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A nonrandomized phase I and biomarker trial of regorafenib in advanced myeloid malignancies

Abstract We conducted a single‐center, open‐label, dose escalation, and expansion phase I trial of the antiangiogenic multikinase inhibitor regorafenib in patients with advanced myeloid neoplasms. We enrolled 16 patients with relapsed/refractory acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), or myelodysplastic syndrome (MDS). A 3 + 3 dose escalation design was used with two planned dose levels (120 or 160 mg daily) and one de‐escalation level (80 mg daily). An additional 10 patients were treated on an expansion cohort. The recommended phase two dose of regorafenib was 160 mg daily, with no dose‐limiting toxicities. The best overall disease response by International Working Group criteria included one partial and stable disease in 11 patients. Tissue studies indicated no change in Ras/mitogen‐activated protein kinase (MAPK) pathway activation in responders. Pharmacodynamic changes in plasma VEGF, PlGF, and sVEGFR2 were detected during treatment. Baseline proinflammatory and angiogenic cytokine levels were not associated with clinical response. Single‐agent regorafenib demonstrated an acceptable safety profile in relapsed/refractory myeloid malignancy patients. Most patients achieved stable disease, with modest improvements in cell counts in some MDS patients. Biomarker studies were consistent with on‐target effects of regorafenib on angiogenesis. Future studies should investigate the role of regorafenib in combination therapy approaches.

Within AML specifically, increased bone marrow angiogenesis is associated with decreased overall survival [7], and microvessel density reverts to levels similar to controls following induction chemotherapy [8]. Angiogenesis and VEGF expression are increased in patients with myeloproliferative neoplasms (MPNs), especially in MF [9], and in advanced MDS and CMML [10].
VEGF inhibitors have been previously evaluated in myeloid malignancies given the essential role of this pathway in tumor angiogenesis. A phase 2 trial of bevacizumab, a recombinant monoclonal antibody targeting VEGF, with standard induction chemotherapy in newly diagnosed elderly AML patients showed no improvements in clinical outcomes [11]. However, a separate phase 2 trial in relapsed/refractory AML utilizing a time-sequential strategy of chemotherapy followed by bevacizumab yielded a modest complete remission (CR) rate of 33% [12]. A phase 2 study of MF patients receiving bevacizumab monotherapy demonstrated significant toxicity such that the trial was closed early, with no objective responses observed [13]. Aflibercept, a decoy receptor and VEGF trap, was tested in a phase 2 trial of MDS and MDS/MPN overlap patients who have failed hypomethylating agents, and was also ultimately halted early due to lack of efficacy [14].  [15][16][17]. Molecularly targeted therapies, including FLT3 and isocitrate dehydrogenase inhibition, have been developed and represent a major step forward in treatment [18,19]. However, these approaches usually have limited responses as single agents, in part due to the clonal heterogeneity of diseases and the existence of redundant pathways leading to oncogenesis. Regorafenib is a multikinase inhibitor that targets angiogenic, stromal, and oncogenic kinases and is approved for the treatment of advanced hepatocellular and colorectal carcinomas. As a result of regorafenib's broad inhibition of kinases and its effects on angiogenesis, regorafenib has the potential to overcome the limitations of more selective/specific VEGF inhibitors in advanced myeloid malignancies.

Study design and procedures
We

Correlative studies
Exploratory analyses of biomarkers were evaluated in participants at baseline (C1D1 prior to first treatment dose) and on C1D8, C1D15, and C2D1. Whole blood was centrifuged and separated into plasma and cellular components. The following proangiogenic and proinflammatory biomarkers were performed on plasma with the Human Angio-

Statistical analysis
The primary objective of this study was to evaluate the safety and tolerability of regorafenib in patients with advanced myeloid malignancies. All patients who received at least one dose of regorafenib were included for analysis. DLTs were assessed during cycle 1, and rates of toxicity were summarized descriptively.
The secondary objectives of this study were to assess responses criteria for MF [20][21][22]. Bone marrow biopsies for disease response assessment were performed at 1, 3, 6, 9, and 12 months and summarized with descriptive statistics.
For biomarker analysis, variables were summarized as median and interquartile ranges at each timepoint. Statistical analysis was performed using R 4.0. A Wilcoxon signed-rank test was used to test the paired differences in biomarkers from C1D1 to C1D8. Plasma biomarkers were excluded if more than 50% of values were out of the range of detection. For the purposes of plasma biomarker testing, responders were defined as patients who obtained a partial response or had stable disease for at least 2 months. A Wilcoxon rank-sum test was used to compare baseline biomarkers and changes in biomarkers from C1D1 to C1D8 between responders and nonresponders. A Bonferroni correction was used to adjust for multiple comparisons.

