BCR-ABL1 like acute lymphoblastic leukemia (ALL); TK inhibitors represent a useful therapeutic choice in these patients who respond poorly to conventional chemotherapy. Here we report a novel peptide biosensor (PABL)-ELISA assay to investigate ABL1 activity in four immortalized leukemic cell lines with different genetic background. The PABL sequence comprises an ABL1 tyrosine (Y) phosphorylation site and a targeting sequence that increases the specificity for ABL1; additional peptides (Y-site-mutated (PABL-F) and fully-phosphorylated (PPHOSPHO-ABL) biosensors) were included in the assay. After incubation with whole cell lysates, average PABL phosphorylation was significantly increased . Cell lines expressing ABL1-chimeric proteins  presented the higher TK activity on PABL; a lower signal was instead observed for NALM6 and REH . Phosphorylation was ABL1-mediated, as demonstrated by the specific inhibition of imatinib  in contrast to ruxolitinib (JAK2-inhibitor), and occurred on the ABL1 Y-site, as demonstrated by PABL-F whose phosphorylation was comparable to basal levels. In order to validate this novel PABL-ELISA assay on leukemic cells isolated from patient’s bone marrow aspirates, preliminary analysis on blasts derived from an adult affected by chronic myeloid leukaemia (BCR-ABL1 positive) and a child affected by ALL (BCR-ABL1 negative) were performed. Phosphorylation of PABL was specifically inhibited after the incubation of BCR-ABL1 positive cell lysates with imatinib, but not with ruxolitinib. While requiring further optimization and validation in leukemic blasts to be of clinical interest, the PABL-based ELISA assay provides a novel in vitro tool for screening both the aberrant ABL1 activity in BCR-ABL1 like ALL leukemic cells and their potential response to TK inhibitors.The pathogenic role of the overactivated ABL1 tyrosine kinase (TK) pathway is well recognized in some forms of  BCR-ABL1 fusion gene with different breakpoints in BCR gene, are well-recognized for their pathogenic role in leukemias, including chronic myeloid leukemia (CML), some forms of acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL). BCR-ABL1 results from the chromosomal translocation t , and encodes for the chimeric BCR-ABL1 protein that leads to an aberrant costitutive activation of the ABL1 proliferation pathway. In the chimeric BCR-ABL1, the N-terminal BCR region interferes with the negative regulation of ABL1 and mediates dimerization of the protein that autophsophorylates the kinase, fully and constitutively activating it  transloctation. These forms are recognized as a high-risk ALL subtype across all clinical studies, being characterized by an unfavorable prognosis and a higher relapse rate in comparison to other B-ALL subtypes , the JAK-kinase genes  or their upstream receptors  -1 tyrosine kinase (TK) belongs to the non-receptor TK family and is an ubiquitously expressed cytosolic enzyme that plays a role in many key processes linked to cell growth and survival as well as to cell differentiation, cell adhesion and migration . Oncogenating it . Moreovewith age ], presensubtypes . BCR-ABL2, EPOR) , resulti2, EPOR) . A third2, EPOR) .Y-X-X-P/F . This sequence has been considered as the base to develop a peptide biosensor to detect ABL1 activity and was found by screening a library of billion distinct peptides for interaction with purified ABL1 (YAAPFAKK (named as “reporter” sequence in this manuscript), was designed. This sequence represents an artificial kinase substrate, not a naturally occurring motif of endogenous proteins (ABLTIDE), nowadays largely accepted as a reference peptide biosensor for screening ABL1 activity. However, PABLTIDE is suitable only for biochemical research use , and is not intended for use in clinics and diagnostics because of the lack of serum stability, of cell-penetrating properties and of specific ABL1 targeting among other kinases in primary cells (“targeting” in this manuscript) has been designed using biocomputing tools on the basis of physicochemical reasoning, and is recognized by the SH3 domain of ABL1 protein  with an increased specificity for ABL1. This peptide was further developed to detect intracellular ABL1 kinase activity in live intact cells  located at the ABL1 N-terminal. SH1 binds and cleaves ATP, and mediates tyrosine (Y) phosphorylation in target substrate proteins; SH3 and SH2 domains function as interaction modules for targets and as allosteric inhibitors of the catalytic SH1 domain . The conied ABL1 . Phosphoproteins  and corrry cells . An addi protein . This sect cells , and wasct cells .BCR-ABL1 positive leukemias . The heterogeneity in their genetic background makes the identification of BCR-ABL1 like ALL very challenging. Currently, the molecular analyses carried out on patient’s blasts at diagnosis include karyotyping, fluorescence in situ hybridization (FISH) or RT-PCR to detect few specific rearrangements. Useful high-throughput genomic approaches such as exome, whole genome and whole transcriptome sequencing are still too expensive to be routinely employed in clinical practice on large patients’ cohorts, particularly in low-income countries  as a biosensor able to quantify the ABL1 phosphorylation activity in whole lysates of immortalized leukemic cell lines, including BCR-ABL1 like ALL cells. Kinase activity was monitored in vitro by an ELISA assay. Long-term goal is to set up a point-of-care device to improve the diagnosis and clinical management of BCR-ABL1 and BCR-ABL1 like leukemias in clinics. Far from overcoming the importance of monitoring the minimal residual disease, these devices could nonetheless boost the therapeutic monitoring by measuring the residual TK activity or the acquired resistance to these drugs in residual blasts.The aim of this study was to investigate the role of PImatinib  was dissolved in DMSO at a concentration of 1 mg/ml (2 mM) and ruxolitinib  in ethanol at a concentration of 10 mg/ml (32.6 mM), according to manufacturer instructions.The study was performed on four human leukemia cell lines purchased from the DSMZ GmbH (Germany): NALM6 (ACC 128), ALL-SIL (ACC 511), K562 (ACC 10), REH (ACC 22). Additionally HEL (ACC 11) and SET2 (ACC 608) cell lines, kindly provided by Professor A. Vannucchi , were included in MTT cell viability assay.ABL-based ELISA assays. The method of extraction has been optimized by Professor Sorio (University of Verona) and requires the use of kinase and phosphatase inhibitors to maintain the integrity of BCR-ABL1 in the K562 cell line -2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue exclusion assay, as described in ABL comprises two tyrosine, one in the “target” and one in the “reporter” region. PABL-F is a site-mutated biosensor with the tyrosine in the “reporter” region replaced by phenylalanine, and was used in order to verify the specificity of the phosphorylation signal on the catalytic site. PPHOSPHO-ABL is a fully phosphorylated version of PABL, used for relative quantification of the phosphorylation level obtained.Biosensor peptides were synthesized by GenScript  with 98% purity, and are shown in ABL-based ELISA assay procedure is shown in 2, 0.1 mM EDTA, 0.01% TritonX) with 100 μM ATP, 1x Roche Inhibitors, 2 mM DTT, 0.1 mM Na3VO4 and MilliQ-water  for 1 h at RT. To evaluate the effect of TKI, cell lysates were pre-incubated for 15 min at RT with drugs  before adding on the biosensor-coated plate. The phosphorylation of the peptides was measured using an anti phosphorylated tyrosine antibody  and detected with a secondary antibody ECL Anti Mouse IgG, HRP from goat . Signal was allowed by adding a solution of citrate buffer pH 6.0, 3% H2O2, 0.5% Amplex Ultra Reagent Invitrogen  and measured 25 times every 0.2 s . PPHOSPHO-ABL was included in each experiment and added to lysates of any cell lines used as an ELISA assay positive control and as a tool to measure the maximal fluorescent signal achievable on the biosensor. PABL and PABL-F phosphorylation levels were expressed as a percentage related to PPHOSPHO-ABL phosphorylation in the same cell line, according to the following formula: (mean PABL)/(mean PPHOSPHO-ABL) × 100.The PBCR-ABL1 negative ALL, data regarding BCR-ABL1 like translocations not known) treated at IRCCS Burlo Garofolo in Trieste . Bone marrow aspirates were collected at onset as part of diagnostic procedures, and used for research purpose only when clinical procedures had been completed. Patients’ bone marrow aspirates (∼3–5 ml) were diluted with PBS to a final volume of 10 ml and loaded on Ficoll-PaqueTM Plus (5 ml); after centrifugation , interphase was recovered and washed twice with PBS ; 20 × 106 mononuclear cells were used for cell lysate preparation.Two patients were included in this study. The first was a 40–50 years old male adult affected by chronic myeloid leukaemia (p210 BCR-ABL1 positive), treated at Azienda Ospedaliero-Universitaria in Udine . The second was a 1–3 years old female child  gene fusion.Four human leukemia cell lines with different genetic background were selected and initially characterized by western blot to confirm the presence of candidate chimeric proteins: NALM6 and ALL-SIL cells were chosen because they harbor the gene fusions 6-PDGFRB  and NUP2tivation . BecauseIn NALM6 the presence of ETV6-PDGFRB was investigated using an antibody directed against human PDGFRB. As showed in ABL1 fusion genes was clearly impaired by imatinib, and differed significantly each other (p < 0.0001) with the ALL-SIL being more sensitive (IC50 = 46.9 ± 6.99 nM) than K562 . Imatinn tested . As expen tested , in cont control . Cytotox control . ImatiniABL-based ELISA analysis showed a significant phosphorylation of the PABL biosensor probe after incubation with all four leukemic cell lines tested . A lower signal was instead observed for NALM6  and REH . To confirm the contribution of ABL1 activity on the probe, cell lysates were pre-incubated with ABL1 specific and non-specific inhibitor . Pre-incubation of lysates with 5 μM imatinib lead to a significant decrease of PABL phosphorylation  to PABLTIDE (“reporter” sequence only). PABL shows a higher phosphorylation compared to PABLTIDE , with a significant decrease after imatinib pre-treatment of lysates . The levels of PABL-F phosphorylation were similar among cell lysates, and did not differ from the basal fluorescence signals detectable in all cell lines in the absence of peptide  and another by ALL , were also used. In both cases, the PABL biosensor probe was phosphorylated after incubation with cell lysates  but not by ruxolitinib), in contrast to what was observed in BCR-ABL1 negative pt#2  but not by imatinib). Cells sensitivity to imatinib and ruxolitinib could not be assessed due to the scanty biological material available.To further validate the novel Pin vitro have been developed  are present in about 1% of BCR-ABL1 like positive patients  except for K562. Some investigators report that NALM6 cell line harbors a different translocation, in particular DUX4-rearranged [t  ]/ERG deletion  could be an issue. However, the PABLtarget region should guarantee an increased specificity for ABL1 compared to ARG. Mass spectrometry studies identified additionally one off-target (non-tyrosine kinase) of imatinib, the NAD(P)H:quinone oxidoreductase NQO2, whose enzymatic function should not influence the peptide phosphorylation (ABL phosphorylation levels at any of the concentrations selected (52 nM and 5 μM). The concentration of 52 nM was chosen in accordance to pharmacokinetic parameters measured in healthy subjects at steady state following a twice-daily administration of ruxolitinib at 15 mg and is 20 times higher than the IC50 of 2.8 ± 1.2 nM calculated in vitro for JAK2 by a biochemical enzymatic inhibitory assay , and the test of patient samples of known genotype. Nonetheless, the results here described represents the first step towards the setup of a point-of-care device for diagnosis and therapeutic drug monitoring of pediatric BCR-ABL1 like patients.Taken together, these observations suggest that the P


