Among patients with non-metastatic pancreatic cancer, 80% have high-risk, borderline resectable or locally advanced cancer, with a 5-year overall survival of 12%. MASTERPLAN evaluates the safety and activity of stereotactic body radiotherapy (SBRT) in addition to chemotherapy in these patients.2 IV), irinotecan (150 mg/m2), 5-fluorouracil (2400 mg/m2 CIV), leucovorin (50 mg IV bolus)) plus SBRT in the investigational arm. Gemcitabine+nab-paclitaxel is permitted for patients unsuitable for mFOLFIRINOX. SBRT is 40Gy in five fractions with planning quality assurance to occur in real time. Following initial chemotherapy ± SBRT, resectability will be evaluated. For resected patients, adjuvant chemotherapy is six cycles of mFOLFIRINOX. Where gemcitabine+nab-paclitaxel was used initially, the adjuvant treatment is 12 weeks of gemcitabine and capecitabine or mFOLFIRINOX. Unresectable or medically inoperable patients with stable/responding disease will continue with a further six cycles of mFOLFIRINOX or three cycles of gemcitabine+nab-paclitaxel, whatever was used initially. The primary endpoint is 12-month locoregional control. Secondary endpoints are safety, surgical morbidity and mortality, radiological response rates, progression-free survival, pathological response rates, surgical resection rates, R0 resection rate, quality of life, deterioration-free survival and overall survival. Tertiary/correlative objectives are radiological measures of nutrition and sarcopenia, and serial tissue, blood and microbiome samples to be assessed for associations between clinical endpoints and potential predictive/prognostic biomarkers. Interim analysis will review rates of locoregional recurrence, distant failure and death after 40 patients complete 12 months follow-up. Fifteen Australian and New Zealand sites will recruit over a 4-year period, with minimum follow-up period of 12 months.MASTERPLAN is a multi-centre randomised phase II trial of 120 patients with histologically confirmed potentially operable pancreatic cancer (POPC) or inoperable pancreatic cancer (IPC). POPC includes patients with borderline resectable or high-risk tumours; IPC is defined as locally advanced or medically inoperable pancreatic cancer. Randomisation is 2:1 to chemotherapy + SBRT  or chemotherapy alone (control arm) by minimisation and stratified by patient cohort (POPC v IPC), planned induction chemotherapy and institution. Chemotherapy can have been commenced ≤28 days prior to randomisation. Both arms receive 6 × 2 weekly cycles of modified FOLFIRINOX (oxaliplatin (85 mg/mMASTERPLAN evaluates SBRT in both resectable and unresectable patients with pancreatic ductal adenocarcinoma.ACTRN12619000409178, 13/03/2019.Australia New Zealand Clinical Trials Registry Protocol version: 2.0, 19 May 2019 Pancreatic cancer (PC) is anticipated to become the second leading cause of cancer death in the United States by 2030 . SurgeryThe current recommended management for patients with operable PC, including patients with high-risk features of size > 4 cm, extrapancreatic extension and node positivity, is resection with consideration of adjuvant chemotherapy –9. Even P < 0.001) [nab-paclitaxel as peri-operative treatment for pancreatic cancer. The authors concluded that there is little evidence that either treatment improved OS to historical standards [For BRPC and LAPC, the recommended treatment is for chemotherapy with or without external beam radiation therapy (EBRT), and consideration of surgical resection in those that become resectable . Some in< 0.001) . The SWOtandards . This putandards , 23. Unttandards , 25; howtandards . Adjuvantandards . The imptandards .A systematic review on the use of SBRT in PC demonstrated encouraging results with a median OS of 17 months . A largeIt is essential that the apparent benefits of this technique be explored in a multi-centre randomised setting to provide solid evidence. The combination of chemotherapy and SBRT, while promising, remains unpublished in a randomised setting. At the time of this publication results from the A021501 Alliance Trial have just been presented, although not published, warranting comparison of trial methodologies , 36. Thenab-paclitaxel (Option 2), and this will need to be specified prior to randomisation. Patients will be randomised in a 2:1 ratio to the investigational arm with six cycles of chemotherapy and SBRT or the control arm with six cycles of chemotherapy alone. Initial chemotherapy can have commenced ≤28 days prior to randomisation. SBRT will be 40Gy in five fractions administered over 2 weeks with central plan storage and pre-treatment quality assurance review through the Trans-Tasman Radiation Oncology Group (TROG).The MASTERPLAN study aims to evaluate the safety and activity of SBRT in addition to chemotherapy for the treatment of POPC and IPC. The primary objective of the MASTERPLAN randomised phase II study is to determine if the addition of SBRT to modern chemotherapy improves locoregional control for patients with POPC and IPC as defined by RECIST criteria v1.1  and AustSecondary outcomes include assessment of 1) Safety (frequency and severity of adverse events as defined by NCI CTCAE V4.03 and RTOG), 2) Surgical morbidity/mortality , 3) Radiological response rates (RECIST v1.1), 4) Progression-free survival (PFS) (disease progression or death), 5) Pathologic response rates (tumour regression grade), 6) Surgical resection rates (rates of attempted resection excluding ‘open and close’ operations), 7) R0 resection rates , 8) Quality of life (QoL) (EORTC QLQ C30 and PAN26 QoL), 9) Deterioration-Free Survival (DFS) (EORTC QLQ C30), and 10) OS (death from any cause).Tertiary objectives for MASTERPLAN are to study associations between clinical endpoints and potential predictive/prognostic biomarkers  including but not limited to ctDNA and protein signatures  and serial buccal and faecal microbial samples . Additionally, radiological assessments of nutrition and sarcopenia will be investigated in the RANDOMS sub-study.or (5) LAPC. In addition, patients must: be aged 18–75 years, have histologic confirmation of pancreatic adenocarcinoma, have measurable disease according to RECIST v1.1, be of ECOG performance status 0 to 1, have study treatment planned to start within 14 days of registration, be willing and able to comply with all study requirements, and provide signed, written informed consent. Patients will need to have adequate renal, haematological and hepatic function defined as bilirubin less than 150% of the upper limit of normal (ULN) and AST/ALT less than five times the ULN. In patients who have had a recent biliary drainage and whose bilirubin is descending, a value of less than three times ULN is acceptable. Major exclusion criteria include: tumour size greater than 70 mm, duodenal invasion seen on endoscopy, prior abdominal radiotherapy, evidence of metastatic disease on baseline radiologic investigations, excluding non-melanomatous skin cancers, pre-invasive cervical cancer or early endometrial cancer, patients with a history of other malignancies are eligible if they have been continuously disease free for at least 2 years after definitive treatment, serological confirmed HIV positivity with CD4 count less than 400, concurrent illness, including severe infection that may jeopardise the ability of the patient to undergo the procedures outlined in this protocol with reasonable safety, neuroendocrine pancreatic carcinoma and life expectancy of less than 3 months. Baseline imaging including a computed tomography (CT) scan of chest/abdomen/pelvis with contrast and appropriate imaging of pancreas using a pancreatic protocol in addition to a 18F-FDG PET is required. Registration requires multidisciplinary confirmation of patient suitability by a medical oncologist, radiation oncologist, surgeon and radiologist. Central review of diagnostic (baseline) images will occur with site feedback of staging planned. Figure After site credentialing, clinicians will be able to screen potential patients for eligibility during clinical review. Eligible patients must have one of the following: 1) T3 (tumour > 4 cm) (2) Extrapancreatic extension, (3) Node positive, (4) Borderline resectable pancreatic cancer  T3  Clinical Trials Centre (CTC), University of Sydney in collaboration with the TROG. This study will be performed in accordance with the Note for Guidance on Good Clinical Practice (Integrated Addendum to ICH E6 (R1): Guidelines for Good Clinical Practice ICH E6(R2) annotated with TGA comments (Therapeutic Goods Administration DSEB July 2000) and in compliance with applicable laws and regulations, NHMRC Statement on Ethical Conduct in Research Involving Humans 2007, NHMRC Australian Code for the Responsible Conduct of Research (updated 2015) and principles laid down by the World Medical Assembly in the Declaration of Helsinki 2008.The study will be conducted in accordance with applicable Privacy Acts and Regulations. All data generated in this study will remain confidential. All information will be stored securely at the NHMRC CTC, University of Sydney. Radiotherapy data generated from the radiotherapy quality assurance activities will be stored securely at TROG. Prior to opening for recruitment, sites will undergo a credentialing process to obtain approval from TROG to confirm SBRT planning and treatment delivery capability. A separate assessment of radiotherapy plan