Karsan Lab

Myeloid ecotropic virus insertion site 1 (MEIS1) is essential for normal hematopoiesis and is a critical factor in the pathogenesis of a large subset of acute myeloid leukemia (AML). Despite the clinical relevance of MEIS1, its regulation is largely unknown. To understand the transcriptional regulatory mechanisms contributing to human MEIS1 expression, we created a knock-in green florescent protein (GFP) reporter system at the endogenous MEIS1 locus in a human AML cell line. Using this model, we have delineated and dissected a critical enhancer region of the MEIS1 locus for transcription factor (TF) binding through in silico prediction in combination with oligo pull-down, mass-spectrometry and knockout analysis leading to the identification of FLI1, an E-twenty-six (ETS) transcription factor, as an important regulator of MEIS1 transcription. We further show direct binding of FLI1 to the MEIS1 locus in human AML cell lines as well as enrichment of histone acetylation in MEIS1-high healthy and leukemic cells. We also observe a positive correlation between high FLI1 transcript levels and worse overall survival in AML patients. Our study expands the role of ETS factors in AML and our model constitutes a feasible tool for a more detailed understanding of transcriptional regulatory elements and their interactome.

Chronic inflammation with aging ("inflammaging") plays a prominent role in the pathogenesis of myeloid malignancies. Aberrant inflammatory activity impacts many different cells in the marrow, including normal blood and stromal marrow elements and leukemic cells, in unique and distinct ways. Inflammation can promote selective clonal expansion through differential immune-mediated suppression of normal hematopoietic cells and malignant clones. We review these complex roles, how they can be understood by separating cell-intrinsic from extrinsic effects, and how this informs future clinical trials.

Background: We explored health professionals' views on the utility of circulating tumor DNA (ctDNA) testing in hereditary cancer syndrome (HCS) management.

Materials and methods: A qualitative interpretive description study was conducted, using semi-structured interviews with professionals across Canada. Thematic analysis employing constant comparison was used for analysis. 2 investigators coded each transcript. Differences were reconciled through discussion and the codebook was modified as new codes and themes emerged from the data.

Results: Thirty-five professionals participated and included genetic counselors (n = 12), geneticists (n = 9), oncologists (n = 4), family doctors (n = 3), lab directors and scientists (n = 3), a health-system decision maker, a surgeon, a pathologist, and a nurse. Professionals described ctDNA as "transformative" and a "game-changer". However, they were divided on its use in HCS management, with some being optimistic (optimists) while others were hesitant (pessimists). Differences were driven by views on 3 factors: (1) clinical utility, (2) ctDNA's role in cancer screening, and (3) ctDNA's invasiveness. Optimists anticipated ctDNA testing would have clinical utility for HCS patients, its role would be akin to a diagnostic test and would be less invasive than standard screening (eg imaging). Pessimistic participants felt ctDNA testing would add limited utility; it would effectively be another screening test in the pathway, likely triggering additional investigations downstream, thereby increasing invasiveness.

Conclusions: Providers anticipated ctDNA testing will transform early cancer detection for HCS families. However, the contrasting positions on ctDNA's role in the care pathway raise potential practice variations, highlighting a need to develop evidence to support clinical implementation and guidelines to standardize adoption.

Objectives: Acute myeloid leukaemia (AML) is a disease of older adults, who are vulnerable to socioeconomic factors. We determined AML incidence in older adults and the impact of socioeconomic factors on outcomes.

Methods: We included 3024 AML patients (1996-2016) identified from a population-based registry.

Results: AML incidence in patients ≥60 years increased from 11.01 (2001-2005) to 12.76 (2011-2016) per 100,000 population. Amongst 879 patients ≥60 years in recent eras (2010-2016), rural residents (<100,000 population) were less likely to be assessed by a leukaemia specialist (39% rural, 47% urban, p=0.032); no difference was seen for lower (43%, quintile 1-3) versus higher (47%, quintile 4-5) incomes (p=0.235). Similar numbers received induction chemotherapy between residence (16% rural, 18% urban, p=0.578) and incomes (17% lower, 17% high, p=1.0). Differences between incomes were seen for hypomethylating agent treatment (14% low, 20% high, p=0.041); this was not seen for residence (13% rural, 18% urban, p=0.092). Amongst non-adverse karyotype patients ≥70 years, 2-year overall survival was worse for rural (5% rural, 12% urban, p=0.006) and lower income (6% low, 15% high, p=0.017) patients.

