Postmortem Study of Organ-Specific Toxicity in Glioblastoma Patients Treated with a Combination of Temozolomide, Irinotecan and Bevacizumab

doi:10.21203/rs.3.rs-1214765/v1

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Research Article

Postmortem Study of Organ-Specific Toxicity in Glioblastoma Patients Treated with a Combination of Temozolomide, Irinotecan and Bevacizumab

https://doi.org/10.21203/rs.3.rs-1214765/v1

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Purpose

Systemic monotherapy with temozolomide (TMZ) or bevacizumab (BEV); two-drug combinations, such as Irinotecan (IRI) and BEV, TMZ and BEV and a three-drug combination with TMZ, IRI and BEV (TIB) have been used in treating patients with progressive high-grade gliomas including glioblastoma. Most patients tolerated these regimens well with well-established sides effects of hypertension, proteinuria, and reversible clinical myelosuppression (CM). However, organ-specific toxicities have never been examined by postmortem studies.

Methods

Postmortem tissues (from all major organs) were prospectively collected and examined by standard institution autopsy and brain cutting procedures from 76 decedents, including gliomas (N=68, 44/M, and 24/F) and brain metastases (N=8, 5/M, and 3/F) between 2009 and 2019. Standard hematoxylin and eosin (H&E) were performed on all major organs and brain samples harvested. Electronic microscopic (EM) study was carried on selected subjects kidney samples per standard EM protocol.

Results

Twenty-four glioma subjects were treated with TIB [median: 5.5 (range: 1-25) cycles] at glioma recurrence. Exposure to IRI significantly increased the frequency of CM (p=0.05). No unexpected adverse events were detected clinically or permenant end-organ damage by postmortem examination among subjects who received TIB compared to subjects who received standard of care (SOC) therapies. Among glioma decedents, the most common causes of death (COD) were tumor progression (63.2%, N=43), followed by aspiration pneumonia (48.5%, N=33). No COD was attributed to acute toxicity from TIB. The study also demonstrated that postmortem kidney specimen is unsuitable for studying renal ultrastructural pathological changes due to autolysis.

Conclusion

IRI, but not the extended use of TMZ, significantly increased CM in recurrent glioma patients. There is no permanent organ-specific toxicity among glioma decedents who received prolonged BEV, TMZ or TIB regimen based chemotherapies except expected occasional myelosuppresson. COD are most commonly resulted from glioma tumor progression and aspiration pneumonia.

Neurology

Oncology

Autopsy

postmortem

end-organ toxicity

chemotherapy

Temozolomide

Irinotecan

Bevacizumab

Glioblastoma

Glioma

Glioblastoma (GBM) is the most common primary malignant brain tumor with an extremely poor prognosis. The median overall survival (mOS) ranges from 14.6¹ to 23.1² months based on phase III clinical trials and population analysis-reported mOS is about 11 months.³ The disease usually recurs within 3.3-8.2 months while on active treatments.⁴ There has been no uniform standard of care (SOC) for recurrent GBM (rGBM). For example, BEV (Avastin®) is commonly used in the USA but rGBM is not an approved indication for BEV in the European Union.⁵ Unfortunately, no new therapeutic drug has been demonstrated improving mOS of GBM patients in phase III clinical studies in the last decade compared to established therapies.^6–8

Two-drug combinations, temozolomide (TMZ)/BEV, irinotecan (IRI)/BEV and TMZ/IRI, have been studied in adult rGBM. However, none of these combinations were further evaluated in phase III studies.^5,9,10 A phase III clinical trial using BEV with lomustine (CCNU) (N=288) for rGBM failed to demonstrate an overall survival advantage over CCNU monotherapy (N=149).⁷ In the pediatric oncology community, the TMZ/IRI combination is adopted as an effective "backbone" regimen.¹¹ Combination therapy with three drugs TMZ/BEV/IRI (TIB) was studied in clinical trials for treating pediatric diseases^12–15 and gliomas: 1) a phase I study of unresectable GBM (N=41);¹⁶ 2) a phase II study of newly diagnosed GBM (N=75);¹⁷ and 3) a phase I study of high-grade glioma (N=12).¹⁸ A retrospective study of two institutions’ results demonstrated an improved mOS among rGBM patients receiving TIB plus Tumor Treating Fields (TTFields) therapy (N=18) compared to those receiving non-TIB BEV-based chemotherapies with TTFields (N=30) without unexpected side effects.¹⁷ All these studies concluded that the TIB regimens were well-tolerated without unforeseen toxicities.^12–19 The safety profiles support the TIB regimen administration in rGBM patients with normal bone marrow function since rGBM has limited treatment options.