quality and treatment compliance will be undertaken by TROG on study completion. No patient will be recruited to the study until all the necessary Human Research Ethics Committee and other approvals have been obtained and the patient has provided written informed consent.nab-paclitaxel (Option 2). mFOLFIRINOX will be six 14-day cycles of oxaliplatin (85 mg/m2 IV), irinotecan (150 mg/m2 IV), 5-fluorouracil (2400 mg/m2 CIV over 46 h), leucovorin (50 mg IV bolus) all given on Day 1. Alternatively, patients can have three cycles of gemcitabine (1000 mg/m2 IV) and nab-Paclitaxel (125 mg/m2 IV) Day 1, Day 8 and Day 15 per a 28-day cycle.Patients will receive either six cycles of mFOLFIRINOX (Option 1) or if unsuitable for mFOLFIRINOX, three cycles of gemcitabine and Patients randomised to SBRT will receive repeat staging with CT chest/abdomen/pelvis with contrast after chemotherapy and prior to SBRT. Patients without progressive disease will receive SBRT as 40Gy in five fractions, 8Gy per fraction, with two to four fractions delivered per week over 2 weeks. Two consecutive days of SBRT are permitted but not 3 days. SBRT is to commence within 4 weeks of completing initial chemotherapy. Given this prescription dose is above threshold dose for nearby organs at risk (OAR), compromise of coverage will be required near these structures. A radiotherapy quality assurance (QA) planning manual has been developed and available to all sites. AGITG and TROG guidelines for contouring and simulation have been published for use in the MASTERPLAN study . To aid During radiation planning and delivery, TROG will provide QA in accordance with TROG Policy Statement TP E6 , a QA technical review will be undertaken for SBRT plans with remote technical audits conducted by an independent reviewer to ensure protocol compliance and appropriate SBRT delivery. The on-trial quality assurance for the trial will consist of Pre-Treatment Radiotherapy QA Technical Review and Post-Treatment Radiotherapy QA Technical Review.Bloods will also be taken after initial chemotherapy +/− SBRT and prior to surgery to assess renal, haematological, and hepatic function.Prior to surgery, all patients require a clinical assessment, routine bloods, CA19.9 and CEA, toxicity assessment and QoL questionnaire completion. CT chest, abdomen and pelvis scan will be mandatory 4 weeks post-completion of chemotherapy +/− SBRT, with images transferred for central storage and batched independent, blinded assessment at a later stage.For all patients deemed resectable, surgery should be performed 6 weeks (±2 weeks) after completion of the initial therapy . The aim of the surgery is to achieve R0 resection. When the tumour is within the head of the pancreas, patients would be offered a standard Whipple’s procedure and level 2/3 dissection , with moThe standard pancreatoduodenectomy resection may comprise of regional lymphadenectomy around the duodenum and pancreas. For head/neck lesions this includes the lymph nodes on the right side of the hepatoduodenal ligament, the right side of the superior mesenteric artery (superior and inferior), and the anterior and posterior pancreaticoduodenal lymph nodes. For body/tail lesions this includes the pancreaticosplenic lymph nodes and lymph nodes to the left of superior mesenteric artery (superior and inferior). A lymphadenectomy beyond the abovementioned area could therefore be considered an extended lymphadenectomy. These anatomical definitions will be used to determine locoregional recurrence. If macroscopic removal of the tumour and/or clinically involved nodes is not possible, these patients will receive ongoing treatment as per trial protocol. If distant metastases are found at surgery, these patients should receive ongoing treatment at discretion of treating team. The method used for all surgical events will be prospectively obtained.Standardised synoptic pancreas cancer histology reporting is required as outlined in Royal College of Pathologists of Australasia . Post-sunab-paclitaxel as initial chemotherapy, adjuvant chemotherapy may be mFOLFIRINOX or three cycles of gemcitabine and capecitabine  after completion of surgery. Patients having surgical resection should receive an additional six cycles (12 weeks) of adjuvant mFOLFIRINOX following surgery. In patients who received gemcitabine and nab-paclitaxel was used initially a further three cycles of gemcitabine and nab-paclitaxel.Patients determined not suitable for surgery after multidisciplinary review, either unresectable or medically inoperable, will continue with the initial choice of chemotherapy within 4 weeks (+/− 2 weeks) of imaging. This is a further six cycles of mFOLFIRINOX or where gemcitabine and Unresectable patients with locoregional progression or metastatic disease (progressive disease (PD)) at CT scan restaging may receive further chemotherapy at the discretion of their treating medical oncologist. Follow-up data until death will be collected.The primary endpoint of 12-month locoregional control will be assessed using RECIST version 1.1 criteria. A CT chest/abdomen/pelvis with contrast will be performed within 7 days prior to registration, prior to SBRT (SBRT arm only), 4–6 weeks following induction treatment (chemotherapy +/− SBRT), 4 weeks post-surgery (if applicable), then at 6, 9 and 12 months following study randomisation, and every 3 months during Year 2, every 6 months during Years 3 and 4, unless new disease is reported.Patients who experience synchronous locoregional and distant progression will be deemed to have a locoregional failure. Local and regional relapse will be determined based on imaging findings and/or biopsy (if performed). The first post-treatment scan cannot be used to determine locoregional relapse due to treatment related changes that may impact imaging assessment. However, locoregional control can be determined and dated back to the first scan only if the subsequent scan confirms locoregional progression. Regional relapse is defined as disease progression to regional lymph nodes that would be included in a resection of pancreatic lesion in the same location  QoL will be assessed using the European Organisation for the Research and Treatment of Cancer (EORTC) Quality of Life Questionnaires - QLQ C-30 and QLQ-PAN26.Sarcopenia will be assessed using CT-assessed body composition analysis and completion by participant and local dietician of the Patient Generated – Subjective Global Assessment (PG-SGA) which will generate scores to be used in addition to objective radiological assessments of sarcopenia. These assessments will be performed by trained dietitians on centrally stored images as part of the RANDOMS sub-study .Baseline assessment of patients includes clinical examination, haematological, serum electrolytes (creatinine / creatinine clearance) and liver function assessment along with tumour marker (CA19.9). Patient reported outcomes commence at this time with quality of life (EORTC QLQ C30 and PAN26 QoL) and dietary questionnaires (PG-SGA). An 18-FDG PET will be performed within 28 days of randomisation to exclude patients with metastatic disease.nab-paclitaxel) and prior to each injection of gemcitabine for patients receiving gemcitabine + Capecitabine. CA19.9 is assessed at every treatment cycle, then every 3 months to 12 months post end of treatment. Toxicity and adverse events are assessed at each cycle.Assessment during chemotherapy includes haematological, biochemistry and liver function assessments, repeated at Days 8 and 15 for patients receiving Option 2 chemotherapy , prior to confirmation of eligibility for surgery and at 3, 6, 9 and 12 months from randomisation. Patients receiving SBRT will have CT 2 weeks prior to SBRT - 2 (±1 week) weeks following initial chemotherapy. CT images will be stored for central radiology review.A CT scan must be performed prior to surgery and a CT chest, abdomen and pelvis scan is mandatory 4–6 weeks post-surgery. All surgical specimens are required for retrospective central histological assessment and reporting of resection outcome (R0 versus R1). Tumour tissue and normal tissue (pancreas and duodenum) for translational research is collected from all surgical patients at time of resection. Surgical complications are assessed per Clavien grading system at discharge, 30 and 90 days post-surgery.Consultation with a radiation oncologist following chemotherapy is required to confirm the patient remains suitable for SBRT. For patients receiving SBRT, late radiation therapy AEs will be assessed every 3 months for the first year then 6-monthly up to 4 years.Quality of life questionnaires are completed during chemotherapy, and prior to SBRT and 4–6 weeks post-completion of initial treatment . For patients eligible for surgery QoL is completed within 3 days prior to surgery. All patients will complete QoL questionnaires 30 days after end of treatment then at 3, 6, 9 and 12 months from randomisation and followed by completion every 6 months during Years 2, 3 and 4.The Patient Generated – Subjective Global Assessment (PG-SGA) questionnaire is completed within questionnaire within 7 days of CT scans performed at baseline, 4–6 weeks post-completion of initial therapy, 12 weeks post-surgery and 6 months post-randomisation. Assessment by a dietician will occur at the same time.Participants will be followed up for a minimum of 12 months and up to 4 years. After completion of study