This cohort study examines the long-term risk of developing alcohol abuse or dependence among a predominantly male bariatric population who underwent either a laparoscopic sleeve gastrectomy or a Roux-en-Y gastric bypass. Is a bariatric surgical procedure associated with unhealthy alcohol use up to 8 years after the operation in patients with or without baseline unhealthy alcohol use?In this cohort study of 2608 US veterans who underwent a bariatric surgical procedure, the mean alcohol screening scores in patients without baseline unhealthy alcohol use and the probability of unhealthy alcohol use both increased significantly 3 to 8 years after the procedure, and the probability of no alcohol use decreased significantly 5 to 8 years after the procedure. In a small sample of patients with unhealthy alcohol use at baseline, the prevalence of postoperative unhealthy alcohol use was higher for patients undergoing gastric bypass than controls.Findings of this study suggest that unhealthy alcohol use was much more common after undergoing a bariatric surgical procedure in patients without unhealthy alcohol use at baseline compared with those who did not undergo a procedure. Bariatric surgical procedures have been associated with increased risk of unhealthy alcohol use, but no previous research has evaluated the long-term alcohol-related risks after laparoscopic sleeve gastrectomy (LSG), currently the most used bariatric procedure. No US-based study has compared long-term alcohol-related outcomes between patients who have undergone Roux-en-Y gastric bypass (RYGB) and those who have not.To evaluate the changes over time in alcohol use and unhealthy alcohol use from 2 years before to 8 years after a bariatric surgical procedure among individuals with or without preoperative unhealthy alcohol use.This retrospective cohort study analyzed electronic health record (EHR) data on military veterans who underwent a bariatric surgical procedure at any of the bariatric centers in the US Department of Veterans Affairs (VA) health system between October 1, 2008, and September 30, 2016. Surgical patients without unhealthy alcohol use at baseline were matched using sequential stratification to nonsurgical control patients without unhealthy alcohol use at baseline, and surgical patients with unhealthy alcohol use at baseline were matched to nonsurgical patients with unhealthy alcohol use at baseline. Data were analyzed in February 2020.LSG (n = 1684) and RYGB (n = 924).Mean alcohol use, unhealthy alcohol use, and no alcohol use were estimated using scores from the validated 3-item Alcohol Use Disorders Identification Test-Consumption (AUDIT-C), which had been documented in the VA EHR. Alcohol outcomes were estimated with mixed-effects models.A total of 2608 surgical patients were included in the final cohort  age of surgical patients was 53.0 (9.9) years and 53.6 (9.9) years for the matched nonsurgical patients. Among patients without baseline unhealthy alcohol use, 1539 patients who underwent an LSG were matched to 14 555 nonsurgical control patients and 854 patients who underwent an RYGB were matched to 8038 nonsurgical control patients. In patients without baseline unhealthy alcohol use, the mean AUDIT-C scores and the probability of unhealthy alcohol use both increased significantly 3 to 8 years after an LSG or an RYGB, compared with control patients. Eight years after an LSG, the probability of unhealthy alcohol use was higher in surgical vs control patients ). Similarly, 8 years after an RYGB, the probability of unhealthy alcohol use was higher in surgical vs control patients . The probability of no alcohol use also decreased significantly 5 to 8 years after both procedures for surgical vs control patients. Among patients with unhealthy alcohol use at baseline, prevalence of unhealthy alcohol use was higher for patients who underwent an RYGB than matched controls.In this multi-site cohort study of predominantly male patients, among those who did not have unhealthy alcohol use in the 2 years before bariatric surgery, the probability of developing unhealthy alcohol use increased significantly 3-8 years after bariatric procedures compared with matched controls during follow-up. Unhealthy alcohol use spans a spectrum from drinking at levels that increase the risk of health consequences to severe alcohol use disorders, which are associated with extensive disability and death.10Nearly 2 in 5 adults in the United States have obesity.13 and uncontrolled cohort studies.16 The only studies that included nonsurgical control patients suggested that Roux-en-Y gastric bypass (RYGB) was associated with increased alcohol use, unhealthy alcohol use, and alcohol use disorder.17 However, these studies in Swedish populations did not include the most common bariatric surgical procedure performed in the US, laparoscopic sleeve gastrectomy (LSG). No US-based study has compared the alcohol-related risks in patients who underwent an RYGB with the risks in nonsurgical control patients. Furthermore, most patients in past studies were women. Understanding alcohol-related risks associated with bariatric surgical procedures in men, who have higher rates of unhealthy alcohol use and alcohol use disorder, is also important.18To date, most research on alcohol-related risks after a bariatric surgical procedure has been limited to laboratory studies21 producing electronic health record (EHR) data for monitoring alcohol use and unhealthy alcohol use over time.24 In this study, we leveraged these unique population-based data on VA patients who underwent an LSG or an RYGB to evaluate the changes over time in alcohol use and unhealthy alcohol use from 2 years before to 8 years after a bariatric surgical procedure among those with or without preoperative unhealthy alcohol use.The present study compared alcohol-related outcomes between patients who underwent an LSG or an RYGB and nonsurgical control patients in the US Department of Veterans Affairs (VA) health system. Annually since 2004, the VA has required screening of all outpatients using the validated 3-item Alcohol Use Disorders Identification Test-Consumption (AUDIT-C),STROBE) reporting guideline.In this retrospective cohort study, we identified 10 653 veterans who underwent a bariatric surgical procedure at any VA bariatric center between October 1, 2000, and September 30, 2016. Patients who underwent reoperations, lacked documented obesity, had cancer or other medical exclusions, received uncommon bariatric procedures, or had an LSG or an RYGB between October 1, 2000, and September 30, 2008 . Race was dichotomized due to the small numbers of Asian, American Indian, and other non-Black racial groups included. Race was determined from VA medical record. Surgical patients and potential control patients were required to be within 5 years of age, have similar BMI measurements  within 6 months before the surgical date, and have similar alcohol screening scores.20 Surgical patients without a representative match were excluded .Control patients often had many BMI measurements and could be matched to multiple surgical patients. The final control cohort included 22 284 individual patients who represented 24 232 matches. Matching was not contingent on future information; thus, 225 nonsurgical control patients (representing 247 matches) who later underwent an LSG or an RYGB contributed person-time to the control group until their operation and were then censored at their surgical date.baseline hereafter). Among patients without unhealthy alcohol use at baseline, we matched 1539 patients who underwent an LSG to 14 555 nonsurgical control patients and 854 patients who underwent an RYGB to 8038 nonsurgical control patients. Among those with unhealthy alcohol use at baseline, we matched 145 patients who underwent an LSG to 1091 nonsurgical control patients and 70 patients who underwent an RYGB to 548 nonsurgical control patients.The final analytical cohort was stratified according to whether patients screened positive for unhealthy alcohol use on their highest AUDIT-C result in the 2 years preceding their surgical date  and validated in diverse populations.20 Patients who scored 0 on the AUDIT-C self-reported no alcohol use in the past year.AUDIT-C scores range from 0 to 12 and are a successful proxy measure for average alcohol consumption.36 Among patients with and without baseline unhealthy alcohol use, separate analyses were conducted for patients who underwent an LSG and patients who underwent an RYGB (pooling open and laparoscopic procedures).Covariate balance at baseline between surgical patients and nonsurgical control patients was evaluated using standardized mean differences (SMD).In main analyses, mean AUDIT-C scores over time were modeled. Preplanned secondary analyses evaluated changes over time in the probability of unhealthy alcohol use. In post hoc secondary analyses, the probability of no alcohol use was modeled to evaluate whether changes in mean AUDIT-C score over time were influenced by changes in the proportion of patients who did not drink alcohol. Models for mean alcohol consumption and no drinking used all available AUDIT-C data from the VA EHR system from 2 years before to 8 years after the surgical procedure. Models for unhealthy alcohol use used only postbaseline values.37 Patients were censored at death, 8 years after the surgical procedure, or the end of the study period , whichever occurred first; control patients were also censored when a bariatric procedure was initiated, if applicable.Mean alcohol use was estimated with a linear mixed model, whereas the proportions of patients with unhealthy alcohol use and of those with no drinking were estimated with logistic mixed models; all models included a patient-level random intercept. After specification testing for the best-fitting mean trajectory, mean alcohol use and no drinking models included prebaseline and postbaseline quadratic time terms, allowing for separate quadratic trajectories before and after baseline. An interaction between each time term and a surgical indicator was also added to assess whether presurgical and postsurgical outcomes differed over time between surgical patients and controls. For mean alcohol use and no drinking analyses, the estimated mean outcomes or estimated proportions (as applicable) were generated at 2 years before (–2 years) and at 0, 1, 3, 5, and 8 years after the bariatric surgical procedure. The unhealthy alcohol use analysis similarly included quadratic time terms and the interaction with the surgical indicator for postsurgical trajectories. Estimated proportions were obtained using the subject-specific to population-average logistic variance transformation.The a priori level of statistical significance was an alpha level of .05 for all analyses. All analyses were conducted with PROC MIXED and NLMIXED in SAS, version 9.4 (SAS Institute Inc). Data were analyzed in February 2020.Of the 10 653 veterans identified, 2608 surgical patients were included in the final cohort . Mean [SD] age of surgical patients was 53.0 (9.9) years and 53.6 (9.9) years for the matched nonsurgical patients. More than 84% of the entire study cohort had AUDIT-C scores documented in each year of the uncensored follow-up period. Most patients (n = 2393 [91.8%]) who underwent a bariatric surgical procedure screened negative for unhealthy alcohol use in the 2-year baseline period . Among tOf the small sample of 215 surgical patients with unhealthy alcohol use at baseline, most underwent an LSG rather than an RYGB (145 [67.4%] vs 70 [32.6%]). Most of the sample with unhealthy alcohol use were male, with 36 (24.8%) and 18 (25.7%) females, in the LSG and RYGB cohorts, respectively, which had a mean (SD) age of 50.0 (11.4) years and 51.3 (9.7) years  compared with control patients (n = 14 555) as well as patients who had an RYGB (n = 854) compared with control patients (n = 8038) were similar in most observed characteristics, except for a few minor differences . For exaIn veterans without baseline unhealthy alcohol use, the adjusted mean AUDIT-C scores documented in the VA EHR system 2 years before the surgical procedure were similar in both surgical and control patients . In the Patients without unhealthy alcohol use at baseline who underwent a bariatric surgical procedure, regardless of which type, had a higher probability of unhealthy alcohol use in all 8 years after the surgical date compared with nonsurgical control patients . The proRegardless of the surgical procedure type, surgical patients had a higher probability than control patients of having no alcohol use documented in the EHRs at the time of the surgery , despite the probability of no alcohol use being similar 2 years before surgery  and control patients (n = 1091) as well as those who underwent an RYGB (n = 70) and