BCR::ABL1 protein. This translocation represents one of the milestones in molecular oncology in terms of both diagnostic and prognostic evaluations. The molecular detection of the BCR::ABL1 transcription is a required factor for CML diagnosis, and its molecular quantification is essential for assessing treatment options and clinical approaches. In the CML molecular context, point mutations on the ABL1 gene are also a challenge for clinical guidelines because several mutations are responsible for tyrosine kinase inhibitor resistance, indicating that a change may be necessary in the treatment protocol. So far, the European LeukemiaNet and the National Comprehensive Cancer Network (NCCN) have presented international guidelines on CML molecular approaches, especially those related to BCR::ABL1 expression. In this study, we show almost three years’ worth of data regarding the clinical treatment of CML patients at the Erasto Gaertner Hospital, Curitiba, Brazil. These data primarily comprise 155 patients and 532 clinical samples. BCR::ABL1 quantification by a duplex-one-step RT-qPCR and ABL1 mutations detection were conducted. Furthermore, digital PCR for both BCR::ABL1 expression and ABL1 mutations were conducted in a sub-cohort. This manuscript describes and discusses the clinical importance and relevance of molecular biology testing in Brazilian CML patients, demonstrating its cost-effectiveness.Chronic myeloid leukemia (CML) is a well-characterized oncological disease in which virtually all patients possess a translocation  that generates the tyrosine kinase  BCR (breakpoint cluster region protein) ABL1  fusion gene, which produces a constitutively activeyrosine-kinase, resulting in the general imbalance found in CML [https://cancerstatisticscenter.cancer.org/#!/cancer-site/Leukemia, accessed on 1 March 2023). In Brazil, the INCA  estimated that 10,070 new leukemia cases were diagnosed in 2017, of which 10% were CML [Chronic myeloid leukemia (CML) is characterized by the presence of the Philadelphia chromosome (Ph) resulting from the reciprocal translocation t9;22) q34;q11). This translocation gives rise to the 1. This t2 q34;q11The largest oncology hospital in the state of Paraná, the Erasto Gaertner Hospital, has a vast center of Hematology and Hemotherapy, which performs therapies of the highest complexity, such as Cellular Therapy with T Cells (CAR T), in addition to Bone Marrow Transplantation for various oncohematology pathologies. The majority of Erasto Gaertner Hospital patients (80%) are assisted by SUS, so data from this hospital is adequate to show the relevance and prevalence of CML, as well as the impact of the introduction of TKIs in the early 2000s. In a survey carried out using audit data from Authorizations for Outpatient Procedures (APAC), there were more than 4800 TKIs emissions between January 2008 and January 2023. A total of 75% of the emissions were for imatinibe, and approximately 35% were for second-generation inhibitors (dasatinib and nilotinib), corroborating the international literature, which presents approximately 1/3 of patients as refractory to the first line of therapy . In the BCR::ABL1 mRNA [CML is divided into three phases, which are distinguished by the number of immature blasts in the blood or blood marrow: chronic (CP), accelerated (AC), and blast phase (BP) . DiagnosBL1 mRNA .BCR::ABL1 quantification in peripheral blood cells at the time of diagnosis is set as 100%, and the subsequent quantifications are expressed in percentage by the BCR::ABL1/ABL1 ratio. The reduction in this ratio in subsequent tests is used to assess the expected response to treatment. When there is a reduction of 2 logs, representing 1% of the original level, there is a complete cytogenetic response (CCyR). Likewise, a reduction of 3 logs (0.1% of tumor cells) is referred to as a major molecular response (MMR or MR3), and a reduction of 4 logs is called deep molecular response  [RT-qPCR is usually performed with cells from the peripheral blood during the initial workflow and can be correlated to the number of CML cells and measured using an international scale (IS) of molecular response . In CML .5, MR5) .BCR::ABL1 transcripts every three months in the first year of treatment and then every six months once MMR is reached. However, if the quantification of BCR::ABL1 remains ≥10% after 3–6 months of treatment, or ≥1% after 12 months, the treatment is considered to have failed. Both cases are indicative of resistance mutations or a high risk of abnormal chromosome aberration in Ph+. In patients who do not reach DMR, treatment may be changed to a second-generation TKI (2GTKI) to improve the depth of response [The recommended procedures for treatment follow-up include the quantification of response .4 and two years in MR4.5 is required [BCR::ABL1 levels.On the other hand, MMR at 12 months is associated with a lower rate of disease progression and a higher probability of reaching DMR—a prerequisite for discontinuation of the treatment, which, when successful, is called treatment-free remission (TFR). To try discontinuation protocol, a minimum 5 years of TKI therapy with a sustained DMR of at least three years in MRrequired , confirmBCR::ABL1 ≤10% IS) in three or six months; 20–59% reach MMR within one year; 60–80% reach MMR in five years; and 35–68% reach DMR in five years [BCR::ABL1 tyrosine kinase, reducing the side effects. Imatinib resistance occurs in 10–15% of cases and in about 10% of patients treated with 2GTKIs. Known mechanisms of resistance include clonal evolution, mutations in the BCR::ABL1 kinase domain, and activation of BCR::ABL1 independent pathways. Many BCR::ABL1 kinase domain mutations have been described. Changes in treatment may be recommended depending on the mutation [Imatinib belongs to the first generation of TKIs (1GTKIs), which substituted the previous treatment with recombinant interferon alpha (IFNα) and low-dose cytarabine. The IRIS study showed higher rates of cytogenetic and molecular responses and better progression-free survival (PFS) and overall survival (OS) in patients treated with imatinib ,15. Betwve years . PFS ratve years ,16. Othemutation ,17,18, aABL1 kinase domain resistance mutations [4.5 (42%). However, PFS and OS were no higher than imatinib .Dasatinib has a 325-fold higher potency in inhibitory activity against some utations . In addiutations . They foBCR::ABL1 tyrosine kinase and also demonstrates clinical activity against some ABL1 mutations. It is recommended as a second-line therapy for CP or AC patients resistant or intolerant to imatinib [4 (66% and 73%), and MR4.5 . The five- and 10-year OS, meanwhile, were not different from imatinib .Nilotinib, another 2GTKI, was developed to have enhanced selectivity and potency toward imatinib . The druimatinib . The fivBCR::ABL1 resistance mutants. The BFORE study found that the early molecular response rate (75%) and one-year MMR rate (47%) with bosutinib were significantly higher than with imatinib [Bosutinib is a 3G TKI that also inhibits several imatinib . Ponatinimatinib . The resimatinib .BCR::ABL1 is essential for all patients [Several clinical studies have evaluated the effectiveness of treating CML patients with different TKIs ,26,27,28patients ,30,31.® [® 400 mg  is priced at around $3870, while generic imatinib is priced at $2515. This amount can be reduced with a subsidy; therefore, considering a positive scenario with a partial subsidy for generic imatinib, the monthly cost per patient is approximately $1200. The cost of treating these 4960 CML patients who do not respond to imatinib therefore represents an annual expenditure of over $70 million on an ineffective treatment. Regarding the other group comprised of around 8150 patients treated with imatinib for more than five years, 66% (5187 patients) will achieve MMR and DMR and can be included in the TFR protocol. Studies show that only 35% of patients in TFR will have a recurrence in six months [Imatinib treatment for CML patients is quite costly, even with the availability of generics to Gleevec® ,33. To i® ,21,28,34CML is characterized by a period of innate and adaptive immune dysfunction at diagnosis, which prevents antileukemia immune responses. Immune suppressor cells, including myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs), contribute to T-cell dysfunction and disease progression in CML, expanding at diagnosis, and reducing following TKI therapy. Clinical data showed that imatinib or dasatinib-treated patients exhibit an expansion of CD8+ CTLs or NK cells, which are associated with an improved response to therapy. Concerning immunotherapies for CML, IFN-α may provide an alternative approach in selected TKI-resistant mutation-positive CML patients, particularly those ineligibles for bone marrow transplantation. A combined therapy with Chimeric Antigen Receptor T-cells therapy (CAR-T) and TKI may confer particular therapeutic benefit to TKI-resistant/intolerant, young, or advanced phase CML patients .In this manuscript, we describe an almost three-year molecular oncology project in CML patients in a single Brazilian treatment center, with several approaches that provide clinically relevant and directly applied data, resulting in positive public health and financial impacts in a complex and expansive scenario.BCR::ABL1 and ABL1. LoQ represents the lowest detected concentration that maintains the linear regression with an r2 equal to or higher than 0.99, ensuring that data extrapolation enters the direct quantification interval. We observed an LoQ of 500 copies/well for both BCR::ABL1 and ABL1  for both and ABL1 . It is iBCR::ABL1 and 87% for ABL1.BCR::ABL1 detection and quantification, as demonstrated in The cross-validation assay was performed by comparing the results obtained from our test with those obtained from a private laboratory. The results for 24 samples were similar, although a slight difference was observed in BCR::ABL1, while all two positive cell lines showed amplification.Analytical specificity and sensitivity were assessed using RNA from 16 human cell lines, of which 14 were negative and 2 were positive t. The assay had a specificity and sensitivity of 100%, with none of the negative cells exhibiting amplification of the BCR::ABL1 of 19 samples were quantified in parallel with DualQuant and Mobius commercial kit, we observed that DualQuant assay provided a significantly lower cycle threshold compared with the commercial kit  were male and 69 (44.6%) were female. The medium age at diagnosis was 50.56 (±14.94) years old. The number of samples per patient varies from 1 to 15 due to several factors such as disease stage, treatment response, recommended