Within the European Treatment and Outcome Study (EUTOS) for CML we established and validated robust RT-qPCR methods for these patients.Approximately 1BCR-ABL1 transcripts were amplified and sequenced to characterize the underlying fusion. Residual disease monitoring was carried out by RT-qPCR with specific primers and probes using serial dilutions of appropriate BCR-ABL1 and GUSB plasmid DNA calibrators. Results were expressed as log reduction of the BCR-ABL1/GUSB ratio relative to the patient-specific baseline value and evaluated as an individual molecular response (IMR).BCR-ABL1 transcripts . Most patients (61%) responded well to TKI therapy and achieved an IMR of at least one log reduction 3 months after diagnosis. Four patients relapsed with a significant increase of BCR-ABL1/GUSB ratios.In total, 330 blood samples  from 33 CML patients  were analyzed. Patients expressed seven different atypical BCR-ABL1 transcripts is essential for adequate patient monitoring and to avoid false-negative results. The results cannot be expressed on the International Scale (IS) and thus the common molecular milestones and guidelines for treatment are difficult to apply. We, therefore, suggest reporting IMR levels in these cases as a time-dependent log reduction of BCR-ABL1 transcript levels compared to baseline prior to therapy.Characterization of atypical The online version contains supplementary material available at 10.1007/s00432-021-03569-8. BCR-ABL1) fusion gene  is characterized by the Philadelphia chromosome (Ph), produced by the balanced reciprocal translocation t leading to the breakpoint cluster region-Abelson  and/or e14a2 (b3a2) BCR-ABL1 transcripts located within the major breakpoint cluster region (M-BCR) associated with a p210 fusion protein. Overall, three breakpoint regions are present in the BCR gene whereby the M-BCR spans from exon 12 to exon 16 (formerly named exons b1–b5). The most frequent breakpoint region of the ABL1 gene is located at a 200 kb segment upstream of exon 2 (a2)  producing a p190 protein was found initially in two-thirds of Ph+ acute lymphoblastic leukemia (ALL) patients. A breakpoint in the micro breakpoint cluster region (µ-BCR) leads to the largest chimeric BCR-ABL1 with an e19a2 junction translated into a p230 protein product (Melo ABL1 exon a2 [e.g. e13a3 (b2a3) or e14a3 (b3a3)].More than 95% of Phuct Melo . Severaluct Melo  or e8a2 uct Melo  as well BCR-ABL1 subtypes remain non-standardised , we suggest a new evaluation criterion for molecular monitoring of these patients—the individual molecular response (IMR) level based on a log reduction from pretreatment levels.The characterization of the precise rearrangement at diagnosis with a qualitative multiplex PCR and sequencing .Peripheral blood samples were obtained from 33 patients  with atypical 7 cells) from peripheral EDTA blood samples were lysed in 1 mL TRIzol®  and stored at −20 °C until RNA extraction. Frozen samples were allowed to thaw at room temperature and RNA was isolated using the acid guanidinium thiocyanate-phenol–chloroform extraction method. Reverse transcription of 7.7 µL total RNA (maximum 4 µg of RNA) was performed immediately after isolation with random hexamer primer and M-MLV reverse transcriptase (Invitrogen GmbH) for 60 min at 37 °C according to the manufacturer’s recommendations.Freshly isolated leukocytes . PCR was carried out with a master mix containing: 1.2 × Taq polymerase buffer with MgCl2 , 0.24 mM desoxynucleoside triphosphate (dNTPs) mix , 0.6 µM of each primer , 0.2 mM MgCl2 (Qiagen) and 0.03 U/µL Taq Polymerase . After adding 1 µL of cDNA to 19 µL PCR mix, the reaction was performed using a thermocycler  with the following conditions: initial denaturation at 96 °C for 2 min, 35 cycles of 96 °C for 45 s, 60 °C for 30 s, 72 °C for 50 s and final extension at 72 °C for 10 min. Finally, reaction products were electrophoresed on a 3% agarose gel for detection of the BCR-ABL1 transcript. As controls for multiplex PCR the cell lines SD1 (e1a2 BCR-ABL1+ ALL cell line), K562 (e14a2 BCR-ABL1+ CML blast crisis cell line) and BV173 (e13a2 BCR-ABL1+ CML blast crisis cell line) were used.For identification of atypical s et al.  which usBCR-ABL1 fusions. Therefore, cDNA was amplified with oligonucleotides for the respective atypical BCR-ABL1 transcript  with the following conditions: initial denaturation for 10 min at 94 °C, 45 cycles of denaturation for 60 s at 94 °C, annealing for 60 s at 60 °C and extension for 60 s at 72 °C followed by the final elongation for 10 min at 72 °C. Purified amplicons were sequenced bidirectionally by standard Sanger sequencing on a 3500 Genetic Analyzer (Life Technologies). Purification was performed as previously described  following the manufacturer’s instructions to generate pCR2.1_e6a2, pCR2.1_e8a2, pCR2.1_e14a3 and pPCR2.1_e19a2, respectively. Furthermore, fragments of the BCR, GUSB, and ABL1 transcripts, 963, 813, and 1803 bp in size, respectively, were generated from K562-derived cDNA and ligated into the vector pCR2.1. Following digestion using EcoR I for pCR2.1_BCR, Xba I/Kpn I for pCR2.1_GUSB and Spe I/Xba I for pCR2.1_ABL1 the resulting fragments were subcloned into pUC18 vector to create pUC18_BCR_GUSB (4556 bp) and pUC18 BCR_GUSB_ABL1 (6275 bp) backbone plasmids .PCR products for the e6a2, e8a2, e14a3, and e19a2 transcripts were generated from patient cDNA samples that expressed the relevant fusions using the oligonucleotides shown in Table Hind III for e14a2, Hind III/Xba I for e6a2 and e19a2, see supplemental figures S3, S5 and S6), while plasmid pUC18_BCR_GUSB_e8a2 was created by amplification of e8a2 from pCR2.1_e8a2 with Sal I tagged primers (tagged sequence in bold and underlined with the enzyme cutting site indicated by the /):To allow amplification of the target and control gene from the same construct, the transcript fragments were subcloned into the respective control gene backbone plasmids. Plasmids pUC18 BCR_GUS_e6a2, pUC18 BCR_GUS_e19a2 and pUC18 BCR_GUS_ABL_e14a3 were generated by digestion of pCR2.1 constructs with their specific enzymes .The resulting e8a2 PCR product was digested with Xba I restriction site. Following Xba I digestion products were ligated into the pCR2.1 plasmid using T4 ligase generating plasmid pME3 .Standard plasmids used for e1a2 measurement were generated as previously described . Each 20 µL reaction mix contained 4 µL LightCycler-FastStart DNA MasterPLUS HybProbe master mix (Roche Diagnostics), 2 µL cDNA template or plasmid dilution, 0.5 µM forward primer , 0.25 μM of each hybridization probe listed in Table BCR-ABL1 transcript was amplified within the PCR. Beta-glucuronidase (GUSB) transcripts were measured as an internal control using the same standard plasmids as for the BCR-ABL1 measurement.Expression analysis of atypical BCR-ABL1/GUSB. This baseline value was set as 1 and log-reduction was calculated for every time of investigation so that one log-reduction means IMR1, two log-reductions IMR2 and so on.Every CML patient had his individual baseline value at diagnosis which was calculated as the ratio BCR-ABL1 fusion genes were determined by multiplex PCR whereby the size of the resulting PCR product depended on the breakpoint in the BCR and ABL1 gene. Figure BCR-ABL1 transcripts detected in patients and cell lines. The typical BCR-ABL1 transcripts e13a2 and e14a2 are shown by the CML cell lines BV173 and K562 with a PCR product size of 310 bp (lane 4) and 385 bp (lane 3), respectively. The small fusion transcript e13a3 with a product size of 128 bp is found in lane 5 of the agarose gel and one sample with the BCR-ABL1 double transcript e13a3 and e14a3 (product size 128 and 203 bp) is shown in lane 6. A much greater DNA fragment of 481 bp presents the e1a2 fusion gene found in the ALL cell line SD1 (lane 2) and rarely in CML patients (lane 8). The largest currently known BCR-ABL1 transcript in CML patients is the e19a2 transcript with a PCR product size of 925 bp (lane 9) which is located above the BCR control band. This BCR fragment with a size of 808 bp serves as an internal control for the RNA quality and was visible for patients without a BCR-ABL1 fusion gene (lane 10). All atypical transcripts were verified by Sanger sequencing.Atypical BCR-ABL1 transcripts was performed over time periods ranging from 3 months to a maximum of 14 years (median follow-up 16 months) by qPCR  followed by the fusion gene e1a2 found in six patients. Fusion of another ABL1 exon could be found in six patients harboring the e14a3 BCR-ABL1 transcript and four patients with the e13a3 fusion gene. Two patients expressed both e13a3 and e14a3. The rare atypical BCR-ABL1 transcript e8a2 was detected in two patients and monitored in median for 2 years (range 18–38 months). We also analyzed two follow-up samples of one patient with the e6a2 fusion gene (data not shown).Molecular monitoring of 33 CML patients with atypical PCR Fig. . A totalBCR-ABL1 transcripts the application of the international scale is not applicable for the assessment of treatment response due to the use of different PCR primers. Therefore, individual molecular response (IMR) level for CML patients with atypical BCR-ABL1 transcript