control patients (n = 548) were similar for most observed characteristics ; higher rates of no alcohol use at the time of the procedure , with convergence between surgical and control patients 1 to 3 years after the procedure; similar rates 5 years after the procedure ; and similar rates 8 years after the procedure   suggest a substantial increase in alcohol use in those who drank alcohol, given that the proportion of patients who reported drinking alcohol decreased from baseline. The estimated prevalence of unhealthy alcohol use 8 years after a bariatric surgical procedure was higher for patients with baseline unhealthy alcohol use (30%-40%) than in those without baseline unhealthy alcohol use (5%-10%), whereas nondrinking was less common (approximately 33% vs 66%). Although similar patterns to those seen in RYGB were observed after LSG, no significant differences were observed in mean alcohol use or unhealthy alcohol use in patients who had an LSG and unhealthy alcohol use at baseline compared with control patients. These results suggest that alcohol consumption changes over time after a bariatric surgical procedure but may differ between RYGB and LSG and may depend on the presence of baseline unhealthy alcohol use.7In addition, this study contributes to the understanding of changes in self-reported alcohol use before bariatric surgical procedures. Although surgical patients were matched to nonsurgical control patients on the basis of the highest AUDIT-C score in the 2 years before the surgical procedure, mean documented alcohol use decreased more during the preoperative period in surgical patients than in control patients, which was not previously reported to our knowledge. In patients without unhealthy alcohol use at baseline, a small decrease in drinking in surgical patients was observed, with no meaningful change seen in control patients. In contrast, surgical and control patients with baseline unhealthy alcohol use both had decreased mean AUDIT-C scores during the 2-year preoperative period, which likely reflected regression to the mean. However, the preoperative decrease in reported alcohol use among surgical patients was steeper, resulting in significantly lower mean AUDIT-C scores by the surgical date. This study cannot determine whether or how often this finding reflects true changes in alcohol consumption or simply a change in reported alcohol use, possibly to increase the likelihood of approval for a surgical procedure, but these preoperative changes in mean alcohol use provide a context to postsurgical changes, especially for uncontrolled nonrandomized cohort studies starting at the time of the procedure.The clinical implications of these results suggest that patients undergoing bariatric surgical procedures should be cautioned that drinking alcohol can escalate after bariatric surgery, even in patients with no previous evidence of drinking alcohol above recommended limits. Although no expert consensus exists on the recommended limits for those who drink alcohol, not drinking alcohol is the safest option after a bariatric surgical procedure, given that blood alcohol concentration  peaks at higher levels after a bariatric operation. Furthermore, all patients who undergo bariatric surgical procedures should be monitored long-term for unhealthy alcohol use, which can be detected with the 3-item AUDIT-C scale.28 enabled the examination of a generalizable sample that was not biased by research recruitment. It also allowed the comparison of outcomes among patients who underwent bariatric surgical procedures and control patients with or without unhealthy alcohol use at baseline. Use of the data allowed the evaluation of changes in documented alcohol use from 2 years before to 8 years after the surgical procedure, building on past US prospective cohort studies.39 Second, this study is the first, to our knowledge, to evaluate alcohol-related outcomes of bariatric surgical procedures in a predominantly male population.This study has several strengths. First, the use of secondary data from the population-based alcohol screening program of the VA40 despite the associations with subsequent health outcomes.22 Therefore, the results provide a minimum estimate of postoperative unhealthy alcohol use in patients who underwent a bariatric surgical procedure and in control patients. In addition, because patients with unhealthy alcohol use often are not accepted for bariatric surgical procedures, self-reporting might be biased. Specifically, surgical patients may have been more likely than control patients to have underreported alcohol use in the 2 years before the operation. Second, residual confounding may persist after sequential stratification because of imbalanced covariates  and lack of data on several patient factors .41 Third, causality cannot be established from this observational study.This study has several limitations. First, it relied entirely on alcohol screening documented in the VA EHR system, which may underestimate the prevalence of unhealthy alcohol useThis multisite cohort study involving predominantly male US veterans found that the probability of developing unhealthy alcohol use increased 3 to 8 years after both LSG and RYGB in patients without previous unhealthy alcohol use, compared with control patients. Alcohol-related risks were somewhat more pronounced after RYGB than LSG, potentially reflecting the alcohol pharmacokinetics changes after RYGB.


The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib potentiated radiation and temozolomide (TMZ) chemotherapy in preclinical glioblastoma models but brain penetration was poor. Clinically, PARP inhibitors exacerbate the hematological side effects of TMZ. The OPARATIC trial was conducted to measure penetration of recurrent glioblastoma by olaparib and assess the safety and tolerability of its combination with TMZ.Preclinical pharmacokinetic studies evaluated olaparib tissue distribution in rats and tumor-bearing mice. Adult patients with recurrent glioblastoma received various doses and schedules of olaparib and low-dose TMZ in a 3 + 3 design. Suitable patients received olaparib prior to neurosurgical resection; olaparib concentrations in plasma, tumor core and tumor margin specimens were measured by mass spectrometry. A dose expansion cohort tested tolerability and efficacy of the recommended phase II dose (RP2D). Radiosensitizing effects of olaparib were measured by clonogenic survival in glioblastoma cell lines.2 daily for 42 days. Fourteen (36%) of 39 evaluable patients were progression free at 6 months. Olaparib radiosensitized 6 glioblastoma cell lines at clinically relevant concentrations of 100 and 500 nM.Olaparib was a substrate for multidrug resistance protein 1 and showed no brain penetration in rats but was detected in orthotopic glioblastoma xenografts. Clinically, olaparib was detected in 71/71 tumor core specimens  and 21/21 tumor margin specimens . Olaparib exacerbated TMZ-related hematological toxicity, necessitating intermittent dosing. RP2D was olaparib 150 mg (3 days/week) with TMZ 75 mg/mOlaparib reliably penetrates recurrent glioblastoma at radiosensitizing concentrations, supporting further clinical development and highlighting the need for better preclinical models. Olaparib penetrates core and margin regions of GBM despite not crossing the intact BBB.Tumor olaparib concentrations range from 100-1000 nM, which achieve radiosensitization in vitro.Olaparib given three times per week can be safely combined with continuous low-dose TMZ.Despite failing to cross the intact blood–brain barrier (BBB) in preclinical models, olaparib penetrated both tumor core and tumor margin regions of recurrent glioblastoma at concentrations similar to those observed in breast cancer patients. The novel observation that tumor margin concentrations of olaparib were similar to those in the tumor core indicates clinically significant enhancement of BBB permeability even in 5-aminolevulinic acid nonfluorescing regions of glioblastoma. Olaparib effectively radiosensitized 6 glioblastoma cell lines in vitro at the median tumor concentration (500 nM) and the lower end of the tumor concentration range (100 nM). While olaparib exacerbated the hematological toxicity of TMZ, as expected, intermittent dosing was tolerated. These observations support further clinical development of olaparib in glioblastoma in combination with radiation therapy and indicate that conventional preclinical models of the BBB do not predict clinical pharmacokinetics.1 Treatment options for recurrent GBM are particularly limited; recent randomized studies have yielded 6-month progression-free survival (PFS) rates of 18–35% and median overall survival durations of 9–10 months.3Improvements in outcomes for brain tumor patients have failed to match those for many extracranial cancers. Glioblastoma (GBM) is the most common malignant primary brain tumor and confers a poor prognosis, with average life expectancy being 12–18 months for patients undergoing neurosurgical resection followed by radiotherapy with concomitant and adjuvant temozolomide (TMZ) chemotherapy.4 The BBB protects the central nervous system from toxic molecules in the blood through ultrastructural features that restrict paracellular diffusion of polar solutes, and efflux mechanisms that export substrate molecules. The human BBB expresses high levels of ATP-binding cassette transporter proteins, notably multidrug resistance protein 1 (MDR1) .5Poor treatment outcomes have been attributed in part to the failure of systemic agents to penetrate brain tumors at therapeutic levels, for which the blood–brain barrier (BBB) has largely been held responsible.7 which cause florid contrast enhancement on magnetic resonance imaging (MRI). While BBB integrity is grossly disrupted in the contrast-enhancing “tumor core” regions of GBM, the extent to which the BBB is compromised in marginal regions, which do not display contrast enhancement, is poorly understood. Whereas most conventional cytotoxic drugs are large, polar molecules that do not penetrate GBM,8 the pharmacokinetic (PK) properties of “small molecule” agents are likely to be more conducive to penetration of the dysfunctional BBB. At the moment, however, there is little clinical PK data with which to substantiate this theory. Small molecule treatments for GBM have been ineffective to date,9 but this may reflect lack of biological efficacy as much as failure of drug penetration.10–12 Hence there is a need for detailed PK studies of candidate small molecule therapies in glioblastoma patients, which should characterize drug delivery to the tumor margins as well as the tumor core.Blood vessels in GBM are grossly abnormal, however, exhibiting microvascular proliferation and increased permeability,13 While single agent activity of PARP inhibitors is restricted to tumors with defects in homologous recombination DNA repair,15 olaparib and other PARP inhibitors sensitize a broad spectrum of cancer cells to both IR and TMZ and potentiate these agents in preclinical glioma models.16 PARP inhibitors also have vasodilatory effects that have been associated with increased drug delivery to tumors in preclinical models.17 Despite these promising preclinical data, clinical development of olaparib for GBM has been hampered by lack of PK information, the absence of a reliable pharmacodynamic (PD) biomarker of PARP inhibition, and the propensity of PARP inhibitors to exacerbate hematological side effects of TMZ.18Olaparib is an orally bioavailable small molecule inhibitor of poly(ADP-ribose) polymerase (PARP), which contributes to repair of DNA damage induced by ionizing radiation (IR) and alkylating drugs, including TMZ.The aims of this study were fourfold: preclinically, to measure penetration of the normal brain and orthotopic GBM xenografts by olaparib; clinically, to characterize olaparib PK in core (5-aminolevulinic acid [5-ALA] fluorescing) and margin (5-ALA nonfluorescing) regions of recurrent GBM; to ascertain whether olaparib affects tumor perfusion and vascular permeability; and to assess the safety and tolerability of combining olaparib with a 42-day, daily low-dose TMZ regimen. This TMZ schedule was selected because it is given concomitantly with IR to patients with newly diagnosed GBM and represents the clinical “line of sight” for the combination with olaparib. Having established the range of olaparib PK in patient specimens, we evaluated the radiosensitizing effects of these clinically relevant concentrations in established and patient-derived GBM cell lines.14C)-olaparib in the presence of ketoconazole (25 μM). Whole body autoradiography was performed on male pigmented rats after single oral administration of [14C]-olaparib . For autoradiography studies, animal care and experiments were carried out in accordance with