BEV is a recombinant humanized monoclonal antibody against vascular endothelial growth factor-A. The most common and predictable side effects from its long-term use are hypertension (HTN) and proteinuria. It is postulated that kidney injury from long-term use of BEV is related to the development of HTN and proteinuria, but few renal biopsies in patients who developed BEV-related HTN and proteinuria have been carried out.²⁰ The standard duration of TMZ chemotherapy for GBM is six weeks of daily therapy with concurrent fractionated radiation followed by six cycles of adjuvant treatment per the Stupp protocol¹. It is not uncommon for some GBM patients to take up to 12 cycles of TMZ (prolonged duration) or even longer if there is no evidence of GBM progression or severe side effects. However, there is concern that prolonged use of TMZ alone or with other chemotherapy drugs may produce more harm than benefit, including alkylating agent-related leukemia.^21,22

At Tufts Medical Center and Memorial Hermann Hospital (MHH)-Texas Medical Center (TMC)/UTHealth, the TIB regimen was offered to rGBM patients with normal bone marrow function. Treatment decisions were based upon the physician's best knowledge and judgment along with close monitoring of adverse events, balancing the need to control the aggressive tumor and manageable side effects. Clinical side effects from individual chemotherapy drugs, including TMZ, BEV, IRI, and a combination of TMZ/IRI, have been well-documented by the Cancer Therapy Evaluation Program (CTEP). However, there are few reports on the side effects from long-term use of TIB, BEV/TMZ, or TMZ monotherapy beyond 12 months. Autopsy studies of 117 GBM²³ and 40 cases of brain stem gliomas²⁴ did not report chemotherapy-related end-organ toxicity. This study aimed to determine any significant end-organ damage from TIB therapy, prolonged use of TMZ monotherapy or TMZ based two-drug therapy. We also wanted to identify pathological changes of the kidney post BEV therapy by standard postmortem examination plus electron microscopic (EM) study of kidney specimens prospectively. From 2009 to 2019, 76 deceased subjects were examined in this study, including 24 patients treated with TIB.

Regulatory approval and consent

The study protocol and consent received approvals from the Health Sciences Institutional Review Board at Tufts Medical Center and the Committee for the Protection of Human Subjects (CPHS) at Memorial Hermann Hospital (MHH) at Texas Medical Center (TMC) and UTHealth (HSC-MS-11-0133), respectively. Consents for autopsy were obtained from patients antemortem or next of kin postmortem.

Autopsy, brain cutting and EM examinations

Twelve decedents from Tufts Medical Center and 64 decedents from the MHH-TMC/UTHealth were enrolled in the study between 2009 and 2019 prospectively. Autopsies and brain cutting were done at the decedents’ respective institution per the institution’s standard of practice (SOP) including unrestricted autopsies (N=69), restricted autopsies to the brain (N=6), and to brain and kidney (N=1). Eight brain metastasis (BM) decedents served as non-glioma references. Most of the decedents’ bodies were transported to the morgue within two to four hours. Bodies were stored at +4°C until autopsy. Postmortem organ studies included gross organ examination and microscopic tissue evaluation with hematoxylin and eosin (H&E) staining per SOP. Kidney tissues from 15 subjects, including ultrasound-guided biopsies in four cadavers before the full autopsy, were analyzed by EM. The tissues destined for EM examination were collected in formaldehyde fixative solution and then prepared per EM examination SOP. Cadaver kidney biopsies were performed with ultrasound guidance by an interventional radiologist (AKP) prior to autopsy to shorten the delay of kidney sample collection. EM slides were reviewed by EM trained nephropathologists (WFG and MP). Three decedents that never received BEV (#31, #49, and #60) were included as controls. The brain was harvested at the time of autopsy and placed in the fixative solution until brain cutting which ranged from 3-6 weeks. Brain cutting was performed per SOP and included gross inspection and microscopic examination with H&E stain. Fresh samples from each major organ and selected locations of the brain were also collected. The samples were frozen with liquid nitrogen and then stored at -80°C for other use.