follow-up milestones, and clinical outcomes. BCR::ABL1 quantification by RT-qPCR for 496 samples obtained from the 155 CML patients. Molecular quantification of BCR::ABL1 aims to evaluate treatment efficacy and adaptation and reveals the undeniable value of this molecular tool for clinical decision and management. Considering BCR::ABL1 molecular monitoring, our cohort was divided into four groups: (1) BCR::ABL1 reduction; (2) Drug alteration; (3) Persistently low BCR::ABL1 patients; (4) Allogeneic Hematopoietic Stem Cell Transplantation . The first group comprises patients presenting a reduction in BCR::ABL1 levels during follow-up, demonstrating the effectiveness of TKI treatment. The second group comprises the patients presenting an increase in molecular BCR::ABL1 levels, demonstrating that TKI treatment is not effective and that there is a need to change the treatment protocol. The third group comprises patients with molecular BCR::ABL1 quantification lower than 1% but never reaching MMR 4.0, showing persistently low levels. This feature may indicate the maintenance of the leukemic clone. The fourth group comprises patients that underwent BMT, either because of resistance or relapse. BCR::ABL1 molecular levels for these different groups of patients.In this clinical cohort, we performed BCR::ABL1 levels to detect relapse after allo-HSCT as early as possible. In this cohort, five patients underwent allo-HSCT. Three of them did not show any of the TKI resistance mutations analyzed here , while two of them presented the T315I mutation. From the three wild-type cases that underwent HSCT, two of them are currently stable and are not being treated with TKIs, while one case presented severe GVHD, the major complication after HSCT. The other two HSCT patients carried the T315I mutation. One of the T315I cases currently has undetectable levels of BCR::ABL1 and is under Dasatinib treatment. Importantly, the T315I mutation was no longer detected after allo-HSCT in this patient. The other T315I case is under Ponatinib treatment but showed a worse response, with the most recently measured levels of BCR::ABL1 being greater than 1%.Allogeneic hematopoietic stem cell transplantation  is recommended in CML when disease resistance occurs, including suboptimal response to two or more TKIs or even failure to respond to Ponatinib. It is crucial to monitor BCR::ABL1 IS% of each year for each unique patient. The number and percentage of patients that reached DMR in each year are also shown and correspond to the sum of patients with MMR of 4.0 or less. The average percentage of patients that reached DMR in each complete year of follow-up was 49.1% .The number and percentages of patients in each level of response to TKIs can be seen in BCR::ABL1 IS% in their last visit to the hospital.The total number of unique patients that reached DMR in the whole 33 months of follow-up was obtained by selecting only the latest sample in the total period for each patient. By doing so, from a total of 178 unique patients, 99 (55.6%) reached DMR, while the remaining patients showed different levels of response, as shown in BCR::ABL1 quantification is an essential requirement for the treatment-free remission protocol, as patients must be on TKI with DMR for two years to be eligible. We subdivided the patients in our study cohort receiving imatinib treatment into those who are within TFR requirements for one- and two-year follow-ups. In total, we found 14 patients in DMR or undetected BCR::ABL1 expression at their one-year follow-up, indicating that they should have one more year of molecular quantification to have TFR requirements. In addition, 19 patients are currently under TFR requirements and eligible for imatinib discontinuation. In our cohort, there are six CML patients in the TFR protocol, with imatinib discontinuation ranging from 294 to 602 days (440.33 ± 98.91 days), all of whom are in DRM or with undetected BCR::ABL1 expression for all clinical samples quantified by RT-qPCR  was performed for the most recently collected samples from 22 patients in DMR. These patients had DMR or undetected ABL1 mutation in CML patients by qPCR. T315I (rs121913459) was detected in four patients, E255K (rs121913448) was detected in one patient, and V359F (RS121913452) was detected in one patient. T315I mutation results in clinical resistance to first- and second-line TKIs such as imatinib, dasatinib, and nilotinib; hence, ponatinib is indicated in these cases. The E255K mutation confers a low response to all TKIs, including ponatinib. The V359F mutation is clinically linked with a positive correlation with dasatinib, which is the most effective treatment for patients with this mutation. We found two patients who carry the T315I mutation, who presented 43.8% and 80.4% of BCR::ABL1 expression by molecular quantification, and were submitted to allo-HSCT. After allo-HSCT, for these two patients, the peripheral blood was screened for rs121913459 (T315I) by qPCR and showed no presence, indicating an effective transplantation and highlighting the importance of screening ABL1 mutations. However, in posterior samples from these patients, BCR::ABL1 increased again, and dPCR analysis showed the presence of T315I . For Brazilian and Latin American laboratories, a secondary reference calibrators panel was developed by Bianchini et al. to provide a harmonization panel for BCR::ABL1 measurement that could be applied to the molecular monitoring response of patients [The current clinical management and guidelines of CML treatment require the close molecular monitoring of epancies ,38,39. Tpatients .4, MR4,5, and MR5) [BCR::ABL1 in the Brazilian Unified Health System (SUS), noting that this approach should be part of the integral treatment of patients with CML since this monitoring will reduce the chances of disease progression, decrease health system costs, ensure compliance with international guidelines, and allow eligible patients to enter the TFR protocol, which will lead to cost savings that more than offset the cost of molecular testing [BCR::ABL1 molecular monitoring in CML patients, with 60 patients followed from June 2005 until September 2008, with hematological, major cytogenetic, and complete cytogenetic responses achieved by 95%, 75%, and 63% patients, respectively, while 40% of patients achieved a major molecular response in a median time of 8.5 months [4, MR4,5, and MR5, respectively, indicating that more than 77% of CML patients in the cohort had a good clinical and molecular response [BCR::ABL1 quantification was compared in 631 paired peripheral blood and bone marrow samples from 283 CML patients, a good overall concordance was observed, but there was a systematic tendency towards higher BCR::ABL1 levels in peripheral blood than in bone marrow, supporting the current practice of using peripheral blood [4, i.e., BCR::ABL1 RT-qPCR < 0.01% IS for 12 months, showed a negligible risk of regressing to MR3, suggesting that MR4 can be characterized as a secure molecular threshold and that patients within this group would need less frequent monitoring [Effective TKI therapy in CML patients requires the achievement of a deep molecular response (MRand MR5) ,42. A Br testing . One Bra5 months . In anotresponse . When BCal blood . It was nitoring . The secnitoring .In a cohort of 208 CML patients who had undergone three months of treatment with imatinib, 137 patients (65.8%) achieved CCyR, and 15 (7.2%) achieved MR3, while 11 patients (5.2%) showed any early molecular response. After 12 months, 83.1% achieved CCyR, and 62% achieved MR3, while after a seven-year follow-up, 64.4% reached MR3. The cumulative incidence of MR4 was 51%, and that of MR4.5 was 34.6% after a median time of 3.8 and 5.4 years .BCR::ABL1 milestones such as MMR could be supported by the acquisition of TKI-resistant BCR::ABL1 kinase domain mutations, though once DMR is achieved, it is relatively stable, and the risk of TKI resistance is low [BCR::ABL1 kinase domain mutation analysis, indicating when and how to perform mutation analysis and how to translate results into clinical practice [BCR::ABL1 quantification, of which just 2 exhibited a mutation of the ABL1 kinase domain (E255K and M351T) [BCR::ABL1 kinase domain mutation, from which 7.2% had T315I and 3.2% had E255K [ABL1 mutation in a sub-cohort of 175 patients who exhibited TKI resistance  and found that just 54 harbored mutations in the kinase domain, with there being a greater frequency of T315I and E255K [ABL1, with the highest frequencies found for T315I and G250E  [ABL1 mutations in 58 CML patients undergoing treatment with imatinib who showed a suboptimal response [ABL1 wild type, while, surprisingly, one-third remained sensitive to imatinib and bind with similar or higher affinity than the wild type, identifying three clinical ABL1 mutations that bind imatinib with wild type-like affinity but with considerably faster dissociation [The loss of TKI therapy effectiveness and e is low . The Eurpractice . A Brazid M351T) . In a read E255K . A recenctively) . Anotherresponse . An inte5 and monitored 69 patients for at least 12 months. A total of 42 patients relapsed . At 12 months, the probability of persistent MR5 for these 69 patients was 41%. All patients who relapsed responded well to the reintroduction of imatinib, suggesting that imatinib can be safely discontinued in patients with MR5 for at least two years [BCR::ABL1 mRNA levels (approximately MR4.5) under TFR protocol and with a median follow-up of 8.6 years (ranging from 5.7 to 11.2 years). Eighteen patients remained in continuous TFR, and no patient progressed to the advanced phase, suggesting long-term safety and remarkable stability of response after imatinib discontinuation in appropriately selected CML patients [4. The median time from TKI discontinuation and the loss of MMR to TKI treatment restarting in the TFR failure group was 4 months and 4.4 months, respectively. This report suggests a threshold value of BCR::ABL1 RNA expression for TKI discontinuation, where an absolute value of <0.0051% at six months was associated with an extremely high chance (over 90%) of maintaining MMR after treatment discontinuation, both for patients stopping imatinib or 2G-TKI [BCR::ABL1 doubling time was associated with a higher rate of TFR failure [The treatment-free remission protocol relies on a social-economic benefit in CML clinical management. In one of the first reports in this field, Mahon et al. recruited CML patients ≥ 18 years of age in MRwo years . A similwo years . A long-patients . A recenpatients . A similpatients . In a rer 2G-TKI . This re failure .BCR::ABL1 and ABL1 mutations is consolidated with real-time