AstraZeneca guidelines, which comply with UK standards. Olaparib PK was assessed in CD1 nude mice bearing intracranial G7 GBM xenografts from which tumor, blood, and contralateral brain specimens were taken and snap frozen 2, 5, and 24 hours after dosing (50 mg/kg). Olaparib concentrations were measured by mass spectrometry. Xenograft experiments were performed under the relevant UK Home Office Project Licence and carried out with ethical approval from the University of Glasgow under the Animal (Scientific Procedures) Act 1986 and the EU directive 2010. Mice were maintained in individually ventilated cages with environmental enrichment and following the guidelines of ARRIVE . Radiosensitizing effects of olaparib (100 and 500 nM) were measured by clonogenic survival assay in established  and patient-derived cell lines . The linear quadratic model was fitted and integrated to determine mean inactivation dose (MID) for each condition and sensitizer enhancement ratios (SERs) calculated as MID ratios between control and olaparib treatments. Ratiometric t-test analysis determined SER confidence intervals and P-values. Further details are available in the Supplementary Material.Canine kidney epithelial cells, stably expressing human MDR1 cDNA (Madin-Darby canine kidney type II [MDCKII]–MDR1 cells) or empty vector (MDCKII control), were used to model the intact BBB and determine permeability coefficients of (OPARATIC (NCT01390571) was a phase I study investigating the pharmacokinetics, safety, and toxicity of olaparib in combination with TMZ in adult patients with recurrent GBM. Stage 1 was a pilot study to confirm tumor penetration; stage 2 was divided into dose escalation and dose expansion cohorts.Eligible patients were aged ≥18 years with World Health Organization performance status 0–2 and had radiological evidence of recurrent GBM (Response Assessment in Neuro-Oncology [RANO] criteria) after primary treatment with chemoradiotherapy and adjuvant chemotherapy. Exclusion criteria included previous chemotherapy for recurrent GBM; radiotherapy, endocrine therapy, or immunotherapy within 12 weeks, or chemotherapy or biological therapy within 4 weeks. Study investigations and treatments were approved by an NHS Research Ethics Committee in accordance with assurances approved by the UK Medicines and Health Regulatory Authority. Informed consent was obtained from each participant and data were anonymized. Isocitrate dehydrogenase (IDH) mutational status was not available for several of the patients  and IDH status was not an eligibility criterion.Stage 1 patients received olaparib tablets 200 mg twice daily for 7 doses prior to resection of recurrent tumor. Stage 2 patients  received different doses and schedules of olaparib and TMZ for 42 days of each 56-day cycle using a 3 + 3 dose escalation design . Up to 3The primary endpoint of stage 1 required olaparib to be detectable above the lower limit of quantification in at least one tumor specimen from up to 6 patients. At neurosurgery, contrast-enhancing tumor regions identified on preoperative MRI scans were targeted for resection. Specimens were assessed by intraoperative cytology; 3 regions of viable, solid tumor from each patient were snap frozen for PK analysis by liquid chromatography mass spectrometry (LC-MS). In the expansion cohort, 5-ALA guided neurosurgery enabled discrimination of fluorescent “core” tissue from nonfluorescent “margin” tissue. Up to 3 tumor margin biopsies were taken from each patient, from each of which half was frozen for PK analysis and half fixed for histology. Blood and tumor specimens for PK analysis were taken 3–5 hours after the final preoperative olaparib dose.The stage 2 dose escalation primary endpoint was maximum tolerated dose (MTD) and schedule of olaparib/TMZ. Toxicity was defined using the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.02. If 2 or more of 6 patients at one dose level experienced a dose limiting toxicity (DLT), this was considered “not tolerated” and designated the maximum administered dose (MAD). Dose escalation and expansion decisions were influenced by the need for dose reductions of olaparib and TMZ as well as DLTs as defined by the protocol. MTD was defined as either reduced dose or less frequent administration of olaparib than MAD and was further evaluated in the dose expansion cohort to generate the recommended phase II dose (RP2D).Secondary endpoints included PFS at 6 months  and radiological measurement of BBB permeability and perfusion by diffusion weighted and dynamic contrast-enhanced (DCE) MRI. Patients underwent 2 baseline MRI scans 24 hours apart before commencing olaparib and a third scan after at least 3 days of olaparib, prior to surgery. Further details of the imaging protocol are provided in the Supplementary Material. Confidence intervals for PFS data were calculated using the exact binomial distribution.Exploratory endpoints included correlation of tumor core and margin olaparib concentrations with plasma levels and histological and immunohistochemical (IHC) features including hematoxylin and eosin (H&E), Ki67, PARP-1, and blood vessel density. Histopathological methods are provided in the Supplementary Material. Additional GBM specimens for pilot poly(ADP-ribose) IHC studies were obtained from Brain Tumour Bank South West  with appropriate ethical approvals.14C]-olaparib  revealed radioactivity to be undetectable in brain or spinal cord at any timepoint (Data from MDCKII-MDR1 cells indicated that olaparib (1–10 µM) was a substrate for MDR1, with the MDR1 inhibitor ketoconazole reducing the apparent permeability of MDCKII-MDR1 cells to olaparib by 40–62% . Whole bimepoint . OlapariThe OPARATIC study enrolled 48 patients: 3 in stage 1, all of whom underwent surgery; 32 in the dose escalation phase of stage 2, of whom 11 had surgery; and 13 in the dose expansion phase of stage 2, of whom all had surgery. Ten of the 13 dose expansion patients underwent 5-ALA guided resections and tumor margin sampling . Sixty p19 Whereas plasma olaparib levels were broadly dose dependent  verified whether H&E stained sections represented genuine tumor margin material and estimated the proportion of tumor cells in each section. IHC staining for 2 putative tumor cell markers PARP-12); this was defined as the MTD and confirmed as the RP2D after evaluation in the dose expansion cohort. Of 16 RP2D patients evaluable for toxicity, 7 experienced grade 3–4 hematological toxicities: anemia (3), lymphopenia (7), thrombocytopenia (4), and neutropenia (2). None was complicated by sepsis or bleeding. Additional grade 2 adverse events in this cohort included pruritus (1), nausea (1), and vomiting (1). Adverse events are summarized in Table 1.Thirty-nine patients  were evaluable for safety. Cohort 1 was expanded to 6 patients because neutropenia (grade 3) and thrombocytopenia (grade 2) necessitated dose reductions in 2 patients. One DLT (grade 3 vomiting) required expansion of cohort 3. One patient in cohort 4 experienced toxic death associated with pancytopenia, septic shock, and renal failure. This schedule was not considered tolerable, because most patients required dose reductions and/or discontinuations. For cohort 5, olaparib was de-escalated to 150 mg once daily (days 1–3 per week) with full dose TMZ  remained progression free at 6 months, of whom 9 were in dose escalation cohorts and 5 in the dose expansion cohort. Of 16 evaluable RP2D patients, 10 received 1 cycle, 3 received 2 cycles, 2 received 3 cycles, and 1 received 5 cycles. Since most patients underwent neurosurgery prior to commencing study treatment, measurable disease was uncommon and radiological response was not a study endpoint.trans value in their baseline scans  (the product of PARP activity) was essentially undetectable in untreated tumor specimens from 5 GBM patients that were obtained from a brain tumor biorepository . In the 21 The modest activity of the olaparib-TMZ combination is at least partly attributable to the reduced olaparib dosing that was required to avoid severe hematological toxicity, and in our view should not be taken as evidence of inadequate tumor penetration by olaparib.This study demonstrates unequivocally that olaparib penetrates tumor core and tumor margin regions of recurrent GBM, despite preclinical evidence indicating failure to penetrate the intact BBB. The absence of a robust PD biomarker of PARP inhibition together with the infeasibility of obtaining pre/post-olaparib tumor specimens in these patients made it impossible to rigorously assess the clinical activity of the drug concentrations achieved. However, olaparib levels were within the range of 100 to 1000 nM in all specimens, concentrations that achieved significant radiosensitization in all 6 GBM cell lines tested. Combining olaparib with daily low-dose TMZ was safe and reasonably well tolerated, but intermittent olaparib dosing was required to mitigate hematological toxicity. The observed PFS-6 rate of 39% compares favorably with recent clinical trials but was deemed insufficient to support further development of the combination in this population. However, the resoundingly positive PK data have underpinned phase I and II studies of olaparib in combination with radiotherapy ± TMZ in patients with newly diagnosed GBM.22 Consistent with studies in other tumor types,22 no correlation was observed between tumor and plasma concentrations. While variability in the time between olaparib dosing and tumor sampling may have influenced these results, we propose that uptake and retention of olaparib in GBM is determined primarily by tumor vasculature characteristics. This is supported by DCE-MRI data showing an inverse correlation between tumor olaparib concentrations and baseline Ktrans values. The in vitro radiosensitization data presented here are consistent with an ex vivo study demonstrating >90% inhibition of PARP activity at 50 nM olaparib23 and support the concept that meaningful radiosensitization of GBM may be achievable with clinically deliverable olaparib doses. Our study also highlights the need for contemporaneous development of robust PD biomarkers during drug development for GBM and other brain tumors.This study provides the first direct evidence that a drug showing no penetration of the intact BBB in preclinical models penetrates GBM in patients at clinically meaningful concentrations. Indeed, olaparib penetrated tumor core and margin regions at concentrations similar to those observed in breast cancer specimens.24To study tumor margin regions, neurosurgeons undertook 5-ALA guided resections to obtain macroscopic clearance of tumor tissue, then sampled adjacent nonfluorescent tissue. These samples were confirmed as tumor margin material by histological analysis: the majority comprised less than 5% tumor cells and only one represented solid tumor. Accurate quantification of tumor cell density was not possible because there are no validated tumor cell markers for IDH wild type GBM, but the surrogate tumor markers PARP-1 and Ki67 supported the histological evaluation. The highly novel finding that tumor margin olaparib concentrations were similar to those in core regions indicates that BBB integrity is compromised even in regions of GBM where tumor cells are sparse and MRI contrast enhancement is not observed. One possible explanation is that co-option of vessels by invading glioma cells enables small numbers of infiltrating tumor cells to disrupt BBB function, as recently demonstrated in orthotopic murine models.trans was observed. We speculate that this reflects increased retention of olaparib within tumors with reduced blood flow and hence reduced drug washout.DCE-MRI investigations were performed to assess the impact of olaparib on tumor perfusion and identify candidate imaging biomarkers of olaparib PK. While olaparib did not reproducibly affect DCE-MRI parameters, a negative correlation between maximum tumor olaparib concentration and KOur findings have important implications for preclinical and clinical development of small molecule treatments for GBM and other brain tumors. They illustrate that the preclinical models currently used to measure BBB penetration fail to predict GBM penetration in patients, and motivate us to recommend direct measurement of GBM PK and PD in early phase evaluation of novel agents, in both core and margin regions of these tumors. Finally, our data support the need for more representative preclinical models of the “blood–tumor barrier” to enable rational selection of compounds for development and clinical testing.OPARATIC was sponsored and funded by the Cancer Research UK Centre for Drug Development and supported by the UK National Cancer Research Network. Olaparib was supplied by AstraZeneca.noaa104_suppl_Supplementary_MaterialClick here for additional data file.


