng per sample of DNA was fragmented to a target length of 350 base pair and bisulfate converted using EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA). Sequencing libraries were constructed from bisulfate converted DNA using Swift Accel-NGS Methyl-Seq (Swift Biosciences, Ann Arbor, Michigan) per the manufacturer’s protocol. Libraries were then pooled and sequenced to a goal depth of 30x using 150 base pair paired-end reads on a NovaSeq platform (Illumina, San Diego, California) and reads were trimmed to remove low-quality bases and adapters using trimgalore²⁵ v0.6.6. Bismark was used to align reads to GRCh38, demultiplex and extract methylation calls²⁶. Data was smoothed across nearby CpG sites and global methylation was calculated with bsseq v1.26.0²⁷. To identify differentially methylated regions, CpGs with less than 5x coverage in all samples were removed to decrease false positives, t-statistics were calculated and thresholded using a cutoff of 4.6. differentially methylated regions were filtered to include at least 5 CpGs and a mean difference of at least 0.2. Statistical analyses were performed using R v.4.0.3.

Serum and urine samples were collected at baseline and on days 7, 14 and 28 of cycle 1 to explore the metabolomic profile of patients with tumors related to Krebs cycle defects. Isotope dilution liquid chromatography-mass spectrometry was performed to measure the concentrations of metabolites in several key cellular metabolic pathways. For detailed description see Supplemental Methods. In brief, all reference target compounds and isotopic standards were purchased from Sigma-Aldrich, Cambridge Isotope Laboratory, Medical Isotopes, Inc. and CDN Isotopic Inc (Supplemental Methods Table 1). Urine samples were processed by dilution in water, except for oxidation and phosphorylation metabolites which were diluted with Tris buffer supplemented with internal standards to optimize pH. Serum samples were extracted in either methanol (central carbon pathway), percholic acid (electron transfer metabolites) or diluted in water (Pyruvate and short-chain fatty acids) and the extraction solutions were supplemented with appropriate isotopic standards. Reversed-phase ion-pairing LC-MS² analysis was performed using a Thermo TSQ^™ triple quadrupole mass spectrometers (Thermo Scientific, San Jose, CA) coupled to either Shimadzu 20AC-XR or Vanquish (Thermo Scientific) liquid chromatographic system for the central carbon pathway and electron transfer metabolites respectively. Pyruvate and short-chain fatty acids were analyzed by reveres phase LC-MS² on Thermo TSQ^™ triple quadrupole mass spectrometers (Thermo Scientific, San Jose, CA) coupled to Shimadzu or 20AC-XR HPLC system. The mass spectrometers were operated in either negative or positive mode and set to monitor parent-product ion transitions using Selected Reaction Monitoring (Supplemental Methods Table 2). Quantitation of targeted metabolites was carried out using Xcalibur^™ Quan Browser (Thermo Scientific). Calibration curves for each metabolite were constructed by plotting reference compound/isotopic peak area ratios obtained from the calibration standards curve and fitting the data using linear regression with 1/X weighting. The analyte concentrations in samples were then interpolated using the linear function obtained from the calibration curve.

Data Availability Statement

Raw data is available as a supplementary table and upon reasonable request.

RESULTS

Patient characteristics

Nine adult patients (3 male and 6 female; median age, 36 years; range, 18–58) were enrolled from August 16, 2017, to March 12, 2019. The race/ethnicity of patients on study included: white / not Hispanic or Latino (6); White / Hispanic or Latino (1); Black or African America / not Hispanic or Latino (1); and Asian (1). Table 1 provides a summary of demographic, clinical, and baseline disease characteristics. Supplemental Table S1 discusses the representativeness of study participants. All patients had dSDH tumors as determined by genomic analysis (one wild-type GIST patient was confirmed to be negative for KIT and PDGFRA mutations). Seven patients had dSDH GIST, 1 patient had paraganglioma, and 1 patient had HLRCC-RCC. The median number of lines of prior therapy was 2 (range 0–6). One patient was ECOG 0, while eight patients were ECOG 1. All patients had evidence of metastatic disease.