Medical record review and side effects grading

Medical records were reviewed, including operative reports, lab results, MRI image reports, pathology and autopsy reports of body and brain cutting, chemotherapy received and documented adverse events. Clinical notes were reviewed to extract age, gender, body weight, diagnosis, overall survival, and adverse events (AEs). Common Terminology Criteria for Adverse Events (CTCAE), version 4.03, was used for grading. Hospitalization and death were considered severe AE (SAE).

Chemotherapy regimens

Each TIB cycle consists of one 5-day period of TMZ (150 mg/M², PO, QD during day 1-5 every 28 days), two BEV (10 mg/kg), and two IRI (125 mg/M²) infusions every 14± 2 days. Each cycle lasts 28-32 days and allows for the delay of either TMZ or IRI or both based on lab results before administration of chemotherapy per treating physicians. No patient was on p450 enzyme-inducing anti-seizure medication. TIB was offered to rGBM patients for up to 12 cycles, depending on the patient's tolerance. Four patients received more than 12 cycles either continuously or restarted following disease progressions. When TMZ was used for rGBM, but not as a component of the TIB regimen, it was first administered at 200 mg/m², PO, QD on days 1-5 of a 28-day cycle, and then the dose was reduced to 150 mg/m² if grade III or higher CM was observed. Detailed chemo/anti-angiogenesis therapy records from each patient are provided in the supplement (Supp Table I).

Grouping based on therapies received

Decedents with glioma were separated into four groups: Group 1: no chemotherapy beyond concurrent chemoradiation (CCRT), or NCBC (N=7), including three subjects never received chemotherapy; Group 2: received 1-5 cycles of TIB (N=12); Group 3: received 6-25 cycles of TIB (N=12); Group 4: SOC group (N=37). Group 4 included eight patients on maintenance TMZ only; the other 29 received BEV alone or BEV/IRI doublet therapies, including three patients who discontinued TMZ during CCRT. Cancer treatment(s) for decedent groups are summarized in Table 2 and detail of drugs for individuals are provided in supplemental Table 1.

Clinical myelosuppression

Clinical myelosuppression score (CMS), the sum of the highest grade of AEs based on white blood cell count, hemoglobin or platelet lab results, respectively. Grade 1 and 2 AEs do not receive scores, grade III, IV or V AEs are assigned with scores of 3, 4 or 5, repectively. For example, subject 36 (supplement table) experienced grade IV AEs of leukocytopenia and thrombocytopenia but without worse than grade II anemia, so her CMS was 4+4+0=8. CMS was analyzed based on the number of TMZ cycles: reference (1-6 cycles) and four subgroups (7-12, 13-18, 19-24, and 25-47 cycles). In addition, CMS was compared in patients who received TIB (cycles: 1-5, 6-25, and 1-25) vs. Group 4, which included various numbers of IRI infusions (0, 1-6, 7-52).

Statistical analysis

Univariable binary logistic regression models were performed to explore the associations between different treatment strategies and incident myelosuppression. Odds ratios (OR) and 95% confidence intervals (95% CIs) are presented. A swimmer plot was utilized to illustrate and compare the timelines of patient history by treatment group. Statistical analyses were performed using Stata IC 15.1 (StataCorp, College Station, TX). P values are two-sided and are considered statistically significant when P < 0.05.

Clinical and pathological characteristics of patients

A total of 76 decedents (49 males, ages 23-80 years old at initial diagnosis, were enrolled prospectively with initial diagnoses as GBM (N=53), grade III glioma (N=8), brain metastasis (BM, N=8), grade II glioma (N=5), and spinal glioma (N=2) (grades III and IV, respectively). At autopsy, 4 grade II and 5 grade III gliomas had progressed to secondary GBMs (Table 1). The swimmer plot is presented in Figure 1. The average time from death to postmortem examination at MHH-TMC/UTHealth was 35.3±3.7 hours (range: 7-100 hours, calculated from 38 cases with accurate documentation of timing of death).