were applied. With the IMR, the molecular response to therapy is assessed based on the individual baseline of every patient at diagnosis, i.e. IMR1 means a 1 log reduction from the baseline diagnostic sample for that individual, IMR2 a 2 log reduction, etc.For patients with atypical BCR-ABL1 transcript e19a2 reached an IMR1 3 months after diagnosis  with the e19a2 transcript had high BCR-ABL1 levels and did not reach any IMR level.Eight patients (67%) with the atypical BCR-ABL1 transcript (n = 6) reached no better than IMR1 (50%) or failed to reach IMR1  already reached IMR2 or better 3 months after diagnosis  had undetectable BCR-ABL1 2 years after diagnosis  and IMR2 (patient #30) level after 3 months, respectively  or e14a2 (b3a2) BCR-ABL1 transcripts corresponding to the major breakpoint cluster region (M-BCR). Alternate breakpoints in either of both genes generate other rare transcripts. These atypical fusion transcripts are only seen in 1–2% of CML patients  with the associated BCR-ABL1 transcripts by RT-qPCR to assess treatment response and residual disease. At first, it is essential to determine the precise rearrangement by multiplex PCR at CML diagnosis prior to the start of TKI treatment  therapy and are candidates for stem cell transplantation  or exon 14 (e14a3). Two CML patients showed a double transcript e13a3/e14a3. Almost all patients (83%) achieved deep molecular remission with BCR-ABL1 values below the detection limit. Only patient #28 with the atypical transcript e13a3 showed increasing BCR-ABL1 levels corresponding to IMR1 16 month after the diagnosis after the previous achievement of IMR4.The fusion of BCR-ABL1 transcripts (n = 12) whereby two patients never achieved IMR1 and three patients lost previously achieved IMR2 or IMR1. Arun et al.  and thus the common molecular milestones and guidelines for treatment discontinuation are difficult to apply. We recommend the evaluation of CML patients with atypical BCR-ABL1 transcripts using the "individual molecular response” (IMR) level presented here.In conclusion, although few patients are diagnosed with atypical BCR-ABL1 transcript type in all patients with suspected CML.At diagnosis, multiplex PCR or equivalent should be performed to detect the underlying BCR-ABL1 transcript sizes direct sequencing should be performed to identify the precise fusion.For cases with unusual BCR-ABL1 should ideally be quantified using GUSB as an independent control gene.In the pretherapeutic sample, Molecular monitoring should be performed at the intervals recommended for the specific treatment situation.BCR-ABL1/GUSB ratios should be compared with the result from the pretherapeutic sample and expressed as individual molecular response (IMR).BCR-ABL1 IMR response levels are scored based on GUSB transcript numbers and similar criteria as for MR scoring on the International Scale  is not feasible and therefore not recommended.Supplementary file1 (PDF 129 KB)Below is the link to the electronic supplementary material.


The role of ABL1 in hepatocellular carcinoma (HCC) is still unclear. Therefore, this study aims to explore the potential role of ABL1 in the progression of HCC using bioinformatics methods.  We analyzed the expression, prognostic potential, and immune cell effect of ABL1 in HCC by using a variety of datasets.  ABL1 is highly expressed in HCC and associated with unfavorable overall survival (OS) and disease-free survival (DFS). Functional network analysis revealed that ABL1 plays an important role in mitochondrial activity, ATP metabolism, protein translation and metabolism, various neurological diseases, nonalcoholic fatty liver disease, and notch signaling pathway. In addition, we found that ABL1 expression was closely correlated with B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophils, and dendritic cells. Furthermore, ABL1 expression was positively associated with the expression levels of immune checkpoint genes, such as PD-1L, TIM3, TIGIT, and CTLA4.  ABL1 is associated with immune infiltration and prognosis of HCC. Hepatocellular carcinoma (HCC) is the most common primary liver tumor and the second most common risk factor for cancer death worldwide . In ChinThe human Abelson tyrosine-protein kinase 1 (ABL1) gene is located on chromosome 9q34, and the ABL1 protein encoded by it belongs to the ABL interacting protein family . ABL1 plSo far, only a few studies have reported the relationship between ABL1 and HCC. Chitsike et al. indicated that ABL1 was abnormally expressed in human liver cancer . Wang etImmune infiltration is known to be closely related to the progression of liver cancer . In thishttps://TCGA data.nci.nih.gov/tcga/). Standardization was performed on the downloaded HCC datasets, and cases that did not contain survival information were excluded. The total number of samples in the original study of the TCGA data was 529, consisting of 369 liver cancer tissue samples and 160 paracancerous tissue samples. Five HCC samples contained no survival information. Using the median of ABL1 mRNA expression as a node, HCC tissue samples with survival information were divided according to ABL1 expression levels into a high expression group and a low expression group, with 182 samples in each group.Datasets with gene expression profiles and clinical information regarding HCC mRNA expression were downloaded from the TCGA database    was usedThe LinkedOmics database  is a freTIMER  is a visCIBERSORT  can asseR 3.5.3, and the P value < 0.05 was considered statistically significant. The survival rate was analyzed by a log-rank test and Mantel–Cox test. Logistic regression was used to analyze the correlation between clinical features and ABL1 expression. The correlation between the expression of ABL1, coexpressed genes, and 22 immune cell types was measured by the Pearson correlation coefficient.The statistical data obtained from TCGA were processed by To evaluate the expression of ABL1 in HCC tissues and normal tissues, we used GEPIA to analyze 369 HCC specimens from TCGA. This analysis revealed that ABL1 expression was significantly higher in HCC tissues than in normal tissues . In addiP=0.013), and survival status (P < 0.01). We further analyzed the prognostic value of ABL1 in HCC. The survival curve from GEPIA showed that high expression of ABL1 predicts unfavorable OS (P=0.014) and DFS (P=0.035). The results are shown in In addition, we analyzed the association between ABL1 expression and clinicopathological characteristics in the TCGA HCC cohort . ABL1 exWe explored the biological interaction network of ABL1 in HCC to clarify the biological function of ABL1. We first selected the genes related to ABL1 and performed an enrichment analysis. The top 50 genes with significant positive and negative correlations with ABL1 are shown in Figures P=0.001623), M2 macrophages (P=0.003429), mast cells resting (P=6.74e − 7), and NK cells resting (P=0.0002681) were most abundant in the high ABL1 expression group, whereas dendritic cells resting (P=4.747e − 05), M0 macrophages (P=3.308e − 12), CD4 cells (P=0.0008109), and CD8 cells (P=0.01354) were significantly reduced.Immune infiltration is closely related to tumor progression. Therefore, we also evaluated the effect of ABL1 expression on immune infiltrating cells in liver cancer using CIBERSORT. The proportions of 22 immune cell subgroups are shown in r = 0.285, P=7.25e − 08), CD8 cells , CD4 cells , macrophages , neutrophils , and dendritic cells  in HCC. These results suggest that ABL1 expression influences liver cancer progression by altering immune cell infiltration.We used TIMER to further study the association between ABL1 and the level of tumor immune cell infiltration . The resImmune checkpoint blockade therapy is a popular immunotherapy method and shows a strong therapeutic effect. We explored the relationship between ABL1 expression and tumor immunotherapy. We found that some immune checkpoints  were positively correlated with ABL1 expression . In addiHCC can occur at any age and is most common in patients with chronic hepatitis . Viral hOur analysis showed that ABL1 expression was significantly higher in liver cancer tissues than in normal tissues. Subgroup analysis further confirmed that ABL1 mRNA was highly expressed in HCC. In addition, we used the GEPIA database to evaluate the prognostic value of ABL1 in HCC. We found that high ABL1 expression was significantly associated with adverse OS and DFS. Multivariate Cox regression analysis further showed that the ABL1 expression level was an independent risk factor for liver cancer prognosis.P=2.48e − 09), the ATP metabolic process (P=2.27e − 05), and the Wnt signaling pathway (P=0.035) [P=1.12e − 08), amyotrophic lateral sclerosis (P=3.0e − 07), Alzheimer's disease (P=3.59e − 07), and Parkinson's disease (P=6.53e − 08). Surprisingly, ABL1 was also associated with nonalcoholic fatty liver disease (P=5.96e − 08). In addition, we found pathways and metabolic processes related to tumors, such as oxidative phosphorylation (P=5.54e − 08), inositol phosphate metabolism (P=0.0035), and notch signaling pathway (P=0.0038). These results indicate that ABL1 is involved in a variety of diseases and may play an important role in them. The results also confirmed that ABL1 regulates the progression of HCC through a complex mechanism, revealing ABL1 as a potential target in HCC treatment.Coexpressed genes act synergistically in strictly regulated biological processes, and thus they can provide alternative pathways to sidestep barriers, providing an adaptive evolution advantage . We perfP=0.035) –27. Funcγ, interleukin 12p40, granzyme B, and TRAIL and acted in cooperation with CD8 (+) T cells to promote tumor immunity and predict good prognosis in HCC [r = 0.285, P=7.25e − 08), CD8 cells , CD4+ cells , macrophages , neutrophils , and dendritic cells  in HCC. Among these immune cells, CD4+ cells were most strongly associated with ABL1 expression. The findings indicate that ABL1 has an important effect on immune infiltrating cells in HCC.HCC is a typical inflammatory-related tumor. Its tumor microenvironment includes a large number of immune cells, inflammatory factors, and extracellular matrix, forming a complex immune microenvironment. Immune cells in the tumor microenvironment of HCC mainly include tumor-infiltrating lymphocytes (TILs), tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), myeloid-derived suppressor cells (MDSCs), and dendritic cells (DCs). TILs are composed of regulatory T cells, cytotoxic T lymphocytes, B cells, and NK cells in HCC. Regulatory T cells are a subset of CD4 + T cells, a group of lymphocytes with a high degree of immunosuppression, which can achieve immunosuppressive effects by inhibiting CD8 + T cells . Studiess in HCC . Howevers in HCC . NK cells in HCC . TAMs cas in HCC . Howevers in HCC . TANs als in HCC . On the s in HCC . MDSCs as in HCC . Anothers in HCC . They maImmune checkpoints have been proven to be effective targets for the treatment of tumors. Studies have shown that the expression of immune checkpoints such as PD-1L, TIM3, TIGIT, and CTLA4 can affect tumor progression and thus change patient prognosis –46. ImmuOur results demonstrated that high ABL1 expression is associated with unfavorable prognosis in HCC. The high ABL1 expression significantly influences the immune cell infiltration and immune checkpoint expression in the tumor microenvironment in HCC. We hypothesize that specific molecular targeting ABL1 expression could affect immune cell infiltration in the tumor microenvironment and improve the prognosis of patients with HCC. Targeting ABL1 expression may effectively strengthen the effectiveness of other immune checkpoint inhibitors in HCC. ABL1 may be a promising prognostic biomarker and therapeutic target for HCC patients. Our research provides a basis for the role of ABL1 in HCC, and further research is strongly recommended.


