Direct nuclear reprogramming has the potential to enable the development of
β
cell replacement therapies for diabetes that do not require the use of progenitor/stem cell populations. However, despite ...their promise, current approaches to
β
cell‐directed reprogramming rely heavily on the use of viral vectors. Herein, the use of extracellular vesicles (EVs) derived from human dermal fibroblasts (HDFs) is explored as novel nonviral carriers of endocrine cell‐patterning transcription factors, to transfect and transdifferentiate pancreatic ductal epithelial cells (PDCs) into hormone‐expressing cells. Electrotransfection of HDFs with expression plasmids for Pdx1, Ngn3, and MafA (PNM) leads to the release of EVs loaded with PNM at the gene, mRNA, and protein levels. Exposing PDC cultures to PNM‐loaded EVs leads to successful transfection and increases PNM expression in PDCs, which ultimately result in endocrine cell‐directed conversions based on the expression of insulin/c‐peptide, glucagon, and glucose transporter 2 (Glut2). These findings are further corroborated in vivo in a mouse model following intraductal injection of PNM‐ versus sham‐loaded EVs. Collectively, these findings suggest that dermal fibroblast‐derived EVs can potentially serve as a powerful platform technology for the development and deployment of nonviral reprogramming‐based cell therapies for insulin‐dependent diabetes.
Hepatic parenchymal disease, including chronic viral hepatitis, has traditionally been considered a relative contraindication to islet transplantation as the islets are infused into the recipient’s ...liver. We present a case study of a patient with treated chronic hepatitis C infection (HCV) who safely received an autologous islet transplant following total pancreatectomy with excellent clinical outcomes. The patient was a 60-year-old woman diagnosed with debilitating abdominal pain secondary to chronic pancreatitis and with preserved islet function. She had previously been treated >10 years prior to surgical evaluation with interferon monotherapy for 1 year that led to sustained virologic response, including at the time of surgical evaluation for total pancreatectomy and islet autotransplantation (TPIAT). She underwent comprehensive preoperative evaluation of the liver, including liver biopsy, which showed no significant portal inflammation or fibrosis. Following a multidisciplinary meeting and discussion of the potential risks for the patient, the decision was made to proceed with TPIAT. The patient underwent a standard total pancreatectomy, and an autologous islet dose of 6638 islet equivalents/kg body weight was infused into the liver via the portal vein. Portal vein pressure was monitored throughout the infusion with a transient peak pressure of 27 cm H2O (basal pressure of 14 cm H2O) and final pressure of 23 cm H20 at 10 min post-infusion. Aside from a transient transaminitis, liver enzymes were normal at the time of hospital discharge. At greater than 1 year of follow-up, the patient has improved quality of life, with reduction in narcotic analgesia, remains insulin independent (with normal islet function), and has normal liver function. This case illustrates that islet autotransplant into the liver can be safely performed and suggests that carefully selected patients with liver disease may be eligible for TPIAT.
Chronic pancreatitis (CP) is a progressive disease that leads to eventual loss of endocrine and exocrine function. Total pancreatectomy and islet autotransplantation (TPIAT) is a treatment option for ...patients with CP; however, predicting postoperative metabolic outcomes remains elusive. In this single-center retrospective study, we report pre-TPIAT characteristics, beta cell function indices, islet yield, and post-TPIAT glucose management data to further understand their relationship. Islet yield, glucose level, and insulin requirement for 72 hours postoperatively were collected for a total of 13 TPIAT recipients between 9-2013 and 9-2018. In addition, their glucose control and basal insulin requirements at 3, 6, and 12 months post-TPIAT were analyzed. All 13 subjects had normal baseline fasting glucose levels. Median islet yield was 4882 IEq/kg (interquartile range 3412 to 8987). Median postoperative total insulin requirement on day 3 was 0.43 units/kg. Pre-TPIAT baseline glucose, insulin, or c-peptide level did not have a significant correlation with the islet yield. Similarly, there was no correlation between islet yield and insulin requirement at 72-hour postoperatively. However, there was an inverse correlation between the absolute islet yield (IEq) and insulin requirement at 6 months and 12 months following post-TPIAT. Further analysis of the relationship between 72-hour post-op insulin requirement and insulin requirement at discharge, 3, 6, and 12 months showed a positive correlation. Despite the finding of inverse correlation of islet yield with long-term basal insulin requirement, this study was not able to detect a correlation between the preoperative parameters to postoperative short-term or long-term outcome as noted in other studies. The 72-hour postoperative insulin requirement is a helpful postoperative predictor of patients needing long-term insulin management following TPIAT. This observation may identify a high-risk group of patients in need of more intensive diabetes education and insulin treatment prior to hospital discharge.