Conclusions: AML incidence in older adults is increasing, and outcomes are worse for older rural and low-income residents; these patients face treatment barriers.

Inflammation is associated with bone marrow failure syndromes, but how specific molecules impact the bone marrow microenvironment is not well elucidated. We report a novel role for the miR-145 target, Toll/interleukin-1 receptor domain containing adaptor protein (TIRAP), in driving bone marrow failure. We show that TIRAP is overexpressed in various types of myelodysplastic syndromes (MDS) and suppresses all three major hematopoietic lineages. TIRAP expression promotes up-regulation of Ifnγ, leading to myelosuppression through Ifnγ-Ifnγr-mediated release of the alarmin, Hmgb1, which disrupts the bone marrow endothelial niche. Deletion of Ifnγ blocks Hmgb1 release and is sufficient to reverse the endothelial defect and restore myelopoiesis. Contrary to current dogma, TIRAP-activated Ifnγ-driven bone marrow suppression is independent of T cell function or pyroptosis. In the absence of Ifnγ, TIRAP drives myeloproliferation, implicating Ifnγ in suppressing the transformation of MDS to acute leukemia. These findings reveal novel, noncanonical roles of TIRAP, Hmgb1, and Ifnγ in the bone marrow microenvironment and provide insight into the pathophysiology of preleukemic syndromes.

Blocked cellular differentiation is a central pathologic feature of the myeloid malignancies, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Treatment regimens promoting differentiation have resulted in incredible cure rates in certain AML subtypes, such as acute promyelocytic leukemia. Over the past several years, we have seen many new therapies for MDS/AML enter clinical practice, including epigenetic therapies (e.g., 5-azacitidine), isocitrate dehydrogenase (IDH) inhibitors, fms-like kinase 3 (FLT3) inhibitors, and lenalidomide for deletion 5q (del5q) MDS. Despite not being developed with the intent of manipulating differentiation, induction of differentiation is a major mechanism by which several of these novel agents function. In this review, we examine the new therapeutic landscape for these diseases, focusing on the role of hematopoietic differentiation and the impact of inflammation and aging. We review how current therapies in MDS/AML promote differentiation as a part of their therapeutic effect, and the cellular mechanisms by which this occurs. We then outline potential novel avenues to achieve differentiation in the myeloid malignancies for therapeutic purposes. This emerging body of knowledge about the importance of relieving differentiation blockade with anti-neoplastic therapies is important to understand how current novel agents function and may open avenues to developing new treatments that explicitly target cellular differentiation. Moving beyond cytotoxic agents has the potential to open new and unexpected avenues in the treatment of myeloid malignancies, hopefully providing more efficacy with reduced toxicity.

Objectives: Non-invasive prenatal testing requires the presence of fetal DNA in maternal plasma. Understanding how preexamination conditions affect the integrity of cell-free DNA (cfDNA) and fetal fraction (FF) are a prerequisite for test implementation. Therefore, we examined the adjusted effect that EDTA and Streck tubes have on the cfDNA quantity and FF.

Methods: A total of 3,568 maternal blood samples across Canada were collected in either EDTA, or Streck tubes, and processing metrics, maternal body mass index (BMI), gestational age and fetal karyotype and sex were recorded. Plasma samples were sequenced using two different sequencing platforms in separate laboratories. Sequencing data were processed with SeqFF to estimate FF. Linear regression and multivariate imputation by chained equations were used to estimate the adjusted effect of tube type on cfDNA and FF.

Results: We found a positive association between cfDNA quantity and blood shipment time in EDTA tubes, which is significantly reduced with the use of Streck tubes. Furthermore, we show the storage of plasma at -80 °C is associated with a 4.4% annual relative decrease in cfDNA levels. FF was not associated with collection tube type when controlling for confounding variables. However, FF was positively associated with gestational age and trisomy 21, while negatively associated with BMI, male fetus, trisomy 18, Turners syndrome and triploidy.

Conclusions: Preexamination, maternal and fetal variables are associated with cfDNA quantity and FF. The consideration of these variables in future studies may help to reduce the number of pregnant women with inconclusive tests as a result of low FF.