Table 1.

Baseline and treatment characteristics

Patient number	Age (years)	Sex	Sites of disease	Diagnosis	Mitotic rate**	Treatment cycles (n)
1	40	F	Liver, peritoneum, pelvis	GIST (SDH-C)	>10	9
2	52	F	Liver	GIST (SDH-C)	<10	17
4	18	F	Liver, stomach, omentum	GIST (SDH-C)	12	4
5	20	F	Liver, stomach	GIST (SDH-A)	82	12
6	46	F	Liver, lymph nodes	GIST (SDH-B)	11	7
7	20	M	Liver, lymph nodes	GIST (SDH-B)	5–6	5
9	57	F	Stomach	SDH-deficient GIST#	11	12
3	41	M	Lung, spine, pelvis	PGL (SDH-B)		3
8	23	M	Kidney, lymph nodes, pelvis	HLRCC-RCC (FH)		1

FH=fumarate hydratase; GIST=gastrointestinal stromal tumor; HLRCC-RCC=hereditary leiomyomatosis and renal cell cancer-associated renal cell carcinoma; PGL=paraganglioma; SDH=succinate dehydrogenase

^*Negative for KIT and PDGFRA mutations:

^#loss of tumor SDHB expression by immunohistochemistry without identified pathogenic mutation in any of the SDH subunits:

^**mitoses per 50 high power fields at initial diagnosis

Toxic effects and duration of treatment

Two patients developed TLT of severe neutropenia (one grade 4, one grade 3) requiring dose reduction following cycle 1, and subsequently tolerated treatment. One additional patient developed grade 2 lung infection requiring hospitalization that delayed the start of cycle 2 (Table 2). No partial or complete responses were observed, and the best response was stable disease (median number of completed cycles, 7; range, 1–12). Three out of seven patients with GIST, the patient with paraganglioma and the patient with HLRCC-RCC were taken off study for progressive disease. Among patients with stable disease, 3/7 patients with GIST were taken off study for patient preference. While not considered a TLT, one patient with GIST was taken off study at investigator discretion due to a combination of unexpected toxicity (posterior reversible encephalopathy syndrome, PRES—reversible and not previously reported in trials of guadecitabine, attributed by sponsor as not related to guadecitabine) and interval surgical tumor debulking.

Table 2.

Number of patients (only highest grade noted for each patient across all treatment cycles) with grade 2–4 toxicities possibly, probably, or definitively attributed to guadecitabine across all 70 treatment cycles in all 9 patients

Toxicity grade CTCAEv5	2	3	4
Cardiovascular toxicity
Hypertension	1
Hypotension	1
Constitutional toxicity
Fatigue	2
Increased weight	1
Gastrointestinal toxicity
Abdominal distension@	1
Abdominal pain@	1
Constipation@	1
Hematologic toxicity
Anemia	1	1
Decreased leukocyte count	4	2
Decreased lymphocyte count	1	1
Decreased neutrophil count	2	2	1
Infectious toxicity
Febrile neutropenia		1
Herpes simplex virus reactivation	1
Sinusitis	1
Neurologic toxicity
Nystagmus	1

CTCAEv5=Common Terminology Criteria for Adverse Events Version 5.0; @=all gastrointestinal toxicities occurred in the same patient

Response evaluation

Because there were no partial or complete responses observed in the first 7 patients with GIST, the GIST stratum was terminated after stage 1 on February 24, 2020. Concurrently, the pheochromocytoma/paraganglioma and HLRCC-RCC stratums were terminated due to low accrual.

6/7 patients with GIST continued therapy for at least 5 cycles (Table 1) with time to progression greater than 6 months (Table 3). The 3 patients with stable disease who discontinued therapy for patient preference completed at least 12 cycles and their time to progression was at least 10 months. The patient taken off therapy for investigator discretion (patient 4) had surgical debulking. All four patients who had stable disease when they were taken off therapy were censored at that time. For the GIST stratum (n=7), using the Kaplan-Meier method as implemented in SAS version 9.4, progression-free survival (PFS) censoring at the dates the patients came off treatment and overall survival (OS) at 12 mo were 53.6% [95% confidence interval (CI) (Fig. 1A), 13.2%-82.5%] and 100% (Fig. 1B), respectively. Median PFS was not reached, and median OS was 38.2 months (CI not estimable).

