On behalf of the Canadian Association of Medical Oncologists, we are pleased to present the abstracts of the 2022 Annual Meeting. The CAMO Virtual Annual Scientific Meeting (ASM) took place on 28 April 2022. Twenty-five (25) abstracts were selected for presentation as oral presentations and poster presentations. Awards for the top three (3) abstracts were presented during the ASM. All of them are marked as “Award Recipient”. We congratulate all the presenters on their research work and contribution. Award RecipientCharacterization of Immune-Related Adverse Events and Their Impact on Outcomes in Metastatic Melanoma (MM) Treated with Anti-PD-1 Alone or in Combination with Anti-CTLA-4 ImmunotherapyAlexander Watson 1, Siddhartha Goutam 2, Igor Stukalin 3, Benjamin W Ewanchuk 4, Michael Sander 4, Daniel E Meyers 3, Aliyah Pabani 1, Winson Cheung 1, Daniel Heng 1, Tina Cheng 1, Jose G Monzon 1, Vishal Navani 11 Tom Baker Cancer Centre, Department of Oncology, University of Calgary, Calgary, AB, Canada2 Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada3 Department of Medicine, University of Calgary, Calgary, AB, Canada4 Cumming School of Medicine, University of Calgary, Calgary, AB, Canadaalexander.watson@albertahealthservices.ca* Correspondence: ObjectiveImmune-related adverse events (irAEs) due to immune checkpoint blockade (ICB) have been shown to correlate positively with survival outcomes in multiple solid tumors. For MM, data have been more conflicting, and combination ICB therapy with ipilimumab/nivolumab, classically associated with increased irAE toxicities, is supported by limited real-world evidence. We sought to characterize irAE and resulting outcomes using the Alberta Immunotherapy Database (AID).MethodsThe AID database is a multi-center, province-wide, retrospective dataset that includes consecutive patients treated with ICB. We included adult patients with MM from August 2013 to May 2020. Baseline characteristics between patients that developed significant irAEs (requiring systemic steroids and/or treatment delay) and those that did not were compared using the chi-squared test. To reduce immortal time bias, a 12-week landmark was applied for overall survival (OS), compared via log-rank test. Cox regression analyses were performed to determine the association of variables with OS.Resultsp = 0.02), with ECOG 0 (p < 0.001), while tumor characteristics were not associated. Patients with irAEs had longer median OS , which was maintained in patients treated with combination ICB . Type of irAE did not impact OS. Favourable association of irAE development with OS was confirmed in multivariable Cox analysis .Of 492 MM patients, 364 received single-agent ICB (pembrolizumab or nivolumab), while 124 received combination ICB. A total of 87 patients (70.2%) on combination ICB experienced irAEs, and 35 (28.2%) required hospitalization, compared to 30.2% and 8.7%, respectively, for single ICB. IrAEs were more common in patients <50 yo  ChemotherapySara Moore 1, Deepro Chowdhury 1, Mathieu Mckinnon 2, Paul Wheatley-Price 11 The Ottawa Hospital Research Institute, Ottawa, ON, Canada2 University of Ottawa, Ottawa, ON, Canadasmoore@toh.ca* Correspondence: ObjectiveExplore the use of second-line therapy for small-cell lung cancer (SCLC) in the real world, with a focus on comparing platinum–etoposide (PE) rechallenge to cyclophosphamide-doxorubicin-vincristine (CAV) chemotherapy.MethodsWe reviewed all patients diagnosed with SCLC at the Ottawa Hospital from January 2014 to December 2018 and identified those treated with second-line systemic therapy. Baseline demographics, diagnostic, and treatment information were collected. The primary outcome was overall survival after platinum rechallenge compared to CAV.Resultsn = 105, 99%). At the time of second-line therapy: 73 (69%) had ECOG performance status (PS) 0–1, and 76 (72%) had chemosensitive disease with a treatment-free interval (TFI) > 90 days. Second-line regimens included platinum–etoposide , CAV , platinum–irinotecan , docetaxel , and other . Median overall survival from the start of second-line chemotherapy was 6.2 months (m).During the study period, 539 patients were diagnosed with SCLC, 446 (83%) received first-line systemic therapy, and 106/446 (24%) went on to receive second-line systemic therapy. Baseline characteristics at initial diagnosis of this second-line cohort: median age 64.5, 60 (57%) female, 105 (99%) former/current smoking history, 90 (85%) extensive stage. First-line treatment was almost exclusively platinum-based (p = 0.004). PE chemotherapy led to higher physician-assessed clinical benefit  and overall survival (median OS 9.0 m versus 3.8 m) compared to those receiving CAV. In a sensitivity analysis, the survival difference persisted in patients with TFI < 180 days.Comparing patients who received PE rechallenge versus CAV, there were no significant differences in baseline characteristics including age, sex, smoking history, stage at diagnosis, and ECOG PS. Patients receiving CAV were more likely to have a TFI < 180 days  resulted in an abrupt transition to virtual oncology care worldwide. This study’s objective was to evaluate the quality of systemic therapy in patients receiving chemotherapy for colorectal cancer before and during the COVID-19 pandemic.MethodsThis observational study evaluated patients treated with intravenous chemotherapy for colorectal cancer consecutively at the Ottawa Hospital Cancer Centre from June 2019 to September 2021. Non-metastatic rectal cancers were excluded. Patients were stratified by whether they started chemotherapy pre-pandemic (June 2019–January 2020) versus intra-pandemic (February 2020–September 2021). Baseline characteristics and treatment data were collected from electronic medical records. Outcomes of interest included chemotherapy delays, dose reductions, emergency department (ED) visits and hospitalizations. We used generalized linear and binary logistic regression modelling to compare outcomes between pre- and intra-pandemic periods.Resultsp < 0.001) and 37% to 84% (p < 0.001), respectively.There were 220 patients included in this study, with 108 (49%) diagnosed with metastatic disease. In total, there were 66 (30%) patients treated in the pre-pandemic and 154 (70%) in the intra-pandemic period. Virtual consultations and follow-up visits increased during the pandemic from 1.5% to 43.5% (p = 0.78), dose reductions , ED visits , hospitalizations  or total length of treatment breaks  between the pre- and intra-pandemic periods by multivariable analysis. Subgroup analysis showed no difference in outcomes independent of the presence of metastases. There was no difference in the incidence of treatment delays , the role of local ablative radiation therapy (LAT) for oligoprogressive disease (OPD) on targeted therapy was first described by our group for EGFR- and ALK-positive cases. Here, we present an enlarged dataset, together with an extension of the analysis to other driver oncogenes.MethodsEGFR mutations, ALK rearrangements, ROS1 rearrangements and BRAF V600E mutations who had OPD on the respective tyrosine kinase inhibitor (TKI) and were treated with LAT was performed. OPD was defined as disease progression in ≤5 sites. Clinical characteristics, treatment regimens and treatment outcomes, including progression-free survival (PFS) and duration of treatment (DOT), were captured. PFS1 was defined as time from initiation of TKI-containing regimen to the first course of LAT for OPD. Subsequent PFS times  were defined as time from prior LAT to subsequent LAT or switch of systemic therapy, whichever occurred first. DOT was defined as time from initiation of TKI-containing regimen to switch of systemic therapy.A single-center, retrospective review of patients with metastatic NSCLC harboring ResultsEGFR mutations, 19 with ALK rearrangements, 6 with ROS1rearrangements and 4 with BRAF V600E mutations. A total of 103 systemic therapies containing a TKI and 159 courses of LAT were recorded. Median line of therapy of TKI was 2 (range 1–6) and median LAT courses per systemic regimen was 1 (range 1–4). There were 50, 108 and 1 LAT treatment courses for central nervous system (CNS) OPD, extra-CNS OPD and both CNS and extra-CNS OPD, respectively. For extra-CNS LAT, the median number of sites of radiation per LAT was 1 (range 1–5). PFS1 was 9.1 months, and DOT was 17.3 months, implying a total extension of ~8 months on systemic therapy with at least one course of LAT. Median PFS per course of LAT was 4.8 months (95% CI 3.3–6.3 months). Subgroup analysis showed a similar benefit for each individual driver oncogene. Median PFS was longer for CNS LAT than extra-CNS LAT . For extra-CNS LAT, 12-month PFS was 12.2%, 6.1%, 0% and 0% for 1, 2, 3 and 5 sites, respectively; 24-month PFS was 8.7%, 0%, 0% and 0% for 1, 2, 3 and 5 sites, respectively.A total of 75 patients were identified, including 46 with ConclusionsLAT is effective in prolonging treatment duration for TKI-containing systemic therapy in oncogene-addicted NSCLC. The PFS extension is greater for CNS LAT compared to extra-CNS LAT. Prolonged PFS can be achieved in a subset of patients who have a single site of OPD.Nivolumab in Squamous Cell Carcinomas of the Head and Neck (SCCHN): A Real-World Outcome Study in Ontario, CanadaArman Zereshkian 1, Ruaa Shafi 2, Gregory Pond 2, Sebastien J. Hotte 21 Department of Medicine, McMaster University, Hamilton, ON, Canada2 Department of Oncology, McMaster University, Hamilton, ON, Canadaarman.zereshkian@medportal.ca* Correspondence: ObjectiveThe CheckMate 141 trial led to the approval of nivolumab in platinum-resistant, metastatic squamous cell carcinomas of the head and neck (SCCHN). We evaluated outcomes of SCCHN patients in Ontario, Canada, treated with nivolumab since its funding through the New Drug Funding Program (NDFP) of Cancer Care Ontario (CCO).MethodsA retrospective review using the provincial treatment registry (CCO NDFP) was undertaken. Patient characteristics, reason for treatment, treatment length, and date of death were collected. The Kaplan–Meier method was used to estimate overall survival and Cox regression to evaluate the prognostic effect of selected factors.Resultsp = 0.12). Patients with a lower BSA (<1.81) had a mOS of 3.9 months versus 9.0 months for those with a higher BSA, HR = 0.12 (CI 0.04–0.39). Differences between indications were statistically significant (p < 0.001). Patients who received nivolumab for I1 had a mOS of 7.2 months versus 11.9 months for I3, HR = 1.73 (CI 0.94–3.16). Patients who received nivolumab for I2 had a mOS of 3.9 months versus 11.9 months for I3, HR = 3.27 (CI 1.80–5.94).A total of 134 patients with SCCHN received nivolumab. Median patient age was 63 years, with 80.6% being male. Nivolumab was used as second-line therapy after disease relapse within 6 months of curative-intent platinum chemotherapy (PC) in 39.6% (Indication 1—I1), used as second-line therapy post PC with non-curative intent in 42.6% (Indication 2—I2), and used as first-line therapy with non-curative intent due to contraindication for PC in 17.2% of patients (Indication 3—I3). Median overall survival (mOS) was 5.8 months, and one-year survival proportion was 28.4%. HPV status had no statistically significant impact on mOS  include first-line immune checkpoint inhibitors with chemotherapy and second-line lurbinectedin. In the ATLANTIS study, lurbinectedin/doxorubicin did not improve overall survival (OS); however, patients with a chemotherapy-free interval (CFI) of ≥180 days appeared to have an improved progression-free survival. The objective of this study was to identify the proportion of real-world SCLC patients who may be suitable for lurbinectedin/doxorubicin and to examine their characteristics.Methods2 and Mann–Whitney U tests.A retrospective study of all SCLC cases referred to BC Cancer between 2012 and 2017 was conducted. BC Cancer is a provincial health care program that oversees cancer management for a population of 5.2 million. Patient demographics, staging, treatment, and survival information was collected. Baseline characteristics were compared using descriptive statistics including the XResultsp = 0.97), male 45%/45% (p = 0.97), median pack years 40/44 (p = 0.14), limited