PCR. However, digital PCR methods are emerging, even for BCR::ABL1 quantification and for subsequent TKI discontinuation [BCR::ABL1 %IS  droplet digital PCR (ddPCR) assay—the first commercially available tool of its type—and found a very strong correlation between this assay and the real-time PCR ipsogen BCR::ABL1 Mbcr IS-MMR , suggesting that dPCR can provide reliable results for CML patients [BCR::ABL1 analysis until the moment no divergence results could be identified, as shown in ABL1 mutations has a great amount of influence on the prognosis.The molecular detection of inuation ,63,64. Apatients . A more patients . About BBCR::ABL1 quantification using an in-house duplex one-step RT-qPCR protocol and detecting ABL1 mutations. As suggested by international guidelines, the molecular monitoring of CML patients provides clinical benefits, allowing clinicians to make assertive and correct decisions regarding TKI choice and the proper follow-up of patients recruited for TFR protocol. Beyond the clinical benefits, the molecular monitoring of CML patients provides socio-economic advantages that warrant its recommendation by the Brazilian public health system. This manuscript describes and discusses the molecular biology usage in CML patients as an advance in clinical practice using known biomarkers such BCR::ABL1 expression and ABL1 mutations. An interesting point left out in this manuscript relies on novel genetic alterations in CML patients that may be represent risk factors for disease progression and treatment response. This topic is widely studied worldwide by several groups, including our research group, in various scientific fields to access information that contributes to the determination and validation of novel biomarkers for these patients.In this manuscript, we describe the molecular management in a single-center Brazilian CML cohort, especially with respect to BCR::ABL1 quantification and ABL1 mutation screening represents an essential and necessary approach for CML treatment follow-up and therapy adaptation. The suggested BCR::ABL1 RT-qPCR quantification protocol [ABL1 mutation screening should be performed to assertively determine the mutation to select the optimal TKI. For this, the digital PCR approach described in this paper is a methodology that we proposed to be used especially for ABL1 mutations when BCR::ABL1 expression shows signs of increased expression, anticipating the ABL1 mutation and performing the TKI change.In conclusion, we demonstrate that molecular biology for protocol  for CML Study cohort—clinical samples from patients with chronic myeloid leukemia were collected from diagnostic and confirmed cases in Erasto Gaertner Hospital, Curitiba, Paraná, for 33 months from May 2020 until February 2023 following approval from the Erasto Gaertner Hospital Ethics Committee (CAAE 08809419.0.0000.0098). Sample collection and experimental design followed Brazilian guidelines and regulations. The project was described in detail for all participants who read, discussed, and signed an informed consent form before sample collection. For each patient, 4 mL of peripheral blood was collected in EDTA tubes and processed within 24 h. Patients could have blood collected several times during treatment and clinical follow-up during the project period. This means that mostly recruited patients had more than one sample collected, resulting in a much greater amount samples than patients analyzed. The blood was centrifuged (500× g/10 min/4 °C), and the buffy coat was collected with a Pasteur pipet in a 15 mL tube. RNA extraction was processed utilizing a Qiamp RNA Blood Mini Kit , while DNA was extracted with a Qiamp DNA Blood Mini Kit , both by following the instruction manual. Personal and clinical data such as age, gender, diagnostic date, and treatment were also accessed from patients’ charts.BCR::ABL1 target, a partial sequence from the fusion transcript b3-a2 was purchased from GenScript , where it was cloned in pGEM®-T Easy Vector Systems . For ABL1, RNA from the Jurkat cell line was extracted with Qiamp RNA mini kit  and synthesized to cDNA with Random Primers and SuperScriptIII . After that, using ABL1 primers, an end-point PCR was performed using Platinum™ Taq DNA Polymerase High Fidelity  and PCR product purified with QIAquick PCR Purification . The purified amplicon was cloned in pGEM®-T Easy vector . Both BCR::ABL1 p210 and ABL1 vectors were transformed into Escherichia coli cells grown in LB-Amp medium for 16–20 h, and plasmid DNA was extracted with a GeneJET kit . The plasmids were measured in NanoDrop One , and concentrations were transformed into copy numbers using the following formula: Copy number = (mass × 6.022 × 1023)/(length × 1 × 109 × 650), where 6.022 × 1023 is Avogadro’s number and 650 is the average weight of a base pair.Standard curves—for the BCR::ABL1 quantification assay—after leukocyte RNA extraction, the molecular quantification of BCR::ABL1 was made using an in-house one-step duplex qPCR. In this assay, two sets of primers were used simultaneously. The first set was designed to detect both BCR::ABL1 b3-a2 and b2-a2 isoforms with a probe labeled with HEX, and the second detected ABL1 in both BCR::ABL1 b3-a2 and b2-a2 isoforms and wild-type transcript, with a probe labeled with FAM. All primers and probes were purchased from IDT  and resuspended in nuclease-free water . The molecular quantification of BCR::ABL1 was performed in a final volume of 20uL using TaqPath™ 1-Step RT-qPCR Master Mix, CG , 2.4uL of oligonucleotides, and 5uL of RNA in LightCycler96 equipment . The reaction conditions were 50 °C for 30 min, 95 °C for 5 min, and 45 cycles of 95 °C for 15 s and 60 °C for 30 s. Linear regressions of the BCR::ABL1 and ABL1 standard curves were generated by plotting concentration and cycle threshold on the x and y axes, respectively, generating slope, Y-intercept, and r2 values. These standard curve parameters were used to assess the absolute concentrations of BCR::ABL1 and ABL1 on the secondary reference calibrator panel for each sample, and a ratio of BCR-ABL/ABL1 was calculated. The laboratory-specific conversion factors were generated using these data. Values were used for each clinical sample’s BCR::ABL1 quantification.6 copies/well to 5 × 102 copies/well.Limit of quantification—nine independent experiments with a standard curve of five points with ten-fold dilution in duplicate were used, ranging from 55 × 10Amplification efficiency and repeatability—to access amplification efficiency without using the standard curve but with a real scenario, we used RNA extracted from the KCL-22 cell line, which is t positive. Three independent experiments were performed, using five points with two-fold dilution in duplicate, from 200 ng until 12.5 ng of RNA. This assay was used to calculate repeatability parameters, which was determined as relative standard deviation percentage in repeatability conditions (rRSD%), calculated by the formula rRSD% = repetitions SD × 100/average of repetitions.Analytical specificity and sensibility—200 ng of extracted RNA from 16 human cell lines were used, of which 14 were negative and 2 were positive for t expression: A549 (lung); Capan, MiaPaCa, Panc1, and AsPC1 (Pancreas); MCF7 and MDA-MB-231 (breast); Huh7.5 (liver); SW20 (colon); HL60, THP1, ReH, and Jurkat (hematopoietic); NHDF (fibroblast); K562 and KCL-22 (chronic myeloid leukemia).BCR::ABL1 quantification: First, 24 samples were quantified in parallel by a private laboratory. Second, to avoid sampling, equipment, and manipulation bias, 19 samples were quantified in our laboratory using the commercially available Kit XGEN MIX p210 .Cross-validation assay—two approaches were evaluated to compare BCR::ABL1 in a ratio of BCR::ABL1/ABL1 between 10% and 0.001%. This secondary reference calibrator panel was necessary for BCR::ABL1 quantification on the International Scale (IS), deriving laboratory-specific conversion factors (CF). The panel could also determine the assay sensitivity of the established TKI clinical response criteria between 10%, 1%, 0.1% (MR3), and 0.01% (MR4). To give a deeper assay performance, the secondary reference calibrator panel presented an additional dilution  to assess the assay limit of detection; however, it was not considered for the estimation of the CF. Each panel duplicate had RNA extracted on different days using the RNeasy mini kit (Qiagen) and following the instructions. The BCR::ABL1 quantification assay was performed four times on different days, with the standard curve ranging from 5 × 106 to 5 × 101BCR::ABL1 and ABL1 copies per well. The BCR-ABL/ABL1 ratio was calculated for all assays to calculate the CF.Assay validation and certification—a secondary reference calibrator panel was purchased from Dr. Michele Bianchini of CIO-FUCA, Alexander Fleming Institute, Buenos Aires, Argentina. This secondary reference calibrator panel is composed of a set of five vials in duplicate, each one containing a mixture of lyophilized K562 and HL-60 cell lines in different proportions, reproducing different percentages of 3 or to assess information about mutation in ABL1. For this purpose, two different master mixes were purchased from Qiagen—Qiacuity OneStep Advanced Probe kit and Qiacuity UCP Probe PCR kit—which were used as recommended. To detect ABL1 mutations, rs121913459 (T315I), rs121913448 (E255K), and rs121913452 (V359F) were used in TaqMan assays (ThermoFisher) in the concentration recommended in the Qiacuity UCP Probe PCR kit manual. The PCR reactions were amplified, and the images were captured by Qiacuity one using Qiacuity Nanoplate 26k 24-well. The analyses were performed in Qiacuity Software Suite. The samples for the detection of ABL1 mutations were chosen based on the qPCR genotyping analyses that were performed before dPCR. We chose samples before the detection of the mutation by qPCR, as well as the point where the mutation was detected. Regarding the BCR::ABL1 analysis, the RNA samples were diluted to 10 ng/µL and used 100 ng in the reaction. For the analysis of ABL1 mutations, the DNA samples were diluted to 10 ng/µL and used 100 ng in the reaction as well.Digital PCR—digital PCR was performed to assess information about the molecular response of patients in treatment-free remission (TFR) or in patients in, at least, MRABL1 mutation—the following ABL1 mutations were screened in CML patients: rs121913459 (T315I), rs121913448 (E255K), rs121913461 (Y253H), rs121913449 (E255V), and rs121913452 (V359F) using TaqMan Genotyping assays (ThermoFisher), TaqPath ProAmp Master Mix , and 50ng of DNA in QuantStudio 5 . These same mutations were evaluated in some CML patients by digital PCR, as described above.


