Cost Effectiveness of Molecular Diagnostic Testing Algorithms for the Treatment Selection of Frontline Ibrutinib for Patients with Chronic Lymphocytic Leukemia in Australia.

BACKGROUND: Clinical indications for ibrutinib reimbursement in Australia should consider the inclusion of patients with chronic lymphocytic leukemia (CLL) harboring prognostically unfavorable TP53/IGHV genomic aberrations. This study assessed the cost effectiveness of five first-line treatment strategies in CLL for young (aged ≤ 65 years), fit patients without significant comorbidities: (1) no testing (fludarabine, cyclophosphamide and rituximab [FCR] for all), (2) test for del(17p) only, (3) test for TP53 gene mutation status, (4) test for TP53 and IGHV gene mutation status and (5) no testing (ibrutinib for all). METHOD: A decision analytic model (decision tree and partitioned survival model) was developed from the Australian healthcare system perspective with a lifetime horizon. Comparative treatment effects were estimated from indirect treatment comparisons and survival analysis using several studies. Costs, utility and adverse events were derived from public literature sources. Deterministic and probabilistic sensitivity analyses explored the impact of modeling uncertainties on outcomes. RESULTS: Strategy 1 was associated with 5.69 quality-adjusted life-years (QALYs) and cost 458,836 Australian dollars (AUD). All other strategies had greater effectiveness but were more expensive than Strategy 1. At the willingness-to-pay (WTP) threshold of 100,000 AUD per QALY gained, Strategy 1 was most cost effective with an estimated probability of 68.8%. Strategy 4 was cost effective between thresholds 155,000-432,300 AUD per QALY gained, and Strategy 5 >432,300 AUD per QALY gained. CONCLUSION: Population targeting using mutation testing for TP53 and IGHV when performed with del(17p) testing specifically in the context of frontline ibrutinib choice does not make a cost-ineffective treatment into a cost-effective treatment.

The clinical and genomic landscape of patients with DDX41 variants identified during diagnostic sequencing.

Deleterious germ line variants in DDX41 are a common cause of genetic predisposition to hematologic malignancies, particularly myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML). Targeted next-generation sequencing was performed in a large cohort of sequentially recruited patients with myeloid malignancy, covering DDX41 as well as 30 other genes frequently mutated in myeloid malignancy. Whole genome transcriptome sequencing data was analyzed on a separate cohort of patients with a range of hematologic malignancies to investigate the spectrum of cancer predisposition. Altogether, 5737 patients with myeloid malignancies were studied, with 152 different DDX41 variants detected. Multiple novel variants were detected, including synonymous variants affecting splicing as demonstrated by RNA-sequencing. The presence of a somatic DDX41 variant was highly associated with DDX41 germ line variants in patients with MDS and AML, and we developed a statistical approach to incorporate the co-occurrence of a somatic DDX41 variant into germ line variant classification at a very strong level (as per the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines). Using this approach, the MDS cohort contained 108 of 2865 (3.8%) patients with germ line likely pathogenic/pathogenic (LP/P) variants, and the AML cohort 106 of 2157 (4.9%). DDX41 LP/P variants were markedly enriched in patients with AML and MDS compared with those in patients with myeloproliferative neoplasms, B-cell neoplasm, and T- or B-cell acute lymphoblastic leukemia. In summary, we have developed a framework to enhance DDX41 variant curation as well as highlighted the importance of assessment of all types of genomic variants (including synonymous and multiexon deletions) to fully detect the landscape of possible clinically relevant DDX41 variants.

Biallelic deleterious germline SH2B3 variants cause a novel syndrome of myeloproliferation and multi-organ autoimmunity.

SH2B3 is a negative regulator of multiple cytokine receptor signalling pathways in haematopoietic tissue. To date, a single kindred has been described with germline biallelic loss-of-function SH2B3 variants characterized by early onset developmental delay, hepatosplenomegaly and autoimmune thyroiditis/hepatitis. Herein, we described two further unrelated kindreds with germline biallelic loss-of-function SH2B3 variants that show striking phenotypic similarity to each other as well as to the previous kindred of myeloproliferation and multi-organ autoimmunity. One proband also suffered severe thrombotic complications. CRISPR-Cas9 gene editing of zebrafish sh2b3 created assorted deleterious variants in F0 crispants, which manifest significantly increased number of macrophages and thrombocytes, partially replicating the human phenotype. Treatment of the sh2b3 crispant fish with ruxolitinib intercepted this myeloproliferative phenotype. Skin-derived fibroblasts from one patient demonstrated increased phosphorylation of JAK2 and STAT5 after stimulation with IL-3, GH, GM-CSF and EPO compared to healthy controls. In conclusion, these additional probands and functional data in combination with the previous kindred provide sufficient evidence for biallelic homozygous deleterious variants in SH2B3 to be considered a valid gene-disease association for a clinical syndrome of bone marrow myeloproliferation and multi-organ autoimmune manifestations.

Enrichment of BTK Leu528Trp mutations in patients with CLL on zanubrutinib: potential for pirtobrutinib cross-resistance.

The covalent Bruton's tyrosine kinase inhibitors (BTKis) are highly effective for the treatment of chronic lymphocytic leukemia (CLL). The dominant resistance mechanism observed with the BTKi ibrutinib is the development of BTK Cys481 codon mutations. Whether a similar resistance mutation profile exists for the newer-generation, more selective BTKi zanubrutinib is unknown. In samples referred for diagnostic next-generation sequencing in patients with progressive CLL, we observed an enrichment in the kinase-dead BTK Leu528Trp mutation in patients treated with zanubrutinib compared with ibrutinib (54%; 7 of 13 vs 4%; 1 of 24, P = .001). We describe 2 patients with BTK Leu528Trp mutations who showed clinical cross-resistance and progressive enrichment of the BTK Leu528Trp mutation over time when treated with the noncovalent BTKi pirtobrutinib. Both patients subsequently responded to venetoclax-based treatment. In summary, we have identified an enrichment of the BTK Leu528Trp mutation arising in patients treated with zanubrutinib that may impart cross-resistance to the noncovalent inhibitor pirtobrutinib and therefore may have implications for sequencing of these treatments in CLL.