stage 25%/50% (p < 0.001), OS from initiation of second line 4.2/8.6 months (p < 0.001).A total of 1062 patients were identified. Baseline characteristics: median age 68, male 53%, never/former/current smoker 23 (2%)/381 (36%)/648 (61%), median pack years 50, limited stage 339 (32%), extensive stage 722 (68%). Systemic therapy was delivered to 821 (77%): first-line treatment was platinum doublet 748 (91%), single agent 65 (8%), CAV 4 (0.5%), other 4 (0.5%). A total of 173 patients received second-line treatment, and 60 received ≥3 lines. A total of 225 patients received first-line platinum doublet followed by ≥2 lines, and the CFI was ≥180 days for 104 patients. Characteristics by CFI < or ≥180 days: median age 66/63.5  are at risk of developing chemotherapy-induced neutropenia (CIN). We assessed survival outcomes in patients who develop CIN in this setting.MethodsWe performed a retrospective chart review of patients with CRC treated with FOLFOX at our institution in Canada from 2013 to 2015. Demographic, treatment, and outcome data were collected. CIN was defined as ANC < 1.5, and all episodes of neutropenia were assumed to be the result of chemotherapy. Median OS was calculated using Kaplan–Meier product limit estimates.ResultsA total of 302 patients were included . In the overall cohort, 174 (58%) patients had ≥1 episode of CIN. CIN occurred in 43% of those with low-risk stage III cancer  and 45% of those with high-risk stage III cancer (T4/N2). Among patients with ≥1 episode of CIN, 79 (45%) received subsequent granulocyte colony-stimulating factor (GCSF).p = 0.43). The median OS for patients with CIN treated with and without GCSF was not reached, HR 1.02 . The 5-year survival rate for patients with and without CIN was 87% vs. 77%. The 10-year survival rate for patients with and without CIN was 70% vs. 64%. A trend toward improved survival in those with CIN remained when results were analyzed by cancer stage.For patients with and without CIN, the median OS was not reached, HR 0.84  ≤60 is Strongly Associated with Shorter Brain-Specific Progression-Free Survival among Patients with Metastatic Breast Cancer with Brain MetastasesMark C. Freeman 1, Marguerite Ennis 2, Katarzyna J. Jerzak 1,3,41 Faculty of Medicine, University of Toronto, Toronto, ON, Canada2 Applied Statistician, Markham, ON, Canada3 Sunnybrook Research Institute, University of Toronto, Toronto, ON, Canada4 Sunnybrook Odette Cancer Centre, Department of Medicine, University of Toronto, Toronto, ON, Canadamarkchaim.freeman@mail.utoronto.ca* Correspondence: ObjectiveTo examine the association between Karnofsky performance status (KPS) and brain-specific progression-free survival (bsPFS) among patients with breast cancer brain metastases (BCBrM).MethodsUsing a previously compiled retrospective cohort of 683 patients who were treated for BCBrM with surgery and/or radiotherapy at the Sunnybrook Odette Cancer Centre from 2008 to 2018, electronic records were reviewed to impute KPS scores at the time of BCBrM diagnosis. Patients were then grouped into KPS ≤60 and KPS >60 cohorts. The dataset was analyzed to identify variables that were prognostic for bsPFS and/or overall survival (OS) under univariable and multivariable Cox proportional hazards models.Resultsn = 622, 91%) had extracranial metastatic disease, and 174 (25%) had leptomeningeal disease. A total of 247 patients (36%) had hormone-receptor-positive/HER2-negative tumors, 189 (28%) had HER2-positive disease, and 153 (22%) had triple-negative breast cancer. Of the 331 patients (48%) who could be assigned a KPS cohort, 102 (31%) had KPS ≤60. Most patients were treated with whole-brain radiotherapy  and/or stereotactic radiosurgery (SRS) . Median bsPFS was 9 months (95% CI 8–10 months), and median OS was not reached. In univariable analyses, KPS ≤60, presence of leptomeningeal disease, neurological symptoms, ≥2 brain metastases, and not undergoing SRS were factors associated with shorter bsPFS. In a multivariable analysis, KPS ≤ 60 was the only statistically significant determinant of bsPFS . Although survival data were limited, KPS ≤60 was associated with shorter OS in both univariable  and multivariable  analyses.The mean age of patients was 57 (range 24–93). Most patients  correlated with radiographic disease response/progression during or after the treatment of non-small-cell lung cancer (NSCLC), regardless of stage, histologic classification, or type of systemic therapy.MethodsA retrospective, single-center review of NSCLC patients at the London Regional Cancer Program between 1 January 2016 and 1 August 2020. This was a planned futility interim analysis to assess the first 50 patients with imaging and tumor markers at baseline and at least one follow-up time-point. Disease response/progression was assessed radiographically using RECIST 1.1 criteria. Statistical analyses were completed using paired nonparametric tests (Wilcoxon signed-rank test).Resultsn = 32) was 2.16  for CEA, 1.46  for CA19-9, and 1.65  for CA-125. The median fold change in the tumor marker from baseline to its level at first response was CEA 0.82 , CA19-9 0.97 , and CA-125 0.84 . The best radiographic response was progression in 18% of patients, partial response in 44%, and stable disease in 38%.A total of 50 patients were analyzed. At baseline imaging, an elevated CEA was seen in 54% of patients, elevated CA19-9 in 32% of patients, and elevated CA-125 in 42% of patients. The median fold change in the tumor marker from its nadir to its level at progression  has been publicly funded in British Columbia (BC) since 2011 for treatment of recurrent glioblastoma (rGBM). We performed a retrospective outcomes assessment of patients with rGBM treated with Bev.MethodsPatients with rGBM treated at BC Cancer centers with Bev between January 2011 and December 2017 were reviewed. Patient demographics, tumor characteristics, treatment regimens, and dates of radiographic progression and death were collected. The Kaplan–Meier method was used to assess survival, and comparisons were made using the log-rank test.Results95 = 2.5–3.5) and an OS of 7 months (CI95 = 6.2–7.8) from Bev initiation. A higher corticosteroid dose prior to Bev initiation was associated with worse OS in univariate and multivariate analyses . Of the patients on corticosteroids, 67% reduced their dose shortly after Bev initiation. Most patients (68%) were treated with multiple lines of therapy prior to Bev, with a median time from chemoradiation to Bev initiation of 8 months (range 1–67). Patients that started on Bev < 6 months from chemoradiation (prior to adjuvant temozolomide completion) had improved PFS (p = 0.02) and OS compared to those who started Bev later (p = 0.05), but there was no association between extent of treatment prior to Bev and outcomes (p = 0.09). The addition of chemotherapy to Bev did not improve survival over Bev monotherapy (p = 0.10).A total of 182 patients were reviewed. There were 180 reported deaths with a median PFS of 3 months  of the geriatric population attending a skin cancer clinic.Methods370-year-old skin cancer patients attending regular visits at the Princess Margaret Cancer Centre since December 2021. Their ability to handle technology was measured through the validated eHEALS tool, which provides overall scores between 8–40, with higher values indicating more technology skills to obtain and apply health information. Correlation between age and eHEALS scores was assessed by the Spearman test . Information regarding steroid use, active treatment, and interruption or discontinuation due to toxicities was also collected.We performed convenience sampling of Results and Conclusionsp = 0.28). Moreover, 11 (22%) individuals did not agree with any of the eHEALS statements. These results suggest that low EHL is a reality among patients 370-year-old and could pose additional hurdles in handling electronic tools otherwise developed to facilitate communication. Adopting eHEALS in our practice may be a time-saving strategy to identify those necessitating more assistance as we move towards broader use of digital tools to improve care.In 50 patients assessed, the median age was 78 (70–87), 34 (68%) were male, 47 (94%) had ECOG-PS 0–1, and 31 (62%) had cutaneous melanomas. Thirty-five (70%) were on active treatment: 14 with anti-PD1 and anti-CTLA4, 16 with anti-PD1 in monotherapy, and 5 with other regimens. Nineteen (38%) patients interrupted or discontinued treatment due to toxicities, and 13 (26%) were on steroids. Twenty (40%) patients did not consider it important to have access to health resources on the internet, and 22 (44%) did not use the internet to make health decisions, relying totally on close relatives and healthcare providers. The mean eHEALS score was 25.24 (SD 9.05), and no correlation between increased age and low eHEALS was identified  calls that require in-person assessment for drugs such as paclitaxel has been seen. This has negative impacts on direct patient care, and, indirectly, patient flow. Our quality improvement project “REsident-led Assessment of ChemoTherapy Infusion reaction Guidelines” (ReACTInG) aims to decrease DRP calls.MethodsWe propose a series of Plan–Do–Study–Act (PDSA) cycles to clarify the need for in-person assessment of DRPs and decrease the number of paclitaxel infusion reactions. Our multifaceted approach includes nursing education (slated to begin March 2022) and a prospective cohort study for breast and gynecological cancer patients receiving paclitaxel using standard versus high(er)-dose dexamethasone prophylaxis. This is planned to start in September 2022, pending ethics approval.ResultsWe report the baseline frequency and nature of DRP calls at BC Cancer, Vancouver, from 30 August 2021 to 22 October 2021. Over the course of 8 weeks, 64 calls were recorded. On independent review, 49 (76.5%) were deemed appropriate (grade 2 or above), while 14 were grade 1 as per BC Cancer guidelines. Paclitaxel accounted for nearly half of the calls . An estimated total provider time of 1269 min was needed to respond to these calls, averaging 19 min per call. During a four-week block, residents in the medical oncology training program were missing, on average, two half-day clinics to manage these calls.ConclusionsOur findings support that the largest proportion of DRP calls are due to paclitaxel hypersensitivity and will be the baseline for our cohort study. Through this resident-led initiative we endeavor to limit the number of (low-grade) DRP calls, specifically paclitaxel infusion reactions, to help decrease patient distress and personnel/resource costs.Award RecipientComparison of Two-Weekly Versus Four-Weekly Durvalumab Consolidation for Advanced NSCLC Treated with ChemoradiotherapyMarie-Hélène Denault 1,2, Shelley Kuang 1, Aria Shokoohi 1, Bonnie Leung 1, Mitchell Liu 1, Eric Berthelet 1, Janessa Laskin 1, Sophie Sun 1, Tina Zhang 1, Barbara Melosky 1, Cheryl Ho 11 BC Cancer Agency-Department of Medical Oncology, University of British Columbia, Vancouver, BC, Canada2 Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec-Département de pneumologie et de chirurgie thoracique, Université Laval, Québec, QC, Canada.mariehelene.denault@bccancer.bc.ca* Correspondence: IntroductionDurvalumab 10 mg/kg every two weeks for one year after chemoradiation has improved overall survival (OS) in unresectable stage III non-small-cell lung cancer (NSCLC) . Subsequently, a 20 mg/kg four-weekly regimen was approved. The study goal was to compare the efficacy and toxicity of the two regimens.MethodsWe reviewed the medical records of all NSCLC patients treated with curative-intent chemoradiation followed by durvalumab from 1 March 2018 to 31 December 2020 at BC Cancer, British Columbia, Canada. The four-weekly dose was introduced in April 2020. The information on the durvalumab dosing schedule, toxicity, progression pattern and survival data was collected. Patients were divided according to the dosing schedule that was used for the majority of treatment. Comparisons were made using chi-square and independent t tests. Kaplan–Meier curves and a log-rank test were used to analyze overall survival.Resultsp = 0.55). Toxicity profiles were similar in terms of sites and severity.A total of 152 patients were included