This article was migrated. The article was marked as recommended.As the number of graduating medical students increases, the number of primary care residency positions is not keeping pace. One solution to this problem is the creation and accreditation of new residencies, although there is little literature describing the perspectives of the residents and educators who found new programs. Seven out of ten resident physicians who served as the inaugural interns in a new residency training program in pediatrics provide their reflection on the distinctive perspective they had from this experience. They have identified consensus themes in topic areas of strengths, challenges, and lessons learned from training in a new program. Themes applying to strengths of participating in a new residency training program were the opportunity to shape the program, individualized learning experience, and enthusiastic faculty. Challenges of a new program included missing upper level residents, diverse faculty expectations, and morale. Themes under lessons learned included resident engagement, expectations and feedback, and wellness. Each theme was then considered in the context of the medical education literature, underscoring the important balance that new program leaders must strike between structure and flexibility. This inaugural resident class has identified key challenges and opportunities to inform education leaders who are planning new GME training programs. Unfortunately, this change has been occurring without available residency positions increasing accordingly . One approach proposed to meet this need is the creation and accreditation of new residency training programs  community in the United States faces a daunting challenge: the Association of American Medical Colleges (AAMC) has repeatedly predicted a significant physician shortage, and thus an increasing enrollment of medical students , with four others coming from Missouri, Nebraska, Rhode Island, and Washington state. All ten matched from within our top 20% of ranked applicants.Seven residents  prepared personal responses to six open-ended questions: (1) What qualities, features, or opportunities made you choose to rank a newly-formed residency program for your training? (2) Following this first year, what do you think has been the greatest strength of training in a new residency program and how has that impacted your training for your future career? (3) Following this first year, what do you think has been the greatest challenge you have faced as a direct result of being in a new training program? (4) How did you cope with or manage the challenge you described in question #3? (5) What advice would you give to residents who are about to join a new or expanding training program? (6) What advice would you give to those leading a new residency program for ways to support their first-year trainees and maximize the educational impact?We individually conducted a keywords-in-context analysis of the pooled, anonymized responses , then meOpportunity to shape the programThe chance to meaningfully influence the development of the residency was the most important draw to our new program. This potential influence made the program stand out from a large pool of other well-established training institutions. Given prior work experiences in rigid systems resistant to change, the opportunity to shape the culture of learning appealed to us. As interns, we felt a greater sense of purpose by not simply maintaining the function of an established system, but instead by helping to create a residency that will impact future generations of pediatric residents. As trainees, we were encouraged to give direct and frequent feedback for a system we were helping create.Individualized learning experienceThe unusual ability of a residency program to offer an individualized learning experience greatly appealed to us. This was an important factor when deciding to rank the program: a personalized curriculum and a focus on individualized resident education and development. Since there were no upper-level residents, we were given the opportunity to rise above the training of an intern. We had more responsibilities and more opportunities for procedures. Through direct interaction with faculty and staff, we identified individual educational needs and clinical teaching felt truly personalized. This fostered an environment of ownership and responsibility for our own training, leading to improved leadership skills. Overall, we perceived the program to have increased attention to our success via encouragement to provide feedback and partake in an educational experience with our specific needs and goals as the focus.Enthusiastic and available faculty Energetic and passionate faculty helped us feel genuinely welcomed and supported during both the recruiting season and intern year. As they were recently recruited themselves to join a new children’s hospital, faculty members were invested in teaching and in encouraging our successes as individuals. With the capability to work one-on-one with attending physicians, we had unfiltered access to their clinical expertise and wisdom while also benefiting from numerous career opportunities and research mentorships. This dedicated group of faculty gave us more confidence in our decision to invest in a new residency training program.Lack of upper level residents An immediate challenge we faced was not having senior residents. Senior residents can directly shape the learning environment and offer much needed guidance to interns. Interns look to upper levels to model their position and establish role identity and expectations. Many of us found that it was difficult to gauge the normalcy of what we were experiencing because there were no upper level residents to guide our perspective. Interns learn much from the residents who are 1-2 years ahead of them in training, and their absence poses a distinct challenge for a new training program.Inconsistent faculty expectations We quickly realized that faculty expectations of us varied widely, from unrealistically high expectations to disappointingly low expectations. It seemed that some attending physicians assumed that we were the “best in the country.” Other attending physicians appeared to assume that we should act as senior-level residents rather than as interns. On the other end of the spectrum, some attending physicians created the impression that they preferred for us to merely observe patient care, with minimal autonomy. Some services may have found it difficult to incorporate an intern into their already established routine, especially those that relied heavily on advanced practice professionals.Resident morale The challenge of sustaining an optimistic attitude while facing a seemingly experimental year of training was a common theme among the new class. We each faced times during our first year when the appeal of starting a new program was challenged by the reality of the growing pains that accompanied it. Morale declined as we realized that we had less of a voice to instill change than we first anticipated. This was compounded by the unknown of whether the program would be successful. Feelings of loneliness arose, which was thought to be related to a lack of upper level and peer support on individual rotations.Coping with challenges Coping with the challenges of the new program requires a unique set of qualities of those recruited. A strong sense of self-motivation drove us to be proactive, rather than passive, in the program. Many of us sought opportunities to augment our clinical education such as proposing quality improvement projects, promoting program development, conducting independent research, and participating in national meetings. Maintenance of good communication, whether it was direct through attending feedback or indirect through our quarterly Voice in the Program sessions  also proved to be crucial in addressing the challenges.Residents who are proactive in pursuing educational goals and those who are more passively guided by their program can all be successful in residency and become competent physicians. However, in the context of training in a new program, we feel that more proactive residents may be more successful. They can contribute the most to a developing program by actively participating in patient care, contributing to scholarship, and crafting smart goals to effect change. They also tend to be open-minded and can think critically through problems and compromise on solutions. The residents who fit best with our new residency program were patient, mindful of the growing pains, and willing to constantly make changes to improve the efficiency and experience of everyone involved.The goals of a new residency program should include standardized and realistic expectations, open lines of communication, and an environment agreeable to change. A new program should provide a clearly defined set of expectations for each rotation agreed upon by all rotation faculty to guide the interns toward the learning objectives expected to be achieved by the end of the rotation. A program should also provide a framework that frequently solicits feedback from the residents and be prepared to implement changes quickly. Feedback can be accomplished with monthly resident feedback meetings, open-door policies, leaders who demonstrate empathy, and frequent updates on prior concerns or ideas brought up by residents. Residents should be flexible, creative, and not afraid to speak up with novel ideas or ways to improve the system. Everyone should be able to give and receive feedback regardless of level of training. A mutual respect as well as a unified effort for excellence must exist between the program faculty and residents for a new program to thrive. All members of the team need to be heard and know that their contribution is valued.We encourage program leaders to implement a resident-run support system that readily integrates incoming residents, requires little maintenance, and requires little administrative effort. We organized three opportunities for resident-run support: housestaff meetings, fireside chats, and wellness/social events. Housestaff meetings allow the residents to regroup and communicate about national meetings, child advocacy opportunities, ongoing research and quality improvement, and hospital initiatives. Fireside chats provide an informal way to debrief in a safe zone amongst co-residents. These chats exclude program leaders and involve a chief resident to mediate the discussion. It is helpful to have a chief resident who is an advocate for the interns. The effective chief resident listens to resident concerns, assists in brainstorming creative solutions to problems, and provides encouragement and an uplifting perspective. In addition to structured housestaff meetings and fireside chats, the emphasis on wellness should also be resident run. One of the challenges we faced in integrating wellness into the curriculum was the perception that there is no room or time; however, opportunities for residents to check in with each other in a residency-sponsored event may be one of the most important aspects of preventing burnout, enhancing resident camaraderie, and eliminating feelings of isolation. Besides camaraderie, residents need to see their leadership caring about them, reassuring them, and guiding them in their development as future physicians.Reciprocal enthusiasm and engagement from the teaching faculty was key to this class’ recruitment and to their morale. We saw significant benefit from