treatment, participants will be followed up at 6, 9 and 12 months from study randomisation then every 6 months during Years 2, 3 and 4.Tissue and blood samples will be collected from all participants for translational research. Archival formalin-fixed paraffin-embedded diagnostic core biopsy will be collected for central histology review. Surgical resection tissue  will be collected and processed into formalin-fixed paraffin-embedded tissue and some stored in preservative (RNAlater) where possible. In addition, core biopsy at time of fiducial insertion (for patients having SBRT) and disease progression will be collected from consenting patients .Serial blood collections (up to seven timepoints) for translational research will occur at baseline, prior to SBRT (Arm B only), insertion of fiducial markers , 4–6 weeks post-completion of initial treatment , at surgery , 6 and 12 months post-randomisation or at progression (whichever comes first). Bloods are processed to recover serum and plasma and frozen and some research bloods may be shipped to central laboratories in real time for analysis. Translational research studies may include the molecular and genetic drivers of PC, prognostic and predictive biomarkers for clinical endpoints including circulating tumour DNA and a funded study of glycoproteins (by mass spectrometry).A microbiome sub-study of 40 to 60 patients aims to assess the effect of chemotherapy on microbial composition in patients with PC. Serial microbial samples will be obtained with patients collecting their own buccal and faecal samples before, during and after chemotherapy. The primary objective is to explore the variation in microbial composition with chemotherapy. Secondary objectives include correlating variation in microbial composition with response, exploring the impact of opportunistic use of antibiotic and healthy diet on the variation microbial composition and correlating buccal and faecal microbial composition.Among the potentially operable patients with high-risk or BRPC (POPC), we expect a LRR rate at 12 months of 40% . LRR is Among inoperable patients with LAPC or medically IPC, we expect a LRR rate at 12 months of 50% , 46 wherA comparative analysis of chemotherapy and SBRT versus chemotherapy alone for the two groups will be performed. The combined analysis will assess 1) LRR rates and 2) PFS. One hundred twenty patients (80 versus 40): Using competing risk analysis the trial will have 80% power and 95% confidence (two-tailed comparison) to detect a HR of 0.469 on LRR. One hundred twenty patients will provide preliminary evidence on PFS. Assuming no difference between the treatment groups across groups A and B, a 95% CI will provide a lower bound for the similarity of the two treatments. The lower bound for a one-sided 95% CI for the HR of 1 is 0.71, based on 90% event rate in the chemotherapy alone group at 24 months [After the first 40 patients have completed 12 months of follow-up an interim analysis for assessment of the rates of locoregional failure, distant failure (metastasis) and death will occur. In addition, the rate of distant failure/death prior to locoregional relapse will be monitored periodically in the pooled cohort and if required the sample size may be re-estimated.An IDMC will meet at least 6-monthly to review patient safety, trial progress, the planned interim analysis results and the periodic assessment of rate of distant failure/death prior to locoregional relapse.The role of radiotherapy, including SBRT, in PC is controversial. To date there is no published randomised evidence exploring the role of SBRT in pancreatic cancer. Institutional experience reports high local control rates and low toxicity, however confirmation in a randomised study remains elusive. Recent improvements in systemic treatment, have placed an increased importance on locoregional control in this difficult to manage disease. In this publication we describe an investigator initiated multi-centre phase II trial conducted by the AGITG in collaboration with TROG and the NHMRC CTC which was developed in collaboration by these groups through a series of clinical and academic meetings and workshops in Australia.MASTERPLAN has emerged as an important trial exploring the utility of SBRT in patients with high-risk, borderline resectable or LAPC. In this patient cohort, locoregional recurrence risk is high and can be potentially mitigated by SBRT. Given the high radiation doses employed with SBRT, and the potential for significant toxicity, it is important this technique is explored in the setting of a prospective trial with appropriate QA and follow-up. Pancreatic SBRT in the context of a clinical trial with RT QA offers a tremendous opportunity to train radiation oncologist and provide constructive feedback to treating teams. A separate assessment of radiotherapy plan quality and treatment compliance will be undertaken by TROG on study completion.This trial assesses whether a clinical improvement in locoregional control can be achieved with pancreas SBRT. Locoregional control remains an endpoint of value to both clinicians and patients. The impact of locoregional progression can be devastating with associated morbidity and/or death. The 2:1 randomisation allows rapid collection of prospective data within the interventional arm, while maintaining an appropriate control arm for comparison. The interim analysis after 40 patients allows early assessment of the primary endpoint, in addition to distant failure and death, and the IDSMC reviews will determine if closure of the study is required due to safety issues or treatment futility. Despite R0 resection rates being a readily accessible and important endpoint, this is not an appropriate endpoint for interrogation during the interim analysis.Published guidelines for SBRT delivery, in addition to real time RT QA ensure that results from this trial will be robust, extrapolatable and generalisable.Secondary endpoints allow for assessment of safety, surgical morbidity, pathological response, tumour control and quality of life, all comparable between treatment groups with or without SBRT. Importantly, a number of sub-studies in the MASTERPLAN trial including translational sciences in addition to radiologically assessed markers of sarcopenia, assessment of microbiome, provide a unique opportunity to collect a wealth of information and data across a range of disciplines.A similar study Alliance for Clinical Oncology Trial A021501 , 36has rResults for Alliance A021501 were presented in early 2021  and are There are some initial concerns regarding the heterogeneity between treatment groups in the A021501 publication. Patients in the interventional group had a baseline Ca19.9 that was nearly 50% higher (248 U/ml versus 171 U/ml). Fewer patients in the interventional arm underwent surgery (58% versus 51%), and an eventual pancreatectomy (48% versus 35%). These two factors have considerable impact on R0 resection status, the primary endpoint in determining the cessation of the interventional arm. Central review of resection margin status prior to cessation of the interventional arm in A021501 is controversial, due to the subjective nature of R0 resection status, the possibility of sterilised tumour cells impacting the R0 margin interpretation, and a low number of patients. Tumour cells considered viable near the resection margin may subsequently senesce and cloud reliability of margin status in patients receiving radiotherapy. Fewer patients going to surgery raises the possibility of discrepant radiological evaluation of patients following SBRT. It is well known that SBRT can make response assessment difficult. The 18-month OS in the interventional arm was 21% lower than the control arm (68% versus 48%) and when assessed amongst patients who underwent pancreatectomy it was 14% lower (93% versus 79%). However, it is noted the Alliance A021501 study was not powered to measure OS and this difference is most likely explained by fewer patients making surgery, a critical component of the treatment paradigm in patients with BRPC with significant impact on survival.Pancreas SBRT is technically challenging even in high volume centres, with risks and consequences of geographical miss and/or exceeding normal tissue constraints. Therefore, MASTERPLAN mandates radiotherapy plan undergoing pre-treatment expert review, permitting corrections to suboptimal radiotherapy voluming and planning prior to treatment delivery. Contouring guidelines including an anatomic atlas, in addition to a planning and delivery manual were developed to further improve the standard of SBRT delivery. Patient eligibility for MASTERPLAN is much broader than that for A021501. MASTERPLAN includes patients with more earlier staging in the ‘high-risk’ cohort, and also those with less favourable anatomic features and with LAPC. Therefore, the applicability of MASTERPLAN patients to the general clinic may be greater.MASTERPLAN addresses an important clinical question in a very challenging and increasingly common disease.This prospective randomised phase II study addresses the efficacy of SBRT in addition to modern chemotherapy in pancreatic cancer patients. Results from this study will inform the need for a larger phase III study. Importantly, MASTERPLAN provides an exciting platform for tissue, blood and microbiome collection for significant translational research into this devastating disease.