in the two-weekly group and 53 patients in the four-weekly group. Median follow-up was 19.7 months and 12.0 months, respectively. Most patients received carboplatin, and over 90% completed both chemotherapy (≥2 cycles) and radiation (≥60 Gy). Median OS was not reached, but 12-month survival rates were 88.4% versus 85.2%  Reactivity and Vaccination Eligibility in Autologous Hematopoietic Stem Cell Transplant (HSCT) RecipientsOlivia Owen 1, Gopika Punchhi 1, Hammad Saif 1, Aman Sehmbi 1, Sharon Pritchard 1, Kayla Negus 1, Caroline Hamm 1,21  Schulich School of Medicine & Dentistry2  Windsor Regional Cancer Centreoowen2024@meds.uwo.ca* Correspondence: ObjectiveWe aimed to assess measles, mumps, and rubella (MMR) reactivity in autologous hematopoietic stem cell transplant (HSCT) recipients and eligibility for live vaccine administration at two years post HSCT in recipients who required MMR revaccination to inform current guidelines on post-HSCT vaccination.MethodsWe completed a retrospective chart review of 69 autologous HSCT recipients at the Windsor Regional Cancer Centre transplanted between June 2016 and January 2020 to assess post-transplant MMR reactivity. In those without reactivity, we assessed eligibility for revaccination based on administration of contraindicated medications at two years post HSCT, as stated in the most recent Canadian Immunization Guidelines.Resultsn = 58), 55%, 72%, and 48% had non-reactive or indeterminate status to measles, mumps, or rubella, respectively. While 67% were reactive to at least one of the three, only 10% of these patients were fully reactive to MMR. Of living patients who required revaccination 2 years post HSCT (n = 47), 47% were on a contraindicated medication with the most common medications being dexamethasone or prednisone (23%), bortezomib (8.5%), and carfilzomib (8.5%).The most common indication for autologous HSCT was multiple myeloma, with 68% of patients having this diagnosis. Of those with post-HSCT MMR reactivity assessments  patients. This study compares survival outcomes in advanced, high-risk EOC patients across British Columbia who received standard treatment with or without first-line bevacizumab.MethodsAll consecutive patients with inoperable or sub-optimally debulked stage III or stage IV EOC (high-risk by ICON7 criteria) diagnosed between 1 January 2018 and 31 December 2020 were identified through the provincial cancer health record. Survival outcomes were stratified based on receipt of upfront bevacizumab, FIGO stage, performance of surgery, disease relapse, and number of chemotherapy cycles. Univariate analysis (log-rank tests and Kaplan–Meier curves) and multivariate analysis were performed.Resultsp = 0.0065 and p = 0.034, respectively). With multivariate analysis, a statistically significant improvement in RFS  and an improvement in OS that was not statistical significance  were noted.A total of 166 patients with high-risk disease were identified; the median age at time of diagnosis was 67 years, with 71% having stage IV disease. With regards to treatment, 11% had upfront bevacizumab , and the remaining 89% received standard chemotherapy alone. Univariate analysis showed that patients receiving first-line bevacizumab had significantly longer relapse-free survival (RFS) and overall survival (OS) compared to controls  in Canada: A National Survey Study1ESMAIL AL-EZZI , Carlos Stecca 1, Rob Hamilton 2, Michael Crump 1, John Kuruvilla 1, Lori Wood 3, Lucia Nappi 4, Christian Kollmannsberger 4, Scott North 5, Eric Winquist 6, Denis Soulieres 7, Sebastien Hotte 8, Srikala Sridhar 1, Di Maria Jiang 11 Division of Medical Oncology and Hematology, Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, ON, Canada2 Division of Urology, Department of Surgery, Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, ON, Canada3 Division of Medical Oncology, Queen Elizabeth II Health Sciences Centre, Dalhousie University, Halifax, NS, Canada4 Department of Medicine, British Columbia Cancer Agency, University of British Columbia, Vancouver, BC, Canada5 Division of Medical Oncology, Cross Cancer Institute, University of Alberta, Edmonton, AB, Canada6 Department of Oncology, London Health Sciences Centre, Western University, London, ON, Canada7 Département Hématologie-oncologie, Centre Hospitalier de l′Université de Montréal, Montréal, QC, Canada,8 Department of Oncology, Juravinski Cancer Centre, McMaster University, Hamilton, ON, Canadaesmail.al-ezzie@uhn.ca* Correspondence: BackgroundAlthough metastatic GCT is highly curable, 10% of patients relapse after initial cisplatin-based chemotherapy and have a poorer prognosis. Salvage chemotherapy options include conventional-dose chemotherapy (CDCT) and high-dose chemotherapy (HDCT). However, definitive comparative data remain lacking. We aimed to characterize the contemporary practice patterns of salvage chemotherapy across Canada.MethodsWe conducted a 30-question online survey in August 2021 on medical oncologists (MO) and hematological oncologists (HO) with experience in treating GCT, assessing treatment availability, patient selection, and management strategies used for relapsed GCT patients.Resultsn = 13), maintenance etoposide (n = 1), and surveillance only (n = 1).Respondents included: 25 staff MO, 3 HO, and 2 who were both; from British Columbia, Alberta, Manitoba, Ontario, Quebec, New Brunswick, Nova Scotia, and PEI; 86% were from academic centers. Reported case volumes for salvage chemotherapy were <1 (18%), 1 (21%), 1–5 (39%), and 6–10 cases/year (21%). No active clinical trials were available at the time of the survey. The most common CDCT regimens used were TIP (64%) and VIP (25%). HDCT was available for 70% and used as first- , second-  or third-line/beyond  salvage therapy. Only some used the IPFSG risk classification for treatment selection . Assuming tolerability and feasibility, only one respondent indicated a clinical scenario precluding HDCT . The HDCT regimen used included carboplatin and etoposide  and the TICE protocol (two centers). “Bridging” CDCT was needed by 63% while waiting to access HDCT. Post-HDCT treatments considered included surgical resection for residual disease  following implementation of in-house molecular biomarker testing as compared to send-out testing for advanced non-small-cell lung cancer (NSCLC) at our institution.MethodsEGFR analysis, and immunohistochemical assays were used for assessment of ALK and PD-L1. TTD was defined as the interval between the pathological diagnosis of NSCLC and the decision for treatment with systemic therapy. We performed a detailed workflow analysis to provide insight on the pre-testing, testing, and post-testing intervals that constituted the total TTD.We retrospectively reviewed patients with newly diagnosed stage IV NSCLC treated at Sunnybrook Health Sciences Centre  between March 2017 and March 2021. Data were compared during the send-out (March 2017–May 2019) and in-house (July 2019–March 2021) testing periods. Polymerase-chain-reaction-based assays were used for Resultsn = 92 for send-out testing, n = 73 for in-house testing). Of the patients with in-house testing, 88% had results available at their initial oncology consultation compared to only 52% of patients with send-out testing (p < 0.0001). Median TTD was significantly faster with in-house testing  vs. 25 days , p < 0.0001). This improvement was largely driven by decreased internal handling/specimen transit times  vs. 4 days , p < 0.0001) and laboratory turnaround times  vs. 12 days , p < 0.0001), with 96% of in-house cases meeting the international recommended guideline of a ≤14-day TAT . In-house testing had no impact on the post-testing interval  vs. 9 days , p = 0.86).A total of 165 patients were included  and annotated using SnpEff. HPViewer was used to identify the specific HPV genotype.ResultsCase 1: An 18-year-old female presented with a six-year history of hoarseness and one-year history of sore throat, difficulty swallowing, and weight loss. She had no comorbid medical conditions and was a non-drinker and a non-smoker. She was diagnosed with T4N2b squamous cell carcinoma of the glottic larynx and, subsequently, underwent a total laryngectomy, bilateral neck dissection, and total thyroidectomy, followed by chemoradiation. She has remained disease free for 15 years. Molecular testing identified HPV45, and exome sequencing identified 312 mutations including FAT1 and FAT2.Case 2: A 24-year-old female presented with a one-year history of a hoarse voice. There was a 10-pack-year smoking history. She was diagnosed with cT3N0 squamous cell carcinoma of the left vocal cord and treated with chemoradiation therapy. There is no evidence of recurrence 19 months post treatment. Molecular testing identified HPV31, and exome sequencing identified 95 mutations including NOTCH1, MAPK1 (ERK2), and HIST1H2AK.ConclusionsLaryngeal carcinoma is rare in young individuals. Our data suggest that these cancers may be quite different biologically from smoking-related cancers in older adults. These data provide a rationale for further investigation into the etiology, prevention, and treatment of these cancers. Specifically, HPV vaccination and organ preservation strategies may be particularly relevant and effective.Clinical Experience of Patients Referred to a Multidisciplinary Cardio-Oncology Clinic in Northwestern Ontario: An Observational Cohort Study1,2,Andres, G.G.G.  Smylie, P. 2, Svyst, H. 3, Docherty, A. 1,4, Roberts, K. 1,4, Melenchuk, K. 1,4, Alaref, A.A. 1,2, Rohani, A. 1,2, Laferriere, N. 1,4 and Aseyev, O.I. 1,41 Thunder Bay Regional Health Sciences Centre, Thunder Bay, ON, Canada2 NOSM University, Thunder Bay, ON, Canada3 Carleton University, Ottawa, ON, Canada4 Regional Cancer Care Northwest, Thunder Bay, ON, Canadaagriborioguzman@nosm.ca* Correspondence: ObjectiveThis study aims to report the experience of a cardio-oncology clinic recently established at the Thunder Bay Regional Health Sciences Centre (TBRHSC) to contribute to the literature on the emerging field of cardio-oncology and compare it with experiences at similar centers.MethodsThis retrospective observational study included cancer patients referred to the TBRHSC Cardio-Oncology Clinic (COC). Patient demographics, cancer type, the reason for referral, cardiovascular risk factors, cancer and cardiac therapies, and clinical outcomes were collected. Between January 2018 and June 2019, 124 patients  were referred to the COC. The median age of the patients at cancer diagnosis was 62 years (range: 23–83 years).Resultsn = 104, 84%) were alive at the time of the last data collection.The most common types of primary malignancy were hematologic (38%) and breast (29%). The most frequent reasons for referral were clinically suspected cardiotoxicity (69%), history of acute coronary syndrome (32%), abnormal blood pressure (26%), and heart failure (26%). Treatment with cardiac medication was given in 58 patients (47%), with 24 (19%) receiving a beta blocker, 7 (6%) receiving an angiotensin-converting enzyme (ACEi), and 12 (10%) receiving an ACEi and a beta blocker. During or before the study period, 29 patients (23%) were able to complete their prescribed cancer therapy with COC co-management, with 74 patients (60%) having ongoing treatment and 21 patients (17%) ceasing therapy. Most of the 124 patients , Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada2 Department of Medicine, Faculty of Medicine, University of Toronto, Toronto, ON, Canada3 Institute of Health Policy, Management and Evaluation, University of Toronto, Toronto, ON, Canada4 Radiation Medicine Program, Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canadachris.mcchesney@utoronto.ca* Correspondence: BackgroundThe COVID-19 pandemic led to the rapid implementation of remote work, but few studies have evaluated how this impacted staff and hospital operations. Utilizing a cross-sectional survey, we evaluated the impact of remote work on clinical staff at the Princess Margaret Cancer Centre.Methods“Remote work” was defined as any work  performed from home. A Qualtrics survey was disseminated via email three times from June 2021 to August 2021 to 1168 physicians, nurses, and allied health and administrative staff normally involved in patient care. The