Kidney ultrastructure examination

EM studies were performed in 14 subjects to understand the ultrastructural changes in those who had HTN and proteinuria due to BEV therapy. An interval from death to tissue collection was not available from five decedents at Tufts Medical Center; the interval from death to tissue collection was 19.8±7.5 hours from nine glioma decedents at MHH-TMC/UTHealth. To differentiate true microscopic findings from possible artifacts from autolysis, interventional radiologist (AKP) biopsied cadaver kidney under ultrasound guidance on 4 decedents #22 (3 hrs), #20 (6 hrs), #17 (7 hrs), and #49 (9 hrs). The EM results of podocyte effacement with basement membrane detachment were observed in subjects exposed to BEV and naïve to BEV. making observation inconclusive due to autolysis.

Clinical myelosuppression on active therapies

Analysis showed that 41.7% (5/12), 16.7% (2/12), and 20% (10/37) of decedents experienced grade III or worse CM during active therapies in groups 2, 3, and 4, respectively. Compared to group 4, ORs were 1.93 (95% CI: 0.50-7.50, P=0.343) and 0.54 (95% CI: 0.10-2.91, P=0.473) for groups 2 and 3, respectively (Table 3). The CM rates were 57.1% and 47.6% in IRI infusion subgroups who received 1-6 cycles (N=14) and 1-12 cycles (N=21), respectively. The ORs in these two groups were 7.67 (95% CI: 1.71-34.33, P=0.008) and 5.23 (95% CI: 1.34-20.45, P=0.018) (Table 3). By contrast, the likelihoods of CM for patients treated with 7-12, 13-18, 19-24, and 25-47 cycles of TMZ were not significantly increased compared to those receiving 1-6 cycles (all P>0.05) (Table 3).

Bone marrow examinations at autopsy

Bone marrow exams were performed in 39 decedents. BM study in 4 decedents (case #9, 46, 65 and 69) showed as hypocellular, but not conclusive if they are appropriate for age or indicative of treatment-related changes. Nine severe hypocellularity cases were identified: two from group 2 (case #17 and 23), three from group 4 (case #29, 52 and 59), and four from the BM group (case #71, 73, 74 and 76). Three severe hypocellular bone marrow specimens (#17, 23, and 52) matched antemortem CM history. Subject # 36's (group 4, SOC) bone marrow, who had a history of leukocytopenia and thrombocytopenia, was hypercellular. There was no evidence of postmortem BM abnormality among decedents in groups 1 and 3.

Five patients (#29, 32, 38, 62, and 63) discontinued TMZ due to severe CM during CCRT. But CM in glioma patients did not develop permanent bone marrow damage as often as seen among BM decedents treated with chemotherapy. Patients exposed to extended TMZ (Figure 2C) or TIB regimens (Figure 2D) still had normal bone marrow.

Other clinical adverse events

The three most common AEs in group 4 were grade III HTN (37.5%), weight loss (29.7%), and venous thromboembolism (VTE, 24.3%). Grade III HTN (41.7%) was also the most common AE among patients who received TIB (groups 2 and 3 combined). The grade III CM was 33.3% and 21.6% among TIB and group 4, respectively. However, no grade IV thrombocytopenia was observed in the TIB group, while the rate was 13.5% in group 4. All AEs grade III or above are listed in Table 2.

Cause of death (COD) for recurrent/progressive glioma patients including GBM patients

Among the 68 glioma decedents, disease progression was the most common COD (N=43, 63.2%) (Table 2). The second most common COD was aspiration pneumonia (N=33, 48.5%) among glioma cases. Herniation with brain swelling was detected in 12 (17.6%) subjects, while cerebral or intraventricular hemorrhages were documented in 7 glioma cases. Uncommon CODs include decedent #67, who died at home from sudden death with autopsy finding of cardiomyopathy; descendants #26 and #76 expired from cerebral herniation soon after decompression surgeries. Three subjects (#52, 57, and 60) passed away in hospital due to severe adverse events while on active anti-cancer treatments.

Postmortem examination is the gold standard to identify end-organ toxicities from chemotherapies.²⁵ In this study, we documented clinical AEs and autopsy findings from 76 decedents, including 68 glioma patients with the majority of diagnosis as de novo GBM, to study the possible sequelae of TIB chemotherapy-related toxicity. Hypertension (41.7%) and combined CM (33.3%) were the two most common AEs among patients who received TIB treatment. In contrast, hypertension (32.1%), weight loss (29.7%), and VTE (24.3%) were the most common AEs among patients treated with SOC. We found that IRI, but not the extended TMZ cycles, significantly increased the likelihood of CM among rGBM patients. Long-term use of TIB (up to 25 cycles) did not cause unexpected toxicities or end-organ injuries among rGBM patients. To limit toxic drug effects from IRI, we recommend screening patients for the polymorphism of the UDP-glucuronosyl-transferase 1A1 (UGT1A1) locus¹⁷ and closely monitoring bone marrow function when an IRI-containing regimen is considered.