Parallel changes of AMs and neutrophils in bronchoalveolar lavage (BAL) correlated with virus load when compared to macaques with mild influenza. Both AMs and IMs in lethal influenza were M1-type inflammatory macrophages which expressed neutrophil chemotactic factors, while neutrophils expressed genes associated with activation and generation of neutrophil extracellular traps (NETs). NETs were prominent in lung and were found in alveolar spaces as well as lung parenchyma. Genes associated with pyroptosis but not apoptosis were increased in lung, and activated inflammatory caspases, IL-1β and cleaved gasdermin D (GSDMD) were present in bronchoalveolar lavage fluid and lung homogenates. Cleaved GSDMD was expressed by lung macrophages and alveolar epithelial cells which were present in large numbers in alveolar spaces, consistent with loss of epithelial integrity. Cleaved GSDMD colocalized with viral NP-expressing cells in alveoli, reflecting pyroptosis of infected cells. These novel findings reveal that a potent interferon and inflammatory cascade in lung associated with infiltration of inflammatory macrophages and neutrophils, elaboration of NETs and cell death by pyroptosis mediates lethal H5N1 influenza in nonhuman primates, and by extension humans. These innate pathways represent promising therapeutic targets to prevent severe influenza and potentially other primary viral pneumonias in humans.Severe influenza kills tens of thousands of individuals each year, yet the mechanisms driving lethality in humans are poorly understood. Here we used a unique translational model of lethal H5N1 influenza in cynomolgus macaques that utilizes inhalation of small-particle virus aerosols to define mechanisms driving lethal disease. RNA sequencing of lung tissue revealed an intense interferon response within two days of infection that resulted in widespread expression of interferon-stimulated genes, including inflammatory cytokines and chemokines. Macaques with lethal disease had rapid and profound loss of alveolar macrophages (AMs) and infiltration of activated CCR2 Influenza can cause acute lung injury and death, but the mechanisms resulting in lethal influenza in humans are not well understood. We used a novel model of lethal influenza in nonhuman primates caused by aerosol infection with highly pathogenic avian influenza virus that closely resembles human disease to define how the virus causes severe pneumonia. We found that a potent innate immune response starting with high-level production of interferons and inflammatory factors in the lung drives severe disease. Inflammatory cells including macrophages and neutrophils were recruited into lung because of this early response, which in turn led to release of neutrophil extracellular traps that blocked lung alveoli. In addition, a particularly inflammatory form of cell death known as pyroptosis occurred in lungs during lethal influenza. These new findings show that an intense interferon response leading to an inflammatory cascade of macrophages and neutrophils, release of neutrophil extracellular traps, and cell death by pyroptosis is responsible for acute lung injury in lethal influenza. These innate pathways could be targeted by drugs to prevent lung injury in critically ill influenza patients. Human infection with highly pathogenic influenza viruses can produce primary viral pneumonia resulting in acute lung injury and death –4. The min vitro studies [in vivo and how this relates to pathogenesis is a central question that remains to be answered. Data on the innate cellular response to severe influenza are also incomplete. Alveolar macrophages (AMs) are an essential component of the innate response of the lung [There are several fundamental factors relating to the antiviral and innate responses to severe influenza in humans that remain ill-defined. Studies in nonhuman primates suggest that the severity of disease caused by infection with highly pathogenic H5N1 virus and the 1918 influenza virus is associated with attenuation of the antiviral interferon (IFN) response ,8, and t studies . In muri studies . Whetherthe lung , and depthe lung ,14. Recrthe lung –17. Howethe lung . Howeverthe lung ,22 and cthe lung –25. Finathe lung . Howeverthe lung .To date it has been difficult to address these fundamental questions relating to influenza pathogenesis in humans as the field has lacked a suitable translational model of lethal influenza. Influenza virus infection in nonhuman primates generally produces only mild clinical disease even when using a strain that is highly lethal in humans such as avian influenza (H5N1), with some exceptions ,10,28–3110 plaque forming units (PFU) of the highly pathogenic avian influenza virus human isolate A/Vietnam/1203/2004 (H5N1) by small-particle aerosol. All animals had severe influenza that progressed to ARDS and died or were humanely sacrificed between 2- and 6-days post infection, as detailed elsewhere [in situ hybridization , day 3 (135) and day 4 (138) , along wCXCL10 and CXCL13 and a cluster of IFN genes  were upregulated in response to infection, including inflammatory chemokines such as FN genes . More inFN genes . There wFN genes  and 2B. TM1 Figs  and S1 [TM1 Figs .++ CD206+, whereas IMs were non-autofluorescent CD163+CD206dim cells. The autofluorescence of AMs and the differential expression of CD206 were confirmed by inclusion of an isotype control in the place of CD206 antibody (10 PFU) and presented with only mild influenza. We found a strong negative correlation between the percent change of AMs in BAL at 2 days post infection and the titer of virus in BAL fluid at the same time point when comparing mild and lethal influenza . Using flow cytometry, we gated viable lung cells based on forward scatter and side scatter and then stained for expression of CD163 and CD206 to define macrophages based on previous studies ,38. In lantibody . AM and antibody . Moreoveantibody . In BAL antibody . To detenfluenza . IMs wernfluenza . To detenfluenza , indicatCCL2, CCL3, CXCL10 and CXCL11, in addition to marked upregulation of genes associated with neutrophil recruitment  and lethally infected animals (n = 5) and performing bulk RNA sequencing. AMs and IMs had differentially expressed genes that were either upregulated or downregulated in response to lethal infection, and between 32 and 44% of these genes were unique to each cell type, confirming that they were distinct macrophage populations . We usedruitment .