Neurodevelopmental psychiatric disorders including schizophrenia (Sz) and attention deficit hyperactivity disorder (ADHD) are chronic mental illnesses, which place costly and painful burdens on patients, their families and society. In recent years, the epidermal growth factor (EGF) family member Neuregulin 1 (NRG1) and one of its receptors, ErbB4, have received considerable attention due to their regulation of inhibitory local neural circuit mechanisms important for information processing, attention, and cognitive flexibility. Here we examine an emerging body of work indicating that either decreasing NRG1–ErbB4 signaling in fast-spiking parvalbumin positive (PV+) interneurons or increasing it in vasoactive intestinal peptide positive (VIP+) interneurons could reactivate cortical plasticity, potentially making it a future target for gene therapy in adults with neurodevelopmental disorders. We propose preclinical studies to explore this model in prefrontal cortex (PFC), but also review the many challenges in pursuing cell type and brain-region-specific therapeutic approaches for the NRG1 system. NRG1 is the best characterized of a four-member gene family (NRG1-NRG4) that all share an epidermal growth factor (EGF)-like domain with ErbB receptor binding activity ; however, none meet genome wide significance for conferring an increased risk for the disorder6. The ErbB4 receptor subtype, in particular, plays a key role in the inhibitory functions of NRG1 in the CNS11. There is an extensive literature on the role of NRG1–ErbB4 signaling in early cortical development15 but see also ref. 16. In the following sections, we critically re-evaluate over two decades of research on the function of NRG1 in the mature central nervous system (CNS). We place particular emphasis on the complex role of NRG1–ErbB4 signaling in local circuit inhibitory neurons on cortical function and plasticity. We also examine the intrinsic and extrinsic factors regulating ED shedding, as this step holds the key to controlling NRG1 bioavailability. Finally, we develop a model for treating adults with Sz via gene therapies targeting NRG1/ErbB4 signaling in local circuit interneurons. Although much data support this model, we discuss the many challenges currently hindering translation of these types of therapeutic approaches.Neurodevelopmental disorders including schizophrenia (Sz) and attention deficit hyperactivity disorder (ADHD) are chronic mental illnesses. For years, scientists have sought more effective treatment options for improving the cognitive outcomes in patients with Sz. The trophic factor Neuregulin 1  activates signaling cascades, which regulate inhibitory neural processes important for executive functions, such as attention and working memory, which are impaired in Sz18. Critical periods are also a time of heightened synaptic remodeling and integration of inhibitory interneurons into local neural circuits in cortex19 are associated with higher brain functions impaired in neurodevelopmental disorders such as working memory and attention24. Patients with Sz exhibit gamma oscillation abnormalities, which underscores the relevance of local circuit function as a therapeutic outcome27.Neurodevelopmental psychiatric disorders such as Sz typically emerge in late adolescence or early adulthood. This developmental epoch is considered to be a ‘critical period’ for maturation of prefrontal cortex (PFC), analogous to the postnatal timeframes defined for visual and other sensory systemsex19Fig. . The bes 20 Fig. . During 18. Along with increased inhibitory tone, these mechanisms are thought to constrain cortical plasticity, in effect limiting the learning and memory gains achievable with aging. Presumably, a persistent reduction in PFC plasticity in adulthood contributes to the failure of current therapies to rehabilitate the cognitive deficits associated with Sz28.The extensive synaptic remodeling that occurs during critical period development decreases excitatory drive in cortex while enhancing inhibitory tone. As a result, neural circuits in cortex are less plastic after critical period closure Fig. . Additio22. Subsequent studies using transgenic and viral tools showed that either activating VIP+ interneurons or silencing SST+ interneurons represent other ways to disinhibit pyramidal cells and re-evoke plasticity29. Likewise, recent evidence indicates that manipulating NRG1–ErbB4 signaling in fast- spiking PV+ basket cells offers an additional option for overcoming reduced cortical excitability30. As discussed below, this and related findings from studies in VIP+ interneurons suggest that NRG1–ErbB4 signaling in interneurons could be poised to provide a tractable target for re-evoking cortical plasticity in PFC11.An exciting body of work in the visual system points to a set of local circuit neuron-based manipulations that can be performed after critical period closure to reactivate cortical plasticity Fig. . Initial31. This discovery set the stage for subsequent work demonstrating that the NRG1 EGF-like domain regulates not only gamma oscillations1, but also a host of related neural and behavioral phenomena including pyramidal cell excitability2, seizure activity34, synaptic plasticity35, and anxiety-like behavior36. The effects were shown in all cases to hinge on expression of ErbB4 receptors on PV+ neurons. The ability of the EGF-like domain peptide to suppress seizure activity and long-term synaptic potentiation (LTP) also implicates NRG1 in homeostatic mechanisms controlling excitatory and inhibitory (E/I) balance in neural circuits34.Several lines of evidence link NRG1 and ErbB4 with local circuit interneuron function. Foremost is the finding showing that a recombinant peptide corresponding to the EGF-like domain of NRG1 stimulates activity-dependent release of the inhibitory transmitter GABA37. As observed for PV+ basket cells, the vast majority of VIP+ interneurons in cortex prominently express ErbB4, whereas expression of the NRG1 receptor in cortical SST+ interneurons is sparse38  neurons as well as in oligodendrocytese38 Fig. . Knockou40. This is relevant for neurodevelopmental disorders as ventral hippocampus-prefrontal synchrony in the theta and delta frequency ranges positively correlates with performance on the 5-choice serial reaction time test (5-CSRTT), which is a well-accepted task of attention41. Studies with ErbB4 ATP binding site mutant knockin mice indicate that ErbB4 receptors in ventral hippocampus and PFC play a role in performance on the 5-CSRTT as well as in ventral hippocampal-prefrontal synchrony40. These studies also showed that ErbB4 constitutive knockout mice (heart rescued) exhibit reduced hippocampal-prefrontal synchrony and deficits in attention40. Note, however, that another group reported no deficits in the performance of the ErbB4 constitutive knockout mice (heart rescued) on the 5-CSRTT relative to controls42. Other electrophysiological studies indicate, however, that interneuronal NRG1–ErbB4 signaling regulates neural synchrony in cortex44.Recent work indicates that NRG1 signaling also modulates the phase locking  of local field potentials in ventral hippocampus and PFC31, electrophysiological studies indicate that ErbB4 signaling in interneurons largely promotes maturation of glutamatergic inputs onto PV+ and VIP+ interneurons45. Consistent with this idea, ErbB4 receptors localize near excitatory inputs onto somatodendritic regions of interneurons47. Molecular studies suggest that NRG1–EGF-like domain binding stimulates ErbB4 receptor phosphorylation, which results in activation of a set of downstream signaling cascades  (reviewed in ref. 48). Several studies indicate that ErbB4 signaling in interneurons stimulates incorporation of AMPA receptors into postsynaptic membranes, which is an established mechanism for potentiating excitatory transmission51. Two studies reported that ErbB4 signaling in interneurons preferentially regulates NMDA receptors. However, one study examined responses in a type I NRG1 transgenic mouse in which levels of ErbB4 in cortical interneurons are significantly downregulated52; the other study investigated the actions of NRG253. Interaction of ErbB4 with PSD95, a postsynaptic scaffolding protein found at mature glutamatergic synapses, could also play a role in the mechanism as NRG1 signaling increases PSD95 levels near excitatory synapses on cortical interneurons in culture56. Altogether, these findings suggest that ErbB4 signaling regulates the molecular composition of excitatory synapses in interneurons via diverse mechanisms.Although NRG1 enhances activity-dependent GABA transmission in cortex45. In contrast, ErbB4 signaling in VIP+ neurons regulates the synchronous firing of cortical pyramidal cells11. As discussed above, reduced pyramidal cell excitability