Islet transplantation continues to be a promising treatment for type 1 diabetes, however, numerous limitations still prevent its widespread use. Many immunosuppressive medications used for islet ...transplantation are known to be diabetogenic. The goal of this study is to evaluate the short-term follow-up (90 day) of a steroid-free maintenance immunosuppression protocol of mycophenolate mofetil (MMF) and cyclosporine (CSA) in non-human primate islet allotransplantation.
Diabetes was induced in the primate Macaca fascicularis via total pancreatectomy. Freshly isolated islets were autotransplanted (n=5) or allotransplanted (n=5) into the portal vein. Immunosuppression consisted of induction with rabbit anti-thymocyte globulin (ATG) and prednisone. CSA (25 mg/daily) and MMF (250 mg daily) were used for maintenance and given orally once daily throughout the 90 day study period. Fasting blood glucose measurements were used to monitor graft function. Intravenous glucose tolerance tests (IVGTT) were performed prior to pancreatectomy and at the study endpoint.
Average fasting blood glucose levels were elevated in diabetic controls (313.6 mg/dl ±51.8) and untreated allograft recipients (257 mg/dl ±61.0) post-transplant. Auto-transplanted animals and allograft recipients treated with immunosuppression, on the other hand, maintained normoglycemia (74.5 mg/dl ±20.9, 62.3 mg/dl ±4.40) throughout the follow-up period. Additionally, beta cell function and first phase insulin secretion did not differ significantly between auto-graft and immunosuppressed allo-graft recipients post-transplant.
We conclude that this steroid-free maintenance immunosuppression regimen is effective in maintaining islet allograft survival in the non-human primate and offers comparable graft function to that of autografts for up to 3 months post-transplant.
To date, the portal vein has been the primary site for clinical islet transplantation. Despite success, potential complications such as portal vein thrombosis still exist. The kidney subcapsule has ...been used successfully in rodent models of islet transplantation. We hypothesized that the kidney subcapsule as a site for islet transplantation in the nonhuman primate model would be as effective as the portal vein. Diabetes was induced in the primate Macaca fascicularis via a total pancreatectomy. Animals were kept under anesthesia during the isolation procedure. Islet isolation was performed using intraductal infusion with Liberase™ HI and mechanical digestion in the Ricordi chamber, and were purified using a continuous Ficoll gradient. Purified islets were autotransplanted either into the portal vein (n = 6) or the left kidney subcapsule (n = 5) of pancreatectomized animals. Intravenous glucose tolerance tests were performed prior to pancreatectomy and 10 days following transplantation. Three animals underwent pancreatectomy and served as diabetic controls. Of the six animals receiving islets in the portal vein, one developed portal vein thrombosis. All remaining autotransplanted animals in this group remained normoglycemic with glucose-induced insulin secretion that was not different from that prior to pancreatectomy. Of the five animals undergoing transplantation into the kidney subcapsule, only one maintained normoglycemia and elicited insulin secretion in response to glucose stimulation. The other four animals remained hyperglycemic. We conclude that the portal vein is superior to the kidney subcapsule as a site for islet transplantation in nonhuman primates 10 days posttransplantation.