–MHC-II–CD11b+ cells . To visualize neutrophils and NETs in lung tissue directly, we stained sections of lung from uninfected macaques and macaques with lethal H5N1 influenza with antibodies to calprotectin and citrullinated H3 histone, respectively. There were abundant neutrophils in the lung of macaques with lethal influenza, and widespread staining with antibody to citrullinated histone H3, some of which co-labeled with calprotectin, both in the lung parenchyma and in airways  and alveolar epithelial cells (cytokeratin+) in uninfected macaques and those with lethal influenza by immunohistochemistry. Negligible staining for IL-1β or cleaved GSDMD was observed in lungs from uninfected macaques. There was widespread expression of IL-1β in lungs from macaques with severe influenza, and much of this expression was in macrophages, whose density was significantly increased with lethal disease relative to uninfected animals  and the Institutional Biosafety Committee (protocol # 055–15) at the University of Pittsburgh. Macaques were observed twice daily and were given a combined clinical score based on body temperature, clinical appearance and respiratory signs at each observation. Samples were collected following sedation with ketamine hydrochloride, and virus infection was carried out under anesthesia with Telazol. If an animal had a combined score that reflected advancing and irreversible disease the macaque was humanely euthanized to ameliorate suffering. Euthanasia was achieved using pentobarbital sodium and phenytoin sodium injection followed by cardiac perfusion.Experiments with nonhuman primates were conducted in accordance with the National Institutes of Health recommendations in the Animal and biosafety level 3 (BSL-3) experiments were performed in the University of Pittsburgh Regional Biocontainment Laboratory, which is a registered entity with both the Center for Disease Control and Prevention as well as the U.S. Department of Agriculture for work with highly pathogenic avian influenza viruses. All personnel were required to wear appropriate personal protective equipment including powered air-purifying respirators (3M Versaflo) when working with infectious virus and infected macaques.Macaca fascicularis) of Mauritius origin from a group of seven that were infected by small-particle aerosol with an average of 6.72 log10 PFUs of highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1) strain and sacrificed or died between 2 and 6 days post infection due to ARDS [Tissues from six adult cynomolgus macaques  with β-mercaptoethanol and cell lysates stored at -80°C. Replicates of cell lysates were shown to be free of infectious virus by plaque assay prior to removal from the containment facility.Flow cytometry and antibody staining of cells in suspension were done as previously described , with thRNA extraction and NGS library preparation was performed in the Yerkes NHP Genomics Core. RNA was isolated using RNeasy Micro Kits (Qiagen) with on-column DNase Digestion (Qiagen). RNA quality and quantity were determined using a Bioanalyzer RNA Pico chip (Agilent). RNA was converted to cDNA and amplified using the Clontech SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio) according to the manufacturer’s instructions. Amplified cDNA was fragmented and appended with dual-indexed bar codes using the NexteraXT DNA Library Preparation kit (Illumina). Libraries were validated by capillary electrophoresis on an Agilent 4200 TapeStation, pooled at equimolar concentrations, and sequenced on an Illumina HiSeq3000 at 100SR, yielding 20–25 million reads per sample.2 FPKM values, cluster maps removed for ease and space.The quality of sequenced reads was assessed with FastQC. The Spliced Transcript Alignment to a Reference (STAR)  aligner Protein assay was performed using the Pierce Rapid Gold BCA Protein Assay kit (Thermo Fisher Scientific) on snap frozen lung lysate or BAL fluid. 25 ug of lysate or equal volumes of BAL fluid (to add 25 ug of the most concentrated sample) were prepared for each of the antibodies to be used in Bolt LDS sample buffer and Bolt sample reducing agent (Thermo Fisher Scientific) and denatured at 70°C for 10 minutes. Samples and SeeBlue Plus2 pre-stained protein ladder were run on Bolt Bis-Tris 4–12% gel (Thermo Fisher Scientific) and transferred to nitrocellulose. Blots were rinsed with water, imaged and de-stained with 1X tris buffered saline with 0.05% Tween 20 (TBS-T). Blots were then blocked with 5% milk and incubated with primary antibody followed by secondary antibody. Immunoblots were exposed to Super Signal West Pico plus or Fempto chemiluminescent substrate (Thermo Fisher Scientific) and exposed to Amersham HyperFilm ECL (Fisher) film for 1 second to 5 minutes and developed using SRX-101A Medical Film Processor (Konica Minolta). The following primary antibodies were used: caspase-4 , caspase-8 ; and GSDMDC1 . Separate gels were run for each of the antibodies using the same sample preparation on the same day with the same loading control.p value of <0.05 was considered significant. Comparisons between groups were two-tailed t-tests with Welch’s corrections. Comparisons of multiple groups was performed with a Kruskal-Wallis ANOVA, and if the null was rejected, further analysis was performed between groups using Dunn’s multiple comparisons test.All statistical analyses were performed in GraphPad Prism v8.0–9.0 (GraphPad Software). A S1 Fig2 FPKM values. Genes are separated by expression value. Grey boxes denote no expression.Heat maps of differentially expressed ISGs in lungs from uninfected macaques (n = 3) and macaques with lethal influenza . The panel of ISGs is based on a previous publication . Heat ma(TIF)Click here for additional data file.S2 FigPercent of total cells in bronchoalveolar lavage that is IM at pre-infection and 2 days post infection in animals with lethal influenza disease.(TIF)Click here for additional data file.S3 Fig2 FPKM values. Grey boxes denote no expression.Heat maps of differentially expressed genes previously identified as M2-macrophage genes in sorted AM and IM from uninfected macaques (n = 3) and macaques with lethal influenza . Genes expressed as log(TIF)Click here for additional data file.S1 TablePathways and associated genes shown in (XLSX)Click here for additional data file.S2 TablePathways and associated genes shown in (XLSX)Click here for additional data file.S3 TableGenes used to generate the volcano plot of (XLSX)Click here for additional data file.S4 TableCanonical pathways and associated genes for AMs shown in (XLSX)Click here for additional data file.S5 TableCanonical pathways and associated genes for IMs shown in (XLSX)Click here for additional data file.S6 TableGenes expressed in AMs used in Figs (XLSX)Click here for additional data file.S7 TableGenes expressed in IMs used in Figs (XLSX)Click here for additional data file.