one-on-one clinical mentorship from faculty throughout the first year, especially in the absence of senior residents. At the same time, faculty expectations of the first intern class need to be set appropriately and consistently so that we do not forget that clinical responsibilities of interns are different from those of senior residents.et al., 2018). The themes identified by our residents have provided essential insight into the experience of learners in a new residency.This resident class was recruited in part based on their pioneering spirit and enthusiasm to help build a new program. Despite the importance of developing new GME training opportunities, there is limited literature addressing the challenges of creating and recruiting residents to new training programs  A discussion of the supervision model used within this training program: While it is clear that there were no senior residents to provide feedback, mentoring and support, this was known prior to launching the program. Arrangements could have been made for other supportive measures to be put in place proactively, for example, one to one or group supervision by the chief resident or junior faculty, creation of action learning sets, etc. Individual residents can take responsibility for morale and wellness but surely at least some of this responsibility lies with the program too? Good supervision needs to be a balance between the formative, normative and restorative elements of supervision.2) The authors say, “Faculty expectations of the first intern class need to be set appropriately and consistently so that we do not forget that clinical responsibilities of interns are different from those of senior residents”. I would have liked to see a fuller discussion of the expectations of the program from the residents versus the expectations of the residents from the program, especially the extent of any potential mismatch between the two.3) A discussion of specific changes made to this residency program in view of the lessons learned - what changes have been put in place based on the authors’ recommendations so that the identified challenges could be minimized for future residents joining this program?In summary, this paper highlights the importance of careful planning prior to setting up a new residency training program for mutual benefit of both the program and the new residents.Reviewer Expertise:NANo decision status is available This review has been migrated. The reviewer awarded 5 stars out of 5 I enjoyed reading this well-written manuscript written mainly by alumni and members of the first batch of residents at the program. I could identify with many of the issues mentioned. During my undergraduate education in Kerala, India we did not have any postgraduates (residents) at our medical school and so we got undivided attention from our faculty and the opportunity to play an important role in patient management and to carry out procedures. We were also the inaugural batch during a health professions education fellowship and we had the undivided attention of our faculty but did miss the guidance of senior fellows and alumni. The methodology of the study has been well described. I would be interested in knowing more about the quarterly Voice in the program sessions. The lessons learned will be of interest to new residency and even new undergraduate medical programs in other settings.Reviewer Expertise:NANo decision status is available This review has been migrated. The reviewer awarded 5 stars out of 5 I started to read this paper with no expectations that it would be useful except in a US environment. I'm very glad I persisted in reading it as I was completely mistaken. This contribution is well written and of interest to any one setting up a postgraduate training programme. Some of the themes reported made me think of areas I might not have considered seriously eg the absence of senior residents and some themes re-confirmed the need for communication and faculty development. Thank you for an interesting and worthwhile contributionReviewer Expertise:NANo decision status is available


Epigenetic remodeling and metabolic reprogramming, two well-known cancer hallmarks, are highly intertwined. In addition to their abilities to confer cancer cell growth advantage, these alterations play a critical role in dynamically shaping the tumor microenvironment and antitumor immunity. Recent studies point toward the interplay between epigenetic regulation and metabolic rewiring as a potentially targetable Achilles’ heel in cancer. In this review, we explore the key metabolic mechanisms that underpin the immunomodulatory role of AT-rich interaction domain 1A (ARID1A), the most frequently mutated epigenetic regulator across human cancers. We will summarize the recent advances in targeting ARID1A-deficient cancers by harnessing immune-metabolic vulnerability elicited by ARID1A deficiency to stimulate antitumor immune response, and ultimately, to improve patient outcome.  Epigenetic alterations and metabolic rewiring are among the well-known cancer hallmarks. Increasing evidence demonstrates that epigenetic remodeling and metabolic reprogramming are highly intertwined and reciprocally regulate each other, and their perturbations often enhance cancer progression and metastasis, as well as reshape the tumor microenvironment (TME) to drive immune escape . On the AT-rich interaction domain 1A (ARID1A) is the DNA-binding subunit of the SWI/SNF (switch/sucrose non-fermentable) chromatin remodeling complex, which regulates chromatin accessibility by using energy provided by ATP hydrolysis and modulates a wide range of cellular processes, including transcription, replication and DNA damage repair . FunctioArid1a in early mouse embryos causes the absence of the mesodermal layer, developmental arrest and embryonic lethality (at about embryonic day 6.5). Specifically, loss of Arid1a impairs the pluripotency and self-renewal of embryonic stem (ES) cells, leading to cell lineage-specific differentiation defects, where Arid1a-ablated ES cells are defective in differentiating into mesoderm-derived cardiomyocytes and adipocytes but not ectoderm-derived neurons. Further mechanistic analysis indicates that ARID1A regulates the expression of genes essential for ES cell self-renewal, differentiation and cell linage decisions through chromatin remodeling [Arid1a is widely expressed across a large majority of cell types during multiple stages of mouse embryonic development . Ablatiomodeling . These rArid1a have been reported in different compartments, displaying context-dependent phenotypes. In microglia, the loss of Arid1a leads to the perturbation of microglial homeostasis through disruption of genome-wide H3K9me3 occupancy and changes of the chromatin landscape of regulatory elements that influence the switching of microglial states [Arid1a ablation enhances M1 microglial polarization and weakens M2 polarization, which may explain anxiety-like behaviors in these animals [Arid1a impairs liver regeneration through down-regulation of chromatin accessibility and gene expression associated with injury-induced liver-progenitor-like cells [Arid1a in T-cell lineage leads to developmental blocks from double-negative to double-positive stages mainly through reducing intracellular TCRβ expression and inducing apoptotic pathways [Arid1a impairs the differentiation of both myeloid and lymphoid lineages through a global reduction in open chromatin and gene transcription of key genes involved in hematopoietic development [Consistently, tissue-specific knockout mouse models of murine l states . Further animals . In the ke cells . Furtherpathways . Finallyelopment . Given the crucial role of ARID1A in multiple tissue types, it is not surprising to see that mutations of ARID1A have emerged in a broad range of cancers of different origins. According to a recent analysis that integrates epidemiological and genomic data to provide real-world estimates for the overall proportion of US cancer patients who harbor a coding exonic mutation in each gene, ARID1A is among the ten most commonly mutated driver genes, highlighting the role of epigenetic dysregulation in cancer . FurtherTo differentiate the context dependence on ARID1A in cancers carrying either wildtype or mutant ARID1A, we analyzed the cell type dependencies on ARID1A across 32 cancer types. As shown in Arid1a manipulation, loss of Arid1a presents resistance to tumor initiation whereas ARID1A overexpression accelerates initiation. However, in established liver tumors, Arid1a loss promotes tumor progression and metastasis. These results provide clear in vivo evidence supporting context-dependent tumor suppressive and oncogenic role of ARID1A [Consistent with the pro-tumor functions of ARID1A under certain contexts, such as hepatocellular carcinoma (HCC), the tumors have higher expression levels of ARID1A than adjacent normal tissues . Moreovef ARID1A . Consistf ARID1A .Synthetic lethality was initially described in Drosophila as recessive lethality . ClassicARID1B, a paralogue of ARID1A, is among the top genes first identified that are preferentially required for the survival of ARID1A-mutant cancer cells . MoleculRecent advancements in cancer immunology have shifted the paradigm of cancer treatment and has become one of the most prominent therapeutics for human cancers. This breakthrough has prolonged the survival of patients with relapsed or refractory metastatic cancers; however, only a subset of patients display favorable responses and most patients with immunologically cold solid tumors do not respond ,47. EmerOgiwara et al. reported the first study linking metabolic dependency and ARID1A deficiency in cancer . In thisThe notion of metabolic dependency elicited by ARID1A deficiency was further affirmed by recent studies. In ARID1A-inactivated ovarian carcinoma clear cell carcinoma (OCCC), the loss of ARID1A increases glutamine utilization and metabolism through upregulation of the expression of glutaminase (GLS1), a key enzyme in glutamine metabolism that is overexpressed in several human cancers. Therefore, this dependence on glutamine metabolism created by ARID1A inactivation could be therapeutically exploited by pharmacological inhibition of glutaminase with an inhibitor CB-839, which selectively suppresses the growth of ARID1A mutant cells. Furthermore, CB-839 synergizes with immune checkpoint blockade (ICB) anti-PDL1 antibody in a genetic mouse model driven by conditional Arid1a ablation, suggesting a novel combination therapy to target ARID1A-deificent OCCC . Given aIn line with context-dependent functions of ARID1A, a recent study in HCC provides additional insights into the link between epigenetic regulatory function of ARID1A to cancer metabolism, which may offer a new possibility of targeting the metabolic vulnerability in certain HCC patients. Somatic mutations of ARID1A occurs in around 10% of patients with HCC . Zhang eFinally, recent studies have implicated mitochondrial and metabolic reprogramming as new vulnerabilities of ARID1A deficiency. Srinivas et al. demonstrated that cells lacking ARID1A are highly sensitive to inhibition of polo-like kinase 1 (PLK1) . PLK1, aDespite the overall impressive clinical effects of ICB in cancer treatment, not all patients respond to immunotherapy. Therefore, there is a substantial and unmet medical need for the development of biomarkers that could predict the response of individual patients to immunotherapeutic agents ,47. ReceShen et al. reported a positive correlation between