Patients with non-small cell lung cancer (NSCLC) harboring ROS proto-oncogene 1 (ROS1) gene rearrangements show dramatic response to the tyrosine kinase inhibitor (TKI) crizotinib. Current best practice guidelines recommend that all advanced stage non-squamous NSCLC patients be also tested for ROS1 gene rearrangements. Several studies have suggested that ROS1 immunohistochemistry (IHC) using the D4D6 antibody may be used to screen for ROS1 fusion positive lung cancers, with assays showing high sensitivity but moderate to high specificity. A break apart fluorescence in situ hybridization (FISH) test is then used to confirm the presence of ROS1 gene rearrangement. The goal of Canadian ROS1 (CROS) study was to harmonize ROS1 laboratory developed testing (LDT) by using IHC and FISH assays to detect ROS1 rearranged lung cancers across Canadian pathology laboratories. Cell lines expressing different levels of ROS1 (high, low, none) were used to calibrate IHC protocols after which participating laboratories ran the calibrated protocols on a reference set of 24 NSCLC cases (9 ROS1 rearranged tumors and 15 ROS1 non-rearranged tumors as determined by FISH). Results were compared using a centralized readout. The stained slides were evaluated for the cellular localization of staining, intensity of staining, the presence of staining in non-tumor cells, the presence of non-specific staining (e.g. necrosis, extracellular mater, other) and the percent positive cells. H-score was also determined for each tumor. Analytical sensitivity and specificity harmonization was achieved by using low limit of detection (LOD) as either any positivity in the U118 cell line or H-score of 200 with the HCC78 cell line. An overall diagnostic sensitivity and specificity of up to 100% and 99% respectively was achieved for ROS1 IHC testing (relative to FISH) using an adjusted H-score readout on the reference cases. This study confirms that LDT ROS1 IHC assays can be highly sensitive and specific for detection of ROS1 rearrangements in NSCLC. As NSCLC can demonstrate ROS1 IHC positivity in FISH-negative cases, the degree of the specificity of the IHC assay, especially in highly sensitive protocols, is mostly dependent on the readout cut-off threshold. As ROS1 IHC is a screening assay for a rare rearrangements in NSCLC, we recommend adjustment of the readout threshold in order to balance specificity, rather than decreasing the overall analytical and diagnostic sensitivity of the protocols.

Next-generation sequencing assays are capable of identifying cancer patients eligible for targeted therapies and can also detect germline variants associated with increased cancer susceptibility. However, these capabilities have yet to be routinely harmonized in a single assay because of challenges with accurately identifying germline variants from tumor-only data. We have developed the Oncology and Hereditary Cancer Program targeted capture panel, which uses tumor tissue to simultaneously screen for both clinically actionable solid tumor variants and germline variants across 45 genes. Validation using 14 tumor specimens, composed of patient samples and cell lines analyzed in triplicate, demonstrated high coverage with sensitive and specific identification of single-nucleotide variants and small insertions and deletions. Average coverage across all targets remained >2000× in 198 additional patient tumor samples. Analysis of 55 formalin-fixed, paraffin-embedded tumor samples for the detection of known germline variants within a subset of cancer-predisposition genes, including one multiexon deletion, yielded a 100% detection rate, demonstrating that germline variants can be reliably detected in tumor samples using a single panel. Combining targetable somatic and actionable germline variants into a single tumor tissue assay represents a streamlined approach that can inform treatment for patients with advanced cancers as well as identify those with potential germline variants who are eligible for confirmatory testing, but would not otherwise have been identified.

As more clinically-relevant genomic features of myeloid malignancies are revealed, it has become clear that targeted clinical genetic testing is inadequate for risk stratification. Here, we develop and validate a clinical transcriptome-based assay for stratification of acute myeloid leukemia (AML). Comparison of ribonucleic acid sequencing (RNA-Seq) to whole genome and exome sequencing reveals that a standalone RNA-Seq assay offers the greatest diagnostic return, enabling identification of expressed gene fusions, single nucleotide and short insertion/deletion variants, and whole-transcriptome expression information. Expression data from 154 AML patients are used to develop a novel AML prognostic score, which is strongly associated with patient outcomes across 620 patients from three independent cohorts, and 42 patients from a prospective cohort. When combined with molecular risk guidelines, the risk score allows for the re-stratification of 22.1 to 25.3% of AML patients from three independent cohorts into correct risk groups. Within the adverse-risk subgroup, we identify a subset of patients characterized by dysregulated integrin signaling and RUNX1 or TP53 mutation. We show that these patients may benefit from therapy with inhibitors of focal adhesion kinase, encoded by PTK2, demonstrating additional utility of transcriptome-based testing for therapy selection in myeloid malignancy.