survey evaluated staff perceptions of productivity and efficiency, patient safety/quality, personal experience, and physical workspace. Results were summarized using descriptive statistics. Free-text responses were categorized into facilitators and barriers using qualitative descriptive analysis.Resultsn = 333; response rate: 28.5%) were female (61.3%) and physicians (23.1%). Very few respondents (1.5%) worked remotely more than half the time prior to COVID; this increased to 66.6% since COVID. Most of respondents reported that working remotely positively impacted productivity (61.8%) and efficiency (57.6%) and expressed interest in continuing (79.0%). While most respondents agreed with the switch to remote work (89.2%), few were provided with the needed equipment (14.1%). In general, respondents indicated that the safety and quality of care delivered remotely were equivalent to that delivered on site, with 13.8% and 18.6% perceiving a negative impact on safety and quality, respectively. Facilitators of remote work included improved efficiency, less commuting, and improved work–life balance. Barriers included a lack of clear expectations for roles, issues accessing applications, and out-of-pocket expenses.Most respondents  with nivolumab has become the standard of care for eligible patients with esophageal and gastroesophageal junction cancers (EGEJC) who had pre-operative chemoradiation (POCRT) and definitive surgery. However, not all patients who receive POCRT are eligible for POI.ObjectiveThe primary objective was to determine the proportion of patients who receive POCRT that are eligible for POI in a real-world population. Further, we aimed to identify the specific barriers to POI to determine factors which could be strategically addressed to maximize access to curative-intent treatment.MethodsAll patients residing in mainland Nova Scotia who received POCRT between July 2016 and April 2019 were analyzed. Disease, patient, and treatment characteristics were collected via chart review. The relative proportion of patients who (1) had definitive surgery, (2) were eligible to receive POI, and (3) would have accepted POI were collected. The specific reasons for non-eligibility or refusal of POI were captured.Resultsn = 11). Of the 21 patients who did not undergo surgery, 12 were found to have new metastatic disease on pre-operative restaging, and nine were felt to be too frail.A total of 65 patients received POCRT during the study period. Of those, 44 went on to have definitive surgery. All 44 of these patients were then screened for eligibility to be enrolled in a trial of POI. A total of 23 were eligible, but 12 declined trial enrolment. A total of 21 were ineligible, with the most common reason being a pathological complete response  for Clinical Trials on the Basis of “Stable” Brain Metastases (BrM)Isabella Kojundzic 1, Rania Chehade 2,3, Mark C. Freeman 2,3, Katarzyna J. Jerzak 1,2,31  Sunnybrook Research Institute, University of Toronto, Toronto, ON, Canada2  Faculty of Medicine, University of Toronto, Toronto, ON, Canada3  Sunnybrook Odette Cancer Centre, Department of Medicine, University of Toronto, Toronto, ON, Canadaisabella.kojundzic@mail.utoronto.ca* Correspondence: ObjectiveDetermine the proportion of patients with MBC who meet common clinical trial criteria for “stable” BrM.MethodsUsing a retrospective cohort of 751 patients treated with local therapy for breast cancer BrM between 2008 and 2018 at the Sunnybrook Odette Cancer Centre, we evaluated the proportion who meet common stipulations for stable BrM in clinical trials. Given that patients are typically evaluated for clinical trials on the basis of systemic disease progression , we identified a subset of patients whose systemic disease progressed within 4 weeks before or after local therapy for BrM with surgery and/or radiotherapy.Resultsn = 189/413) had a Karnofsky performance status (KPS) ≥ 60, and 16.7% (n = 69/413) received stereotactic radiosurgery (SRS) for BrM. A total of 22.8% (94/413) had triple-negative breast cancer, 22.5% (93/413) had HER2+, 39.7% (164/413) had hormone receptor (HR)+/HER2− disease, and 15% (62/413) had unknown receptor status. The proportion of patients who failed to meet common criteria for “stable” BrM varied by the definition used to define stability. A total of 312 (72.5%) of patients had symptomatic BrM, 210 (50.8%) required local therapy within 4 weeks of extracranial progression, and 1 (0.2%) had “active”/progressive BrM that was not amenable to local therapy.Of 751 patients, 413 (55%) had extracranial disease progression, and 300 (40%) had stable extracranial disease at the time of treatment of BrM; the status of extracranial disease was unknown among 38 (5%) of patients. Among 413 patients with concurrent intra- and extracranial disease progression, the median age at BrM diagnosis was 57 (range 24–93); 45.7%  in the Canadian Health Care SystemLuisa Cardenas 1, Naveen Basappa 2, Sunita Ghosh 2, Aaron Hansen 3, Christian Kollmannsberger 4, Lori Wood 5, Daniel Heng 6, Jeffrey Graham 7, Georg Bjarnason 8, Neil Reaume 9, Simon Tanguay 10, Denis Soulieres 11, Vincent Castonguay 12, Eric Winquist 13, Michel Pavic 14, Rodney H. Breau 15, Aly-Khan Lalani 11 McMaster University, Hamilton, ON, Canada2 Cross Cancer Institute, Edmonton, AB, Canada3 Princess Margaret Cancer Centre, Toronto, ON, Canada4 BC Cancer Agency, Vancouver, BC, Canada5 Dalhousie University, Halifax, NS, Canada6 Tom Baker Cancer Centre, Calgary, AB, Canada7 University of Manitoba, Winnipeg, MB, Canada8 Sunnybrook Health Sciences Centre-Odette Cancer Centre, Toronto, ON, Canada9 The Ottawa Hospital Regional Cancer Centre, Ottawa, ON, Canada10 McGill University, Montreal, QC, Canada11 CHUM-Centre Hospitalier de l’Université de Montréal, Montreal, QC, Canada12 Centre Hospitalier Universitaire Pavillon l′Hôtel-Dieu de Quebec, Quebec City, QC, Canada13 London Regional Cancer Program-London Health Science Center, London, ON, Canada14 Centre Hospitalier Universitaire de Sherbrooke, Sherbrooke, QC, Canada15 University of Ottawa, Ottawa, ON, Canadaluisa.cardenas@medportal.ca* Correspondence: BackgroundOver the last decade, standard of care therapies for mRCC have greatly evolved. However, the availability of these emerging options in global health care systems can vary. We sought to describe the integration and usage of systemic therapies for mRCC in Canada since 2011.MethodsWe included mRCC patients enrolled in the Canadian Kidney Cancer Information System (CKCis), which includes 14 academic centers across Canada, from January 2011 to December 2020. Baseline demographic and tumor characteristics were identified, and patients were stratified by line of therapy (1L to 4L+), year of therapy initiation, and type of therapy (VEGF targeted therapy (VEGF-TT), mTOR inhibitor, anti-PD(L)-1-based monotherapy, combination, or other). Descriptive statistics were used: mean (SD) for continuous variables and frequency (%) for categorical values.Resultsn = 2502): median age was 64 years, 74% were male, 65% were Caucasian, 75% had clear cell histology. IMDC risk scores  were 17%, 56%, and 27% respectively. Among patients who discontinued therapy, 1269/2175 (58%) received 2L, and 590/1057 (56%) received 3L. Distribution of systemic therapies are shown in Table 1 by timepoints reflecting the general integration of anti-PD(L)-1-based therapies .We identified 3613 patients with mRCC, of whom 1097 (30%) did not receive systemic therapy . Among patients who received 1L systemic therapy and had complete clinical data  help in triaging clinical problems and providing psychosocial support to patients and care givers as they navigate a complex multi-disciplinary oncology team. The benefits of effective PSLs include reduced morbidity and depression and increased functional capacity. While providing support and training to nursing staff who operate these lines is key, there is limited data on their experience to guide this.MethodsWe invited oncology nurses with experience on The Ottawa Hospital Cancer Centre (TOHCC) PSLs to participate in an anonymous, online survey. Measures collected included nursing experience, characteristics of questions addressed by the PSL, patient and nursing satisfaction with the service, common challenges faced, and initiatives to improve patient and nursing experience.ResultsSeventy-one percent (30/42) of eligible nurses responded to the survey. The most common disease site, stage, issue, and symptom addressed were breast cancer, metastatic disease, treatment-related toxicity, and pain, respectively. Despite the majority of nurses reporting personal and patient satisfaction with the care provided by the PSL, there was variance in the perceived appropriate use of PSL by physicians and patients. As such, fifty-nine percent (17/29) of nurses recommended redefining the responsibilities of the PSL to achieve its maximal potential. Eighty percent (24/30) of nurses denied experiencing a reduction in PSL call volumes with increased patient access to their electronic medical record. Sixty percent (18/30) of nurses reported that having TOHCC-specific management plans for common issues would improve their experience and the quality of care provided by the PSL.ConclusionsThere is need for TOHCC-standardized management algorithms for common issues addressed on the PSL, as well as increased physician and patient education to redefine the goals of the PSL to help address problems with high volume and inappropriate calls. These data represent a first step in a planned quality improved study for the TOHCC PSL, with future initiatives including patient surveys and implementation of quality improvement initiatives with patient and staff feedback.An Assessment of Extended Pembrolizumab Dosing and Outcomes in Advanced Non-Small-Cell Lung Cancer Patients during the COVID-19 PandemicGordon Taylor Moffat 1, Lilian Hanna 1, Wilma Hopman 2, Andrea S. Fung 1, Pierre-Olivier Gaudreau 1,31 Department of Oncology, Queen′s University2 Department of Public Health Sciences, Queen’s University3 Canadian Cancer Trials Group, Cancer Research Institute, Queen’s Universitygtmoffat@gmail.com* Correspondence: BackgroundThe COVID-19 pandemic led to Cancer Care Ontario’s approval of extended dosing (ED) of pembrolizumab in the management of metastatic non-small-cell lung cancer (mNSCLC) as a means of reducing the risk of exposure through fewer visits to the cancer center. Pembrolizumab ED has been evaluated through pharmacokinetic studies and simulated, model-based analyses; however, clinical data are lacking. This study aims to evaluate real-world outcomes and safety of ED vs. standard dosing (SD) of pembrolizumab.Methodst-tests.This retrospective cohort study included patients with mNSCLC  from the Cancer Centre of Southeastern Ontario  who received at least one cycle of single-agent pembrolizumab with SD (2 mg/kg IV up to a maximum of 200 mg q3 weeks) or ED (4 mg/kg IV up to a maximum of 400 mg q6 weeks) from January 2018 to December 2020. Data were collected from a review of clinical, pathological, and radiological reports. Study outcomes included the proportion of patients alive at data cut-off, the median number of treatment cycles, immune-related adverse events (irAEs), and time to toxicity (TTT). Statistical analyses included chi-square, Mann–Whitney, and Resultsp < 0.01). The ED group also received more cycles of therapy  compared to the SD group (median = 3.5). The median TTT was 130 (ED) vs. 105 (SD) days, and the rate  and severity of irAEs (≥grade 3 events: 50% vs. 52%) were similar. A higher proportion of ED patients discontinued treatment due to toxicity , and 5/8 (62.5%) of the irAEs from the ED group occurred after switching (majority of events resulted in treatment discontinuation).Of the 90 patients evaluated, 18 (20%) switched to ED. The proportion of those alive at the data cut-off was significantly higher in the ED vs. SD group . The rate and severity of irAEs were similar between the two groups; however, a high proportion of irAEs in the ED group occurred following the switch, which justified treatment cessation. These results are limited by small patient numbers, and the possible impact of ED on toxicity should be further validated in prospective cohorts.