ARID1A mutations and enhanced antitumor immunity in ovarian tumors . ARID1A ClinicalTrials.gov, NCT02478931) [Furthermore, in pancreatic cancer, where ICB has shown little efficacy, a more recent study showed that a small subset of patients with ARID1A alterations achieved an objective response, including a complete remission after immunotherapy treatment, thus warranting prospective trials (2478931) . In cont2478931) . Mechani2478931) . Taken tA number of studies have investigated the combination of immunotherapy with agents targeting the synthetic lethal interactions with ARID1A ,69,70. FGiven the emerging roles of ARID1A in the DNA damage response , accumulFinally, until recently, the direct role of ARID1A in CD8+ T cell exhaustion remains elusive. Through an in vitro CRISPR-Cas9 screen, Belk et al. showed that elimination of ARID1A confers CD8+ T cells proliferative and cytotoxic function in vivo, leading to better anti-tumor activity . DepletiARID1A has emerged as a tumor suppressor in a broad array of human malignancies. Over the years, mutation of ARID1A has been extensively explored for synthetic lethal targeting several fundamental cellular functions including DNA damage response and cellular metabolism. The recent discovery of the SWI/SNF complex as an essential player in determining the therapeutic efficacy of cancer immunotherapy further highlights great promise for harnessing the cancer-specific vulnerability elicited by ARID1A deficiency to potentiate immunotherapy. Despite the promise, several challenges remain to be tackled before we realize the full potential of ARID1A-based precision oncology. Accumulating evidence has demonstrated promising preclinical and clinical results of combinational therapy to target cancers harboring ARID1A mutations. We summarize the recent clinical trials of targeting ARID1A-deficient cancers in In addition to cancer cells, multiple cell types in the TME, including immune cells may be differentially influenced by the metabolic rewiring and epigenetic alterations caused by the driver mutation, such as ARID1A deficiency . Given t


Increased experiential learning within pharmacy education has highlighted the need for quality preceptors, leading to a recent focus on their development and training. This study describes the design, development and implementation of a pharmacist preceptor training program in a regional Australian University. The design of this program utilized the results of a previously conducted scoping review of preceptor training programs and a comprehensive preceptor training needs analysis. In addition, participation was sought from an expert advisory group, comprising preceptors and pharmacy professional organizations. Program materials were developed and transferred onto a suitable online learning platform for implementation. Post-training program evaluation will follow Kirkpatrick’s model of training evaluation criteria, gaining feedback from both preceptors and students. The four flexible online modules that were designed are aligned with the main preceptor roles: role model, educator, mentor and assessor. The program also included a small-group online interactive networking session and an online discussion forum. Following implementation, 28 pharmacist preceptors completed the training. Preliminary feedback on the training program was extremely positive, particularly to the interactive networking activity. Future programs will continue to be adapted to reflect stakeholder feedback and optimize student placement experiences. Preceptors play a vital role in pharmacy education by teaching and mentoring student and intern pharmacists in practice settings as they develop their professional knowledge and skills. Globally, the focus on experiential learning within pharmacy education has highlighted the increasing role of pharmacist preceptors and the need for preceptor training and development to ensure the provision of quality teaching. Training and support for pharmacist preceptors is thus regarded as essential and considered to be the professional responsibility of the university. The Australian Pharmacy Council (APC) Accreditation Standards for Pharmacy Programs require all professionals who are involved in assessing students or interns to be ‘suitably qualified, experienced and prepared for the role, and ‘provided with appropriate guidance and support’ . In the Two global reviews of preceptor training programs revealed limited documented examples of training programs and a wide variation in the design, delivery and content of these programs ,7. SimilRecent interest has focused on preceptor competencies to provide an underpinning structural framework for preceptor development, promoting consistency of preceptor performance and potentially enhancing the student learning experience. Preceptor competencies are closely associated with a range of ideal preceptor roles, skills and attributes, with their identification being necessary to inform the development of competency standards. Several studies have investigated the ideal skills and attributes of pharmacist preceptors from the perspective of both preceptors and students ,11,12,13In addition to competency standards and adherence to the requirements of professional accreditation bodies, the design of a training program for preceptors needs to consider available resources, the nature of the precepting environment, the background knowledge of the preceptor, their specific roles and responsibilities and their individual training needs ,7,17. WiExperiential learning provides the opportunity for students to link learned theory to the realities of pharmacy practice and is considered by the JCU program as an integral component of their curriculum, with students undertaking 600 h of placement during their degree, under the supervision of a pharmacist preceptor. JCU is a regional university with a focus on rural and remote practice; therefore, preceptors and placement sites are geographically distributed across the state of Queensland, covering an area of approximately 80,000 square kilometres. Placement blocks vary in length, ranging from two to six weeks, with all students required to attend at least one rural placement. This training program was designed to prepare pharmacists for their role as student preceptors, with a particular focus on community pharmacy preceptors, who represent about 70% of JCU’s cohort of volunteer preceptors.The 5-point ADDIE  instructional design model , which hA participatory approach was employed to inform the design of this training program, enabling the program to be tailored to those directly involved in experiential teaching and learning. This approach included a needs analysis of preceptors, academic staff and students, using mixed methods, and collaboration with an expert advisory group. Following a scoping review of preceptor training , both quThe curriculum framework for this program was based on a preceptor training program developed by Condrey , which oAfter the preparation of a draft curriculum for training, the research team established an expert advisory group to provide oversight, especially in relation to the practice, and to contribute to the program design. Group members were chosen by purposive sampling and invited to participate in two online meetings, one prior to program development and one following the completion of the training program by preceptors. The advisory group included the three members of the research team (the authors), two members from key professional pharmacy bodies (PSA and APC) and two preceptor representatives, from both a rural and an urban JCU placement site. The research team are all academic staff members in pharmacy at JCU, with the principal researcher (GK) being an experienced pharmacist and academic, BG being a pharmacist with extensive academic and research experience and MM having a degree in education as well as significant academic experience. To minimize the risk of bias in the program evaluation, the two preceptors involved in the expert advisory group did not participate in the preceptor training program. Advisory group meetings were recorded and minutes were taken, which allowed the researchers to fully examine member comments and suggestions for program improvements.Overall, advisory group members were complimentary of the proposed training program, in terms of both structure and content. Various issues were discussed, including student motivation, the challenges of providing feedback, preceptor competencies and linking preceptor training to career progression. Additional content topics suggested by group members included leadership and teamwork, an expansion of cultural considerations, reflective practice and different student learning styles. Based on feedback from group members, the preliminary draft of the training program was modified and further developed into its final form. A second advisory group meeting was held following completion of the program to report on the program implementation and to discuss any future improvements to the program.The program materials were developed by the principal author and reviewed by the research team. A suitable platform for delivery was selected and prepared and program materials were built as modules within this learning platform. Program implementation was timed to coincide with the schedule of student placements, allowing preceptors to complete the full program prior to hosting their next student. A certificate of completion was provided to all preceptors who completed the program.For quality assurance, the program will be evaluated, guided by Kirkpatrick’s framework model for evaluation of educational effectiveness, which consists of four levels of evaluation criteria: reaction, learning, behaviour and results. The reaction level measures the trainee’s perception of training, i.e., did the learner (the preceptor) enjoy the training. The learning level is a measure of the degree of learning achieved by the trainee, based on the learning outcomes., i.e., was the program informative to preceptors. The behaviour and results section are more difficult to measure and involve ascertaining the effect of the program on work habits and performance outcomes, i.e., did it improve precepting ability and confidence and lead to improved student learning .Student evaluation of the preceptor survey which is routinely conducted by JCU Pharmacy following each student placement. Student post-training surveys for trained preceptors will be compared to untrained preceptors, to identify any changes to precepting behaviour because of training, thus adding to the accuracy of the level 3 evaluation.This evaluation will involve a preceptor self-completion survey immediately following completion of the program, as well as preceptor post-training interviews, conducted shortly after a student has completed a placement in their community pharmacy. The preceptor post-training survey will address the reaction and learning levels of Kirkpatrick’s evaluation of educational effectiveness model. The post-training interviews will seek to identify any self-reported changes to precepting behaviour resulting from the training, thus addressing level 3 of Kirkpatrick’s model. To provide a balanced perspective, the program will also be evaluated from the viewpoint of the student by making use of the standard The scoping review identified a range of preceptor training programs, for both hospital and community preceptors as well as residency preceptors. Modes of delivery of training included face-to-face