TMZ-induced myelosuppression is a known side effect and was reported predominantly in female patients.²⁶ Five (7.7%) decedents had to discontinue TMZ permanently after brief exposure to low daily doses during the CCRT period because of severe myelosuppression. Most oncologists discontinue TMZ for rGBM based on the assumption that TMZ will no longer be effective. However, 101²⁷ and 108²⁸ cycles of TMZ had been used to treat recurrent glioma patients without reports of severe myelosuppression. In the current study, up to 47 cycles of TMZ and 25 cycles of TIB were not associated with an increased risk of developing CM among recurrent glioma patients.

Bone marrow from 3 (#17, 23, and 52) glioma decedents demonstrated chemotherapy-related hypocellularity with documented clinical histories of CM. An analysis revealed that infusion with 1-6 (N=14) and 1-12 (N=21) IRI cycles increased the risk of CM by 7.67 (P=0.008) and 5.23 (P=0.018) times. CTEP-issued side effects from the TMZ/IRI combination include a 4-20% chance of developing into irreversible bone marrow damage with the observation that most of the CM events occurred soon after a new treatment drug was started. Genetic variants of the UGT1A1 gene and NADPH dehydrogenase quinone 1 might predispose susceptible patients to develop CM from IRI.^29,30,31 These genetic factors may explain why some patients developed CM soon after IRI was given, while other patients rarely experienced CM, even with prolonged usage. It is also likely that previous TMZ treatment as adjuvant therapy predisposes patients with an elevated risk for CM when IRI is added into salvage treatment. To reduce overlapping side effects on myelosuppression, TMZ dosage in the TIB regimen was set at 150 mg/M² and subjected to further reduction if a given patient could not tolerate the dosage.

According to forensic studies, fast morphological changes (autolysis, degenerative) at the microscopic and ultrastructural level have been reported in human³² and animal³³ kidneys within hours of death. Variable degrees of autolysis in kidney specimens (supplement Figure S1) prevented us from studying glomerular ultrastructural changes at autopsy. Meanwhile, ultrastructural changes such as unique hyaline occlusive glomerular microangiopathy were demonstrated through renal biopsy from patients actively treated with BEV.³⁴ There was no evidence of fibrin polymers or extensive loss of podocyte foot processes,³⁴ which was quite a difference from acute or chronic thrombotic microangiopathy cases.

The current study concluded that rGBM patients predominantly died directly from respiratory dysfunction, secondary to incapacitation from tumor progression (supplement Table 2). Death from brain herniation is not as common today as was reported in the 1990's²⁶ due to treatment advances, such as repeated debulking surgery, ventriculoperitoneal shunt placement, BEV, and steroid use.

This study has its limitations: 1) Toxicity to male and female reproductive organs was not evaluated; 2) The cohort was not selected by pre-specified criteria. Medical records review and AE/SAE assessment are retrospective analyses which may have introduced selection bias; 3) There were variable intervals from death to tissue collection 4) Personalized and targeted treatments in addition to TMZ, BEV, and/or IRI are potential confounding factors; 5) There may be patient selection bias since subjects received the TIB regimen dictated by normal or near-normal bone marrow function before TIB therapy.

In summary, the TIB regimen might have caused a higher rate of manageable CM than those seen from patients on SOC therapy. This difference is likely due to IRI involvement rather than the extended use of TMZ or BEV. There were no unexpected clinical adverse effects or organ-specific toxicities by postmortem examinations, even with the long-term use of TIB. We demonstrated that GBM disease progression and aspiration pneumonia are the most common CODs among glioma decedents. This result will allow physicians to plan patients’ treatment regimens and design clinical trials with BEV and BEV-based therapies without the fear of the high risk of end-organ damages, besides side effects clinically known.

Funding source: This study is partially funded by the Dr. Marnie Rose Foundation to J-J.Z.

Disclosure of potential conflicts of interest: The authors have no relevant financial or non-financial interests to disclose.