Low phosphate (Pi) availability in soils severely limits crop growth and production. Plants have evolved to have numerous physiological and molecular adaptive mechanisms to cope with Pi starvation. The release of Pi from membrane phospholipids is considered to improve plant phosphorus (P) utilization efficiency in response to Pi starvation and accompanies membrane lipid remodeling. In this review, we summarize recent discoveries related to this topic and the molecular basis of membrane phospholipid alteration and triacylglycerol metabolism in response to Pi depletion in plants at different subcellular levels. These findings will help to further elucidate the molecular mechanisms underlying plant adaptation to Pi starvation and thus help to develop crop cultivars with high P utilization efficiency. Phosphorus (P), as an essential macronutrient for plants, is a vital component of nucleic acids, membrane lipids, and adenosine triphosphate (ATP), and is involved in various metabolic processes ,2. The pArabidopsis thaliana), rice (Oryza sativa), oat (Avena sativa), tomato (Solanum lycopersicum), and soybean (Glycine max) . In. In31]. ficiency . In addificiency . In Arabnditions . Howevery stress . There af OsPHR2 . Additiof OsPHR2 . In addiodiester .The compensatory increase in levels of non-phosphorus lipids such as galactolipids and sulfolipids is an important strategy for plants’ adaption to P deficiency . It is kAtMGD1 belongs to type A and is mainly expressed in green tissues and localized in the envelope of chloroplasts [AtMGD2 and AtMGD3, both of which belong to type B, are highly expressed in nongreen tissue and are localized in plastids [AtMGD2 and AtMGD3 are induced by P deficiency  in Pi-deficient roots in Arabidopsis. In rice, three MGD members  were identified, and all of them were shown to be up-regulated in roots through Pi starvation, especially OsMGD3 [OsMGD3 significantly inhibited or enhanced P utilization efficiency and lateral root growth in response to Pi starvation [OsMGD3 was directly regulated by the OsPHR2 transcription factor under P-deficiency conditions [MGDG is synthesized from DAG by MGDG synthase; subsequently, DGDG can be further synthesized from MGDG by DGDG synthase . Three Mroplasts . AtMGD2 plastids . AtMGD2 ficiency  but AtMG1 is not ,36. P de content , suggesty OsMGD3 ,56. The arvation . Furthernditions .AtDGD1 and AtDGD2, and both of them are induced by P deficiency , UDP-sulfoquinovose synthase (SQD1), and sulfolipid synthase (SQD2) were reported to participate in the synthesis of SQDG in Arabidopsis [sqd2 could reduce Arabidopsis growth under Pi-starvation conditions [SQGD, as an anionic lipid, is also an important non-phosphorus membrane lipid that is significantly increased by low-P stress. Under P-deficiency conditions, SQDG is synthesized to substitute for PG to maintain Pi homeostasis in Arabidopsis . The P-dbidopsis ,42,44,57nditions .diacylglycerol acyl transferase 1 (DGAT1), which catalyzes DAG and acyl-CoA to synthesize TAG and is regulated by the ABA insensitive 4 (ABI4) transcription factor [DGAT1 nor ABI4 was induced by Pi starvation in Arabidopsis [DGAT gene (SlDGAT2) was reported to be up-regulated in leaves after seven days of P-deficiency treatment [P deficiency also induces significant changes in triacylglycerol (TAG) metabolism. It was reported that the content of TAG increased more than 13-fold in both Arabidopsis shoots and roots upon P deficiency . Similarn factor ,61. Howebidopsis , suggestreatment , which ssqd2 mutant were severely impaired compared to the wild-type control, suggesting that the biosynthesis of GlcADG shares the same pathway with SQDG synthesis in chloroplasts [Glucuronosyldiacylglycerol (GlcADG) is a novel lipid that is essential to the protection of plants against P deficiency . The Pi-roplasts . Thus, Groplasts . In soybroplasts , which eroplasts .LPAT members of Arabidopsis, AtLPAT2 was significantly induced by Pi starvation in roots and localized in ER [AtLPAT2 significantly increased the root length and the amount of PC in Arabidopsis root under Pi starvation [AtLPAT2 functions in mediating de novo phospholipid biosynthesis and root development under Pi starvation in Arabidopsis [The glycerolipid de novo biosynthesis pathway (the Kennedy pathway) includes two steps: G3P acyltransferase (GPAT) converts G3P to lysophosphatidic acid (LPA), then the LPA is further converted to PA by lysophosphatidic acid acyltransferase (LPAT) ,45,63. TAtNCP5, AtPLDZ2, AtMGD2/3, and AtDGD1 were partially attenuated and AtNCP4 was evenly and completely suppressed in phr1-mutant Arabidopsis [AtMGD3 [phr1-mutant Arabidopsis [AtMGD2/3 expression, and was indispensable in the Pi-starvation response, while cytokinin played a negative regulatory role [TIR1; the auxin transport from shoot to root could induce the degradation of IAA14 to release ARF7/19, thus regulating the cell-cycle activities and other responses to mediate gene expressions and membrane lipid remodeling [MGD3 and IPS1, in Arabidopsis under P-deficiency conditions [AtMGD3 [OsJAZ11, a Pi-starvation-responsive gene regulated by OsPHR2 regulated root growth and Pi homeostasis by suppressing JA signaling in rice roots [P-deficiency-responsive membrane lipid remodeling has been reported to be an important adaptive strategy that involves several lipid species’ metabolites and diverse Pi-starvation-responsive genes. However, the key regulators and regulatory mechanisms involved in these responses remain largely unknown. Phosphate starvation response regulator 1 (PHR1) is known as the global regulator of the P-deficiency response . Phosphobidopsis ,65. Furt [AtMGD3 . The chabidopsis . These rbidopsis , suggestbidopsis . In addiory role . Furtherory role . A low Pmodeling ,67,68. Hnditions . In Arab [AtMGD3 . However [AtMGD3 . For exace roots . These rNPCs, PLDs, and GDPDs (MGD, DGD, and SQD levels identified in the synthesis of MGDG, DGDG, and SQDG, respectively (PI-PLC genes that function in phosphoinositide hydrolysis remain unknown, although several Arabidopsis PI-PLC family member genes, such as AtPLCs, were expressed in response to other environmental stresses [phr1-mutant Arabidopsis, the Pi-starvation-induced, lipid-metabolism-related genes such as AtNCP5, AtPLDZ2, AtMGD2/3, and AtDGD1 were partially attenuated [To cope with P-deficiency stress, diverse strategies have evolved in plants to enhance their external Pi uptake capacities and improve their internal P use efficiencies ,73,74,75nd GDPDs . Similarectively . These cstresses ,20,21. Sstresses ,16,58, atenuated , indicattenuated . Thus, t


Eleusine coracana, finger millet, is a multipurpose crop cultivated in arid and semi-arid regions of Africa and Asia. RNA sequencing (RNA-seq) was used in this study to obtain valuable genomic resources and identify genes differentially expressed between Al-tolerant and Al-susceptible genotypes. Two groups of finger millet genotypes were used: Al-tolerant  and Al-susceptible . The analysis of the RNA-seq data resulted in 198,546 unigenes, 56.5% of which were annotated with significant hits in one or more of the following six databases: NR (48.8%), GO (29.7%), KEGG (45%), PlantTFDB (19.0%), Uniprot (49.2%), and NT (46.2%). It is noteworthy that only 220 unigenes in the NR database had significant hits against finger millet sequences suggesting that finger millet’s genomic resources are scarce. The gene expression analysis revealed that 322 genes were significantly differentially expressed between the Al-tolerant and Al-susceptible genotypes, of which 40.7% were upregulated while 59.3% were downregulated in Al-tolerant genotypes. Among the significant DEGs, 54.7% were annotated in the GO database with the top hits being ATP binding (GO:0005524) and DNA binding (GO:0003677) in the molecular function, DNA integration (GO:0015074) and cell redox homeostasis in the biological process, as well as cellular anatomical entity and intracellular component in the cellular component GO classes. Several of the annotated DEGs were significantly enriched for their corresponding GO terms. The KEGG pathway analysis resulted in 60 DEGs that were annotated with different pathway classes, of which carbohydrate metabolism and signal transduction were the most prominent. The homologs of a number of significant DEGs have been previously reported as being associated with Al or other abiotic stress responses in various crops, including carboxypeptidase SOL1, HMA3, AP2, bZIP, C3H, and WRKY TF genes. A more detailed investigation of these and other DEGs will enable genomic-led breeding for Al tolerance in finger millet. RNA-seq data analysis also yielded 119,073 SNP markers, the majority of which had PIC values above 0.3, indicating that they are highly informative. Additionally, 3,553 single-copy SSR markers were identified, of which trinucleotide SSRs were the most prevalent. These genomic resources contribute substantially to the enrichment of genomic databases for finger millet, and facilitate future research on this crop. Eleusine coracana (L.) Gaertn) is a highly nutritious and multipurpose cereal crop cultivated in arid and semi-arid tropical regions of Africa and Asia  on the plasma membrane, then activates the transcription of genes that encode proteins involved in the metabolism of organic acids and their transport across the plasma membrane  toxicity and phosphorus (P) deficiency are the primary factors that directly affect crop yield and productivity by disrupting plant physiology and phenology . This abmembrane .Transcription factors (TFs) have been identified in different crops playing a vital role in activating the expression of Al-tolerant genes to tolerate Al-toxicity . AlthougSaccharum officinarum L.), sweet potato and noug (Guizotia abyssinica) (Transcriptome sequencing has opened a new way for the development of DNA markers such as simple sequence repeat (SSR) or microsatellites, and single nucleotide polymorphism (SNP). Microsatellites are co-dominant DNA markers of short tandem repeats consisting of 2–6 bp nucleotides that are extensively present across whole genomes, including within genes . They arssinica) . DNA marssinica) .RNA-sequencing (RNA-seq) is an efficient and less expensive next-generation sequencing (NGS) method that has broad applications, including the identification of differentially expressed genes and determining