Every year, over 65,000 Australians experience an acute coronary syndrome (ACS) and around one-third occur in people with prior coronary heart disease. Cardiac rehabilitation (CR) aims to prevent a repeat ACS by supporting patients’ return to an active and fulfilling lifestyle. CR programs are efficacious, but audits of clinical practice show variability of program delivery, which may compromise patient outcomes. Core components, quality indicators and accreditation of programs have been introduced internationally to increase program standardisation. With Australian quality indicators (QIs) for cardiac rehabilitation recently introduced, we aimed to conduct a survey in one state of Australia to assess the extent to which programs adhere to the measurement of QIs comparing country, metropolitan, telephone and face to face programs.A cross- sectional survey design with face validity testing was used to formulate questions to evaluate cardiac rehabilitation program and personnel characteristics and QI adherence. Between October 2020- December 2021, 23 cardiac rehabilitation programs across country and metropolitan areas were invited to participate. Quality improvement was defined as adherence to the Australian Quality Indicators, and we developed an objective score to calculate program performance categorised by quartiles. Significance of CR completion and time to enrolment between program type (telephone versus face to face) and location .Among the 23 CR programs, 15 were country and 8 metropolitan-based and 22 were face to face and 1 telephone-based. Median wait time from discharge was 27.0 days, (interquartile range 19.3–46.0) across all programs and country completions of enrolled were 76.9% versus metropolitan 56.5%, A survey of 23 cardiac rehabilitation programs, showed variability in adherence to measurement of the Australian Cardiovascular and Rehabilitation Association and Australian Heart Foundation Cardiac Rehabilitation Quality Indicators.ACTRN12621000222842, registered 03/03/2021.Australia New Zealand Clinical Trials Registry (ANZCTR), The online version contains supplementary material available at 10.1186/s12913-022-07667-2. Every year, over 65,000 Australians experience an acute coronary syndrome (ACS) (heart attack or unstable angina) and around one-third occur in people with prior coronary heart disease (CHD)–3. In AuGlobally and in Australia, core components –24 and q2 (5 times the size of the United Kingdom) with a population of 1.7 million in 2020, with 22.4% living in country areas  19.3–46.0) across all programs, with country having a median of 33.0 days (IQR 21–45) and metropolitan a median of 23.0 days (IQR 18–37), Program length and sessions.Mean total program length was 7.0 weeks SD ± 1.11) with country slightly longer than metropolitan programs  2004 CR framework,  NHF PathAll the primary coordinators were registered nurses with 78.2% completing a postgraduate qualification relating to cardiovascular care (Masters or Graduate Diploma). Eighty seven percent belonged to the Australian Cardiovascular and Rehabilitation Association (ACRA) peak body. All participated in some type of professional development in 2019 with the majority choosing cardiovascular-based conferences, webinars, workshops, and seminars.Australian Quality indicator (QI) adherence.All QIs except QI 1 were measured by some programs and program adherence is presented in Table Seventy four percent of programs were ranked at a medium level of performance with a mean performance score of 11.4/16, (SD ± 0.79). No programs were ranked as poor, 13% ranked low and 13% high Table . Comparip < 0.001) and the telephone program had higher completions than face to face programs . We were unable to measure total patients eligible in this dataset and this is something that our quality improvement initiatives hope to address in the future. However, an audit conducted between 2013- 2015 amongst the same programs showed that approximately 16,600 patients per year are eligible and of these 5,000 are referred. [The aim of this study was to assess the extent to which programs met the national QIs. This study surveyed 23 cardiac rehabilitation services in South Australia, Australia for program and personnel characteristics and quality performance adherence, comparing country and metropolitan, telephone and face to face programs. In 2019, of the 69.5% of programs that could provide data, there were 1,972 patients enrolled with 66.7% of these patients completing. Country and metropolitan enrolments were similar with 42.8% of country patients attending the telephone program. Country completions were higher than metropolitan  across country and metropolitan, telephone and face to face. Program length and sessions were similar amongst country and metropolitan, telephone and face to face programs (mean 7.0 weeks or 10.5 sessions) and indeed 6–8 weeks (12–16 sessions) is the norm across Australian programs. However, if we look at international benchmarks, program duration is far less in Australia than our international counterparts in the UK and Europe, who have a standard of ≥ 24 -36 sessions or around 12 -18 weeks, raising the question of what ‘dose’ of CR is most effective to improve outcomes? , 36 TotaThe ideal cardiac rehabilitation program consists of a multidisciplinary team. In South Australia where programs do not have all types of professionals as part of their team, this is enabled by referral pathways. The multidisciplinary team amongst country, metropolitan, telephone and face to face programs consisted of a nurse and either a physiotherapist or exercise physiologist and to a slightly lesser degree a dietician or pharmacist. Where they were not part of the team, they were accessible via a referral pathway, with the exception of the telephone program Table , which cThe most measured QI was comprehensive assessment across country, metropolitan, telephone and face to face programs, followed by depression screening, smoking assessment and counselling thereof Table . There iA key finding of this survey was the higher completion rates in country compared to metropolitan, and telephone compared to face to face programs. This was likely driven by 42.8% of patients accessing country programs attending the telephone option. While this measure reflects a process indicator, we also know from the survey that the telephone program delivers only one exercise session, where efficacy evidence tells us that exercise is a significant driver of improved CR outcomes , 14, 23.Quality improvement measurement and comparisons often require a multi-component approach of standardisation, measurement, reporting and change management. We were not able to conduct a validation process because of the variability of data entry into the CATCH database. This identifies the need for universal data entry to accurately measure program quality and will require a change management strategy to achieve. Accreditation can be a tool to promote change management among clinicians to drive standardisation of care and deliver evidence-based scope of practice, qualifications, and program content delivery .This survey was self-reported for each program and thus is subject to reporting bias. We were not able to objectively validate the questionnaire responses nor correlate these with clinical outcomes, thus we don’t know whether QI adherence is associated with better quality program content. Further, as the performance score is derived from program adherence to the QIs, the discriminatory capacity of the score may be limited. Since data entry into the CATCH database is not mandatory across the 23 programs, not all survey items measured have 23 programs as the denominator. Further enrolment and completion numbers are underestimated. We were not able to measure enrolment against eligible or referred patients for 2019, though we know from our previous audit work that approximately 5,000 patients are referred to cardiac rehabilitation each year in SA. (16) The sample size of 23 programs is small to measure any meaningful differences between country and metropolitan, telephone and face to face programs. Further the Australian Quality Indicators were only published in 2020 and therefore programs have not had time to implement improvements.(31) Despite this we have surveyed 23 CR programs across SA, evaluating program and personnel characteristics and determining adherence with the Australian Quality indicators, giving us an indication of the level of program quality to inform development of an accreditation system.A survey of 23 cardiac rehabilitation programs in one state of Australia, showed variability in adherence to measurement of the Australian Cardiovascular and Rehabilitation Association and National Heart Foundation Cardiac Rehabilitation Quality Indicators as measured by a survey. These data indicate that there are gaps in the delivery of evidence-based content in South Australian cardiac rehabilitation programs, which could be addressed by quality improvement initiatives such as an accreditation system. A state-wide engagement and education program will be required as an essential preliminary step towards an accreditation program.Additional file 1. Cardiac Rehabilitation Program Performance Scoring Criteria.Additional file 2. The South Australian Cardiac Rehabilitation Program Accreditation Survey.