Table 3.

Patient outcomes and time to progression

Patient number	Diagnosis	Outcome	Change from baseline (best response, %, time in mo)	Time to progression (mo)	Overall survival (mo)
1	GIST (SDH-C)	PD	NA*	8.6	38
2	GIST (SDH-C)	SD—PP	−2.2 (12)	15.9	>32
4	GIST (SDH-C)	SD—ID	−13 (2)	>7.2	>27
5	GIST (SDH-A)	SD—PP	−27.27 (5)	>10.3	>27
6	GIST (SDH-B)	PD	NA*	6.7	>25.8
7	GIST (SDH-B)	PD	NA*	5.0	>25
9	Wild-type GIST	SD—PP	−5 (2)	>10	>22
3	PGL (SDH-B)	PD	NA*	3.7	>31
8	HLRCC-RCC (FH)	PD	NA*	1	4

^*NA indicates no tumor reduction of any amount was seen at any time point.

FH=fumarate hydratase; GIST=gastrointestinal stromal tumor; HLRCC-RCC=hereditary leiomyomatosis and renal cell cancer-associated renal cell carcinoma; ID=investigator discretion; PD=progressive disease; PGL=paraganglioma; PP=patient preference; SD=stable disease; SDH=succinate dehydrogenase

Figure 1: Survival outcomes for gastrointestinal stromal tumor (GIST) stratum.

A. Progression-free survival (PFS) and B. Overall survival (OS). CI=confidence interval.

The outcomes for the 1 patient in the pheochromocytoma/paraganglioma stratum (patient 3) and the 1 patient in the HLRCC-RCC stratum (patient 8) are presented in Table 3.

Quality of life evaluation

All 9 patients completed the baseline PROMIS and DT measures with 7 of 8 completing evaluation at the end of cycle 4 (one patient off treatment prior to end of cycle 4) and 8 of 9 completing the end of therapy evaluation. While there was a trend for patients to report less pain at baseline compared to end of treatment on both the DT and the PROMIS pain interference measure, the small number of patients and lack of responses in any stratum precluded evaluation of significant changes in the DT or in the t-scores over time in any of the PROMIS domains.

Pharmacokinetic evaluation

All nine patients had sufficient pharmacokinetic sampling and were thus included in this analysis. The median absolute dose was 80.7 mg, based on a median body surface area of 1.79 m². Following a subcutaneous dose of guadecitabine, decitabine plasma concentrations demonstrated an approximate 30 minute lag time prior to rising to quantifiable levels. Decitabine concentrations peaked on average at 4 hours, consistent with the desired formulation and route of administration. Summarized exposure parameters are presented in Table 4. Only two of nine patients had sufficient data to estimate an elimination rate. Given that t_1/2, V_z/F and CL/F are derived from measure, these parameters were only reported for the following two patients: t_1/2=1.73 hr, 1.35 hr; V_z/F=1127 L, 1147 L; and CL/F= 452 L/hr, 588 L/hr.

Table 4.

Summary of Pharmacokinetic Parameters of Active Metabolite, Decitabine

	Mean (n=9)	%CV
Cmax (ng/mL)	45.0	42.7
Tmax (hr)*	4.0*	1.0 – 5.5**
AUC0–5hr (hr*ng/mL)	114.1	48.1

^*Reported as median (min – max)

Abbreviations: AUC=area under the curve; C_MAX= maximum plasma concentration; CV=coefficient of variation; T_MAX=time to C_MAX,

Pharmacodynamics: Global demethylation and metabolomic analysis

Two patients had sufficient samples collected to perform methylation analysis; the other seven patients did not return for collection on day 14. Both patients, patient 2 (GIST, stable disease but taken off treatment for patient preference after 17 cycles) and patient 3 (paraganglioma, progressive disease after 3 cycles) demonstrated significant global demethylation on cycle 1 day 14 compared with baseline (−42.9%, −47.0%, Supplemental Figure S1). In all, 1,734 differentially methylated regions with at least five CpGs, and a mean difference of greater than 20% were identified between day 14 and baseline white blood cells (Supplemental Table S2).