The GLP-1 receptor agonist, exenatide, has previously been shown to improve insulin secretion, protect beta cells from apoptosis, and promote beta cell regeneration. We propose that pretreatment with ...exenatide will promote islet graft survival and improve graft function. Pancreatectomized cynomolgus monkeys underwent islet allotransplantation and were treated with exenatide beginning on day 0 or day −2. A third group of animals was treated with an immunosuppressive regimen while a fourth group remained untreated. Fasting blood glucose (FBG) was used to evaluate graft function along with intravenous glucose tolerance tests (IVGTTs) performed at study endpoint (day 10 for untreated and posttransplant exenatide or day 90 for pretreatment exenatide and immunosuppression). The average FBG for pre-treated animals day 5 following transplant was 52.7±14.8 mg/dl, compared to 154.3±105.5 mg/dl for animals treated only following transplant, 59.4 mg/dl ±12.1 for animals treated with immunosuppression, and 265.5±172.3 mg/dl for untreated animals. IVGTTs performed at study endpoint showed normal glucose and insulin curves in the pre-treated exenatide and immunosuppression groups only, with beta cell function actually improving after transplant in the pre-treated group. We conclude, therefore, that exenatide pre-treatment can successfully maintain islet graft survival in nonhuman primates.
Background: The discovery of IDH1 and IDH2 mutations in AML, and the accompanying functional implications of the resulting neomorphic activity of these mutated enzymes, has resulted in FDA approved ...targeted therapies. Similarly, the changes brought about in methylation due to IDH mutations in the AML genome have ushered in treatment regimens of venetoclax with hypomethylating agents. In this setting, where traditional chemotherapy regimens also are applied for treatment of primary diagnoses, the available outcomes data for all three therapeutic approaches should now provide clear metrics of predicted response. However, given a lack of direct comparison studies, no guidance exists as to which treatment choice will provide best response in IDH-mutated patients. Furthermore, emerging data about the importance of the biologic context on the response to different agents, including co-existing gene mutations and patient age, pose additional questions that need to be systematically addressed in order to provide patients with the best treatment.
Study Design and Methods: This is a randomized, multi-site Phase 2 study (NCT05401097) comparing overall treatment failure rates at 24 months between two sequences of first-line and second-line therapies in patients ≥ 18 years with newly diagnosed IDH1/2 mutated AML who are not candidates for intensive induction therapy (Figure 1). Patients are randomized into one of the two arms in a 1:1 ratio with stratification based on the type of IDH mutation. Randomized patients are then assigned to receive treatment sequence A (IDHi+Aza followed by ven+aza) or treatment sequence B (ven+aza followed by IDHi+aza). Five patients have been randomized and enrolled on study to date.
Patients randomized to first-line IDHi will receive Ivosidenib 500mg po orally daily on Days 1-28 ( IDH1) or Enasidenib 100mg po orally daily on Days 1-28 ( IDH2), each of 28-day cycle. Aza will be given at 75mg/m 2 daily on days 1-7 or days 1-5/8-9 of each 28-day cycle. Patients can receive a maximum of 5 cycles to attain CR/CRh/CRi. Patients who achieve CR/CRh/CRi will continue IDHi+aza until unacceptable toxicity, withdrawal of consent for further protocol treatment, relapse, or death (henceforth referred to as 'cessation'). If a patient does not meet these parameters, progresses during the first 5 cycles, does not attain CR/CRh/CRi by the end of 5 cycles, attains CR/CRh/CRi by the end of 5 cycles and subsequently relapses, or has unacceptable toxicity, then the patient will stop therapy with IDHi+aza. For patients on second line Ven+aza, they may receive a maximum of 3 cycle of Ven+aza to achieve CR/CRh/CRi. Patients who achieve CR/CRh/CRi will continue Ven+aza until 'cessation.' If a patient progresses during the first 3 cycles, does not attain CR/CRh/CRi by the end of 3 cycles, attains CR/CRh/CRi by the end of 3 cycles and subsequently relapses, or has unacceptable toxicity, they will then come off study treatment.