Preterm birth (PTB) is a major cause of neonatal mortality and health complications in infants. Elucidation of its genetic underpinnings can lead to improved understanding of the biological mechanisms and boost the development of methods to predict PTB. Although recent genome-based studies of both mother and fetus have identified several genetic loci which might be implicated in PTB, these results suffer from a lack of consistency across multiple studies and populations. Moreover, results of functional validation of most of these findings are unavailable. Since medically indicated preterm deliveries have well-known heterogeneous causes, we have reviewed only those studies which investigated spontaneous preterm birth (sPTB) and have attempted to suggest probable biological mechanisms by which the implicated genetic factors might result in sPTB. We expect our review to provide a panoramic view of the genetics of sPTB. Globally, about 15 million preterm babies are born per year, among which around 3.5 million (23.4%) PTBs take place in India alone2. Neonates born early preterm (<34 weeks) have higher mortality and morbidity risks while the ones born late preterm (between 34 and 37 weeks) are at increased risk of health complications, such as, respiratory disorders, cardiovascular disorders, infections, feeding difficulties, visual and hearing problems and learning disabilities as compared to early term (37 to <39 weeks) and full-term (39–40 weeks) neonates4, which are likely due to incomplete development of fetal organs and systems. They are also at increased risk of late-onset disorders, such as obesity, metabolic syndrome, hypertension, and type 2 diabetes in early life5.Preterm birth (PTB) is defined as any live birth before 37 completed weeks of gestation and is the major cause of mortality in neonates and infants6.Clinically, PTB is classified into several subtypes Fig. . About 47, we have restricted our discussions to sPTB only.Being a multifactorial trait, both environmental and genetic risk factors contribute to the pathophysiology of PTB Fig. . Here, w9, twin-based11 and intergenerational studies13 have provided evidence of genomic factors underlying PTB. Estimates of heritability of PTB identified from twin studies range between 17% and 36%11. PTB is a clinical entity that is defined by the genomes of two individuals—the mother and the fetus. Findings of multiple epidemiological studies have suggested increased maternal genetic contribution to PTB either through the mother’s genome or maternally inherited variants in the fetal genome compared to those inherited paternally14. Another study reported that between the parents, mothers born preterm had a higher risk of delivering the first child at preterm : 1.42, 1.67) than the preterm-born fathers 15.In addition to the risk factors of PTB identified in various epidemiological studies (Supplementary Table Three main approaches have been used to identify genetic factors that contribute to the risk for sPTB which are discussed below.IGF1R) gene on 15q26.3 locus to be linked to sPTB16. A haplotype was also identified in this gene which showed co-segregation with sPTB in six out of the seven families and this was further validated in the Lund–Malamö dataset16. However, a subsequent association study based on SNPs in this candidate gene in the Finnish population (334 cases and 197 controls) could not identify any SNP associated with sPTB after permutation correction16. This might be due to the small sample size used. Although the investigators concluded that ligands of IGF1R that regulate IGF1R dependent signaling cascades are associated with sPTB16, how dysregulation of IGF1R signaling itself can be involved in sPTB was not addressed. A candidate gene-based association study on the Chinese population identified an SNP in IGF1R (rs2229765) which is associated with reduced risk of sPTB in women with GA or AA genotype as compared to the women with GG genotype17. A study in mouse and human breast tumor epithelial cell lines reported that downregulation of IGF1R expression increases cellular stress and cytokine (IL-6 and CCL2) production18. Since oxidative stress19 and proinflammatory cytokine-mediated inflammatory pathways20 have been previously reported to be involved in sPTB, hence downregulation of IGF1R expression might lead to sPTB through either or both of these pathways.Initially, genome-wide studies on mothers and infants were conducted using linkage analysis to identify genetic factors contributing to spontaneous PTB. However, none of these could identify any maternal genomic locus significantly linked to sPTB. Genome-wide linkage analysis of infants using autosomal markers in seven clinically characterized sPTB multiplex families from Northern Finland identified three intronic markers rs2684811, rs1521480, rs4966936 (heterogeneity LOD or HLOD > 3) within insulin-like growth factor 1 receptor  in exon 1 of androgen receptor (AR) gene in Xq12 to be over-represented among the preterm infants (P = 0.0006)21. Interestingly, it had been suggested earlier that molecular crosstalk between IGF1R and AR might lead to sPTB21. Later studies have shown that a longer polyglutamine chain in the N-terminal transactivation domain of AR hinders transactivation activity22 resulting in decreased AR signaling which leads to apoptosis via activation of Caspase-323. Since apoptosis in the placenta is involved in sPTB24, decreased AR signaling might lead to sPTB via apoptosis in the placenta.A significant linkage signal was identified on Xq13.1  for sPTBPAI-2) gene was suggested as an important candidate gene in this region25 as this gene was found to be significantly associated with sPTB in the Australian population earlier26. Subsequent studies in mice models revealed that Pai-2 plays an important role in inducing autophagy by enhancing the expression of Beclin1 which induces autophagosome formation28. Autophagy being associated with reduced PTB29, alteration of PAI-2 expression might modulate sPTB risk by effecting change in autophagy.Genome-wide linkage analysis using microsatellite markers in 288 Mexican-American families found 18q21.33-q23 locus (LOD = 3.6) to be significantly linked to sPTB. The plasminogen activator inhibitor type 2  and were either replicated or functionally validated  and EEFSEC (rs201450565 and rs200745338) genes, to be significantly associated (P < 5 × 10−8) with sPTB. The results were replicated in 8643 women in three Nordic datasets among which 2565 women delivered preterm. Association of variants in AGTR2 (rs201386833 and rs5950506) with sPTB achieved genome-wide significance (P < 5 × 10−8) in a joint analysis of discovery and replication sets. Along with loci in these three genes, the association of WNT4 variants with gestational duration also achieved genome-wide significance level both in the discovery and replication datasets31. The investigators concluded from the functional studies that the implicated variant in WNT4 results in longer gestational duration, suggestive of protecting women from delivering preterm31. However, functional analyses of none of the variants associated with sPTB were performed. The biological relevance of the genes found associated with sPTB is discussed below.A two-stage genome-wide association study, which used data from 43,568 women of European ancestry of whom 3331 women had spontaneous preterm deliveries, found the maternal loci in EBF1, is essential for normal B-cell development31. Low EBF1 mRNA in the second and/or third trimester is significantly associated with an elevated risk of PTB which might be due to the involvement of maternal-fetal immune pathway and/or apoptosis32 of the fetal membranes leading to their early rupture.Early B-cell factor 1 encoded by EEFSEC encodes eukaryotic elongation factor selenocysteine tRNA-specific protein, which incorporates essential micronutrient selenocysteine, into selenoproteins. Selenoproteins maintain cellular redox status by antioxidant defenses and also modulates inflammatory responses31. Moreover, it plays an important role in reproduction33. Earlier studies have linked these physiological processes to PTB31. Alteration in EEFSEC expression may lead to sPTB via any of these pathways.AGTR2 encodes angiotensin II receptor type 2 which modulates uteroplacental circulation31. rs7889204 is an intergenic variant located near AGTR2. Decreased binding of CEBPB (CCAAT Enhancer Binding Protein Beta) and HOXA10, a homeobox protein, to rs7889204-C allele results in decreased AGTR2 expression which might lead to sPTB34. Since higher uterine and umbilical artery resistance indices across gestation were found to be associated with sPTB35, decreased AGTR2 expression might result in compromised uteroplacental circulation ultimately leading to sPTB.SLIT2) gene to be strongly associated with sPTB (P = 1.6 × 10−6). It was also overrepresented in 172 very preterm born infants (less than 32 weeks) (P = 1.55 × 10−7). This SNP also exhibited a highly significant association with gestational age at delivery (P = 3.1 × 10−7). Silencing of SLIT2 and its receptor roundabout homolog 1 (ROBO1) gene in trophoblasts resulted in the downregulation of proinflammatory cytokines36. As upregulation of proinflammatory cytokines stimulates uterine myometrial contractions, it was proposed that upregulation of SLIT2 and ROBO1 might result in premature activation of uterine myometrium and sPTB36.A GWAS conducted on 247 infants with spontaneous PTB (less than 36 weeks) and 419 term controls (38–41 weeks) of Finnish descent identified rs116461311 in Slit homolog 2 ) which even surpassed Bonferroni corrected p value37. CR1 encodes complement C3b/C4b Receptor 1 which is located on the surface of the erythrocytes. Although normally erythrocyte sedimentation rate (ESR) increases in pregnancy to clear immune complexes from the walls of blood vessels, this