Research involving human participants: This study used postmortem human specimens and it was performed in line with the principles of the Declaration of Helsinki. It is approved by the Health Sciences Institutional Review Board (IRB) at Tufts Medical Center. The same study protocol was also approved by the Committee for the Protection of Human Subjects (CPHS) at the University of Texas Health Science Center at Houston (UTHealth) (HSC-MS-11-0133).

Informed consent: Consents for autopsy examination were obtained from all participants, either from patients when patients were alive or from the next of kin after subjects had deceased [full autopsies (N=69), restricted autopsies to the brain (N=6) and restricted to the brain and kidney (N=1) ].

Author Contributions: All authors contributed to the study. J-J. Zhu initiated the project and mobilized resources for the study. JK. Wu and J-J Zhu were investigators at two institutes. Autopsies and tissue collections were performeded or supervised by M. Buja, MB. Bhattacharjee, LY. Ballester, X. Tian, M. Pilichowska, GW. Hergenroeder, R. Zhang, and RL. Hunter. Ultrasound guided kidney biopsy in cadaver was performed by AK. Pillai. Slide review and pathology reports were issued by M. Buja, MB. Bhattacharjee, LY. Ballester, X. Tian, M. Pilichowska, RL. Hunter. WF. Glass and L. Chen. Clinial data collection and analysis were preformed by G. Lu, M. Rao, P. Zhu, N. Linendoll and J-J. Zhu. The first draft of the manuscript was written by G. Lu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Acknowledgment: We are very grateful to the pathology attending physicians and residents from both Tufts Medical Center and UTHealth McGovern Medical School/Memorial Hermann Hospital at Texas Medical Center, respectively. We appreciate the technical assistance of sample preparation for electron microscope examination by Ms. Patricia Navarro at the Pathology department of UTHealth. We are very thankful to Drs. Bihong Zhao and Joanna S. O'Leary at UTHealth for their editorial assistance with this manuscript.

Missing Data Availability statement

This manuscript has processed data included as electronic supplementary material. The datasets (raw data) generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Table 1

Summary of patient characteristics
Characteristics	BM	DMG	Grade II Glioma	Grade III Glioma	GBM
Number of patients at autopsy	8	3*	1	2	62**
Number of patients at initial diagnosis	8	2 (spine)	5	8	53
Age at diagnosis (median, range)	60 (39-75)	28 (23-33)	48 (31-56)	38.5 (27-76)	61 (25-80)
Gender (Male/Female)	5/3	0/2	4/1	5/3	35/18
Race
Caucasian	7	1	3	8	44
African American	0	0	0	0	4
Latino	1	0	1	0	0
Asian	0	1	0	0	2
Unspecified	0	0	1	0	3
One anaplastic glioma (grade III) and 2 spine glioma cases were confirmed to be diffuse midline gliomas (DMG) at autopsy based on anatomic location and histone H3F3A K27M mutation. *Nine cases were confirmed of progression to secondary GBMs at autopsy.