Patients randomized to first-line Ven+aza will receive ven dosing per the FDA-label. Aza will be given at 75mg/m 2 daily on days 1-7 of each 28-day cycle. Patients can receive a maximum of 3 cycles to attain CR/CRh/CRi. Patients who achieve CR/CRh/CRi will continue Ven+aza therapy until 'cessation.' If a patient progresses during the first 3 cycles, does not attain CR/CRh/CRi by the end of 3 cycles, attains CR/CRh/CRi by the end of 3 cycles and subsequently relapses, or has unacceptable toxicity, then the patient will stop therapy with the Ven+aza. For patients on second line IDHi+aza, they may receive a maximum of 5 cycle of IDHi+aza to achieve CR/CRh/CRi. Patients who achieve CR/CRh/CRi will continue IDHi+aza until 'cessation.' If a patient progresses during the first 5 cycles, does not attain CR/CRh/CRi by the end of 5 cycles, attains CR/CRh/CRi by the end of 5 cycles and subsequently relapses, or has unacceptable toxicity, they will then come off study treatment. Longitudinally embedded, comprehensive correlative studies will provide integrated genomic profiling of the leukemic clone during different disease stages and assess for potential resistance mutations or clonal evolution that may be predictors of relapse in this difficult to treat patient population. In addition, ultra-sensitive measurable-residual disease testing (ctDNA) and single-cell based combined transcriptional and mutational profiling will complement the detailed assessment.
Background: AML is a highly aggressive hematologic malignancy, but the AYA pts aged 18-39 years (y) have improved survival, with ~50% of pts achieving long-term cure. Pt outcomes are known to be ...affected by disease-related factors including cytogenetic findings and gene mutations, which were established in large genomic studies. In addition, pt-related factors, such as self-reported race and socioeconomic contributors have been found to affect survival in population-based studies. Whereas population-based studies are performed on large pt cohorts to establish meaningful conclusions, they usually lack details with respect to additional pt-related factors, including complete comorbidities, insurance status as well as information on disease presentation, such as time from the first provider contact to confirmation of AML diagnosis (time to diagnosis) and treatment, which might also influence pt survival.
Rationale and Methods: Thus, we set out to establish recent survival rates of AML AYA pts at the population level using pts reported to one of 12 Surveillance Epidemiology End Results (SEER) Program registries of the National Cancer Institute n=1119; median age, 30 y,1992-2014; n=239; median age, 29 y, 2015-2020, and analyze possible factors influencing survival. In parallel, we performed an in-depth analysis of treatment response and survival, including time to diagnosis, and additional pt-associated variables pertinent to AYA pts (employment status, housing, insurance, comorbidities, distance to treatment center, research study enrollment, biobanking) based on retrospectively collected data of AYA AML pts cared for at a major US Comprehensive Cancer Center (CCC) in 2015-2020 (n=96, median age, 31 y), for whom detailed clinical information was available.
Results: Generally, overall survival (OS) of AYA AML pts significantly improved over time, with a marked improvement of 3-y OS rates from 47% in pts diagnosed prior to 2009, to 63% in pts diagnosed in 2010-2014 and 69% in pts diagnosed in 2015-2020. Analysis of possible associations between demographic parameters and risk of death among AYA AML pts reported to SEER Program registries did not show any statistically significant impact of sex, age, county-level median household income or metro/rural status. The only factor affecting OS of AYA AML pts was race. Specifically, Black AML pts had a higher risk of death compared with that of White AML pts HR 2.13 (95% CI 1.03 - 4.39), with 3-y OS rates of 47% and 80% ( P=.04), respectively. Survival disparities did not improve over time. CCC AYA AML pts had similar OS rates to those seen in the population-based analyses, with a 3-y OS rate of 70%. Ninety-three percent of pts achieved a complete remission, and 31% of pts relapsed. Analyses of CCC AML pts allowed for preliminary assessment of additional parameters. Ninety-one percent of pts (77/85) received a diagnosis in ≤2 weeks after first provider contact. Although pt numbers were small, there was a trend for a longer diagnosis time in Black pts, 27% (3/11) of whom were diagnosed >2 weeks after first presentation, compared with only 8% (5/61; P=.10) of White pts. Interestingly, analysis of possible additional outcome modulators in CCC AYA pts suggested that presence of other medical conditions at diagnosis associated with lower relapse rates (16% vs 34%, P=.04), and unemployment tended to associate with higher relapse rates (34% vs 17%, P=.06). In addition, of pts approached for biobanking, fewer Black pts consented to sample collection than White pts (60% vs 87%, P=.06).