SNP is associated with decreased ESR indicating that the risk promoting allele might lead to greater systemic inflammation due to non-clearance of immune complexes37.Whole exome sequencing (WES) of two mother-daughter pairs from Finnish multiplex families with sPTB identified novel missense variants in genes belonging to the complement cascade pathway. This pathway was also found to be associated in exome analysis of six additional mothers delivering preterm babies. Genes from this pathway were investigated for association analysis in Finnish nuclear families (237 cases and 328 controls), resulting in the identification of three SNPs in P < 1.7 × 10−8). This pathway was replicated in ninety-three Danish sister pairs. Missense variants in HSPA1L had a population-specific and family-specific effect in the manifestation of sPTB, which is generally a characteristic feature of rare variants with large effect sizes. A non-synonymous variant rs34620296 (Ala268Thr), identified in four Finnish families, generates an additional phosphorylation site near the nucleotide-binding site of HSPA1L (a member of the HSP70 family) affecting its interaction with ADP or its own stability38, reducing its chaperone activity39. The Hsp70 and Hsp90 chaperones as well as their co-chaperones form a heteromeric complex. The binding of this complex to the GR is essential for the maturation of GR40. GR signaling pathway cross-talks with estrogen signaling41 and progesterone signaling42 pathways for efficient regulation of the pro- and anti-inflammatory microenvironment in reproductive tissues during pregnancy. Impairments in the GR signaling pathway might result in a shift from anti-inflammatory to the pro-inflammatory microenvironment, causing premature activation of labor43. Interestingly, a nonsynonymous HSPA1L SNP, rs2075800, was previously found to be associated with sPTB in African-American women44. Furthermore, a meta-analysis using pathway analysis indicated an association of HSPA1L with sPTB45. This indicates an important role of this gene in spontaneous PTB via the glucocorticoid signaling pathway.A recent WES on seventeen mothers from seven Finnish multiplex families with recurrent sPTB, identified rare variants in the genes in the glucocorticoid receptor (GR) signaling pathway (RAB31 and RBPJ with very early PTB (<28 weeks) in European and American population respectively even after adjusting for population stratification46. Possible mechanisms by which alterations of these genes lead to PTB are as follows:With the advancement in “omics” technologies, significant progress has been achieved in the identification of biological correlates of complex phenotypes. Since individually each of these technologies is unlikely to capture the entire biological complexity of complex phenotypes, integration of the findings from multi-omics approaches is recommended. Integration of whole-genome sequencing, RNA-sequencing, and DNA methylation results from more than 600 families of mixed ancestry resulted in the identification of association of 47. EGFR signaling plays an important role in cellular proliferation, differentiation, migration, and also angiogenesis48. Since disruption of placental angiogenesis leads to PTB49, altered RAB31 expression may lead to altered EGFR signaling resulting in PTB via disrupted placental angiogenesis.RAB31, a member of Ras GTPases, plays a key role in the internalization of ligand-bound EGFR  from the cell membrane into endosomes through receptor-mediated endocytosis50, ultimately leading to preterm labor51.RBPJ  is an important transcriptional regulator of the Notch signaling pathway. Premature activation of Notch signaling might increase the inflammatory response upon the enhancement of NF-ĸβ (nuclear factor kappa beta) activity16. Although a case-control study of mothers and infants belonging to Danish and Norwegian populations failed to detect any association between polymorphisms in mitochondrial genome and PTB52, a retrospective cohort study on Dutch women found that those harboring the mitochondrial mutation mt.3243A>G in MT-TL1 have a higher risk of PTB (25.3%) as compared to the general Dutch population (7.4%). Out of 25.3% of women who delivered preterm, about half of them experienced spontaneous preterm delivery53. MT-TL1 encodes for a tRNA (Leu-UUR) which plays a critical role in the translation of proteins essential for the assembly and function of the mitochondrial complexes in the oxidative phosphorylation pathway54. Increased ROS production due to dysregulation of respiratory chain complexes might lead to placental dysfunction56 resulting in adverse birth outcomes58.Since the mitochondrial genome is maternally inherited, the enhanced maternal genetic contribution to PTB suggests an association of mitochondrial DNA variants with PTBP = 0.046) in the GPN dataset. This finding was replicated in BPD/HRS dataset where 1724 cases were from the BPD dataset and 12,507 controls were from the HRS dataset59. However, it could not be replicated in the GENEVA dataset (1035 cases and 508 controls)59. Since mtDNA and nuclear genomic polymorphisms that are involved in the formation of multi-subunit complexes of electron transport chain have co-evolved, mitochondrial haplogroup containing markers of one ancestry in the background of a nuclear genome with different ancestral origin might result in impaired mitochondrial potential which may partially explain the elevated risk of PTB in African-American population59.Another study found a greater degree of divergence of mitochondrial ancestry in infants born preterm as compared to those born at term  in subcutaneous adipose tissue. This study was conducted on 1733 African-American women (698 cases and 1035 controls) from the Boston Birth Cohort60. It is the first study that not only explored genome-wide gene–environment interactions in PTB by offering insights into the “missing heritability” of sPTB but was also able to replicate the findings in the same ethnic population from the GPN dataset deposited in dbGaP60. However, this finding could not be replicated in the Caucasian population60 which might suggest a population-specific role of this SNP in sPTB. Women with normal body weight and harboring rs11161721-AA genotype and obese women with rs11161721-CC genotype were found to be at elevated risk for sPTB. rs11161721-A allele was reported to be associated with higher COL24A1 expression than rs11161721-C allele in subcutaneous adipose tissue (P = 8.2 × 10−8). COL24A1 encoded collagen type XXIV alpha 1 protein regulates type I collagen fibrillogenesis during fetal development. Type I collagen being a component of the extracellular matrix (ECM), influences the functions of many cells and organs. Altered COL24A1 expression may impact the proper functioning of ECM resulting in pathological events which might lead to sPTB60.Analysis of genome-wide gene–environment interactions identified maternal 63 and the results were found to be inconsistent across different studies conducted even in the same population. In some cases, the association did not reach genome-wide significance65 possibly because of a lack of statistical power. In addition, most of the studies which have analyzed genomics of sPTB as a dichotomous trait have excluded the borderline gestational ages to avoid misclassification. This might have led to inconsistency in defining preterm and term births across these studies.sPTB being a multifactorial trait involving both the mother and the fetus, genetic studies of sPTB should consider contributions of both maternal and fetal genomes, as well as gene–environment interactions since individual’s genotypes interacting with both the external and in utero environmental factors, may have the potential to modify the risk. Although multiple genomic variants have been reported to be associated with sPTB, many of these associations could not be replicated66. As a result, the utility of extrapolation of the findings in animal models to the human system is uncertain. Alternatively, appropriate cell lines of human origin might provide a better choice. For evaluation of the effect of genotypes, specific analytes can be measured in maternal peripheral blood or umbilical cord blood and amniotic fluid as well.Functional validation of these discoveries is essential for a deep insight into the role of the implicated variants in sPTB since the biological effects of the variants may not be always mediated through the nearest genes. The human placenta is a unique organ due to the presence of numerous human-specific molecular features67. Such integrative-omics approaches might help to identify the biological mechanisms through which inter-individual variation results in sPTB.Although PTB is a global phenomenon, most studies have been conducted on families and individuals belonging only to populations of European and African-American ancestries. These studies can be expanded to other populations, emphasizing those who have a substantial prevalence of sPTB. Recently, for improved prediction of a person’s genetic susceptibility to common chronic diseases, polygenic risk scores (PRS) are being calculated using genome-wide genetic data which can be applied to sPTB as well. But the main concern with PRS is that it cannot yet predict effectively in non-European populations. GARBH-Ini, a cohort of pregnant women has been established in India to provide insights into the biological correlates of PTB using an integrative-omics approachEarly detection of the risk of PTB is invaluable to combat this global health burden of adverse pregnancy outcome. As compared to biochemical markers, genomic markers can identify high-risk women even prior to pregnancy. The studies discussed by us were those where findings were either reproducible in a different set of participants or were validated functionally. Our selection criteria, while ensuring that we reviewed only those results which are unlikely to be false positive, might have also resulted in exclusion of findings that are of biological relevance without either attaining statistical significance or which could not be replicated or biologically validated. With improved phenotyping of sPTB viz. premature onset of labor, PPROM, cervical insufficiency and further functional validation of genetic associations, the findings of the studies as those included in the review are expected to contribute towards better prediction of sPTB and development of targeted clinical interventions to reduce the same.Supplementary Information