Metabolites from the glycolytic and pentose phosphate pathway and the tricarboxylic acid cycle, as well as oxidation and phosphorylation metabolites and short chain fatty acids were evaluated in serum and urine at baseline and on days 7, 14, and 28 (+/−3 days, when available) from all 9 patients. No significant changes in any of these metabolites could be detected consistently across patients, and the lack of responses precluded correlation with clinical activity (Supplemental Figures S2–S9). Metabolites not displayed in supplemental figures were below the limit of detection at each measured timepoint.

DISCUSSION

While tyrosine kinase inhibitors such as imatinib have been transformative in the treatment of KIT and PDGFRA mutated GIST^28,29, no active targeted therapies have been identified in the subset of patients with dSDH GIST. Several small studies using various tyrosine kinase inhibitors have not demonstrated responses^7,30–33, highlighting the need for novel agents for these patients. Given the global hypermethylation observed in patients with dSDH GIST¹⁶, we hypothesized that targeting this pathway, common amongst dSDH tumors, with guadecitabine could result in therapeutic benefit. Unfortunately, guadecitabine did not achieve the target response rate of 30% in dSDH GIST. No definitive conclusions can be drawn in dSDH pheochromocytoma/paraganglioma or HLRCC-RCC as only one patient was enrolled in each of these strata, and larger studies perhaps through a cooperative group or consortium may be necessary to truly determine the activity of guadecitabine in these rare tumors. Nevertheless, we were able to gain experience with this novel therapy and to evaluate meaningful biologic correlates which could be used in future trials.

Interestingly, from a pharmacokinetic standpoint the active metabolite, decitabine, achieved higher C_max than was previously reported in the phase 1 trial of this dose (45ng/mL compared to 26.3ng/mL), with similar elimination rates (t_1/2=1.54h compared to 1.25–1.3h)²². The reason for this may be multifactorial: 1) our trial used guadecitabine as a single-agent rather than in combination with carboplatin; 2) specificities related to the underlying diseases; or 3) C_max was only evaluated in a small number of patients in both trials. Our pharmacodynamic analyses suggest that guadecitabine was able to execute its mechanism of action as demonstrated by relative global demethylation in PBMCs on day 14. The lack of responses may be due to: 1) inadequate tumor penetration of guadecitabine; 2) inability of guadecitabine to perform its demethylating action within the tumor; 3) while global hypermethylation is seemingly relevant in tumorigenesis for dSDH tumors, reversal may be insufficient to cause regression of an already-established tumor; 4) while global demethylation appears to occur, it may be that the target genes where hypermethylation drives tumorigenesis (which are unknown) were not demethylated. On-treatment tumor samples would be valuable in determining the action of guadecitabine within the tumor. Furthermore, while we determined that it was feasible to collect serum and urine metabolites over the course of the first month of a small phase 2 clinical trial in this patient population, we were unable to clearly show that guadecitabine consistently altered the metabolites present in patients with dSDH tumors over one month of therapy.

Toxicities of guadecitabine observed in other clinical trials^18–22, predominantly hematologic, were also observed in our trial and required a dose reduction or delay in 3/9 patients. An additional patient developed a severe unexpected toxicity of PRES which was not attributed to guadecitabine, but did contribute to that patient being taken off study. This unique toxicity has not previously been reported in trials of guadecitabine.