Conclusion: Survival of AYA pts with AML significantly improved over time, likely driven by availability of allogeneic stem cell transplantation and improved supportive care. Black race still represents the most important negative survival prognosticator, without marked signs of improvement. An exploratory analyses of additional pt-associated variables suggested less recognized factors that may impact survival, such as trends for longer time to diagnosis and reduced participation in biobanking protocols in Black pts compared with White pts, and a higher relapse rate in pts who are unemployed. Although these differences were not yet statistically significant , we are currently in the process of increasing the sample size to further evaluate these potentially important trends.
Support: American Cancer Society, Leukemia Lymphoma Society
INTRODUCTION:
Pancreas enzyme replacement therapy (PERT) is used to improve nutrient digestion, especially of lipids, in patients with exocrine pancreas insufficiency who otherwise may experience ...maldigestion, malabsorption, malnutrition, and steatorrhea. Although underdosage of PERT is common, adequate dosing is particularly important in patients who undergo total pancreatectomy and islet autotransplantation (TPIAT). Current PERT dosing guidelines do not explicitly provide recommendations following TPIAT so we determined the adequacy of PERT dosing in a cohort of patients with TPIAT.
METHODS:
The TPIAT database from 2009-2018 was searched to determine PERT dosing in subjects before TPIAT (n = 14), immediately after TPIAT and at 6 and 12 months (n = 11). Adequate weight based dosing was established using Cystic Fibrosis Foundation guidelines. Average PERT dose was calculated and the ranges were reported,
P
< 0.05 was significant.
RESULTS:
Data was available in 14 patients, and 6 and 12 month data was available in 11 patients. Indication for TPIAT included 6 idiopathic chronic pancreatitis (CP), 4 genetic CP (4 PRSS1, 1 CFTR), 3 idiopathic recurrent AP, 1 alcohol induced CP. 3 patients were not using PERT before TPIAT, 7 were underdosed (average 62,000 U/day, range 6,000–108,000), and 4 were adequate (205,250 U/day; 125,000–228,000). At discharge 9 patients were underdosed (72,222 U/day; 6000–108,000), and 5 were adequate (248,200 U/day; 125,000–360,000). At 6 months 3/11 pts (27%) were underdosed (56,666 U/day; 30,000–80,000) and 8/11 pts (73%) were adequate (228,000 U/day; 144,000–432,000). At 12 months 2/11 pts (18%) were underdosed (108,000 U/day each) and 9/11 (82%) pts were adequate (430,222 U/day; 144,000–384,000). PERT dose increase was significant for the period post-TPIAT to 6 months (
P
= 0.04). The graph shows the trend of PERT during the study period.
CONCLUSION:
Underdosing of PERT was common before and immediately after TPIAT. It is possible that poor oral intake dictated the immediate low post TPIAT dose. Significant PERT dose increase was noted in the first 6 months, and was sustained at 12 months. PERT dosing needs to be monitored closely and weight based dosing should be considered to achieve optimal nutritional benefit.