classes and web-based modules, supplemented by a preceptor manual, assignments and recommended readings. While online training was convenient and often considered a necessity due to the geographic distribution of preceptors, face-to-face training was thought to better facilitate preceptor networking. Program flexibility was important to preceptors due to time and workload constraints. Areas of interest for training content included teaching skills, how to be a good role model, conducting assessment and the provision of student feedback. Various useful support measures were identified, including regular communication with preceptors, recognition of the work of preceptors and the provision of continuing professional development (CPD) credits for participation in training. Training program evaluations were mostly positive and based on post-training qualitative feedback from preceptors, although some programs conducted pre- and post-preceptor surveys or interviews, with only a small number of programs obtaining student feedback .The preceptor training needs analysis reinforced the benefits of preceptor training and made recommendations that all preceptors undertake training. It was agreed that preceptors play many roles, with their key role being to link theory to practice. Important preceptor qualities include good communication and counselling skills, being a good role model, interest and enthusiasm about precepting and providing effective, and timely and constructive feedback. It was felt that training should be predominantly provided online with flexibility of training a key requirement. In addition to the online component, some form of interactive session was desirable, as well as a supplemental preceptor manual .Training program content should include an overview of the university curriculum, preceptor and student expectations, teaching tips and strategies, conflict management and how to provide feedback. As support for preceptors, the university should communicate regularly and involve preceptors in current and future curriculum development. The most significant barrier to preceptor training was time, although lack of training and the voluntary nature of precepting were also considerations when choosing to become a preceptor. The main benefit of training was the delivery of a consistent approach to precepting, which was thought to lead to an improved student placement experience ,10.The decision to conduct an online program was influenced by practicality, with the geographic distribution of preceptors as well as time pressures being a major impediment to simultaneous face-to-face training. While face-to-face programs are relatively common in the United States and are often preferred due to their interactive nature, they can be a challenge in circumstances where there are limited resources and poor preceptor accessibility ,17. ThisThe individual modules contain instructional materials which address the four main preceptor roles outlined in the Condrey program: role model, educator, mentor and assessor . AlthougWhile Condrey’s program included an online component for theory-based information and a four-hour face-to-face training class, the JCU program was conducted fully online and therefore these two components were integrated across the four modules. Whereas in Condrey’s program, face-to-face training concluded with small group and class discussions of case studies based on the four preceptor roles, in the JCU model, this session was conducted as a small group online synchronous case-based interactive session, following the completion of the online modules.To identify and describe the roles of the pharmacist preceptor and their associated skills and attributes.To have a knowledge of the skills required to be a student role model and to promote the profession of pharmacy and the importance of lifelong learning.To recognize the different teaching and learning styles which may impact on the education of pharmacy students during placement and to be able to implement a variety of teaching strategies.To describe the principles of mentoring or coaching to motivate, guide and manage pharmacy students during their placements.To understand the principles of student assessment and strategies for the provision of student feedback in the clinical environment.The module materials incorporate pedagogical approaches, and teaching and learning theories which are relevant to the experiential learning context. Preceptors are clinical educators who teach students in a practice environment. They are also adult learners; therefore, it was considered that andragogical teaching methods featuring a constructivist approach would be most applicable for preceptor training. The preceptors are introduced to a range of educational theories and concepts, such as deep and surface learning, intrinsic motivation, the VARK learning style classification, Miller’s pyramid for clinical teaching and assessment, and the ACPE preceptor teaching methods ,22,23,25A welcome message and introductory materials are also included. Each module contains a set of individual learning outcomes, along with information relevant to the preceptor role, online activities, a range of brief supporting video clips, a list of references and a ten-item multiple choice quiz to provide immediate feedback and allow the preceptor to reflect on what they had learnt. The modules are designed to be flexible and able to be completed in the preceptor’s own time over a five-week period, with the estimated overall completion time for the modules being six hours. It was felt that for an optimal outcome, preceptors should be advised to complete all modules and their associated quiz prior to attending a 1.5 h online synchronous networking session. The networking session was designed to allow preceptors to interact with other preceptors in a small group, thus encouraging the sharing of information and ideas. A series of six case studies were prepared to promote discussion. Cases were linked back to various aspects of precepting which were covered in the four modules. This included pre-placement preparation, setting expectations, teaching styles, one-minute preceptor, student assessment and conflict management. As examples, one case provided a student scenario from which preceptors could use the one-minute preceptor framework to provide student education and feedback and one case presented a situation where a student is lacking motivation and behaving in an unprofessional manner, asking preceptors to discuss their response to the situation.The decision to establish an optional online discussion forum within the organizational site was intended to facilitate further asynchronous communication between preceptors to share ideas and strategies for precepting at their own convenience.JCU Pharmacy Preceptor Training Hub was developed as part of the JCU Blackboard® Learn educational platform, and with the help of a digital support assistant. As the already established educational platform for JCU, this was convenient and was able to be easily accessed at no cost to the preceptors. Program modules were prepared and loaded into this online organization site. An image of the preceptor training organizational site is provided in An organizational site called the Following the preparation of the organizational site, a list of email addresses of current and past preceptors was obtained from the JCU pharmacy placements administrative officer. All 240 current and past preceptors were invited by email to participate in the program, with 62 pharmacist preceptors (25.8%) registering for the program. After completion of the four online modules, seven small-group online networking sessions were conducted with preceptors. A maximum number of nine preceptors per group was set, to ensure that there was ample opportunity for all group members to participate in the discussion. These sessions were facilitated by the main author, with each of the six case studies being presented and discussed among the group members. Opportunities for general discussion were also provided within the session. Preceptors who had registered for the program were also invited to contribute to the online discussion forum. Several comments were added to the forum by the facilitator to promote conversation.Of the 62 registrants, 33 preceptors commenced the modules, and 28 preceptors (46.8% of registrants) completed the entire program and were therefore eligible to receive a certificate of completion.® survey platform .Following participation in the program, and completion of all components, including the module quizzes and the preceptor networking session, preceptors will be invited to complete a 10 min post-training online feedback evaluation survey, developed using the QualtricsStudent Evaluation of the Preceptor post-placement survey, which is routinely conducted online via Qualtrics after each placement experience. All student surveys in the year following the program will be collected, with the surveys of trained preceptors being compared to untrained preceptors, to identify any changes to preceptor evaluations following program participation. The post-training evaluation of this preceptor training program will be the subject of a future article.As a follow-up to the survey, a sample of preceptors will be invited to participate in a post-training interview, to be conducted shortly after hosting their first post-training placement student. The aim of the interviews will be to ascertain the outcomes of the training program in terms of changes in their knowledge and attitude towards precepting and the impact on their behaviour during the placement. Interviews will be conducted by telephone and audiotaped, with an estimated completion time of 15 to 20 min. Program evaluation will also be undertaken from the perspective of the students through the The goal of this training program is to prepare and support JCU pharmacist preceptors in their role as teachers and mentors of JCU students during experiential placements. The program design was informed by JCU preceptors, students, and academic staff, with additional input from key industry stakeholders. In line with stakeholder requirements, the program provided individualized training via a set of flexible online modules, together with a small group interactive online networking session. Curriculum content was tailored to suit the identified needs of JCU preceptors, and the program was evaluated from the perspective of both preceptors and students. This discussion will follow the five stage ADDIE Model of Instructional Design which guided program development.A needs analysis is a commonly used method of identifying trainee requirements to develop an effective and evidence-based training program ,30. A raCommon modes of delivery in recent preceptor development programs have included electronic methods such as on-demand or live webinars, online modules, and face-to-face classes, with many programs choosing a blended delivery approach incorporating online and face-to-face methods ,7,19. ThFrom this study, important preceptor skills and qualities included good communication, being a professional role model, enthusiasm for precepting and being able to provide good feedback. This aligns well with the literature ,13 and rThe second phase of the ADDIE model focused on the design of the training program, which considered both the overall structure and the proposed curriculum. It was decided that for this program, an online format was most appropriate due to the wide geographical