Purpose: Underrepresentation of racial and ethnic minorities in the health care workforce is a local and national issue. We describe and report on outcomes of a longitudinal service-driven prehealth pathway program in a low-income community intended to address this disparity and increase health equity.Methods: The Bronx Community Health Leaders (BxCHL) is a prehealth pathway program for socioeconomically disadvantaged and underrepresented minority students seeking careers in health care. The program enrolls students in college or college graduates and provides longitudinal near-peer mentorship, exposure to the health care environment, and supports professional development. An academic federally qualified health center serves as the program's home site and learning environment. We conducted surveys and tracked the career advancement of program participants over a 6-year period, 2014–2020.Results: One hundred sixty-eight students participated in BxCHL for >3 months. Of these, 76 students advanced into professional health career programs with 39 direct acceptances and 15 conditional acceptances to medical school programs, 9 nursing, 4 physician assistant, 9 health-related masters level programs, 1 respiratory therapy, and 1 optometry. The direct and overall acceptance  rate of medical school applicants is 59% and 86%, respectively. The first 11 BxCHL alumni obtained their medical degree.Conclusions: BxCHL's longitudinal service-driven and near-peer mentorship program design represents a replicable model to address health equity by supporting prehealth students from communities with limited access to mentors and professional learning environments in entering the health care workforce and serving their communities of origin. For instance, the Bronx Health Opportunities Partnership-Einstein (Bronx-HOPE), a Health Careers Opportunity Program-funded program established by the Department of Family and Social at the Albert Einstein College of Medicine, sponsors prehealth summer enrichment programs.14 Most programs that offer these opportunities are longitudinally constrained by administrative costs and support for students.16Despite these and other national efforts, access to longitudinal mentorship networks and professional learning environments for URM prehealth students in low-income communities is severely limited.In response to the limited opportunities for exposure to medical environments and access to role models experienced by students in the Bronx community, Bronx Community Health Leaders (BxCHL) was created to serve as a longitudinal prehealth pathway program in a Federally Qualified Health Center (FQHC) setting sustained by service-driven student and characterized by near-peer mentorship to fill these training gaps and support needs. As a primary aim, we describe the program's design and report preliminary program and student outcomes. As a secondary aim, we examine the replicability and benefits of the longitudinal and flexible design of the BxCHL program.The program was founded in 2014 by prehealth students serving as volunteers at a community health center. The students self-organized and developed a program characterized by near-peer mentorship, community service, and professional development. The program is primarily student-driven, sustained by the following: (1) four student “coordinators” with a 6-month nonpaid commitment to organize and schedule educational and community activities, and (2) teams of four to five members that sustain year-round educational and wellness programs for health center patients.ignite the fire,” “illuminate the path,” “create the toolkit,” and “sustain the desire.”17 The program's capacity is to support 50–60 students at any one time. This design evolved out of need, as there was no site administrative support or resources to run the program. This design embraces the four pillars of successful longitudinal mentoring programs proposed by Mains and colleagues, including the ability to “18BxCHL enrolls students currently in college or college graduates in their “gap” year(s) before entering professional health career programs. Outreach occurs primarily through peer referral and community partners. Students submit an application that undergoes a peer selection process. It is a “rolling” admissions process year-round. The selection process seeks students with a strong commitment to community service and does not consider academic metrics. This selection process was agreed upon by the students. This approach aligns with health equity and holistic admission processes supported by other prehealth programs and medical school admission committees.Accepted students can remain in the program until they enter professional health career programs or can no longer regularly attend the program, but can re-enter the program at any point later in their prehealth journey.The program's home site is the Montefiore Family Health Center (FHC), an academic family medicine FQHC and the training site for the Montefiore Medical Center/Albert Einstein College of Medicine Department of Family and Social Medicine Residency Program. The FHC serves a large immigrant, refugee, and underserved population in the Bronx. This location is easily accessible to the students by public transportation.19BxCHL focuses on applying evidence known to contribute to the success of socioeconomically disadvantaged and URM students pursuing health care careers. The program enhances competency-based skills identified by the American Association of Medical Colleges (AAMC) and most medical schools' holistic admission process, including interpersonal and communication skills, knowledge of health care systems, participation in scholarly inquiry, cultural competency, service orientation, and teamwork.15 Also, programs that expose students to health care environments have a positive impact on their decision to pursue health care careers.20–22In addition, exposure to near-peer mentors is known to contribute to the success of socioeconomically disadvantaged and URM students pursuing health care careers.The program's near-peer mentorship component connects college, postbaccalaureate, and medical students. Students access mentors through a multidisciplinary and diverse health center and academic program staff. Students receive individualized support from program alumni, including writing personal statements, mock interviews, and letters of recommendation.We conducted a survey  over a 12-week period. Students were sent a survey link via e-mail, and participation was voluntary and confidential. An Institutional Review Board approval (IRB) (number 2019-10769) was issued by the Albert Einstein College of Medicine for this study. Using enrollment and attendance data, we identified all students who participated in the program for at least 3 months. We keep records of the students' application process to professional health career programs (cycles 2014–2020). We reached out via e-mail to students who had left the program to confirm their career progress.active—currently enrolled in the program, (2) graduate—student who persisted in the application process to enter health care professional programs while still in the program, (3) associate—students who transitioned through the program (were enrolled at least 3 months), and were not participating in the application process when they left the program.We grouped students into three categories to examine the potential impact of length of participation in the program and their career advancement outcomes: (1) active members (currently enrolled in the program), 50 graduates , and 73 associates . Of these, 168 (87%) remained in the program for at least 3 months. The survey response rate was 38% (64/168), with active and graduate students representing almost 90% of the respondents.A total of 194 students enrolled in the program from 2014 to 2020. There are 45 students identified as N=64).Students are predominantly female (66%). The main age ranges are 18–25 (52%) and 26–30 (39%); they self-identified mainly as black/African American (28%), Latinx (41%), and Asian (17%). The group is primarily economically disadvantaged. The majority of students had work-related responsibilities while in school. At least 83% of the students had attained/or were enrolled in bachelor's degree when they entered the program. The majority attended a public city college (45%) and a private liberal arts college (non-Ivy league) (34%). Their intended career when they entered the program was mainly medicine (83%).. The majority indicated they took or were planning on taking “gap” years primarily for more exposure to health care-related activities and improving academic metrics. Less than half reported having a professional mentor or peer-mentor before entering the program, and a mentor was one of the top motivations for program enrollment. Of particular note, over half reported feeling “lost and lonely” in the process of applying to health care careers.The students' prehealth journey is highlighted in Table 2Notable socioeconomically disadvantaged measures in this group include growing up in a low-income family and being a first-generation college student. Other challenges faced in the last 12 months (before the COVID-19 pandemic) included the following: having to help their family financially (52%), help with child care or elderly/or sick adult in family (45%), lack of support from family or other people (38%), and at least 19% lacked transportation to attend school, work, or volunteer responsibilities.Combined, 76 students advanced into professional health career programs, with 39 direct and 15 conditional acceptances to medical programs, 9 nursing, 4 physician assistant, 9 masters level programs, 1 respiratory therapy, and 1 optometry . The dirassociates were in the program for a shorter period of time compared with the graduates. Of the students who left the program, many still remained on the prehealth path. Most students who were offered direct acceptance (21/27) and/or conditional acceptance (8/10) to medical programs were in the BxCHL program for more than 1 year.1 These results are comparable or better than outcomes from similar pathway programs in advancing students to graduate programs and medical school acceptance.23 Eleven BxCHL alumni have obtained their medical degree and the majority will be training in primary residency programs. The BxCHL model was successfully replicated for another program.Since 2014, the BxCHL program has served as a support system and advanced many URM and socioeconomically disadvantaged prehealth students into professional health care programs. Medical school applicants have a direct accepted rate (59%) comparable or better than the national direct acceptance rate of 41.1% .The survey findings highlight the need for BxCHL-like programs to support URM and socioeconomically disadvantaged students in their prehealth path. While students face challenges that require significant societal intervention and resources, the BxCHL program addresses concrete needs such as providing funds for transportation and internships. It also addresses many students' sentiment of isolation (“lost and lonely”) during their journey. Students in these communities are at higher risk of not advancing due to the lack of social support or mentoring.25Data suggest that supporting these students would increase workforce diversity in health care professions and directly impact health care access and care of marginalized populations as they are more likely to practice in underserved areas.27The data presented suggest that the longitudinal mentorship and flexible design of the BxCHL may increase the students' retention and success in their prehealth path, and some students may find a better occupational fit within the health care field. This longitudinal mentorship component is