Background
The gene upstream binding transcription factor ( UBTF ) encodesa nucleolar protein that is a key regulator of the epigenetic status of ribosomal DNA and RNA transcription. Tandem ...duplications (TDs) of the UBTF gene have been initially identified in pediatric patients (pts) with acute myeloid leukemia (AML) and were recently also reported as relatively rare, but recurrent genetic events in adult AML based on targeted re-sequencing efforts (1.2%-3%). Their presence was associated with dysplastic bone marrow features, and co-occurrence of select genetic features, including FLT3-internal tandem duplications (ITDs) and WT1 mutations, and inferior outcome. However, associated biologic features and affected pathways have not yet been thoroughly investigated, and may provide further insights into leukemogenesis and identify potentially targetable vulnerabilities. Thus, we set out to assess the frequency of UBTF-TDs in a large cohort of adult AML pts (n=807) treated on frontline protocols of the Alliance for Clinical Trials in Oncology with complete RNA sequencing data, comprehensive targeted mutation profiling, centrally reviewed cytogenetic data and clinical information available, and to gain further insights into UBTF-TDs via comprehensive integrated genomic profiling.
Methods
UBTF-TD detection was performed via the RNAseq-based RNAmut and EnFusion algorithm. To gain insights into associated molecular features, all UBTF-TD + pts underwent paired tumor-normal whole-exome sequencing. To understand associated pathways and downstream effects, differential gene expression (DGE) and gene set enrichment analyses (GSEA) were performed.
Results
UBTF-TDs were detected in 6/807 pts (0.7%). In line with previous studies, these 6 UBTF-TD + pts were young, with a median age of 35y (range: 19-59y). Three pts had extramedullary disease, 2 with skin and 1 with gingival involvement. All but 1 of the 6 pts were cytogenetically normal. Clinically, pts did poorly after treatment with intensive chemotherapy induction, with a CR rate of only 33%. No pt was alive 2.5 y after diagnosis.
In paired tumor/normal exome sequencing, UBTF-TD + pts had a median mutation burden of 21 non-synonymous variants in coding genes (range: 17-27), with a notable presence of larger insertions/deletions (indels), loss of heterozygosity (LOH) or TDs as co-occurring somatic genetic lesions (median: 4, range: 2-5). There was a 100% co-occurrence of UBTF-TDs with FLT3-ITDs. In addition, pts harbored larger indels in WT1 (n=2), BPTF, CEBPA, Corf53, KRTAP5-7, NFE2, PTPRN2, RNF144B, SOWAHC and ZCCHC14. Also, analysis of copy-number variations and LOH revealed LOH in 5 pts, in chromosomes 13 (n=3), 15 (n=1) and 18 (n=1), suggestive of a propensity to the acquisition of structural variants.
In unsupervised clustering analysis of the RNAseq data, UBTF-TD + pts clustered with FLT3-ITD + pts but formed a relatively distinct subcluster in principal component and t-distributed stochastic neighbor embedding analyses. In supervised clustering comparing 6 UBTF-TD +/ FLT3-ITD + and 196 UBTF-TD -/ FLT3-ITD + pts, 2 UBTF-TD - pts clustered together with the UBTF-TD +/ FLT3-ITD + group, suggesting either presence of an undetected TD or genetic phenocopy. UBTF expression was higher in UBTF-TD pts. DGE analysis of UBTF-TD +/ FLT3-ITD + and UBTF-TD -/ FLT3-ITD + pts (Fig 1) showed distinct upregulation of the locus of the stem cell and chemoresistance marker ALDH1A3, including the associated long non-coding RNAs AC015712.7/AC015712.2 in UBTF-TD + pts, which was confirmed by a comparison of those pts to AML pts who had neither TD (n=605). GSEA indicated a downregulation of inflammation pathways and several metabolic pathways of UBTF-TD +/ FLT3-ITD + compared to FLT3-ITD + pts (Fig 2).
Conclusions
UBTF-TDs are rare, but recurrent genetic events in adult AML, associated with high chemotherapy resistance. Our results highlight a susceptibility of those pts to the acquisition of structural variants and suggest a distinct associated biology, with upregulation of ALDH1A3 as possible contributing resistance mechanism.
Support: U10CA180821, U10CA180882, U24CA196171; Clinicaltrials.gov Identifiers: NCT00048958 (CALGB 8461), NCT00899223 (CALGB 9665), NCT00900224 (CALGB 20202); https://acknowledgments.alliancefound.org
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