This work also illustrates some of the challenges in evaluating new therapies for rare cancers where more indolent behavior can occur. While several patients had a prolonged period of stable disease with a median duration of therapy of 7 cycles (compared, for example, to a median number of 4 cycles⁷ or median duration on treatment of 7.7 months⁸ on previous studies), it is difficult to clearly attribute this to guadecitabine, especially given the indolent nature of dSDH GIST and the small study size. A more complete understanding of the natural history of patients with SDH-deficient GIST may help to facilitate the design of future trials. No improvement in quality of life was identified. Our study highlights the difficulty of developing effective therapies in a relatively indolent and rare disease with few relevant preclinical models. Yebra and colleagues reported the derivation of in vitro patient-derived dSDH GIST models and showed a response to the alkylating agent temozolomide in these studies³⁴. Recently, Flavahan et al identified methylation in insulator regions for fibroblast growth factor 4 (FGF4) and KIT followed by the establishment of a patient-derived xenograft model which was sensitive to combined fibroblast growth factor receptor (FGFR) and KIT inhibition³⁵. The lack of activity of guadecitabine does not exclude the possibility that other agents acting on the hypermethylation state of the tumor could be effective. However, such studies highlight the importance of preclinical models, and in this case, suggests that specific methylation of FGF4 and KIT-insulator regions may be more relevant and targetable than global hypermethylation. Recurrent gene duplication of FGF4 has also been reported in “quadruple wild-type GIST”³⁶, suggesting that FGFR inhibition may be a common targetable pathway for these two different subtypes of GIST. These preclinical studies suggest that FGFR inhibition should be further studied in early-phase clinical trials and highlight the importance of developing clinically relevant preclinical models of dSDH GIST.

In summary, while we were able to demonstrate biologic activity of guadecitabine in terms of global demethylation on PBMCs in a subset of patients, this was insufficient to result in clinical disease regression of dSDH tumors. Larger studies in diseases which respond to guadecitabine are needed to discover whether global demethylation in PBMCs as well as changes in serum and urine metabolite are relevant biomarkers for clinical outcomes. Several avenues of investigation remain open for new treatment modalities for patients with these difficult to treat dSDH tumors.

STATEMENT OF TRANLATIONAL RELEVANCE.

Succinate dehydrogenase (SDH)-deficient tumors exhibit global hypermethylation, suggesting a role for demethylating agents in the treatment of these difficult to treat tumors. We tested the hypothesis that guadecitabine, a subcutaneously administered dinucleotide containing the DNA methyltransferase inhibitor decitabine, is clinically active in these tumors. Despite evidence of biologic activity, evaluated as a decrease in global methylation seen on peripheral blood mononuclear cells, no significant changes in metabolite concentrations (including from glycolytic and pentose phosphate pathway, tricarboxylic acid cycle, and oxidation and phosphorylation metabolites and short chain fatty acids) and no complete or partial responses were observed. We show the feasibility of conducting a phase II trial with incorporation of meaningful biologic correlates for a demethylating agent such as guadecitabine. Given the lack of activity for this agent, novel therapies remain to be identified for these patients.

List of Abbreviations

AUC

Area under the plasma concentration vs time curve

CI

Confidence interval

CL/F

Apparent clearance

C_MAX

Maximum plasma concentration

dSDH

Succinate dehydrogenase deficient

DT

Distress thermometer

ECOG

Eastern Cooperative Oncology Group

FGF4

Fibroblast growth factor 4

FGFR

Fibroblast growth factor receptor

GIST

Gastrointestinal stromal tumor

HLRCC-RCC

Hereditary leiomyomatosis and renal cell cancer-associated renal cell carcinoma

MIBG

¹³¹I-meta-iodobenzylguanidine

OS

Overall survival

PDGFRA

Platelet derived growth factor receptor alpha

PBMC

Peripheral blood mononuclear cell

PFS

Progression free survival

PRES

Posterior reversible encephalopathy syndrome

PROMIS

Patient-Reported Outcomes Measurement Information System

SDH

Succinate dehydrogenase

TLT

Treatment-limiting toxicity

T_MAX

Time to maximum plasma concentration

t_1/2

Half-life

V_z/F

Volume of distribution