is a key mechanism in critical period closure, as well as in the subsequent maintenance of reduced cortical plasticity  can reactivate cortical plasticity after critical period closure. Neither strategy is intended to rescue any potential disease-related abnormalities in the NRG1 gene or anomalies its expression. The main driver of these strategies is the evidence (1) showing that cell-specific manipulation of ErbB4 receptors in either PV+ and VIP+ interneurons provides a means of tuning excitability of mature cortex51, and (2) indicating the differential cellular and subcellular distribution of NRG1 isoforms66  or lentivirus (LV) carrying a floxed cDNA or siRNA into PFC of adult transgenic mice in which CRE expression is conferred via a cell-specific promoter. AAVs and LVs are frequently the vectors of choice for gene therapy due to their stable long-term expression (up to years) and low risk of pathogenesis or cytotoxicity69. Use of transgenic CRE mice would enable testing of cell-type-specific predictions of the local circuit interneuron model. Regional specificity is critical because both the NRG1 and ErbB4 genes are expressed in a variety of cortical and subcortical brain regions70. Undesired side-effects resulting from global manipulation of a particular isoform in brain, even if cell-type-specific, could overshadow any potential benefits on prefrontal cortical plasticity. In support of this possibility, Sz-relevant behavioral and neuronal abnormalities were detected in transgenic mice that overexpress either the full length or just the bioactive ED of type I NRG1 in principal neurons throughout the brain73. Behavioral and neural deficits were also detected in transgenic mice with cell specific but brain-wide upregulation of other NRG1 isoforms  of type III is transported to axon terminals following ED cleavage in the stalk segment of the pro-protein66. Indeed, one study showed that inclusion of the EGF-like domain is necessary for presynaptic localization of the BACE1-cleaved type III fragment, highlighting the possibility that interaction with trans-synaptic ErbB4 receptors could play a role in the anchoring mechanism66. For single transmembrane NRG1 isoforms like type I and type II, sheddase cleavage in the linker liberates the EGF-like domain containing N-terminus  agonist of ErbB4 tyrosine kinase activity89. Studies of KO mice indicate that NRG3 functions in excitatory terminals to promote synapse formation on interneurons, and to suppress glutamate release—however, neither function requires ErbB490. Although none of the available data suggests that the functions of type III NRG1 and NRG3 are interdependent93, much remains to be learned about these different NRGs in local circuits. Below, we propose an alternate strategy for reactivating plasticity in adult PFC by manipulating NRG1–ErbB4 signaling in VIP+ interneurons.In addition to contacting local circuit interneurons via axon collaterals, PFC pyramidal cells project to a range of more distant cortical and subcortical targets that regulate cognitive and emotional processing, action selection, and reward behavior61. In visual cortex at least, stimulating VIP+ neuronal activity is sufficient to disinhibit pyramidal cells, which is the central mechanism underlying reactivation of cortical plasticity29  are required for neural activity-stimulated shedding of the bioactive ED63. More detailed mechanistic studies in non-neuronal cells show that PKC stimulates ED shedding (1) by increasing cell surface levels of ADAM17, and (2) by phosphorylating key residues in the intracellular domain (ICD) of NRG1102. These studies also showed that PKCδ-mediated phosphorylation of serine residue 286 in the NRG1 ICD is critical for ADAM17 cleavage104. ICD phosphorylation is proposed to initiate conformational changes in the NRG1 polypeptide that are relayed through the plasma membrane, and ultimately increase accessibility of the ADAM17 cleavage site in the ED106. Like NRG1, many proteins expressed in brain are substrates of ADAM17 or PKC , so upregulation of either enzyme would likely produce “off-target” side-effects110. On the other hand, knowledge of mechanisms promoting NRG1 shedding could be useful in the design of ED shedding-prone variants carrying mutations mimicking phosphorylation of key PKC sites in the ICD.Two studies have looked at mechanisms regulating NRG1 shedding from neurons30. One advantage of this circuit-based approach is that it provides a framework for investigating alternate targets or signaling pathways. Another option would be to manipulate ErbB4 either in PV+ or VIP+ interneurons. Ideally, the biology underlying the illness will dictate which brain area should be targeted111. Justifications for targeting PFC include its well-established role in executive functions , which are significantly impaired in patients with Sz113. Further, stronger activation of PFC positively correlates with better functional outcomes for patients with Sz114. However, the primary neuroanatomical deficits of Sz are not well established. Indeed, meta-analyses suggest that in addition to PFC, improved outcomes correlate with increased resting-sate activity in several other brain regions 117. It will be important to compare the relative efficacy of gene therapies injected into PFC versus these other fronto-limbic areas in improving executive functions. Postmortem neuropathological findings suggest that alterations in the cellular organization of cortex could be associated with Sz. Abnormalities reported range from a decreased density of inhibitory neurons to an increased number of proinflammatory cytokine-secreting microglia118. It is unclear whether these types of abnormalities would limit or accelerate possibilities for neural rehabilitation via the strategies proposed.Here we review data implicating NRG1–ErbB4 signaling in cortical plasticity and executive functions. We also explore the possibility of remediating cognitive and executive function deficits associated with Sz using gene therapy directed at NRG1–ErbB4 signaling in local circuits neurons in PFC. The therapeutic model that we develop takes advantage of cutting-edge information on local circuit mechanisms for reactivating excitability in cortex after critical period closure11. The manipulations that we propose should serve as a starting point for intervention. Better alternatives could very well arise as new discoveries are made that deepen our understanding of the NRG family in local circuits. Another concern is the absence of clinical trial data on the feasibility of stereotactic injection into brain of patients with Sz or other type of psychiatric illness. While a number of clinical studies have been carried out or are underway for a variety of neurological disorders119, use of these approaches for psychiatric disorders could potentially be thwarted by a number of obstacles due to the unique nature of these illnesses. For example, symptoms like paranoia and command auditory hallucinations are common in these disorders. Whether this patient population would be amenable to intracerebral surgical injection is unknown. Other issues that would need to be resolved relate to treatment duration and dosing given the chronic and heterogeneous nature of psychiatric illnesses. Phase I and II clinical trial data indicate cargo expression from intracerebrally injected recombinant AAV can persist for at least 2 years58. Although development of this therapeutic modality is still emerging, evidence from preclinical studies suggests both longer term expression and inducible expression are possible. For example, studies in primates show that expression from intracerebrally injected virus is maintained for up to 15 years without cytotoxicity or Lewy body formation68. Likewise, progress has been made on incorporating inducible genetic elements into AAV vectors, which would enable tighter control of cargo expression levels120.Even if the proposed preclinical studies validate the promise of these novel approaches, there are a number of issues that would still need to be addressed before gene therapy for Sz could be considered a near term possibility. One issue is that there is still much to be learned about the localization and function of the different members of the NRG gene family in the local circuits. Further so far only one study has looked at the impact of NRG1–ErbB4 signaling in VIP+ interneurons on cortical plasticityIn summary, despite over 50 years of research and development, the drugs available to treat Sz largely fail to remedy the cognitive and executive function deficits. Although there are still a number of open questions regarding feasibility as discussed above, gene therapy is a therapeutic alternative that should be explored given the intractability and chronic nature of these impairments.