pathways in crops grown under different stress . There hIn this study, leaves collected from six finger millet accessions were used for RNA-seq-based transcriptome analysis. Originally, the accessions were collected from locations representing different agro-ecologies in Ethiopia. These are accessions 215836 (from Gojjam), 215845 (from Bahir dar), 229722 (from Metekel), 212462 (from Hararge), 215804 (from Wellega), and 238323 (from Tigray). They were selected for sampling their representative genotypes based on the results of a preliminary hydroponic experiment, which was carried out as described in . The res3, 500 μM CaCl2, 500 μM NH4NO3, 150 μM MgSO4.7H2O, 10 μM KH2PO4, 2 μM FeCl3, and 100 μM Al2 (SO4)3.18H2O, which was prepared according to For this study, the germination of the seeds sampled from the selected accessions followed by the exposure of their seedlings to Al-toxicity was carried out as described in The three genotypes sampled from accessions: 215836, 215845, and 229722 were confirmed to be Al- tolerant, and hereafter will be referred to as AT1, AT2, and AT3, respectively. Whereas, the other three genotypes sampled from accessions 212462, 215804, and 238323 were confirmed to be Al-susceptible, and hereafter will be referred to as AS1, AS2, and AS3, respectively. The seedlings of each Al-tolerant and Al-susceptible genotypes were transferred to separately labeled 5 L soil-filled plastic pots for further growth. The seedlings were allowed to grow for about four weeks in a greenhouse adjusted to a temperature of 18°C and a humidity level of 65% at the Swedish University of Agricultural Sciences .The leaf tissue of five-weeks-old seedlings of each genotypes was snap-frozen in liquid nitrogen and then kept at −80°C until RNA was extracted. A total RNA sample was extracted from ca100 mg leaf tissue of each genotype using the RNeasy Plant Mini Kit , and subsequently treated with DNase using the Ambion Turbo DNA-Free Kit . An Agilent Bioanalyzer 2100 system , a NanoDrop ND-1000 spectrophotometer , and agarose gel electrophoresis were used to evaluate the quality and concentration of extracted RNA. The high-quality RNA samples were then shipped to CD Genomics  on dry ice for RNA sequencing. Upon arrival at the CD Genomics, the samples were checked for degradation and contamination using agarose gels (1%), purity was checked using the NanoPhotometer spectrophotometer , concentration was measured using the Qubit RNA Assay Kit in Qubit 2.0 Flurometer , and integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system . The workflow for RNASeq data acquisation and analysis is provided in To construct the sequencing library, 1.5 mg of RNA was utilized per sample, and index codes were added to assign sequences to each sample using the NEBNextR UltraTM RNA Library Prep Kit for Illumina  according to the manufacturer’s instructions. A Beckman Coulter AMPure XP system  was used to purify the library fragments in order to facilitate preferential selection of cDNA fragments with 150-200 bp size. The amplified products were purified using the AMPure XP system following adaptor ligation and polymerase chain reaction (PCR), and then quality of the purified library was assessed with the Agilent Bioanalyzer 2100. The index-coded samples were then clustered using a cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. The clusters were subsequently paired-end sequenced using Illumina HiSeq™ 2500 v2 platform .de novo reference transcriptome using Trinity v. 2.1.1 with default settings , Non-Redundant Protein  cluster analysis of the expressed genes from the finger millet genotypes was carried out using the UPGMA algorithm implemented in the “vegan” package in R  using Eup-values below 0.01 and log2 fold change (log2FC) above 2 were classified as significantly differentially expressed.To quantify the abundance of the transcript in the two (Al-tolerant and Al-susceptible) groups, the sequenced pair-end reads were mapped onto the assembled transcriptome, and the read count for each gene was obtained from the mapping results. RNA Sequencing by Expectation Maximization package  on the y-axis, and the expression fold change between the two experimental groups on the x-axis. The regions of interest in the volcano plot are those found towards the top  and at the extreme left or right . Furthermore, two-way hierarchal cluster analysis of the genes and genotypes was performed using the R software package pheatmap v.1.0.8 . Likewise-value cutoff = 10-10, minimum identity = 40, and minimum query coverage = 50%.GO functional enrichment analysis of DEGs was carried out using topGO. To identify the transcription factor genes among the DEGs, all DEGs were searched against the plant transcription factors database  with anThe European Molecular Biology Open Software Suite (EMBOSS) packages “getorf” and “transeq” were used to identify the exact start and stop points of translation and to translate cDNA sequences in to amino acid sequences, respectively . 3D prothttp://pgrc.ipk-gatersleben.de/misa;  at least in one of the databases: NR , GO , KEGG , PlantTFDB , Uniprot  and NT   had significant hits  Table 3.Setaria italic; 23.7%), perennial Hall’s panicgrass , sorghum , maize , rice , and perennial Heller’s rosette grass  (Eleusine indica), respectively, available in the genomic databases.The analysis based on the non-redundant protein database revealed that more than 97% of the annotated unigenes had homology to protein sequences of the Poaceae family, such as foxtail millet  Figure 3-5). The number of unigenes that were annotated under biological process (BP), molecular function (MF), and cellular component (CC) were 65,230, 62,920, and 59,280, respectively  were annotated with at least one GO term in the GO database  in the KEGG database, 36,696 (41.1%) unigenes were annotated under 201 pathways. These were classified under five major KEGG categories: cellular processes, environmental information processing, genetic information processing, metabolism and organismal systems  resulted in significant hits for 37,828 unigenes across 60 different TF family proteins. The top three most frequent transcription factor protein families were bHLH, MYB-related, and NAC, which corresponded to 3580 (9.46%), 2747 (7.26%), and 2588 (6.84%) unigenes, respectively. TFs from 160 plant species were significant matches for the unigenes, with apple , Manila grass (Zoysia matrella), and tef (Eragrostis tef) being the top three  was also used  among the two groups (2FC< -1 or > 1) with corresponding adjusted p-values below 0.05, and hence they are regarded as significantly differentially expressed. Among the 322 unigenes, 131 (40.7%) were significantly upregulated while 191 (59.3%) were significantly downregulated in the Al-tolerant group in comparison with the Al-susceptible group  genes showed either an up-regulation  or a down-regulation  trend. The further filtering of the 162,974 genes based on their adjusted Hierarchical cluster analysis of the significantly differentially expressed genes revealed eight clusters , DNA and ATP binding activity, integral components of membrane, Acety-CoA carboxylase activity, signaling pathway, glyoxylate and dicarboxylate metabolism, catalyitic activity, starch and sucrose metabolism  were annotated with one or more GO terms in the gene ontology database. Among the annotated significant DEGs, 83 of them were annotated to GO terms in MF, 83 of them with GO terms in BP, and 36 of them with GO terms in CC  were annotated with 71 pathways belonging to 21 pathway classes (-5) against functionally annotated TF genes (Fragaria vesca), Manila grass, and apple, being the top three with eight, seven, and seven matching significant DEGs (The BLAST search of the 322 significant DEGs in the TF database resulted in that 137 (42.5%) DEGs had significant hits  did not have significant matches in the GO, KEGG, or TF databases  showed GH3-1 auxin conjugating enzyme, pollen allergen CJP38, and carboxypeptidase GP 180 residues 503-882 at 68%, 52% and 58% similarity levels, respectively (The unigenes assembled in this study were scanned for both perfect and compound SSRs using MIcroSAtellite (MISA) software. SSRs that contain two or more SSRs of the same or different types separated by up to 100 nucleotides were referred to as compound SSRs. Of the total 198,546 scanned unigenes, 30,561 unigenes (15.4%) contain SSRs of which 6,147 had more than one SSR. In total, 38,090 SSRs were identified across the 30,561 unigenes, of which 35,897 and 2,193 were perfect SSRs and compound SSRs, respectively. Of the 35,897 perfect SSRs, 21,150 (58.9%), 5,694 (15.9%), 8,681 (24.2%), 264 (0.7%), 82 (0.2%), and 26 (0.1%) were mono, di, tri, tetra, penta, and hexa-nucleotide repeat SSRs, respectively Table 4.A/T and G/C repeat SSRs accounted for 93.5% and 6.5% of the mononucleotide repeat SSRs, respectively. Among the di-nucleotide repeat SSRs, the most frequent repeats were AG/CT , and GA/TC , whereas CG/CG  and GC/GC  were rare. Among tri-nucleotide repeat SSRs, the top three most common motifs were CGC/GCG , CCG/CGG  and GCC/GGC  while all others accounted for 61.6% Table 4.In the case of the number of repeats of each SSR type, their frequency generally decreased as the number of repeats increased. For instance, the number of mononucleotide repeats with a repeat of 10 was 9476, whereas those with a repeat of 11 were 4012, an over twofold decrease. The number of dinucleotide repeat SSRs with repeats of six and seven was 1786 and 950, respectively, which is a decrease of almost twofold. Trinucleotide repeat SSRs with a repeat of five (5462) were almost three times more frequent than those with a repeat of six 1883, Table 4.Following the exclusion of mononucleotide repeat SSRs, 14,747 SSRs comprising di, tri, tetra, penta, and hexa-nucleotide repeat SSRs were further analyzed in order to identify single-copy SSRs for their various uses in the future. Following this, primers were designed for the identified single-copy SSRs using the Primer3 program. Overall, primer pairs were successfully designed for 3553 single-copy SSRs in 3275 unigenes , particularly RNA-seq is a highly effective tool for generating high quality transcriptome sequence reads in several crops such as coracana , oat  had statistically significant hits at least in one of the databases. Nevertheless, a large proportion (43.5%) of the unigenes did not have significant hits. Possible reasons includes lack of sequenced databases for finger millet, short sequence length of some of the unigenes, the presence of non-coding sequences or sequences of untranslated regions, and a high sequence variation that results in an E-value above the cutoff. This is in line with the findings of Among the finger millet unigenes identified in the present study that had significant hits in NR database, 23.7% of these hits were for foxtail millet protein sequences, whereas only 0.2% were for finger millet protein sequences. The high proportion of hits against foxtail millet is expected given that foxtail millet is one of the closest species to finger millet among the plant species that are well represented in the database. In their inter-genome collinearity analysis, Gene ontology functional classification may help us understand the distribution of gene functions, and to predict the role of each unigene. The GO analysis performed in this study classified the genes that had significant hits into 19, 13, and 4 functional subcategories of BP, MF, and CC gene ontology classes. The BP class was dominated by cellular process, metabolic process, biological adhesion, and response to stimulus. MF was dominated by catalytic activity and binding, and CC was dominated by cellular anatomical entity and intracellular entity. The results indicate that the identified genes are engaged in diverse functional, regulatory, and structural activities. Previous studies have shown that cellular processes, metabolic processes, binding of molecules, response to stimuli, as well as metabolism of organic anions are highly correlated with stress tolerance and disease resistance .-5) in the KEGG database, 36,696 (41.1%) unigenes were annotated under 201 pathways. These were classified in to cellular processes, environmental information processing, genetic information processing, metabolism, and organismal systems. Most of the unigenes were annotated with the metabolism pathway. The majority of the unigenes predicted were involved in either signal transduction, carbohydrate metabolism, translation, folding, transport and catabolism, lipid metabolism, or sorting and degradation pathways. Being active of these pathway agreed with previous study on Al-stress in Stylosanthes guianensis  and multidrug and toxin extrusion transporters (MATE), respectively , metal ion transport (GO:00300001), defense response to bacteria (GO:0042742), lipid metabolism (GO:0006629), regulation of meristem development (GO:0048509), and seedling development (GO:0090351).Panicum halli, Setaria italica, Sorghum bicolor, Oryza sativa, Lolium rigidum, Zea mays, and Triticum aestivum. Therefore, the unigene shared sequence homolog with carboxypeptidase SOL1 gene. As a result of the gene ontology annotation, the gene has been identified as being involved in metallocarboxypeptidase activity (GO:0004181) as well as the regulation of root meristem growth (GO:0048509). In this study, this gene is downregulated in all Al-tolerant genotypes (log2FC = -7.6). The SOL1 gene encodes a putative zinc carboxypeptidase (The unigene DN38351_c0_g1_i1 (GO:0048509) shared a sequence similarity of over 83% with the carboxypeptidase SOL1 gene found in a variety of Poaceae species, including eptidase . CasamitSetaria italica, Sorghum bicolor, Oryza sativa, and Zea mays. HMA is a member of the ATPase protein family involved in metal ion transport in plants (The unigene DN40309_c0_g2_i3 (GO:0019829) shared sequence similarity of above 82% with cadmium/zinc-transporting ATPase HMA3  gene of various grass species, including n plants . In thisThe unigene DN40527_c0_g1_i4 (GO:0042742) shared a sequence identity of over 93% with the plastid-lipid-associated protein 6 (PAP) gene found in different crops. This unigene shared homolog with plastid-lipid-associated protein 6 (PAP) gene in various crops . In thisArabidopsis thaliana, Panicum hallii, Panicum virgatum, and Setaria italic. The lipase-like PAD4 gene increase immunity by stimulating the production of the defense hormone salicylic acid (SA) and anti-microbial molecules, which limit pathogen growth (A unigene DN44795_c0_g1_i3 (GO:0006629) shared 80% sequence similarity with the lipase like Phytoalexin deficient4 (PAD4) gene found in n growth . In thisn growth .Among the top hits for DN43070_c0_g1_i4 (upregulated = Log2FC = 1.4) in the NT and NR databases are putative 1-phosphatidylinositol-3-phosphate 5-kinase FAB1Cs from Poaceae species such as maize and sorghum. The GO enrichment analysis showed that this gene is enriched for phosphatidylinositol phosphate kinase activity (GO:0016307). Similarly, KEGG pathway analysis revealed it is involved in the Phosphatidylinositol signaling system (ko04070) and therefore plays a crucial role in cell physiology through its involvement in signal transduction pathways. The inhibition of phosphatidylinositol phosphate kinase activity by aluminum has been demonstrated in coffee , and in The plant hormone signaling system (ko04075) comprised two unigenes (DN39933_c0_g2_i4 and DN43311_c0_g5_i10) and are considered significant players in mediating plant stress response. Moreover, activation of metabolic pathways such as glyoxylate and dicarboxylate (ko00630) as well as starch and sucrose (ko00500) metabolisms, indicating that the plants might use those specific compounds to survive or promote its growth. Activation of glyoxylate and dicarboxylate as well as starch and sucrose were also described in cotton plants . Thus, tTranscription factors, such as activators, inhibitors, or both, which regulate the expression of genes have a vital role in various aspects of plant growth and development . Most ofAmong the DEGs, DN42426_c0_g1_i16 had significant hits in the plant TFDB which corresponds to the AP2 TF of Poaceae species. This gene is downregulated in Al-tolerant genotypes (Log2FC = -7.3) compared to Al-susceptible genotypes. Different transcriptome-based studies have demonstrated that AP2 family proteins play a role in Al-tolerance in plants. Based on their root transcriptome studies, Gossypium hirsutum (Gh_D05G0105), Trifolium pretense (TP57577_TGAC_v2_mRNA6397), Zoysia japonicum (Zjn_sc00045.1.g01150.1.am.mk) and Fragaria vesca (mrna13716.1-v1.0-hybrid), respectively. The upregulation of bZIP genes in this study agreed with previously pulished findings under abiotic stress conditions has been documented (via abscisic acid (ABA).The DN44845_c0_g1_i2, DN43468_c1_g2_i9, DN38637_c1_g1_i2, and DN43333_c0_g4_i1 unigenes were significantly upregulated in the Al-tolerant genotypes with Log2FC of 6.8, 2.6, 1.9, and 1.13, respectively. DN44845_c0_g1_i2, DN43468_c1_g2_i9, DN38637_c1_g1_i2, and DN43333_c0_g4_i1 unigenes shared sequence similarity with TFs in cumented . Yang etTriticum Urartu (TRIUR3_28246-P1). DN28807_c0_g1_i1 and DN41816_c0_g2_i6 shared sequence similarity with WRKY TF in Phyllostachys heterocycle (PH01003485G0070) and Ananas comosus (Aco009877.1), respectively. On the other hand, DN40895_c2_g1_i15 and DN40895_c2_g1_i13 were upregulated unigenes with Log2FC values of 7.06 and 9.05 shared sequence similarity with WRKY TF. The involvement of WRKY TFs in conferring Al tolerance to plants has been reported in different studies. In Arabidopsis, WRKY7 has been shown to confer Al tolerance by regulating the expression of genes involved in the modification of cell walls, implicated in a number of developmental processes as well as a defense response against biotic and abiotic stresses  were among the downregulated unigenes with Log2FC value of -2.4, -4.7, -6.5, and -7.1, respectively. The top hit for DN28660_c0_g1_i3 unigene in the plant TFDB is the transcription factor gene of WRKY of stresses .Among the significant DEGs, the predicted 3D protein structure of the downregulated unigene DN39933_c0_g2_i4 shared 68% sequence similar with that of GH3-1 auxin conjugate enzyme. Auxin is mainly produced at the tip of the mature leaf through IPyA pathway in most species and then channeled through polar auxin transport (PAT) into root to produce adventitious root and establish new root to replace damaged roots .On the other hand, the upregulated DN42426_c0_g1_i16 unigene was 64% similar with dihydrolipolyl dehydrogenase multienzyme complex. Dihydrolipolyl dehydrogenase is required to maintain the metabolic fluxes through the TCA cycle, photorespiration and branched side chain amino acid degradation . In addiArabidopsis thaliana  is higher than the G+C content of the SSRs derived from these unigenes (34.1%). A similar pattern was reported in Populus  and nougPopulus . The G+CPopulus . SimilarPopulus , which iIn this study, highly informative SNP markers were generated based on the transcriptome data of the finger millet genotypes. The PIC of 92.9% bi-allelic SNPs and 97.4 tri-allelic SNPs was above 0.30 indicating that the vast majority of the markers are highly informative. As finger millet is an orphan crop and has not been well studied at the genomics level, this data is highly suitable for use in various applications, including population genetic analyses for conservation and breeding, marker-trait association studies as well as genomic predictions for desirable traits. Marker-trait association studies using these markers targeting desirable traits will eventually lead to the identification of a core set of markers for the development of an SNP-chip that can be used to facilitate genomics-led breeding of finger millet. As revealed through cluster and principal component analyses conducted based on randomly selected SNPs as well as SNPs within DEGs, there was high genetic variation among the finger millet genotypes, particularly among the Al-susceptible genotypes. The result is in line with previous SSR and SNP marker based studies on Finger millet . DespiteAmong the 322 significant DEGs, six of them showed genotypic differences between the Al-tolerant and Al-susceptible genotypes  can be found in the article/Conceptualization and methodology, HB, MG, TH, KT, CH and RO. Data collection, HB, MG, CH and RV. Data analysis, HB under guidance of MG, CH and KBA. Original draft preparation, HB. Review and editing, HB, TH, KT, CH, RO, KBA, RV and MG. Project funding acquisition and administration, TH, KT, RO, CH and MG. All authors contributed to the article and approved the submitted version.