Background: In 2020 ASCO recommended that all women with epithelial ovarian cancer have germline testing (GT) for BRCA1/2 mutations, and those without a germline pathogenic variant (PV) should have somatic tumor testing (TT), to determine eligibility for PARP inhibitor (PARPi) therapy (GT-TT strategy). An alternate strategy is to start with tumor testing first, and to conduct germline testing only in those with a PV in the tumor, or a significant family history (TT-GT strategy). The objective was to conduct a cost-effectiveness analysis comparing the 2 testing strategies. Methods: A Markov Monte Carlo simulation model compared the costs (USD) and benefits of the 2 testing strategies. According to local empiric data, a sufficient tissue sample for TT was available in 99% of cases, otherwise the patient would only have GT. Sensitivity of TT was 99% for detecting germline PV. Only those with BRCA1/2 PV were eligible for PARPi. Primary outcomes included the number of women eligible for PARPi, with progression-free years of life (PFLY) gained based on SOLO1 data, and the incremental cost-effectiveness ratio (ICER). Monte Carlo simulation estimated the number of women who would have GT and TT, and the total with germline or somatic BRCA1/2 PV eligible for PARPi. Sensitivity analyses accounted for uncertainty around various parameters. Results: The GT-TT strategy was more effective but more costly than TT-GT in identifying patients eligible for PARPi. Table summarizes the average lifetime costs, benefits, and Monte Carlo simulation estimates for 10,000 women diagnosed with advanced epithelial ovarian cancer annually in the USA. The incremental benefit from the GT-TT strategy would be achieved at substantial cost to the health care system, with an ICER of $119,340 per PFLY gained relative to the TT-GT strategy. The results were highly sensitive to the sensitivity of TT to detect germline PV, and the costs of GT and TT. Assuming that GT was less than 50% of the cost of TT, the sensitivity of TT had to exceed 98% for the TT-GT strategy to be cost-effective. Conclusions: Although the ASCO recommended strategy of BRCA germline testing followed by tumor testing for those without a pathogenic variant may be more effective in identifying ovarian cancer patients for PARP inhibitor therapy, it is more costly. The ASCO strategy is justified if the sensitivity of tumor testing is not sufficiently high. However, assuming high tumor testing performance rates, tumor testing first followed by germline testing if there is a PV in the tumor and/or family history is a cost-effective strategy.

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Objectives: MET exon 14 skipping is a potentially targetable molecular alteration. The goals of this study were to identify patients treated in British Columbia with MET exon 14 skipping to understand prevalence, biology and response to treatment, and to identify molecular signatures that may predict for response or resistance to targeted MET therapy in the setting of advanced disease.

Materials and methods: A retrospective review was completed of patients found to have MET exon 14 skipping alterations between January 2016-September 2019. Information was collected on baseline characteristics, response to systemic treatments, and outcomes.

Results: Out of 1934 advanced, non-squamous and never-smoking squamous NSCLC patients tested, 41 patients were found to have MET exon 14 skipping (2.1 %). MET alteration types: 2% CBL binding-domain mutations, 34 % poly-pyrimidine tract deletions, 63 % splice donor mutations or deletions. The most common co-mutation was TP53 (22 %). Thirty-three patients received systemic therapy. Physician-assessed disease control was 68 % among 19 evaluable patients treated with crizotinib, 80 % among 10 evaluable patients treated with platinum-based chemotherapy, and 70 % among 10 evaluable patients treated with immunotherapy. Median time to treatment discontinuation was 3.0, 2.8, and 2.4 months, respectively. Median overall survival for metastatic patients treated with any systemic therapy was 15.4 months. In this small cohort, there were no clear correlations between molecular aberrations and response, time to treatment discontinuation, or survival for crizotinib, chemotherapy, and immunotherapy.