Dear Editor,1. Two main criteria used to diagnose STS include the documentation of shortened telomere lengths (TLs) by a Clinical Laboratory Improvement Amendments (CLIA)-certified flowFISH (fluorescence in situ hybridization) assay2 and the presence of pathogenic variants in genes related to telomere maintenance identified through next-generation sequencing (NGS). These genes include, but are not limited to, hTERT (human telomerase reverse transcriptase) and hTERC (human telomerase RNA component), the two main components of the telomerase holoenzyme complex that is responsible for creating new telomeric DNA , idiopathic pulmonary fibrosis (IPF), cryptogenic cirrhosis, nodular regenerative hyperplasia, vascular malformations, immunodeficiency, and structural brain abnormalities, all of which are included under the umbrella term of short telomere syndromes (STSs)3. In addition, in some of these cases, the TL values were not conclusively shortened  complicating the diagnosis. At Mayo Clinic, we established a dedicated BMF clinic in collaboration with the center for Individualized Medicine and the division of Hematology, so as to leverage the latest NGS technologies and functional assays to assist patients with unexplained BMF syndromes, including STS-related marrow failure4. We have developed a systematic algorithmic approach to assess STS patients that includes TL testing by flowFISH and genomic assessment of STS-related gene mutations using an in-house designed targeted NGS panel (Supplementary Table hTERT (four variants), hTERC (one patient), RTEL1 , TINF2 , and NAF1 . Patients with hTERT VUS  of uncertain significance (VUS)US Table 5,6.TablehTERT variants in patients 1–4 and a hTERC variant in patient 5. To increase our interpretive resolution for hTERT variants, we created a computational 3D protein model of the human telomerase complex that we used to assess the intra- and inter-molecular interactions within the complex. Our current model includes all amino acids of hTERT and a contiguous section of hTERC with the bound DNA heteroduplex. This model was generated using homology-based methods8 and the experimentally solved structure of TERT from Tetrahymena thermophila [PDB 6d6v9]. The human Reverse Transcriptase thumb domain of telomerase was experimentally solved [5ugw10] and added to our model using threading. Genomic variants were assessed in their 3D context using FoldX11. Recent model fitting to a cryogenic electron microscopy map of human telomerase core particle was published12 and was used here in comparison to our current model of full-length TERT.All patients Table  had BMF 1: hTERT c.2768 C > T; p.Pro923Leu (patient 1) is located near the helix required for oligomerization and is adjacent to p.Arg901 and p.Lys902—sites where bona fide loss-of-function mutations have been described  is present at the end of the reverse-transcriptase thumb domain at a position where the peptide chain crosses back against residues 999–1003. Further, hTERC wraps around one side of this thumb and the DNA winds across the other side, so that any changes to the internal organization/arrangement of this thumb may significantly alter function  affects a residue located in the helix near the hTERC interface possibly affecting stability of this interaction , where we considered the evidence already available, including a published functional report, to be strong enough to consider this variant as being pathogenic13 (more information in ref. 3). For the remaining patients with hTERT variants, we evaluated their telomerase activity using the TRAP assay5. TRAP semi-quantitatively measures the capacity to elongate a telomere-imitating oligonucleotide via hTERT processing in a patient’s samples over several amplification cycles, akin to a quantitative PCR reaction. The commercially available TRAPeze Telomerase Detection Kit  was located in the stem loop in the CD4/5 region and our 3D model analysis indicated a likely reorganization of the stem loop as a consequence of this change, thereby altering the secondary structure of the RNA. Additionally, a previous report had indicated decreased telomerase activity in patients carrying this variant when tested by the TRAP assay14. We felt that given the clinical context, this information was sufficient enough to classify this variant as pathogenic without the need for additional testing.The hTERT holoenzyme. By using these methods, we were able to provisionally re-classify five VUS identified in patients with STS, an important step forward in mutational nomenclature. Importantly, pathogenic variants in hTERT/hTERC comprise 52% of STS-related mutations described in the Human Gene Mutation Database . For the remaining 48% of mutations that encompass several genes involved in TL regulation, there currently are no reliable functional assays . Thus, variants identified in our clinic impacting RTEL1, TINF2, and NAF1 still remain unclassifiable and their causal impact on TL in these patients remains unknown. TRAP assays also demonstrate inherent variabilities between tissue samplings and between individuals within the same age range, limiting the routine implementation of these assays in clinic. The development of cell line-based assays utilizing genetic engineering of variants and assessing their impact on TL could be one potential way of overcoming these limitations. In summary, while we were able to assess the pathogenicity of hTERT/hTERC variants using 3D modeling and the TRAP assay, a lot more work is needed to help develop accurate in silico approaches and functional testing for variants in other genes regulating TL.In conclusion, we demonstrate the importance and feasibility of using 3D molecular modeling and functional assays to classify variants identified in the Supplemental Figures legendsSupplemental MethodsSupplemental Table 1Supplemental Figure 1Supplemental Figure 2


Cisplatin is a platinum-based first-line drug for treating ovarian cancer. However, chemotherapy tolerance has limited the efficacy of cisplatin for ovarian cancer patients. Research has demonstrated that cisplatin causes changes in cell survival and death signaling pathways through its interaction with macromolecules and organelles, which indicates that investigation into the DNA off-target effects of cisplatin may provide critical insights into the mechanisms underlying drug resistance. The multifunctional protein p62 works as a signaling hub in the regulation of pro-survival transcriptional factors NF-κB and Nrf2 and connects autophagy and apoptotic signals, which play important roles in maintaining cell homeostasis. In this review, we discuss the role of p62 in cisplatin resistance by exploring p62-associated signaling pathways based on current studies and our work. Insights into these resistance mechanisms may lead to more effective therapeutic strategies for ovarian cancer by targeting p62. Carboplatin, a second-generation platinum drug, showed equivalent therapeutic effects as cisplatin but with fewer toxic side effects [paclitaxel and carboplatin neoadjuvant chemotherapy [Ovarian cancer is a gynecologic cancer with a high mortality rate. In developed countries, the mortality rate of ovarian cancer is three times that of breast cancer and the 5-year survival rate of patients with stage IV ovarian cancer is only 28% . In 1965activity . In the  effects , 5. At pPlatinum is an electrophilic reagent characterized by its ability to form covalent linkages with nucleophilic residues of nucleobases such as guanine and adenine. Because a variety of cellular macromolecules contain nucleophilic residues, platinum drugs have the potential to interact with various cellular components, such as ribosomes, spliceosomes, and the RNA in telomerase, as well as proteins through Met, His and free Cys side chains , 11. GalAlthough the signaling networks that determine cell survival and death are extensive, only a small number of molecules have been identified that function in coordinating these signaling pathways. The multifunctional protein p62/SQSTM1  integrates both survival and death signaling by regulating the ubiquitination of key cell signaling molecules that control survival and death –19. p62 In this review, we discuss the changes in p62-mediated signaling pathways in ovarian cancer during cisplatin treatment based on our work and current research. We describe a role for p62 in cisplatin resistance of ovarian cancer, providing a theoretical basis for potential strategies for overcoming chemotherapy resistance in ovarian cancer.During macroautophagy, hereinafter referred to as autophagy, autophagosomes isolate components targeted for autophagy by forming a closed membrane structure and transporting them to lysosomes for degradation. Autophagy serves a protective function against malignant transformation and maintains homeostasis in normal tissues. However, once cells undergo transformation, autophagy provides cancer-protective functions to deal with stress from the worse survival environment , 23. Prep62 is an autophagy receptor involved in the recognition of ubiquitin-labeled substrates targeted for autophagy . MatsumoCha-Molstad et al.  found siMitochondrial autophagy (mitophagy) maintains mitochondria mass and function by clearing damaged or overloaded mitochondria. Kingnate et al.  proposedRecent studies have indicated that p62 is not only found in the outer mitochondrial membrane but also localized to the inner mitochondrial membrane, which may be involved in mitochondrial function maintenance and morphology regulation . VDAC  promotes degradation of the NF-κB inhibitor IκB through the proteasome pathway, which subsequently activates NF-κB signaling. Many studies showed that the activated NF-κB pathway functions in promoting cell survival in ovarian cancer. Yang et al.  found thisplatin , 46. TheRecent studies found that p62 inhibition significantly suppressed activation of the NF-κB pathway . We founThe TRAF6/NF-κB pathway was originally identified in immune cells . The E3 The RIP1 serine/threonine protein kinase is involved in the regulation of inflammatory signaling and various cell death pathways such as apoptosis and programmed necrosis , 55. RIPMitochondria are the main location of cellular reactive oxygen species (ROS) production. Previous studies showed that cisplatin enters into cells and directly binds to mitochondria, leading to cytochrome C release, calcium-dependent mitochondrial swelling and production of ROS, which induces oxidative stress and reduces genomic stability , 61. TheRecent studies have also confirmed that p62 is involved in regulation of the Keap1-Nrf2 pathway. Phosphorylation of p62 at serine 349 in the KIR domain increased its binding affinity to Keap1 and promoted Nrf2 activation . ConsistMG132, which indicates that p62 may be involved in mitochondrial dynamics through Keap1-Nrf2 signaling regulation  is structurally similar to caspase 8 and forms a dimer with caspase 8 to inhibit its activity. cFLIP is highly expressed in ovarian cancer , 84. Li cisplatin induces formation of the necrosome containing the RIP1/RIP3/MLKL complex as core, which initiates programmed necrosis via ROS production [In the presence of RIP3, oduction . Liu et oduction  showed toduction . These rWhile early studies suggested that DNA was the main target of cisplatin , later rp62 functions as a key receptor for autophagy. Increasing studies have demonstrated that autophagy is involved in chemotherapy resistance and several compounds have been identified that regulate autophagy in ovarian cancer Table .Table 2INotably, clarifying how p62 functions in chemotherapy may provide benefits for ovarian cancer clinical diagnosis and prognosis. Most ovarian cancer patients treated with cisplatin eventually develop resistance and show poor outcome. Iwadate examined p62 expression in tumor tissues of 266 patients with primary ovarian cancer and found that patients with high expression of p62 had poor prognosis . AnotherSeveral questions remain to be answered. For example, the role of accumulated p62 caused by autophagy blockage in NF-κB or Keap-1/Nrf2 signaling activation is unclear. Furthermore, the function of accumulated p62 induced by increasing transcription or degradation suppression is also unknown. Future studies should focus on alterations in the binding partners of p62 and the post-translational modifications that enhance p62 functions in ovarian cancer cells with cisplatin treatment. Together these findings may provide new strategies to overcome cisplatin resistance in ovarian cancer by targeting p62.


Mechanisms driving immunosuppression in chronic myeloid leukemia are mostly unknown. We show that leukemic extracellular vesicles (EVs) target lymphocytes and amplify suppressive function of thymic regulatory T cells, by driving expression of Foxp3 transcription factor. This could facilitate expansion of leukemic cells outside the bone marrow, leading to blast crisis. Extracellular vesicles (EVs), including exosomes and microvesicles, are one of the key factors controlling immune function, both in steady state and pathological conditions Our data presented here strongly indicate that CML‐derived EVs can regulate Foxp3 expression and suppressive activity of Foxp3+ thymic regulatory T cells (tTregs). To study effect of CML‐derived EVs specifically on the tTregs population, we have sorted tTregs directly from murine thymi , as CML model, or the parental 32D cells. EVs were characterized based on their morphology, size, and expression of specific markers, according to the guidelines of ISEV  and the EV‐TRACK consortium + responder cells, as demonstrated by two parameters – increased percentage of inhibition by tTregs and decreased expansion index of CD8+ responder cells , cytokines , or ectoenzymes CD39 and CD73, which in majority are either directly or indirectly regulated by Foxp3 transcription factor + responder cells. This may seem crucial for immunosuppression in CML, as cytotoxic CD8+ T cells constitute one of the main lines of immune system defense against leukemia In summary, we demonstrate that CML‐derived extracellular vesicles upregulate Foxp3 expression in thymic regulatory T cells, leading to increased suppressive activity of these cells, preferentially (to some extent) toward CD8The authors declare no commercial or financial conflict of interest.CMLchronic myeloid leukemiaEVsextracellular vesiclesFoxp3forkhead box P3Tregsregulatory T cellstTregsthymic regulatory T cellsSupporting informationClick here for additional data file.