known to be important for the persistence and success of URM students in the prehealth path and has been recommended a critical element needed to address the national crisis of lack of black men in medicine.In general, the participation of students self-identifying as black or African American has ranged from 28% to 33% since BxCHL was established, which is higher than the current makeup of the physician workforce. Nonetheless, the ratio of female-to-male black or African American participants has been 3:1, which is similar to other prehealth programs in the community. This report also highlights challenges and opportunities in the delivery of prehealth programs in low-income communities. Notably, the program's attrition rate for the first 3 months of a student's participation in the program is about 13%.27However, we have addressed this by enhancing near-peer mentorship and striving for an inclusive and safe environment to reduce stereotype threats and imposter syndrome. This promotes camaraderie among students from marginalized communities and combats the feeling of being “lost and lonely,” increasing confidence and sense of belonging. The students are exposed to mentors from similar URM backgrounds who have traveled similar paths, allowing the students to envision themselves in health care careers, similar to those influences proposed for science, technology, engineering, and mathematics identity formation.graduate vs. associate) more robustly. Another limitation in this report is that it only includes self-reported academic metrics. Metrics is not a competency the program was designed to address directly, but might indirectly impact the students' academic performance considering that they are encouraged to maintain high academic standards as part of the program's expectations. Similarly designed programs with less rigid entrance metrics than traditional prehealth pathway programs have shown high success in advancing students into professional health care programs.14The limitations in data include low student survey completion, which limited the ability to compare outcomes of the subgroups  was successfully replicated at another intrainstitutional health center. A BxCHL replication guide and toolkit were produced and utilized as the framework for the new program. Several existing BxCHL students with experience in the program design and eager to replicate the sister program volunteered at the SBxCHL program pilot class. A family physician along with a global health fellow took on the role of program champions. SBxCHL has now been in existence for 2 years and currently supports 25 students.Can and should health care and academic institutions commit to investing in programs such as BxCHL? Can these initiatives yield better outcomes in increasing diversity in the health care workforce than the existing efforts? Short term, the preliminary outcomes of the BxCHL program support this. Long term, we strongly believe that an initiative that produces homegrown health care professionals will address equity and improve health outcomes for underserved communities. This has already been on display as several BxCHL students have entered residency programs focusing on primary care.BxCHL-like programs are more important than ever to help close the gap in educational opportunities and health disparities, especially during challenging times brought on by the COVID-19 pandemic.28 We could not agree more with this assessment; BxCHL's service-driven near-peer mentorship approach could be part of the solution. We envision a program model that can be replicated and made accessible to students in low-income communities to increase diversity in the health care workforce.To quote Emery CR and colleagues at the National Academy of Medicine, “To date, the majority of interventions to increase underrepresented minority participation in medicine have been undertaken within the existing systems and structures of academic medicine. To address the urgent need for a diverse physician workforce, we must use new and innovative tools to meet the current needs.”We believe that the BxCHL program represents an innovative and viable program designed to recruit, retain, and advance prehealth students from communities with limited access to support systems and professional learning environments. It is a grassroots effort fueled by service-driven URM prehealth students that has resulted in tangible outcomes. Supporting BxCHL-like programs should be cost affordable for most health care and academic institutions. BxCHL programs are often the only support system for many students in this low-income community. Therefore, we call for the sponsorship and commitment of community and academic stakeholders. After all, this is consistent with the program's motto that “it takes a village to raise a healthcare professional.”


Are there synergies between primary care organization participation in voluntary delivery system and payment reform programs  and improved health services outcomes ?This observational longitudinal study of 47 880 primary care organizations including approximately 5.61 million Medicare beneficiaries found that participation in all 3 reform programs was associated with better performance for only diabetes guideline adherence. This outcome and 2 others (ambulatory care sensitive admissions and spending) also showed benefit from dual and single program participation.These findings suggest that reform programs do not demonstrate consistent evidence of synergies, likely because program requirements and goals are not sufficiently aligned. Policy makers envision synergistic benefits from primary care reform programs that advance infrastructure and processes in the context of a supportive payment environment. However, these programs have been operationalized and implemented separately, raising the question of whether synergies are achieved.To evaluate associations between primary care engagement in voluntary delivery system and/or payment reform programs and health services outcomes.This was an observational longitudinal analysis of US ambulatory primary care organizations (PCOs) with attributed Medicare fee-for-service beneficiaries  using data for 2009, 2010, and 2015 to 2017; PCOs included multispecialty practices that delivered primary care. Data analyses were performed from January 2020 to December 2021.Annual PCO participation in or recognition by (1) the Centers for Medicare & Medicaid’s meaningful use (MU) program, (2) the National Committee for Quality Assurance’s Patient-Centered Medical Home (PCMH) program, and/or (3) the Medicare Shared Savings Program (MSSP), an Accountable Care Organizations program.Independent and joint associations between an additional year of participation by a PCO in each of the 3 reform programs, and 3 types of outcomes: (1) hospital utilization ; (2) evidence-based diabetes guideline adherence ; and (3) Medicare spending .The study sample comprised 47 880 unique PCOs  and approximately 5.61 million unique Medicare beneficiaries  across the study years . Of the hospital utilization measures, only ambulatory care sensitive admission was associated with improved performance, showing a statistically significant marginal effect size for joint participation in MU and MSSP  and MSSP alone . For diabetes adherence, joint participation in PCMH and MU was associated with 0.06 more measures met  while participation in all 3 programs was associated with 0.05 more measures met . Stand-alone PCMH and stand-alone MU participation were also associated with improved performance. Joint participation in MU and MSSP was associated with $33.89 lower spending  as was stand-alone MSSP participation .This longitudinal observational study found that participation by PCOs in single or multiple reform programs was associated with better performance for only a subset of health services outcomes. More consistent and larger synergies may be realized with improved alignment of program requirements and goals. This was a longitudinal observational study of participation by primary care organizations in voluntary delivery and payment reform programs and its associations with hospital utilization, adherence to evidence-based care, and spending. For example, the Centers for Medicare & Medicaid Services (CMS), through its Meaningful Use (MU) incentive program, requires that core primary care data on patient health issues, allergies, smoking status, and other elements, be documented in electronic health records (EHRs).3 Other approaches focus on medical practices’ processes that promote care coordination, a prime example being the National Committee for Quality Assurance’s Patient-Centered Medical Home (PCMH) recognition program.4 Other approaches, such as the Medicare Accountable Care Organizations (ACOs), focus on tying practice reimbursement to the value of care delivered.5Given the central role of primary care in high-performing health care systems, much experimentation to improve health care quality and reduce spending has centered on transforming primary care practices.6 Policy makers hoped for these synergies when they launched MU, the PCMH model, and ACOs, such as the Medicare Shared Savings Program (MSSP), but did not explicitly design these expectations into the programs.7 For example, key dimensions of the PCMH model, such as systematic tracking and follow-up of laboratory test results, are more likely to be effective when recorded in an EHR vs a paper-based patient chart. Moreover, effective use of new infrastructure and processes is likely intensified by increased financial rewards for better outcomes. Yet, these programs have been operationalized and implemented separately, raising the question of whether the intended synergies are achieved in terms of improved outcomes.In concept, these primary care focused approaches should be synergistic, with investment in infrastructure and processes occurring in the context of a supportive payment model.15 A small number of studies have found synergistic benefits from hospital participation in multiple reform programs.17 However, no prior work has examined the participation of primary care organizations (PCOs) in multiple programs over time to determine the extent to which these programs are working synergistically.18The literature on this critical question is limited. Individual programs have been extensively studied, generally suggesting that specific programs have achieved better outcomes, eg, lower spending associated with ACO participation.19 our findings may be highly relevant to the open question of whether policy efforts should pursue more substantive alignment and integration or whether ongoing experimentation with more targeted but disconnected programs is effective for improving outcomes.Creating what is, to our knowledge, the first national longitudinal data set , this study sought to ascertain the relationship between PCO participation in 3 major voluntary reform programs—MU, PCMH, and MSSP—and various health services outcomes, assessing the independent and joint associations for each program. Although the programs changed during the study period and were partially combined under the Medicare Access and CHIP Reauthorization Act,STROBE) reporting guidelines.This observational longitudinal analysis was reviewed and approved by the institutional review board of the University of California, San Francisco. Informed consent was waived because the analysis used only deidentified data. The study followed the Strengthening the Reporting of Observational Studies in Epidemiology  of their primary care during that calendar year. We followed this process for each of the 5 calendar years  for which we had Medicare claims files. We then limited the study sample to the 47 880 TINs that had 1 or more attributed beneficiaries in all 5 years to reflect the