Patient characteristics
Six patients were enrolled during the dose escalation phase, three patients were enrolled at 120 mg daily, and an additional three were

Safety
At the 120-mg dose level, a single grade 3 event occurred and was Adverse effects are summarized in Table 2.

Biomarkers
We first examined the changes in ERK phosphorylation (a downstream RAF factor) using bone marrow aspirates and PBMCs collected before and during regorafenib treatment from patients with RAS-mutant disease (n = 3) and RAS-wild type disease (n = 2). We found high levels of ERK activation in all samples, which were maintained after regorafenib treatment irrespective of RAS-mutation status (Figure 2).
We also examined the changes in soluble blood biomarkers Pro-inflammatory biomarkers that showed significantly increased concentration from C1D1 to C1D8 included TNFα (5.8-8, p = 0.03).
There were also increases in plasma IL-8, IL-6, and IFN-γ but these changes were not statistically significant when adjusted for multiple comparisons.
There were no significant associations between biomarkers at C1D1 and clinical response. An increase in plasma IL-8 from C1D1 to C1D8 was associated with improved regorafenib response (p = 0.03), although this did not retain significance when corrected for multiple comparisons.
Finally, we examined the changes in cellular blood biomarkers after regorafenib treatment (Table 3). We found a decrease in the fraction of circulating CD3 + /CD4 + /CD8helper T-cells from C1D1 to C1D8 (59.3-50.6, p = 0.02), although this was not significant when corrected for multiple comparisons. We detected no other significant changes in the fractions of circulating lymphocyte populations.

DISCUSSION
We report the first clinical trial to evaluate feasibility and biomarkers of regorafenib in patients with advanced myeloid malignancies. Regorafenib is an oral multikinase inhibitor that is food and drug administration (FDA) approved for treatment of multiple solid tumor types and has broad inhibitory activity of multiple kinases involved in proliferation, tumor immunity, and the tumor microenvironment. This makes regorafenib an appealing drug candidate in advanced myeloid malignancies, which is characterized by abnormalities in multiple signaling pathways.
We found that regorafenib was well tolerated at a starting dose of 160 mg daily. Phase I data showed no DLTs and mostly grade 1/2 AEs. LFT abnormalities, fatigue and anorexia occurred frequently in our study, consistent with reported adverse events with regorafenib in other tumor types [24][25][26]. Hematologic toxicity was also similar compared to other safety data [24][25][26]. However, hematologic events tended to be more severe, and attribution was difficult because of the underlying diseases being treated.
Interestingly Ras-mutated CMML in a melanoma patient treated with vemurafenib [27,28]. However, none of the patients in this cohort, including five with RAS mutations, experienced signs of leukemic disease proliferation, and one patient with RAS-mutated MDS remained on regorafenib for over a year with stable disease. We also did not see any signals for an association between RAS mutation status and disease response or AEs. However, further evaluation of regorafenib in RAS-mutated leukemias should include close hematologic monitoring for signs of disease acceleration caused by potential paradoxical activation of ERK signaling upon RAF inhibition. Interestingly, we found no inhibition of MAPK pathway in samples from three RAS mutant or two RAS wild-type cases. Moreover, of these three patients, one had prolonged stable disease on regorafenib for over 1 year, and one patient had initial partial response, suggesting that MAPK activation may not be the mechanism of benefit or indicate regorafenib treatment failure.
The results of the pharmacodynamic biomarker studies were consistent with regorafenib's known anti-angiogenic activity. We did not find associations between baseline angiogenic or proinflammatory cytokines with clinical outcomes when correcting for multiple comparisons. However, we found an increase in IL-8 was associated with TA B L E 3 Circulating lymphocyte populations. Median and interquartile ranges of circulating lymphocyte populations fractions by flow cytometry during regorafenib treatment in for patients with advanced myeloid malignancies. Units are in percentage of CD3 + total lymphocytes, and total CD3 + percentage is out of gating of all peripheral blood mononuclear cells clinical response. IL-8 is an inflammatory chemokine that has known associations with disease severity and overall survival in advanced myeloid malignancies such as MF [29]. Its significance in the context of regorafenib therapy in AML is unclear and should be further In this small and heterogeneous cohort, our correlative analysis was exploratory and hypothesis generating.
Although the primary objective of our study was safety, we found

ETHICS STATEMENT
All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975.