Mobility is important to sustain for older adults to live independently. The purpose of this project was to evaluate teach back and ask me 3 interventions with a health education program that included Otago strength and balance exercise and a walking program The pilot program, Mobility Matters, was completed with 16 older adults , 69% African American, 94% female. Older adults with moderate fall risk were recruited from community centers and participated in a 3-month program where they were paired with physical therapy students for pre- and post-intervention assessment. Participants were randomly assigned to a health literacy intervention group (HLG) (n=9) and received teach back and ask me 3 intervention twice a month for three months. The control group (n=7) received the same program of balance exercises/ walking program and after 3 months was given the health literacy intervention. Groups were not significantly different on age, gender and REALM scores. Assessment measures included: timed up and go, 30 second chair rise, 4 stage step test, 6 minute walk test, and activity balance confidence scale (ABC). Paired t-test analysis revealed mean significant differences on the measures of four stage balance test (p =.008), six-minute walk test (p=.026) and approached significance on ABC (p=.054). No significant differences were found for the non-health literacy group on all measures. The results suggest that health literacy intervention may improve outcomes for health education interventions with balance and aerobic exercise.


Scientific Reports 10.1038/s41598-017-17577-7, published online 12 December 2017Correction to: This Article contains an error in the Data Availability section.“The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.”should read:“Summary data generated during and/or analysed during the current study are available from the corresponding author on reasonable request, but restrictions apply to the availability of the raw data, which is owned by Monash Health, as it details personal patient information.”


Amyotrophic lateral sclerosis (ALS) is a rapidly disabling and fatal neurodegenerative disease. Due to insufficient disease-modifying treatments, there is an unmet and urgent need for elucidating disease mechanisms that occur early and represent common triggers in both familial and sporadic ALS. Emerging evidence suggests that impaired DNA damage response contributes to age-related somatic accumulation of genomic instability and can trigger or accelerate ALS pathological manifestations. In this review, we summarize and discuss recent studies indicating a direct link between DNA damage response and ALS. Further mechanistic understanding of the role genomic instability is playing in ALS disease pathophysiology will be critical for discovering new therapeutic avenues. Amyotrophic lateral sclerosis (ALS) is a lethal degenerative motor neuron disease with a median survival of 2–4 years after diagnosis  and no aAlthough ALS is a mainly sporadic disease (90–95% of patients) , attentiWithout excluding the importance and relevance of other molecular mechanisms that have been extensively covered by others , in thisDDR is starting to be recognized as a unifying mechanism in neurodegenerative disorders . DNA damSince mature neurons are post-mitotic non-replicating cells that are difficult to replace ,31, unsaIn ALS, the endogenous sources contributing to deleterious accumulation of GIN are from both impaired removal of reactive metabolic genotoxins  that can overwhelm DDR  and fromATM; a core DDR gene ..FUS mutaFollowing oxidative damage, in a poly(ADP-ribose) polymerase (PARP1)-dependent manner, FUS facilitates the recruitment of XRCC1/LIG3 to SSBs and enhances LIG3 ligation activity thus promoting base excision repair BER; b 80–83]80–83]. TThese data build a model in which FUS controls the choice between SSB repair and DSB repair pathways in healthy neurons. Further research will be required to specifically understand the connection between FUS and TDP43 in DDR as well as the requirement of HR versus microhomology mediated end-joining . In some patients, ALS is evidenced to manifest on the basis of oligogenic rather than monogenic alterations, with summative effects from several DDR pathologies , as indiC9ORF72 ALS/FTD is caused by the expansion of a hexanucleotide GGGGCC (G4C2) track in the first intron of the C9ORF72 gene [Nucleotide repeat expansion (NRE) disorders encompass more than 20 human genetic diseases, most of which affect the nervous system, that arise from an expansion of a particularly unstable tandem of 3–12 DNA bases ,89. The F72 gene .senataxin (SETX) and HNRNPD also lead to fALS [G4C2–NREs are pathogenic through several non‐exclusive mechanisms that can all influence the accumulation of DNA damage lesions and affect their repair A. Initia to fALS ,96. Subs to fALS ,97–105.  to fALS  and can  to fALS  and sequ to fALS ,112. Mor to fALS  and imba to fALS . Further to fALS .Consequence of these pathologic mechanisms, C9ORF72 ALS/FTD patients show increased GIN accumulation both in the brain  and spinAnother important link between G4C2 expansion and DDR is the observation that DPR accumulation leads to imbalanced chromatin states with impact on DNA repair . Poly-PRIt must be highlighted that neurons do not live in isolation, and neurodegeneration is associated with microglial reactivity and activation of innate immune responses. Neuroinflammation is a common characteristic of ALS and comprises the stimulation of microglia, astrocytes and inflammatory T cells . Upon acNeuroinflammation with subsequent neurodegeneration can also result from a glia autonomous problem in dealing with DDR. Mutant human TDP43 expressed specifically in Drosophila glial cells causes DNA damage, elevated replication of retrotransposable elements (RTE) , and GypALS is one of the most common adult-onset neurodegenerative disorders. Currently, ALS is fatal and incurable with patients expected to survive ∼2–4 years after diagnosis, revealing an urgent need for effective therapeutic strategies. Proof of DNA damage accumulation and DNA repair deficiency in both ALS initiation and progression is amassing, highlighting the fact that genomic instability is a hallmark of disease pathogenesis. Shedding light on the specific DDR mechanisms at play has important therapeutic potential.Genomic instability is a hallmark of both sporadic and familial ALS with many ALS genes involved in recognition or repair of DNA damage.Outside of the nucleus SOD1 works to impede ROS accumulation and in the nucleus to influence DNA damage response via transcription regulation.TDP43 and FUS work mainly to balance the pathway choice between SSB repair and DSB repair.C9ORF72 gene leads to impaired ATM signalling.Expansion of a repeated G4C2 track in the Genomic instability may be a starting point for neuroinflammation in ALS.


We investigate the feasibility of obtaining multiple spatially-separated biopsies from a single lesion to explore intratumor heterogeneity and identify actionable truncal mutations using whole exome sequencing (WES). A single-pass radiologically-guided percutaneous technique was used to obtain four spatially-separated biopsies from a single metastatic lesion. WES was performed to identify putative truncal variants (PTVs), defined as a non-synonymous somatic (NSS) variant present in all four spatially separated biopsies. Actionable truncal mutations—filtered using the FoundationOne panel—were defined as clinically relevant PTVs. Mutational landscapes of each biopsy and their association with patient outcomes were assessed. WES on 50 biopsied samples from 13 patients across six cancer types were analyzed. Actionable truncal mutations were identified in 9/13 patients; 31.1 ± 5.12 more unique NSS variants were detected with every additional multi- region tumor biopsy (MRTB) analyzed. The number of PTVs dropped by 16.1 ± 17.9 with every additional MRTB, with the decrease most pronounced (36.8 ± 19.7) when two MRTB were analyzed compared to one. MRTB most reliably predicted PTV compared to in silico analysis of allele frequencies and cancer cell fraction based on one biopsy sample. Three patients treated with actionable truncal mutation-directed therapy derived clinical benefit. Multi-regional sampling for genomics analysis is feasible and informative to help prioritize precision-therapy strategies. Intratumor heterogeneity is a key challenge in precision cancer therapy, contributing to treatment resistance, therapeutic failure and poor prognosis ,2. With The mutational diversity attributed to ITH limits our ability to resolve the full spectrum of cancer pathway aberrations through a single biopsy of the tumor lesion and may under/overestimate driver alterations ,8,9. Then = 1), non-small cell lung cancer , ovarian carcinoma , breast carcinoma , uterine carcinoma , hepatocellular carcinoma , or cervical cancer  were recruited to the study. A single-pass radiologically-guided percutaneous biopsy technique was used to obtain MRTBs from a dominantly-progressing metastatic lesion in each patient with core biopsies taken at least 2 mm apart within the same metastatic lesion. Two patients  were excluded from analysis as all the MRTB samples collected from them failed quality control (QC). One patient (UC)  P11;  had two The investigation pipeline adopted for analyzing whole-exome sequencing (WES) data that passed QC is illustrated in p = 0.691).The mutational landscape of each patient was examined to evaluate the extent of ITH across different cancer types. The non-synonymous somatic mutational load (nssML)—defined as the total number of non-synonymous somatic (NSS) variants present—was scrutinized for each biopsy sample A. Resultp < 0.05). When copy number alterations (CNAs) were interrogated, a moderate degree of diversity  was observed  and branch  variants were analyzed B. Phylogobserved .p = 0.177). Truncal somatic cancer driver mutations across this study cohort include AKT1, ATM, BCOR, CHD4, KRAS, MAP3K1, and PIK3CA; conversely, branch somatic cancer driver mutations include ERBB2, FOXA1, and PPM1D. In our cohort, EGFR mutations were confined to lung cancers, with three out of four (75%) NSCLC patients found to harbor a truncal variant in at least one reportable mutation in EGFR [Statistically significant somatic cancer driver mutations (ssCDMs) were juxtaposed with non-synonymous somatic variants identified in our study cohort. Results indicate that detectable somatic cancer driver mutations were more likely to be truncal variants 68.2%; ; however in EGFR .The relationship between truncal variants and the number of MRTB samples analyzed was examined. A unique variant in this case refers to a distinct non-synonymous somatic variant that appears in at least one of the MRTB samples analyzed simultaneously. In general, with an increasing number of MRTB samples, a monotonically increasing trend in the number of unique variants and correspondingly decreasing number of truncal variants can be observed . At the exome level , on average 31.1 ± 5.12 more unique non-synonymous somatic variants were detected with every additional MRTB sample analyzed. Conversely, using the number of MRTB samples analyzed simultaneously as the baseline reference to determine putative truncal variants (PTVs), a monotonically decreasing number of PTVs can be observed with an increasing number of MRTB samples. The number of PTVs dropped by 16.1 ± 17.9 on average with every additional MRTB sample, with the decrease most pronounced (36.8 ± 19.7) when two MRTB samples were analyzed compared to just one. Similar trends were observed from filtered variants when the WES data was mapped to genes matching four cancer gene panels—namely COSMIC Cancer Gene Census (CGC), Ion AmpliSeq™ Cancer Hotspot Panel v2 , TruSight® Cancer panel  and FoundationOne™ cancer gene panel  . HoweverTo quantitatively corroborate the minimum number of MRTB samples required across different gene panels, statistical saturation analysis was conducted. Results  indicateTo evaluate the ability to identify truncal variants (defined based on four MRTB samples) using a single biopsy sample, two