subset of organizations that were continuously eligible to participate in primary care reform programs. Therefore, the sample included primary care practices and multispecialty practices delivering primary care. We included this breadth because the programs under study applied to both types of practices. The final sample was at the beneficiary level and ranged from an annual total of 1.6 to 1.9 million unique beneficiaries.The study samples included all Medicare fee-for-service beneficiaries in the standard Medicare 20% research identifiable files  who were assigned to PCOs (identified by Taxpayer Identification Numbers [TINs]) in January to December of 2009, 2010, and 2015 to 2017. Specifically, we replicated the MSSP−ACO methodology for primary care attribution using the Medicare fee-for-service claims files.21 The MU and PCMH programs reward clinicians for processes that are likely to be associated with improved outcomes.23 We examined the requirements of each program and identified 3 outcomes that 2 or more programs were designed to target: (1) adherence to evidence-based practices; (2) avoidable hospital utilization ; and (3) spending  for diabetes: at least 1 annual glycated hemoglobin test, low-density lipoprotein cholesterol test, nephropathy screening, and eye examination. We identified beneficiaries with diabetes based on the 2016 specifications,30The final measures included total annual Medicare spending determined by summing Medicare-paid amounts for a beneficiary’s care across all inpatient and outpatient files and adjusting for medical inflation to 2017 US dollars. We assessed 2 components of total spending—acute inpatient and skilled nursing facility—because reducing avoidable acute and postacute inpatient spending is a focus of ACOs and is enabled by capabilities in the 2 other programs.31 We used the TINs as the organizational unit of program participation for 2 reasons: (1) MSSP participation is at the TIN level and, although MU and PCMH attestation occurs at the National Provider Identifier (NPI) level, both programs require substantial organizational investment, and therefore, individual NPI-level participation within larger organizations is rare; and (2) to identify organizations providing primary care to Medicare beneficiaries, we sought to replicate an existing definition based on Medicare claims to capture organizations that provide a plurality of primary care services to Medicare beneficiaries during multiple years. Because individual NPIs can switch among PCOs over time, this definition is logically operationalized at the TIN level.We measured TIN-level participation in each of the 3 programs at the calendar year level.We measured TIN-year level program participation using MU participation data from Medicare attestation files and Medicaid attestation files, PCMH recognition data, and MSSP participation data from the provider-level research identifiable file . To address heterogeneity across TINs, we adjusted for PCO characteristics  and characteristics of the county in which the PCO was located, per Medicare Data on Provider Practice and Specialty data, the Area Health Resource file, and the American Community Survey (described in our prior study).31We adjusted for beneficiary-level factors that might confound the relationships between program participation and outcomes, including age, sex, and race and ethnicity (using definitions and categories from the Master Beneficiary Summary file), as well as the individual Elixhauser comorbidity categories33 For diabetes adherence and spending, we used multilevel mixed-effects linear regressions. In all models, beneficiary, TIN, and community characteristics were specified as fixed effects. TINs were specified as the random intercept and in clustering standard errors. We used interaction terms between the main program variables  to test the joint associations of an additional year of participation in each program for each outcome. After estimation, we computed marginal effect sizes for 1 year of program participation in individual programs and in all possible combinations  compared with no participation in any program. Treatment effects were considered statistically significant for P < .05; tests were 2-tailed.At the beneficiary-year level, the analytic data set linked beneficiaries to their attributed TIN in the given calendar year and TINs to the running sum of years of program participation in the given calendar year. For hospital utilization outcomes, we evaluated treatment effects using multilevel mixed-effects negative binomial regressions based on goodness-of-fit statistics.Before selecting the analysis models, we tested the linearity assumption of each outcome as a function of the number of years of program participation, and we did not observe any nonlinear relationships  and approximately 5.61 million unique Medicare beneficiaries  across the study years . The most common comorbidities among the beneficiaries were uncomplicated hypertension (45.30%) and uncomplicated diabetes (21.43%). Additional key characteristics of the PCOs and the beneficiaries are shown in Among the 3 programs, participation in MU was the highest, with 23.5% of PCOs having participated for 1 to 2 years, and an additional 36.9% for 3 to 6 years during our study period. . ParticiThere was no statistically significant marginal effect size for 1 year of program participation—not in 1 stand-alone program nor for any combination of the programs—for any measure of all-cause hospital utilization as compared with no program participation . For ACSP = .01), and stand-alone MU participation was associated with an increase of 0.02  and participation in all 3 programs increased adherence by 0.05 measures . Participation in MSSP, alone or in a paired combination, was not associated with higher adherence. The mean adherence in the sample was 2.24 measures per beneficiary per year, such that these marginal effect sizes represent a 0.9%  to 2.7%  improvement if all PCOs had participated for 1 year vs all not participating. For the individual measures, at least 3 of the 4 demonstrated a statistically significant positive 1-year marginal effect size consistent with the composite measure . Mean annual spending per beneficiary was $6679.27; these marginal effect sizes represent savings of 0.51% (for MU−MSSP joint participation) and 0.55%  if all the PCOs had participated for 1 year vs all not participating.Stand-alone MSSP participation was associated with $37.04 lower spending per beneficiary per year  and participation in both MU and MSSP , participation in PCMH and MSSP , participation in MU and MSSP , and participation in all 3 programs , but it is not for either MU or PCMH. Thus, there may be a need to specifically align the goals with the participation requirements for each program.Indeed, prior work has pointed to the lack of aligned requirements as an important policy priority for decreasing participation burden and enhancing effectiveness.17 A best case interpretation of these findings may be that the focus of any of the programs helps to realize benefits from a second program. For example, PCMH models, specifically the National Committee for Quality Assurance’s accreditation criteria, emphasize adherence to guidelines for chronic disease management, which may be reinforced by EHR workflows or alerts that are an option for meeting MU criteria.36 As another example, for the 3-year marginal effect estimates for all-cause ED visits, MU participation was present in all statistically significant combinations associated with lower utilization. Participation in MU may be associated with reduced ED utilization, particularly after multiple years of participation during which the progression of MU stages requires more advanced health information exchange. Specifically, having a robust EHR enables better health information exchange, such as admission-discharge-transfer notifications that alert primary care clinicians when their patient is in the ED. These alerts, in turn, allow the clinician to intervene in a timely manner, which can avoid a hospitalization37 and the associated costs. These relationships offer hypotheses that can be explored in future work to generate targeted guidance on how best to operationalize alignment of program goals and requirements.Nonetheless, we did observe benefits from multiprogram participation, which is consistent with similar work in the hospital setting.38 Without more explicit efforts to synchronize EHR capabilities with the activities needed to deliver high-quality, low-cost primary care, it will be difficult for PCOs to realize substantial benefits from multiprogram participation. Similarly, there is a need to achieve greater incentive alignment between programs  and ensure that PCOs have a clear set of common spending and quality targets to work toward.For policy makers, these study findings should prompt reflection on the overall strategy to drive primary care practice transformation. Although program design has evolved, during and since the study period, federal programs have continued to struggle to achieve this alignment. In particular, programs separate uses of technology from processes involved in primary care transformation and accountable care. For example, at the programmatic level, CMS Innovation models have a category for accountable care and another category for primary care transformation in addition to several others. Even within the Merit-based Incentive Payment System, the health information technology component is separate from the other score components.27 In addition, it is likely that PCOs with an interest in improving quality and resources to invest were more likely to participate. Despite taking several steps to improve the robustness of the study results—eg, using a large set of covariates to capture PCO characteristics and beneficiary demographic information—the associations we observed could be owed to unmeasured factors related to program participation and duration.This study was limited because it included participation data from only 3 programs, thereby missing programs such as Pioneer ACOs and other PCMH programs. However, these other programs are either small or state-specific, such that the magnitude of potential bias is likely small. Similarly, it is possible that the PCOs that did not participate or stopped participating in a given program continued to meet the requirements, although this is unlikely given the financial penalties. In addition, the study’s Medicare claims data were limited to 5 years . Because we relied on these data to identify PCOs and measure outcomes, it is possible that some organizations would not have met our definition of a PCO or had systematically different outcomes in the intervening years (2011-2014), although we suspect either scenario is rare. In terms of outcomes, our diabetes-focused process measures may not generalize to other conditions, although the programs did not focus on any particular disease or condition. We also only captured outcomes for Medicare fee-for-service beneficiaries, which may not generalize to Medicare Advantage, dual eligible, or other beneficiary populations. Finally, these findings are observational because program participation was voluntary. Prior work has shown that estimates of the effect of ACOs can be biased by nonrandom attrition of patients and physicians.This longitudinal observational study of PCOs during 9 years of participation in 3 large-scale delivery and payment reform programs failed to observe consistent synergistic benefits across outcomes compared with no program participation. These findings suggest that efforts to more explicitly bring program requirements and goals into alignment may produce greater gains through consistent synergistic benefits across a range of health services outcomes.