Table 2

Occurrence of adverse events (grade III and above)
	BM (N=8)	Recurrent gliomas, treatment based (N=68)
	BM (N=8)	NCBC (N=7)	1-5 TIB (N=12)	6-25 TIB (N=12)	SOC (N=37)
Groups		1	2	3	4
Treatments
TMZ/BEV/IRI	NA	0	12	12	0
TMZ first, then BEV	NA	0			18
TMZ	1^£	0			6
BEV	NA	0			1
BEV/IRI	NA	0			5
NCBC	NA	7
SOC chemo for primary disease	8	NA	NA	NA	NA
Median TMZ cycle, cycles (range)	NA		12.5 (2-47)	20 (7-40)	3 (0-30)
Median BEV, Infusions (range)	NA		10 (2-13)	30 (12-59)	8 (0-49)
Median IRI, Infusions (range)	NA		4 (1-11)	23 (12-52)	0 (0-19)
Adverse Events
Body weight Change (%)	-3.9±7.7	-2.0±7.7	-8.6±4.1	-3.3± 5.1	-19.6±2.4
Grade III Weight loss N, (%)	1 (12.5%)		1 (8.3%)	1 (8.3%)	11 (29.7%)
Grade III Weight gain N, (%)	1 (12.5%)	1 (14.3%)		2 (16.7%)	1 (2.7%)
Seizure, grade III		1^# (14.3%)		1 (8.3%)	4 (10.8%)
Grade III, hypertension			5 (41.7%)	5 (41.7%)	12 (37.5%)
Grade III, Proteinuria			1 (8.3%)	2 (16.7%)	2 (5.4%)
Grade III, Anemia	1 (12.5%)	1 (14.3%)	1 (8.3%)		4 (10.8%)
Grade III, thrombocytopenia		1 (14.3%)	2 (16.7%)	2 (16.7%)	2 (5.4%)
Grade III, neutropenia			3 (25%)		2 (5.4%)
Grade IV, hypertension					1 (2.7%)
Grade IV, thrombocytopenia	1 (12.5%)	1 (14.3%)			5 (13.5%)
Grade IV, neutropenia	1 (12.5%)				3 (8.1%)
Grade III, VTE	1 (12.5%)		2 (16.7%)	1 (8.3%)	9 (24.3%)
Grade V, Perforated diverticulitis					1 (2.7%)
Grade III, Kidney failure		1 (14.3%)
Grade IV, Kidney failure		1 (14.3%)			1 (2.7%)
Abnormal liver enzymes, grade IV			1 (8.3%)		1 (2.7%)
CK and LDH elevation, grade III					1* (2.7%)
Abnormal electrolytes, grade III.		1 (14.3%)		1 (8.3%)	2 (5.4%)
Meningitis, grade III				1 (8.3%)
PCP, grade IV					1 (2.7%)
CMV pneumonitis, grade III	1 ^Ω (12.5%)
Shingles, grade III		1 (14.3%)			1 (2.7%)
Cellulitis, grade III		1 (14.3%)			1 (2.7%)
^# Pseudo-seizure, chorea history from Huntington’s disease. *Subject participated in clinical trial and study drug is known to cause creatine kinase (CK) and lactate dehydrogenase (LDH) elevation. ^£Patient #72 received low dose TMZ for 42 days consecutively. PCP: Pneumocystis carinii pneumonia.

Table 3

Risk of myelosuppression rate in relation to varied chemotherapy modalities*.
	Myelosuppression rate
Predictors	Total	Myelo- suppression	OR	95%CI	P
Standard care vs. TIB, N=61
Standard care	37	10	1.00	-	-
TIB	24	7	1.11	0.36 - 3.48	0.856
Standard care vs. TIB (cutoff: 6), N=61
Standard care	37	10	1.00	-	-
TIB: <6	12	5	1.93	0.50 - 7.50	0.343
TIB: >=6	12	2	0.54	0.10 - 2.91	0.473
TMZ cycles: continuous, N=58
TMZ cycles	58	15	0.97	0.91 - 1.03	0.312
TMZ cycles: cutoff - 6, 12, 18, 24; N=58
1-6	28	8	1.00	-	-
7-12	10	4	1.67	0.37 - 7.53	0.507
13-18	8	1	0.36	0.04 - 3.39	0.370
19-24	4	1	0.83	0.08 - 9.25	0.882
25-47	8	1	0.36	0.04 - 3.39	0.370
TMZ cycles: cutoff, 12; N=58
<12	35	11	1.00	-	-
>=12	23	4	0.46	0.13 - 1.67	0.238
TIB cycles: cutoff, 6; N=24
<6	12	5	1.00	-	-
>=6	12	2	0.28	0.04 - 1.88	0.190
IRI cycles: cutoff, 0; N=61
0	27	4	1.00	-	-
>0	34	13	3.56	1.00 - 12.64	0.050
IRI cycles: cutoff, 0, 6; N=61
0	27	4	1.00	-	-
<=6	14	8	7.67	1.71 - 34.33	0.008
>6	20	5	1.92	0.44 - 8.31	0.385
IRI cycles: cutoff, 0, 12: N=61
0	27	4	1.00	-	-
<=12	21	10	5.23	1.34 - 20.45	0.018
>12	13	3	1.72	0.32 - 9.17	0.523
Abbreviation: OR, odds ratio; 95%CI, 95% confidence interval; TIB, Temozolomide+ Irinotecan+Bevacizumab; TMZ, Temozolomide; IRI, Irinotecan. * Univariable binary logistic regression models.

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Postmortem Study of Organ-Specific Toxicity in Glioblastoma Patients Treated with a Combination of Temozolomide, Irinotecan and Bevacizumab

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