Conclusion: The prevalence of MET exon 14 skipping in a North American population was 2.1 %. Unlike other targetable mutations, patients were older and more commonly current or former smokers. Patients with MET exon 14 skipping alteration demonstrate disease control with crizotinib, platinum-based chemotherapy and immunotherapy. Co-mutations with TP53 were commonly noted, but correlation between co-mutations and efficacy of therapy were not identified in this cohort.

Introduction: The initial point in the diagnostic workup of solid tumors remains manual, with the assessment of hematoxylin and eosin (H&E)-stained tissue sections by microscopy. This is a labor-intensive step that requires attention to detail. In addition, diagnoses are influenced by an individual pathologist's knowledge and experience and may not always be reproducible between pathologists. Methods: We introduce a deep learning-based method in colorectal cancer detection and segmentation from digitized H&E-stained histology slides. Results: In this study, we demonstrate that this neural network approach produces median accuracy of 99.9% for normal slides and 94.8% for cancer slides compared to pathologist-based diagnosis on H&E-stained slides digitized from clinical samples. Conclusion: Given that our approach has very high accuracy on normal slides, use of neural network algorithms may provide a screening approach to save pathologist time in identifying tumor regions. We suggest that this new method may be a powerful assistant for colorectal cancer diagnostics.

Understanding how patient-reported quality of life (QoL) and socioeconomic status (SES) relate to survival of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) may improve prognostic information sharing. This study explores associations among QoL, SES, and survival through administration of the Euro-QoL 5-Dimension, 3-level and Functional Assessment of Cancer Therapy-Leukemia and financial impact questionnaires to 138 adult participants with newly diagnosed AML or MDS in a longitudinal, pan-Canadian study. Cox regression and lasso variable selection models were used to explore associations among QoL, SES, and established predictors of survival. Secondary outcomes were changes in QoL, performance of the QoL instruments, and lost income. We found that higher QoL and SES were positively associated with survival. The Lasso model selected the visual analog scale of the EQ-5D-3L as the most important predictor among all other variables (P = .03; 92% selection). Patients with AML report improved QoL after treatment, despite higher mean out-of-pocket expenditures compared with MDS (up to $599 CDN/month for AML vs $239 for MDS; P = .05), greater loss of productivity-related income (reaching $1786/month for AML vs $709 for MDS; P < .05), and greater caregiver effects (65% vs 35% caregiver productivity losses for AML vs MDS; P < .05). Our results suggest that including patient-reported QoL and socioeconomic indicators can improve the accuracy of survival models.

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PURPOSE

Plasma detection of EGFR T790M mutations is an emerging alternative to tumor rebiopsy in acquired epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor resistance. Validation of analytical sensitivity and clinical utility is required before routine diagnostic use in clinical laboratories.

PATIENTS AND METHODS

Sixty-three patients with advanced EGFR-mutant lung cancer at 7 Canadian centers, who were being screened for the ASTRIS trial (ClinicalTrials.gov identifier: NCT02474355), participated in this companion study. Plasma T790M mutation was detected using droplet digital polymerase chain reaction, Cobas (Roche Diagnostics, Indianapolis, IN), or next-generation sequencing in 4 laboratories. T790M concordance was assessed between plasma and tumor samples.

RESULTS

Assessment of T790M in tumor biopsy tissue was successful in 81% of patients; 49% had confirmed T790M results (tumor or plasma) for ASTRIS. Plasma testing in this companion study yielded T790M results in 97% of patients; 62% had T790M-positive results, 36% had negative results, and 2% had indeterminate results. Of 38 patients with negative or indeterminate biopsy results, 55% had positive plasma T790M results, increasing the proportion with T790M-positive results to 73%. Sensitivity of plasma T790M testing was 75%. Overall concordance between tissue and plasma was 64%, and concordance among laboratories was 90.3%. Response to osimertinib and duration of therapy were similar irrespective of testing method (overall response rate, 62.5% for tissue, 66.7% for plasma, and 70.6% for both).

CONCLUSION

This multicenter validation study demonstrates that plasma EGFR T790M testing can identify significantly more patients than biopsy alone who may benefit from targeted therapy.

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