Increasingly, literature has focused on community participation in places visited by persons living with and without dementia. Earlier research indicates that multiple factors, including socio-demographic aspects and risk perception may influence community participation.n = 70) in Switzerland. Data was collected through face-to-face interviews with questionnaires . First, we investigated whether the number of places visited was correlated with self-rated participation; then we added socio-demographic and risks perception factors with a bivariate analysis; and searched for a model using multinomial logistic regressions.This cross-sectional, explorative study aims to inquire into how places visited, socio-demographic aspects and risks perception are associated with self-rated community participation for persons living with and without dementia (p = .014) and of getting lost (p = .037) were significantly associated with self-rated participation. For the group of participants living without dementia, visiting places outside the home was significantly associated with self-rated participation, especially visiting places in domain D/places for recreational and physical activities (p = .005).For the group of participants living with dementia, risks of falling (The results of exploring multiple factors and searching for a model highlights the complexity of community participation as a construct. Risks and visiting places for recreational and physical activities seem to play a role in self-rated participation. Mobile interviews might be better suited to gain in-depth understanding on community participation for persons living with dementia. Dementia is a condition which includes progressive memory impairment and loss of other cognitive functions , 2. CognPlaces are understood here as being relational , 15. ThrAdditionally, socio-demographic factors have been highlighted as influencing out-of-home participation and how it is perceived by persons living with dementia, showing that a multitude of factors may have an impact . These iLimiting factors for out-of-home participation include old age, female gender, low socioeconomic status, social deprivation of the living environment, comorbidity, lifestyle, lack of motivation, weak social network and limited social activities , 30. BeiFurthermore, how risks are constructed and perceived have been identified as playing a role in community participation and dementia care , 34. ResStill, getting lost is a commonly perceived risk by persons living with dementia and their families. It is often associated with wandering behaviours and used as a reason to limit community participation , 43. FalA study on risk construction and perception conducted in Switzerland with triads of persons living with dementia, their significant others and home-health professionals, has shown that risks need to be understood as dynamic and co-constructed . Coping In summary, it is hypothesised that the number of places visited outside the home might have an influence on how older adults rate their community participation. As out-of-home participation is a complex, dynamic, and multi-faceted phenomenon, other factors than visiting places may influence how persons living with and without dementia experience and perceive it. Such additional factors are included in socio-demographic variables and perceived risks. To our knowledge, these factors have not yet been investigated in relation to community participation.What associations existed between the number of places visited and self-rated community participation among a sample of persons living with and without dementia?What factors  might have been associated with the self-rating of community participation?The second aim (2) of this study was to explore how these factors were associated with the self-rating of community participation for a sample of persons living with and without dementia in Switzerland. This study addressed the following research questions:This cross-sectional, explorative study reports on research undertaken within the “Life outside home for people with dementia” (OUTDEM) setting as part of a larger project led by Karolinska Institutet in Sweden. Standardised questionnaires were used in interviews with participants living with and without dementia in both rural and urban regions of the French-speaking region of Switzerland.n = 70) were community-dwelling older adults (aged > 65 years old). Recruitment started in December 2015 and ended in May 2017. Participants with dementia (n = 35) were recruited through memory clinics, day hospitals, and the Swiss Alzheimer’s Association. Diagnosis of dementia was established by physicians at memory clinics. Participants in the comparison group (n = 35) were recruited through senior associations and advertisements in grocery stores. We aimed to match, but not pair, the comparison group of persons living with no known cognitive impairment (n = 35) with the dementia group regarding age, gender, education level, living areas and settings. Thus, recruitment strategies for the comparison group targeted specific regions, age groups, or living areas, for example, to bring the distribution of the comparison group closer to the dementia group on those variables.All participants  . The samThe interviews were conducted by two registered occupational therapists, one of whom was the first author. Both interviewers had prior knowledge of using the ACT-OUT and they had harmonised with each other the way they conducted the interviews . The intn = 7); B/ places for medical care (n = 5); C/ social, cultural, and spiritual places (n = 6); and D/ places for recreational and physical activities (n = 7). The interview uses Part I to ask questions about whether respondents visit these places in the past, present, and future. Using Part II, the interviewer poses detailed questions about factors potentially influencing participation in places retained and abandoned, such as the types of activities performed, means of transportation, the presence of accompanying persons, risk perception, and familiarity. Part III consists of general questions about perceived out-of-home participation, life satisfaction, and attitudes towards risk-taking and stress factors.The ACT-OUT has three parts. Part I includes a list of 25 pre-determined types of places, grouped into four domains: A/ consumer, administrative, and self-care places   was usedA socio-demographic questionnaire was used to collect data regarding age, gender, education, living arrangement, setting , and access to using a car.For the purposes of this study, data from the ACT-OUT (Part I and III), the MoCA total score and socio-demographic questions were used.How do you perceive your participation in all situations outside home to be?), to which participants responded with . Only one participant with dementia responded, “I do not participate as I wish”, the lowest level, thus allowing the aggregation of levels 1 and 2.The dependent variable was the data collected by the question from Part III of the ACT-OUT (n = 5) and Part III: risks (n = 4)) and socio-demographic questionnaire (n = 6); and are described in Table The fifteen independent variables were derived from the ACT-OUT questionnaire ; .3–.5 (medium); and .5–1.0 (large) . To testp < .001) in the full sample analyses, we performed the regressions for the dementia and comparison groups separately. We used logistic regressions for the comparison group, since the dependent variables had two categories, and multinomial regressions for the dementia group, since the dependent variable had three categories. At this point, we selected all independent variables that were significant at the bivariate level, and we entered them together into multivariate regressions.Second, we used bivariate regressions to explore the associations between each of our independent variables and self-rated community participation. As the group variable was significant  with 95% confidence intervals for the multinomial regressions, and as odds ratio (OR) with 95% confidence intervals for the logistic regressions. We performed all analyses in the Statistical Package for Social Sciences (SPSS) computer software, version 25.p = <.001), significantly lower access to a car (p = <.001), visited significantly fewer places outside their home (p = <.001), and perceived significantly more risks, like getting lost (p = <.001), compared to the group of participants living without dementia. The group of participants living with dementia also rated their community participation as significantly lower (p = <.001) than the comparison group  between self-rated participation and the total number of places visited. The strength of the association was small (rs = .251) and positive, meaning that the more places that a person visited, the better they rated their community participation. The comparison group showed a significant correlation (p = .043) between self-rated participation and the total number of places visited, and the strength was medium (rs = .344) and positive. However, in the group of participants living with dementia, the correlation was not significant. Figure p = .044), compared to the dementia group which shows non-significant fluctuations  and falling (p = .014) were significantly associated in the bivariate multinomial regressions and were included in the multivariate regression. In the multivariate regression, no independent variable was significant. Both getting lost and falling were reported as hindering the self-rated community participation. These results may indicate that few personal socio-demographic variables play a role in the rating of community participation and that participants living with dementia recognise some risks but might not be influenced by the number of places visited when rating their community participation.Table p = .005) was significantly associated with self-rated participation. Since only one predictor was significant for the comparison group at the bivariate level, no additional regression was required. These results indicate that visiting places related to recreational and physical activities (domain D) may increase the rating of community participation for the comparison group of participants living without dementia. This comparison group did not seem to link their rating of community participation to perceived risks.Table The aim of the study was to explore how various factors were associated with the self-rating of community participation for a sample of persons living with and without dementia in Switzerland. Our results suggest that living with dementia is a significant factor that seems to change not only the number and types of places visited, but also community participation. This result is aligned with earlier research using the ACT-OUT , 20, whiIn response to aim 1, the results showed that for the comparison group, the number and diversity of places visited seems to be associated with self-rated participation. It is possible to infer that the more the persons living without dementia visit a diversity of places, the more they perceive their community participation to be satisfactory for themselves. However, for the group living with dementia, there was no such association. Research indicates that persons living with dementia tend to become more dependent on a significant other, and this may influence how, when, and where they go outside as well as what types of activities they engage in . Out-of-However, by contextualising the experience of community participation, for instance in relation to the availability of support or interventions to address barriers such as social exclusion and stigmatisation, it might refine our understanding of the difficulties and resources persons living with dementia have in their communities . The wayp = .062). Domain D contains a range of places that are linked to physical activity, contact with nature, travelling and vacation, and the neighbourhood. Hence, it would be reasonable to expect an association between visiting these places, especially the neighbourhood [In response to aim (2), the results on the exploration of multiple variables revealed few significant associations with self-rated community participation. This may be partially due to the small samples in this study and the education variable had to be removed from the analyses as there were an insufficient number of participants in some categories. Still, places especially in domain D/places for recreational and physical activities, were associated with self-rated participation for the comparison group, but not the group of persons living with dementia. However, for the latter group, places visited in domain D was the closest of all types of places to being significant  of the four risk variables included in this study were significantly associated with self-rated community participation, among the group of persons living with dementia. However, given that neither the risk of getting lost nor falling were significant in the search for a (multivariate) model for that group, it seems that participants who perceive getting lost as a risk also perceive falling as one. Perceiving space outside the home as fraught with risks  might beThe small sample size is a limitation of this study that warrants attention. Although 70 participants were included in the entire sample, the factor of living with a dementia was shown to be significant in the analysis, supporting the idea of conducting the exploration of multiple factors separately for both groups. As the two groups were comprised of small samples, we first used bivariate analysis before looking for a model to mitigate the effect of having a small sample size. Looking for a model with small samples is tentative, as the number of factors and steps in the scales adds to the number of degrees of freedom and might make the results less reliable. Therefore, our results need to be considered with respect to this limitation. Still, this exploratory study provides insights into community participation for persons living with dementia.Another limitation of this study is that we did not collect data on characteristics of the participants’ community, such as the availability of amenities, commodities, transportation, community support, dementia-friendly initiatives, or community interventions, which could potentially have been included in the factors under exploration. It is uncertain how the absence of these variables might have changed our results. In addition to socio-demographic variables, self-rated community participation is dependent on individual preferences as well as contextual availability and support, making this construct very complex to apprehend. There is a need to better specify the impact of socio-demographic and contextual factors and include individual preferences on community participation in future longitudinal studies for persons living with dementia.Collecting data with persons living with dementia through the use of face-to face interviews raises the question of the reliability of the data , 72, espWe found that community participation was associated with the number of places visited but only for the comparison group of persons living without dementia. Our exploration of multiple factors and search for a model, highlights the complexity and situatedness of community participation as a construct. Visiting many diverse types of places seemed to play a role in participation outside home and future research would benefit from exploring this using a variety of methods, including qualitative research and mobile interviews.