metrics were used as classification thresholds, namely allele frequency (AF) and cancer cell fraction (CCF). Computation of CCF values—the proportion of cancer cells within which the variant is present—for patient P07 (OV) was unable to be performed due to inadequate information related to somatic copy number alteration. Hence, patient P07 was excluded for the purpose of this analysis.p = 0.021).First, the threshold value (for each respective metric) that produces the best average prediction accuracy across all patients was examined. Results, as illustrated in p = 0.031).Next, the threshold value (for each respective metric) that produces the best average prediction accuracy across patients with the same cancer type was scrutinized. Results, as demonstrated in Three (23.1%) patients  received an inhibitor targeting an actionable truncal mutation based on molecular profiling while another six (46.2%) patients were treated with non-actionable truncal mutation-directed therapy either because they did not have any actionable truncal mutations or there was no available therapy to target the actionable truncal mutation at our center . An illuUsing each patient as his/her own control as a strategy to attenuate confounding factors resulting from the diverse patient population and tumor types, we assessed the clinical efficacy of actionable truncal mutation-directed therapy by comparing progression free survival (PFS) on actionable truncal mutation-directed therapy  or non-actionable truncal mutation-directed therapy with the PFS for the most recent prior therapy (PFS-A) in each of these patients . Two NSCPatient P10 with breast cancer and a phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA)_H1047R truncal mutation was enrolled into a highly selective PI3Kα/β inhibitor phase 1  trial , but proIt is noteworthy that the CCF metric performed relatively well in predicting the truncal status of the variants targeted. The median PFS-actionable truncal mutation-directed therapy for the small number of patients treated with actionable truncal mutation-directed therapy was 6.1 months, with a median PFS ratio of 1.5. These findings do suggest that the truncal status of tumors influences response and, if validated, could potentially be used in personalized cancer treatment to help prioritize therapeutic strategies.ITH represents a significant challenge to precision medicine and contributes to drug resistance. Several studies employing multi-region tumor sampling from post-surgical samples have greatly increased our understanding of tumor evolution and highlighted the importance of tumor sampling from spatially distinct areas in order to avoid erroneous interpretation of genomic data from single sampling bias ,9,10. InResults in our small cohort demonstrate a high degree of ITH (>65% branch mutations) across the majority of patients, with private mutations dominating the mutational landscape. Clearly, this indicates that ITH is a ubiquitous issue that would confound the ability to identify bona fide truncal variants. Statistical saturation analysis demonstrates that for small targeted cancer gene panels like CGC, AmpliSeq™ and TruSight®, a minimum of two MRTB samples are required to identify PTVs; for a larger cancer gene panel like FoundationOne™, at least three MRTB samples are needed. The determination of the minimum number of MRTB samples required is highly valuable as it enables clinicians to find the equilibrium between cost and accuracy , and allows the choice of which cancer gene panel to use with the amount of tumor tissue available.p < 0.001). This suggests minimal intratumoral variation in the ML based on our series and that mutational burden is less likely to be impaired by sampling bias. Examination of the nssML of each patient indicates that individual intratumor biopsy samples comprise a similar amount of NSS variants A while tGiven the coveted utopia of making informed clinical decisions based on a single biopsy sample, in silico methods for predicting truncal variants are of particular interest. AF and CCF are two favored metrics commonly used. Empirical experiments indicate that when AF was used as the threshold to classify truncal variants, it achieved comparable, if not better, accuracy compared to CCF; although both approaches were less compelling for some cancer types. Of note, different cancer types (at the whole-exome level) favor different threshold values for segregating truncal from branch variants, suggesting that each tumor type exhibits distinct biological characteristics that require dedicated data analytics.Improved clinical outcomes were observed in two out of three patients whose truncal mutations were selectively targeted. Remarkably, all patients in our series treated with actionable truncal mutation-directed therapy derived symptomatic benefit with improvement in their performance status. The small patient numbers across a diverse spectrum of tumors limits our ability to draw significant conclusions within each tumor type, but nonetheless demonstrates its applicability in a variety of tumor types and preliminary evidence of clinical benefit when used for therapeutic prioritization in selected patients. It is noteworthy that patient P05—who had an EGFR_T790M branch mutation that was targeted—did not respond well to the treatment . This reIn our study, we only analyzed single nucleotide variants (SNV) which may potentially underestimate the frequency of clinically actionable mutations and the mutational load of the tumor. We focused solely on SNV mainly because they make up the majority of pathogenic variants relevant in solid tumor malignancies (59.39%) compared to other genomic alterations such as indels, structural variants and copy number loss . Indeed,All patients were recruited and treated at the National University Cancer Institute (NCIS), Singapore, between December 2014 and May 2016. WES and data analytics were performed at the National University of Singapore (NUS), Singapore. All procedures were conducted in accordance with the approved protocols and written informed consent was provided by the patients . EligiblEach patient had 5 tumor biopsy samples obtained from one metastatic lesion using a single-pass radiologically-guided percutaneous biopsy technique. This technique involves the insertion of a coaxial needle together with its trocar into a lesion. The trocar is subsequently removed to allow for the introduction of a biopsy device that is composed of a needle with a 1.5 cm throw to facilitate multiple passes along acute angles from a single lesion via a single percutaneous access. Each biopsy sample was obtained at least 2 mm apart. One biopsy core was sent to the histopathology lab as part of routine clinical management while the remaining four tumor biopsy samples were analyzed using WES. Four patients had biopsies obtained from the lung , four had peritoneal nodes biopsied , three patients underwent a liver biopsy  and one patient each had a bone (P13) and supraclavicular lymph node (P2) biopsied. Germline samples were collected from each patient in the form of a buccal swab. If the germline sample failed quantitative QC, whole blood would be used as replacement. Samples with DNA concentration <4 ng/μL would be deemed to have failed QC. Radiological images were obtained as part of clinical care. Extraction of genomic DNA from tumor samples was carried out using the Qiagen Allprep DNA/RNA Micro Kit . The MasterAmp Buccal Swab Kit  was used to extract DNA from buccal swab samples while the Qiagen EZ1 DNA Blood 350 µL Kit  was used to process whole blood samples. An Illumina NextSeq 500 Sequencing System  was utilized to perform 150 base pair paired-end WES. All experiments were conducted in accordance to manufacturer guidelines at the Cancer Science Institute of Singapore, NUS. Full patient data can be found in the National Centre for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under accession number SRP137039Sequence reads were aligned to the hg19 reference genome using the Burrows–Wheeler Aligner (BWA) v0.7.7  and realSomatic CNAs were detected using several algorithms. Succinctly, the computation of raw copy number calls and the adjustment of GC content of the raw copy number calls were performed using VarScan2 v2.3.9 . Re-centn = 29), NSCLC (n = 30), OV (n = 10), BC (n = 52), EC (n = 58), and HCC (n = 26) is available in Five gene panels—WES NSS, COSMIC Cancer Gene Census (CGC), the Ion AmpliSeq™ Cancer Hotspot Panel v2, the TruSight® Cancer panel and the FoundationOne™ cancer gene panel—were examined. The WES NSS gene panel comprises of all protein-coding genes (with NSS property) in the genome, while the CGC gene panel consists of all statistically significant cancer-specific genes curated from the COSMIC Cancer Gene Census (CGC) ,29 and TAll NSS variants identified in the WES NSS gene panel were subsequently juxtaposed with individual targeted cancer gene panels  and variants from mismatched genes were winnowed out. The resulting list of variants for each cancer gene panel was used for subsequent downstream analysis.The construction of phylogenetic trees was carried out based on a binary table that represents the presence or absence of variants across all MRTB samples. Using the PHYLogeny Inference Package v3.695 (PHYLIP)  and matci’ refers to the number of variants that are present in at least one of the MRTB samples examined in combination set ‘i’. Similarly, the average number of PTVs was calculated based on the formula above but with ‘xi’ defined as the number of variants present in all MRTB samples scrutinized in combination set ‘i’. The computation of the average number of unique variants present when ‘k’ number of MRTB samples were analyzed concurrently was performed based on the following Formula (1):i,k’  refers to the number of variants present in all ‘k’ number of MRTB samples examined simultaneously in combination set ‘i’.PPV, on other hand, was computed based on the following Formula (2):p-value is less than 0.05.All statistical hypothesis tests were conducted using the Wilcoxon signed rank test unless otherwise stated. Statistical significance is considered when the The CCF value was estimated using the ABSOLUTE algorithm  for eachAF was calculated by dividing the number of alternative sequence read counts with the total number of  sequence read counts. Likewise, the clonality of variants was determined by comparing the variant’s AF with a range of thresholds (from 0.01 to 0.55 incremented at a step size of 0.01). The respective thresholds used for both the cancer cell fraction and allele frequency analysis according to the individual panels are shown in i,k’ represents the number of correct classification made for biopsy sample ‘i’ using threshold value ‘k’, and ‘yi’ refers to the total number of variants assessed for biopsy sample ‘i’.To stratify variants into either truncal or branch based on a single biopsy sample, different (AF and CCF) threshold values were investigated. For each threshold value examined, the following formula was employed to compute the average classification accuracy, by which 4 MRTB samples were used as the baseline reference for defining bona fide truncal variants (3).Treatment decisions were made using molecular profiling results from one core biopsy, as reported in the Intergrated Molecular Analysis of Cancer (IMAC) study  while thrvention .In conclusion, this study has demonstrated: (i) the importance of performing multiple biopsies despite extant in silico prediction methods, (ii) the minimum number of MRTB samples required to alleviate challenges related to ITH is dependent on the tested hypothesis and the examined gene panel, but that at least two biopsies should be submitted for analysis to achieve a PPV of >90% identifying AT mutations, and (iii) the feasibility and clinical efficacy of adopting the proposed approach for strategic therapeutic intervention. Further validation of this approach for identifying and targeting AT mutations in larger cohorts will be required to fully assess its potential value as a precision medicine strategy to circumvent the challenges of intratumoral heterogeneity in cancer therapy.