distribution of preceptors and placement sites. There has been some debate over the most effective way to teach online, with studies finding that while online learning is a valuable tool for teaching clinical skills, a blended approach to learning was preferred ,5,34. ThThe instructional design of this program considered the nature of the learners (the preceptors) who are clinical teachers facilitating student learning and skill development in the practice setting. Clinical teaching can be complex; while practicing in a dynamic working environment, preceptors must not only create a safe learning space for students, but also ensure that they are managing their workload and providing optimal patient care . PharmacGiven that this program was designed to teach preceptors how to precept students, an understanding of both learning and teaching strategies needed to be included, with a range of educational theories, tools, and frameworks being introduced or integrated into the program. In the Educator module, preceptors were introduced to the VARK learning style classification, which describes four learning modes for students: visual, aural, read/write and kinaesthetic, reflecting the different ways in which students learn. Research has found that by identifying the preferred learning style of the student and adapting their teaching style to suit the student, learning can be improved . It is wThe ACPE identify four main preceptor teaching methods: direct instruction, modelling, coaching and facilitating, which may be used by preceptors at various stages of student development to enhance learning and progress their professional skillset ,38. BecaProfessionalism, Structuring the student’s experience, Motivating the student in the clinical arena, Achieving synergy in clinical teaching and Assessment and evaluation [Principles of learning and teaching, Planning for experiential learning, Teaching and instructional strategies, Student assessment and feedback and Communication skills for effective preceptorship and conflict resolution [A range of pharmacist preceptor training programs have been developed in recent years, including small, site-specific programs, university-wide programs and large consortium-based programs ,6,7. Thealuation ,45. In taluation . An intesolution . On closOne of the most desired requirements for a pharmacist preceptor training program is the provision of continuing professional development (CPD) credits for training. Since preceptors are time poor and frequently contribute in a voluntary capacity, they will often prioritise their time based on potential rewards. CPD credits for training are a feature of many current training programs and the integration of preceptor development tools into professional CPD portfolios may also encourage preceptor participation in training in the future ,47.Following the development of the course materials and review, the JCU online program was made accessible to participants, with 28 preceptors completing all components of the program. It was thought that this relatively low participation rate was due to the current high workload of community pharmacists in Australia, considering also the impact of the COVID-19 pandemic on workload and staff absences . Of noteLearning settings can be enriched through collaboration and interaction, with preceptors valuing opportunities to network and interact. ,5,39. WhPreliminary results from the post training evaluation survey indicate that preceptors were overall very positive about the program, particularly appreciating the online networking session. The full results of the post training evaluation will be the subject of a future article.Limitations of this study include the low number of preceptor training program completions, which was thought to be due to the already high workload of pharmacist preceptors, as well as the fact that only 39.6% of preceptors invited to participate had indicated a willingness to host a student within the past 12 months. There was potential for a positive response bias with the researchers being known to most preceptor participants.While the overall response rate for the needs analysis was low, the mixed methods study design with a participatory approach provided breadth and depth to the analysis, thus increasing the validity of the findings. This approach was also instrumental in ensuring that the program was able to be tailored to the needs of JCU preceptors. The broad experience of the research team in both teaching and pharmacy practice was advantageous in providing the most appropriate educational design for the program.It is anticipated that this program will continue to be offered on an annual basis, with refinements being continually undertaken in response to stakeholder feedback. Considering preceptor feedback from previous studies, the wide variation in preceptor experience in both practice and precepting may warrant the streaming of preceptor development programs to cater for both the novice and the more experienced preceptors ,17.It is expected that the existing professional requirements for preceptor development will necessitate mandatory training in the future, which will further promote the development of training programs across Australia. While this program is currently focused on training community pharmacy preceptors, there is potential for it to eventually be expanded to include all preceptors across the broad range of experiential placement sites. The general principles of precepting are similar across a range of health professionals and with the increased emphasis on interprofessional practice in healthcare systems, interprofessional training for preceptors may be a practical and cost-effective approach to facilitate preceptor development, with potential for collaboration with other health professional programs such as medicine and nursing ,51.Although preference for preceptor development tailored to institutions and the individual preceptor has been identified ,5, thereThe training and development of pharmacist preceptors is considered crucial to the success of experiential placements. This study provides an example of a tailored training program which has been informed by the previous literature, as well as through a comprehensive needs analysis and direct consultation with the profession. The development and implementation of this program has been strategically planned and structured to provide preceptor flexibility, with evidence-based and educationally focused content, and linked to a national pharmacist competency framework. Ongoing modifications and extensions to this program in response to program feedback should ensure that it continues to meet the individualized needs of all preceptors. The further development of specific preceptor competencies for pharmacists is recommended to provide and maintain quality and consistency for future preceptor training programs. An increased focus on preceptor development in pharmacy programs will ensure that student placement experiences are of high quality and provide maximum benefit for both the preceptor and the student, promoting the achievement of optimal graduate outcomes.


Combined dual HOPE–NMP has the potential to substantially decrease the high rates of deceased donor liver discard.Hypothermic oxygenated machine perfusion (HOPE) reduces ischaemia–reperfusion injury of donor livers and thereby improves outcomes after transplantation. End-ischaemic normothermic machine perfusion (NMP) enables assessment of hepatobiliary viability and selection of livers that would otherwise have been declined for transplantation. We advocate the combined use of  HOPE and NMP for livers that are considered high risk, but may still be transplanted safely after  Liver transplantation has become a victim of its own success. Shortage of organs is limiting access to this life-saving treatment. Despite this, many organs are refused for transplantation based on suboptimal quality and risk of early graft failure. Most deceased donor livers that are currently declined for transplantation are functioning well in the donor, but this can change after transplantation due to ischaemia–reperfusion injury that occurs during the procurement, preservation, and transplant procedures. The current standard of donor organ preservation is static cold ischaemic preservation after flush-out with a preservation solution. Although this method is sufficient for organs of optimal quality, it does not provide adequate protection against ischaemia–reperfusion injury in suboptimal donor organs. In particular, livers from donation after circulatory death (DCD) donors are more prone to substantial ischaemia–reperfusion injury that will result in graft-related complications after transplantation, such as primary graft non-function and non-anastomotic stricture (NAS) of the donor bile ducts. Both types of complication are more frequent after transplantation of DCD organs compared with donation after brain death (DBD) liversex situ oxygenated machine perfusion. Machine perfusion can be performed at various temperatures, and both hypothermic (8–12°C) and normothermic (35–37°) types of machine perfusion have entered clinical practice. Hypothermic and normothermic machine perfusion are not competing techniques. In fact, they serve different goals and can even be applied sequentially with additional benefits, as discussed below. A comparison of the characteristics of hypothermic and normothermic machine perfusion is provided in Table 1.A promising tool to reduce the risk of ischaemia–reperfusion injury after liver transplantation is ,An essential component of hypothermic machine perfusion is hyperoxygenation of a donor liver at low temperaturesperfusion (back to base) and normothermic machine preservation . During NMP, the donor liver is metabolically active, which allows assessment of hepatobiliary viability before transplantation. In the first RCTpreservation, NMP was not primarily used for graft assessment and selection, but rather to study the potential preservation benefits of NMP compared with static cold storage. Recipients of a NMP-preserved liver had lower peak serum aspartate aminotransferase levels and less early allograft dysfunction than recipients of a liver after static cold storage. This technique brings logistical challenges, as a donor surgeon needs to be able to make arterial reconstructions and connect the donor liver safely to the perfusion device. Furthermore, during transportation, device failure could lead to warm ischaemia of the liver and the risk of discard. These logistical difficulties have favoured the end-ischaemic or ‘back-to-base’ application of NMP,The term normothermic machine perfusion (NMP) is used for two preservation techniques: end-ischaemic normothermic machine As it has become evident that a short period of end-ischaemic (D)HOPE protects against ischaemia–reperfusion injury, whereas end-ischaemic NMP enables viability assessment, but does not provide similar protection against ischaemia–reperfusion injury, we have proposed the combination of these two machine perfusion modalities. If (D)HOPE and NMP are considered as complementary techniques, the sequential use of these two types of machine perfusion could maximize the utilization of high-risk donor liversFig. 1). DHOPE is used for all DCD livers, as well as for DBD livers where the cold ischaemia time threatens to become too long HOPE and NMP for livers that are considered high risk, but may still be transplanted safely after Disclosure. The authors declare no conflict of interest.