Tritium gas is the preferred source of tritium for the preparation of labelled molecules because it is available in high isotopic purity2. The introduction of tritium labels from tritium gas is commonly achieved by heterogeneous transition-metal-catalysed tritiation of aryl (pseudo)halides. However, heterogeneous catalysts such as palladium supported on carbon operate through a reaction mechanism that also results in the reduction of other functional groups that are prominently featured in pharmaceuticals3. Homogeneous palladium catalysts can react chemoselectively with aryl (pseudo)halides but have not been used for hydrogenolysis reactions because, after required oxidative addition, they cannot split dihydrogen4. Here we report a homogenous hydrogenolysis reaction with a well defined, molecular palladium catalyst. We show how the thianthrene leaving group—which can be introduced selectively into pharmaceuticals by late-stage C–H functionalization5—differs in its coordinating ability to relevant palladium(II) catalysts from conventional leaving groups to enable the previously unrealized catalysis with dihydrogen. This distinct reactivity combined with the chemoselectivity of a well defined molecular palladium catalyst enables the tritiation of small-molecule pharmaceuticals that contain functionality that may otherwise not be tolerated by heterogeneous catalysts. The tritiation reaction does not require an inert atmosphere or dry conditions and is therefore practical and robust to execute, and could have an immediate impact in the discovery and development of pharmaceuticals.Tritium labelling is a critical tool for investigating the pharmacokinetic and pharmacodynamic properties of drugs, autoradiography, receptor binding and receptor occupancy studies The isotopic label tritium can be selectively added into aromatic organic compounds by a homogenous hydrogenolysis reaction using aryl thianthrenium salts, tritium gas and a molecular palladium catalyst. However, many reliable hydrogenation or hydrogenolysis reactions cannot be suitably used for tritium labelling owing to a lack of reagents, low molar activity, restricted functional-group tolerance or safety concerns. Tritiated water (3H2O) is problematic owing to fast washout of the label from omnipresent water and safety concerns regarding the potential fast uptake of radioactive water by experimentalists. The preferred source of tritium labels is tritium gas (3H2), which is available in high isotopic purity and practical to handle with commercially available manifolds on the small scale typically used for labelling7.Tritium halides, dihydrogen oxidative addition is commonly faster, which results in metal hydrides in higher oxidation states that are not suitable for aryl (pseudo)halide oxidative addition14. Therefore, for the hydrogenolysis of carbon–heteroatom bonds15, chemists select heterogeneous catalysts, such as palladium supported on carbon, which can effectively reduce aryl(pseudo)halides through a mechanistically distinct pathway17. The reactivity of the active hydrogen chemisorbed on the catalyst surface results in low chemoselectivity, and the undesired reduction of other functional groups typically found in pharmaceuticalshalides through oxidative addition do not engage with dihydrogen in a second oxidative reaction to form metal hydrides. Heterolytic cleavage of the hydrogen–hydrogen bond can also result in the formation of metal hydrides29. For example, electrophilic transition-metal complexes of iridium30 and ruthenium31 can substantially acidify dihydrogen through complexation owing to a strong σ donor–acceptor interaction32, which can result in the formation of metal hydrides through deprotonation of the metal η2-dihydrogen complexes33. Only a few examples of palladium (Pd) dihydrogen complexes are known4, and no electrophilic Pd(II) catalysts—generated through oxidative addition of an aryl (pseudo)halide to low-valent palladium—have been shown to heterolyse dihydrogen36. Upon oxidative addition to a ligated Pd(0), no appropriate coordination site is available at square planar d8 Pd(II) tetracoordinate complexes for dihydrogen coordination because (pseudo)halides outcompete dihydrogen from coordination to Pd(II)  Fig. . Aryl thlts Fig. . The abilts Fig. .2H2 as a more convenient and safer 3H2 surrogate. Palladium-catalysed hydrogenolysis of biphenyl-derived thianthrenium salt 1-TT with 2H2 afforded the desired 2H-labelled product [2H]1 with more than 99% deuterium (D) incorporation , which is consistent with an open coordination site on the metal centre for interaction and coordination of H2 during catalysis. As measured by infrared spectroscopy, even triflate outcompetes thianthrene from coordination to Pd(II)  additives did not result in a productive reaction as observed with the arylthianthreniums, which may be due to the incompatibility of oxidizing Ag(I) and the presumed in situ-generated palladium hydride , electron-neutral  and electron-rich arenes , as well as arenes bearing ortho substituents  successfully participate in Pd-catalysed reductive deuteration , carbamates (5), amides , heterocycles  and acid-sensitive glycosidic linkages (21). In contrast to heterogeneous catalysis, the homogeneous catalyst system tolerates reducible functional groups such as aryl (pseudo)halides , nitriles (3), alkenes , esters , aldehydes (23), sulfones (26), nitro groups (19) and benzyl ethers (23). Primary amines must be protected. No 1H–2H scrambling was observed when the reaction was performed in the presence of water, and high levels of deuterium incorporation were detected throughout. When compared with heterogeneous deuterodehalogenation, where significant isotopic scrambling can occur43, the new catalytic platform with aryl thianthrenium salts provides a viable approach for the synthesis of deuterated and tritiated drug molecules (vide infra) with high isotopic purity in a practical setting that does not require the rigorous absence of water or oxygen.(Tetrafluoro)thianthrenium  salts of electron-deficient , pyridines , pyrazoles (18), quinolines (22), anilines  and thiophenes (17) is non-trivial. We observed that the appropriate choice of counterion for the arylthianthrenium salt starting material can reduce its solubility in tetrahydrofuran (THF), with the reservoir of starting material in the solid phase, leaching material for conversion as the reaction progresses. The ability to tune solubility is another yet-unappreciated advantage of the arylthianthrenium salts when compared with arenes with conventional leaving groups that lack such a handle.Given our design of electrophilic, cationic Pd(II) species for H3H2 gas to reduce the risk of tritium gas leakage and with a higher loading of catalyst to achieve faster reaction rates . In all cases, high, predictable positional selectivity enables single, well defined, labelled molecules that are generally not accessible with hydrogen isotope exchange reactions unless directing groups are used.Translation of the hydrogenolysis reaction to tritium labelling was accomplished on micromole scales at a subatmospheric pressure of tes Fig. . No spectBu3)]2 during catalysis is consistent with the observed reaction order of 0.5 in Pd[(PtBu3)]2. We determined a primary kinetic isotope effect (KIE) of kH/kD = 3.1, where k is the rate constant, by measuring the initial rates of independent reactions with H2 and 2H2, respectively, at the same pressure, and an equilibrium isotope effect of 1.1 from an intermolecular competition experiment between H2 and 2H2 present at the same partial pressure, which is inconsistent with Ar–TT+ oxidative addition preceding dihydrogen binding but consistent with reversible dihydrogen association before oxidative addition to Ar–TT+. The observed first order in Ar–TT+ and positive order in H2 are in agreement with this proposal. Although we cannot exclude dihydrogen oxidative addition from A based on our kinetic data, it would need to be reversible, as indicated by the competition KIE, and the Pd(0) intermediate A could be expected to react with Ar–TT+ faster in concerted oxidative-addition or single-electron-transfer pathways than a putative off-cycle Pd(II) dihydride. Irrespective of the exact mechanism of irreversible—measured zero-order in TFT—oxidative addition, which may also differ depending on the ancillary ligand(s), intermediate B distinguishes the proposed pathway from those that would be accessible with other aryl(pseudo)halides. No H/D scrambling into the product was observed when hydrogenolysis was performed in the presence of H2O halides, the thianthrenyl group can be introduced selectively into complex small molecules. We show here how the lack of strong coordination to palladium and the intrinsic solubility properties enables thianthrenium chemistry to solve the challenge of homogeneous palladium-catalysed hydrogenolysis for the chemoselective synthesis of tritium-labelled small molecules.See Supplementary Information for further methods.Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-021-04007-y.Supplementary InformationThis file contains detailed experimental procedures and spectroscopic data, and includes 11 supplementary tables and 33 supplementary figures.Peer Review File