Metformin may offer benefits to certain cancer populations experiencing metabolic abnormalities. To extend the anticancer studies of metformin, a tumor model was established through the implantation of murine Lewis Lung Carcinoma (LLC) cells to Normal Diet (ND)-fed and High-Fat Diet (HFD)-fed C57BL/6 mice. The HFD-fed mice displayed metabolic and pro-inflammatory alterations together with accompanying aggressive tumor growth. Metformin mitigated tumor growth in HFD-fed mice, paralleled by reductions in circulating glucose, insulin, soluble P-selectin, TGF-β1 and High Mobility Group Box-1 (HMGB1), as well as tumor expression of cell proliferation, aerobic glycolysis, glutaminolysis, platelets and neutrophils molecules. The suppressive effects of metformin on cell proliferation, migration and oncogenic signaling molecules were confirmed in cell study. Moreover, tumor-bearing HFD-fed mice had higher contents of circulating and tumor immunopositivity of Neutrophil Extracellular Traps (NETs)-associated molecules, with a suppressive effect from metformin. Data taken from neutrophil studies confirmed the inhibitory effect that metformin has on NET formation induced by HMGB1. Furthermore, HMGB1 was identified as a promoting molecule to boost the transition process towards NETs. The current study shows that metabolic, pro-inflammatory and NET alterations appear to play roles in the obesity-driven aggressiveness of cancer, while also representing candidate targets for anticancer potential of metformin. The prevalence and incidence of being overweight or obese are increasing rapidly worldwide. Beyond their direct negative effects on human health, obesity certainly leads to a steep rise in related health complications and poor outcomes. Type 2 diabetes mellitus and cancer are common health complications surrounding obesity. Additionally, an obese population with accompanying type 2 diabetes mellitus is particularly threatened by cancer, being predisposed to suffering from its incidence and progression ,2. The iObesity is a metabolic disorder concerning a dysregulated metabolism in glucose, lipids and macromolecules. Hyperglycemia, hyperinsulinemia, dyslipidemia and low-grade inflammation, along with an increase in adipokines, total White Blood Cells (WBCs), neutrophils and Neutrophil Extracellular Traps (NETs), as well as platelet activation, are common biochemical hallmarks of obesity ,5,6. SucObesity-oriented therapeutic compounds such as Sodium-Glucose Cotransporter-2 (SGLT2), statins and metformin all show tumor growth inhibitory effects, and reverse obesity-driven cancer aggressiveness ,11,12. BStudies have been performed showing the cancer-preventive effects of metformin in tumor-bearing rodents with obese or diabetic backgrounds involving targeting insulin, AMPK, fat metabolism, antiapoptotic molecules, Signal Transducer and Activator of Transcription 3 (Stat3) and Extracellular Signal-Regulated Kinase (ERK) ,29,30,31Ectopic tumor growth was produced via the implantation of LLC cells into male C57BL/6 mice that were pre-fed with a Normal Diet (ND) or High-Fat Diet (HFD) over a period of 10 weeks. Within a period of 3 weeks, tumors had grown more aggressively in the HFD-fed obese mice than in the ND-fed lean mice, as evidenced by an increase in both tumor volume A and tumTo correlate with malignant progression and systemic alterations, changes in malignance-associated signaling molecules were examined from resected tumor tissues. Tumors seen in HFD-fed obese mice had an apparent increase in proliferation-promoting cycline D1, β-catenin, HMGB1, Receptor for Advanced Glycation End-Products (RAGE), Akt phosphorylation, Smad2/3 phosphorylation, aerobic glycolysis-associated Glucose Transporter-1 (GLUT1) and Pyruvate Kinase M2 (PKM2), glutaminolysis-associated Solute Carrier Family 1 Member 5 (SLC1A5), SLC7A5, glutaminase, platelet-associated CD62P and neutrophil-associated Myeloperoxidase (MPO). Those elevations were all mitigated by metformin . TherefoBeyond the tumor growth inhibitory evaluation in tumor-bearing mice, tumor cellular responses to metformin were further examined in vitro. Metformin caused a slight reduction in cell viability when exposed to higher concentrations A, while In order to uncover any anticancer actions occurring due to metformin, proliferation and metabolism relevant signaling molecules were further examined in LLC cells. Decreased levels of cyclin D1, β-catenin, HMGB1, RAGE, Akt phosphorylation, Smad2/3 phosphorylation, GLUT1, PKM2, SLC1A5, SLC7A5 and glutaminase were revealed in cells treated with metformin A. MoreovNeutrophils and neutrophil-derived NETs have each been implicated in the aggressiveness of malignancy ,25. To eTo gain further insight into the suppressive effects of metformin on NETs, in vitro human neutrophils were modeled for investigation. Similar to previous relevant studies ,34,35,36To determine the presence of NETs in the resected tumor tissues, a crosstalk between cancer cells and neutrophils was investigated in an in vitro cell model. Human bladder cancer cell line T24 and human lung carcinoma cell line A549 were both cultivated, with the cultured conditioned medium then harvested for evaluation. Based on flowcytometric detection of citrullinated histone H3, conditioned medium of the A549 and T24 cells was relatively inert to the induction of NET formation from human neutrophils. However, both conditioned media promoted additional NET formation in responding to PMA stimulation . AlthougAlthough the cancer-preventive effects of metformin remain controversial, its usage and subsequent reduction of cancer risk has been seen in certain cancer patients, particularly those subjects possessing a diabetic background ,15,37,38Beyond acting as a metabolic integrator, insulin coordinates cancer cell metabolism, proliferation and motility through its action on Akt/mTOR axis, lipogenesis and mitochondria. Therefore, both insulin and AMPK are two well-reported targets for the anticancer actions of metformin ,17. CancBoth obesity and cancer are chronic diseases involving low-grade inflammation. Lymphocytes, neutrophils, platelets, TGF-β1 and HMGB1 have all been implicated in both pathogenesis and obesity-driven cancer ,42,43,44Neutrophils are key component cells in cancer progression and metastasis, having either cancer-promotion or cancer-inhibition effects. Accumulating evidence has highlighted that circulating and infiltrating neutrophils are proposed to be key mediators in neoplastic transformation, cancer cell proliferation and metastasis, malignant progression, angiogenesis, matrix remodeling and tumor immunity through NET structure ,45,46. UHMGB1, a nonhistone DNA-binding protein, possesses pleiotropic biological activities, including cell proliferation, autophagy, inflammation and NET formation ,36,43,47Metformin has displayed anticancer effects towards different types of cancer cells in an in vitro cell study, with Nrf2, YAP, HIF-1α, HMGB1, TGF-β1, glycolysis and glutamine metabolism being proposed action targets at a cellular level ,21,22,23Despite the current anticancer findings, there are limitations in the interpretation of the mechanistic actions of metformin. First, the effects of metformin on tumor growth without including any diet variables were not investigated. To have a better understanding of metformin’s anticancer actions and its application, a study involving a comparable group consisting of tumor-bearing mice that have been fed with a normal control diet and supplemented with metformin is of importance. Second, the metabolic actions may be a prerequisite to the anticancer effects of metformin. It should be noted that the improvements may also be secondary to weight loss or tumor reduction. Lastly, although the current study identified that NETs could be an action target of metformin’s anticancer effects, the detailed mechanistic actions towards NETs inhibition were not characterized. A deeper investigation centered on NETs in both tumor-bearing mice and neutrophil cell models is encouraged.To extend the anticancer study of metformin, as it is centered on certain metabolic situations, a syngeneic tumor model was established through the implantation of LLC cells in HFD-fed obese C57BL/6 mice. Metformin treatment mitigated tumor growth, with accompanying alleviations seen in body mass, fasting glucose, fasting insulin, insulin resistance, circulating WBCs, lymphocytes, neutrophils, plasma leptin, soluble P-selectin, TGF-β1 and HMGB1, as well as the tumor signaling molecules of cyclin D1, β-catenin, HMGB1, RAGE, Akt phosphorylation, Smad2/3 phosphorylation, GLUT1, PKM2, SLC1A5, SLC7A5, glutaminase, CD62P and MPO, while elevation occurred in circulating glutamine. In vitro, metformin inhibited LLC cell proliferation and migration, while decreasing parallel oncogenic signaling molecules. Moreover, tumor-bearing HFD-fed obese mice displayed higher biochemical markers of the NET structure in the bloodstream and tumor tissues, with the suppressive effect being caused by metformin. Data taken from a human neutrophil study confirmed the inhibitory effect that metformin has on NET formation. Furthermore, HMGB1 was identified as a promoting molecule coming from cancer cells to boost the transition process toward NETs. Despite these remaining limitations, our current study has revealed that metabolic, pro-inflammatory and NET alterations appear to play pivotal roles in the obesity-driven aggressiveness of cancer, and represent candidate targets for metformin with regard to its anticancer abilities.2 incubator at 37 °C. For conducting experiments, cells were placed in their corresponding media containing 5% FBS. Cell viability was evaluated using a Cell Counting Kit-8 , according to the manufacturer’s instructions.Human bladder carcinoma T24 (#60062), human lung carcinoma A549 (#60074) and murine LLC (#60050) cells were obtained from the Bioresource Collection and Research Center . Upon propagation, T24, A549 and LLC cells were maintained in McCoy’s 5a medium, Ham’s F12K medium and Dulbecco’s Modified Eagle Medium (DMEM), respectively, with additions of 1% nonessential amino acids and 10% fetal bovine serum (FBS) in a 5% CO3 cells in 100 μL of serum-free DMEM) were implanted subcutaneously into the right flanks of the mice. Three days after cell implantation, the ND group (n = 6) and half of the HFD group (n = 6) were intraperitoneally administered daily doses of normal saline, while the remaining half of the HFD group (n = 6) received an intraperitoneal metformin (50 mg/kg) injection. All mice were continuously fed their appropriate ND or HFD for an additional 18 days. The dosages and administration routes of metformin were according to the previous study /22.5, and used as the reported method [Prior to being sacrificed, the mice were not allowed access to any food for 8 h. Under anesthesia with isoflurane, blood was withdrawn from the left femoral artery. WBCs, lymphocytes and neutrophils were each measured using routine complete blood counts. Plasma levels of insulin  in a DMEM, supplemented with 5% FBS over the course of 6 days. To visualize the grown cell colony, cells were stained with crystal violet.4) in 200 μL DMEM containing 2% FBS with the presence of various concentrations of metformin (0–1 mM) were added to the Transwell inserts . The Transwell inserts were put into a 24-well plate and the lower chambers were filled with DMEM containing 10% FBS for a course of 24 h. Cells that trans-migrated to the lower surfaces of the Transwell inserts were stained with Giemsa. During visualization under a light microscope, six random fields per insert were picked up, with cell numbers then being counted for evaluation.Cell migration was evaluated using a method based on a 24-well Transwell apparatus according to reported procedures . LLC celx 380 nm and Em 460 nm).Caspase 3 activity in the resected tumor tissues was measured using a Caspase Fluorometric Assay Kit  according to the manufacturer’s instructions. Equal amounts of extracts were subjected to the reaction, with the yielded fluorescence signals measured using a fluorometer (Ex 480 nm and Em 520 nm) and calibrated with a standard curve.To measure the plasma levels of dsDNA, samples were incubated with Sytox Green  at a concentration of 1 μM for 5 min. The fluorescence signals were measured with a fluorometer  were incubated with anti-citrullinated histone H3 , anti-neutrophil elastase , anti-PAD4  and anti-MPO  antibodies. After further incubation with biotinylated secondary antibodies and avidin–biotin–peroxidase complex, sections were developed with diaminobenzidine and counterstained with hematoxylin. The immunoreactive intensity in the sections was then quantified using Image J software .g for 30 min. The high-density fraction pellets were then subjected to red blood cell lysis [g for 10 min. The supernatants were named CM. For the induction of NETs, neutrophils were treated with a vehicle, either PMA (100 nM), HMGB1 (1 μg/mL), metformin (500 μM), A549 CM , T24 CM  or HMGB1-neutralizing IgG , or with combinations of vehicles for a period of 3 h. For the neutralization study, A549 CM and T24 CM were incubated with HMGB1-neutralizing IgG (5 μg/mL) for 15 min prior to being added to the neutrophils. After fixation and permeabilization, neutrophils were then incubated with either an anti-citrullinated histone H3 antibody or isotype IgG , followed by an Alexa Fluor 488-labeled secondary antibody . The levels of citrullinated histone H3 immunopositivity were measured using the FACSCalibur and analyzed by Cell Quest Pro software .To determine NETs induction in vitro, human neutrophils were prepared from 10 healthy volunteers of both genders after obtaining written informed consent. The study protocol  was approved by the Taichung Veterans General Hospital Institutional Review Board. Peripheral blood (10 mL in an EDTA tube) was drawn from each individual participant and diluted with an equal volume of dextran 500 (3%). After red blood cell sedimentation, the upper layer was gently nestled on top of a Histopaque-1077 solution, followed by centrifugation at 400× ll lysis . The obtp value less than 0.05.Graph preparation and data analyses were performed with SigmaPlot (12.3) and GraphPad Prism 8.0 Software . For multiple groups and variables, one-way analysis of variance (ANOVA) with a Bonferroni or Dunnett post hoc test was performed. For the in vitro NETs study, data are representative of three biological replicates from each individual participant. All values are listed as mean values ± standard deviation (SD), with differences between groups being statistically significant at a