Health economic evidence for the use of molecular biomarker tests in hematological malignancies: A systematic review.

OBJECTIVES: Molecular biomarker tests can inform the clinical management of genomic heterogeneous hematological malignancies, yet their availability in routine care largely depends on the supporting health economic evidence. This study aims to systematically review the economic evidence for recent molecular biomarker tests in hematological malignancies. METHODS: We conducted a systematic search in five electronic databases for studies published between January 2010 and October 2020. Publications were independently screened by two reviewers. Clinical study characteristics, economic methodology, and results were extracted, and reporting quality was assessed. RESULTS: Fourteen studies were identified, of which half (n = 7; 50%) were full economic evaluations examining both health and economic outcomes. Studies were predominantly conducted in a first-line treatment setting (n = 7; 50%) and adopted a non-lifetime time horizon to measure health outcomes and costs (n = 7; 50%). Five studies reported that companion diagnostics for associated therapies were likely cost-effective for acute myeloid leukemia, chronic myeloid leukemia, diffuse large B-cell lymphoma, and multiple myeloma. Four studies suggested molecular biomarker tests for treatment monitoring in chronic myeloid leukemia were likely cost-saving. CONCLUSIONS: Although there is initial confirmation of the promising health economic results, the present research for molecular biomarker tests in hematological malignancies is sparse with many applications of technological advances yet to be evaluated.

Integration of tumour sequencing and case-control data to assess pathogenicity of RAD51C missense variants in familial breast cancer.

While protein-truncating variants in RAD51C have been shown to predispose to triple-negative (TN) breast cancer (BC) and ovarian cancer, little is known about the pathogenicity of missense (MS) variants. The frequency of rare RAD51C MS variants was assessed in the BEACCON study of 5734 familial BC cases and 14,382 population controls, and findings were integrated with tumour sequencing data from 21 cases carrying a candidate variant. Collectively, a significant enrichment of rare MS variants was detected in cases (MAF < 0.001, OR 1.57, 95% CI 1.00-2.44, p = 0.05), particularly for variants with a REVEL score >0.5 (OR 3.95, 95% CI 1.40-12.01, p = 0.006). Sequencing of 21 tumours from 20 heterozygous and 1 homozygous carriers of nine candidate MS variants identified four cases with biallelic inactivation through loss of the wild-type allele, while six lost the variant allele and ten that remained heterozygous. Biallelic loss of the wild-type alleles corresponded strongly with ER- and TN breast tumours, high homologous recombination deficiency scores and mutational signature 3. Using this approach, the p.Gly264Ser variant, which was previously suspected to be pathogenic based on small case-control analyses and loss of activity in in vitro functional assays, was shown to be benign with similar prevalence in cases and controls and seven out of eight tumours showing no biallelic inactivation or characteristic mutational signature. Conversely, evaluation of case-control findings and tumour sequencing data identified p.Ile144Thr, p.Arg212His, p.Gln143Arg and p.Gly114Arg as variants warranting further investigation.

Single-cell sequencing demonstrates complex resistance landscape in CLL and MCL treated with BTK and BCL2 inhibitors.

The genomic landscape of resistance to targeted agents (TAs) used as monotherapy in chronic lymphocytic leukemia (CLL) is complex and often heterogeneous at the patient level. To gain insight into the clonal architecture of acquired genomic resistance to Bruton tyrosine kinase (BTK) inhibitors and B-cell lymphoma 2 (BCL2) inhibitors in CLL, particularly in patients carrying multiple resistance mutations, we performed targeted single-cell DNA sequencing of 8 patients who developed progressive disease (PD) on TAs (either class). In all cases, analysis of single-cell architecture revealed mutual exclusivity between multiple resistance mutations to the same TA class, variable clonal co-occurrence of multiple mutations affecting different TAs in patients exposed to both classes, and a phenomenon of multiple independent emergences of identical nucleotide changes leading to canonical resistance mutations. We also report the first observation of established BCL2 resistance mutations in a patient with mantle cell lymphoma (MCL) following PD on sequential monotherapy, implicating BCL2 as a venetoclax resistance mechanism in MCL. Taken together, these data reveal the significant clonal complexity of CLL and MCL progression on TAs at the nucleotide level and confirm the presence of multiple, clonally independent, mechanisms of TA resistance within each individual disease context.

Clonal hematopoiesis, myeloid disorders and BAX-mutated myelopoiesis in patients receiving venetoclax for CLL.

The BCL2 inhibitor venetoclax has established therapeutic roles in chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). As BCL2 is an important determinant of survival of both myeloid progenitor and B cells, we investigated whether clinical and molecular abnormalities arise in the myeloid compartment during long-term continuous venetoclax treatment of CLL in 89 patients (87 with relapsed/refractory CLL). Over a median follow-up of 75 (range 21-98) months, persistent cytopenias (≥1 of neutropenia, thrombocytopenia, anemia) lasting ≥4 months and unrelated to CLL occurred in 25 patients (28%). Of these patients, 20 (80%) displayed clonal hematopoiesis, including 10 with therapy-related myeloid neoplasms (t-MNs). t-MNs occurred exclusively in patients previously exposed to fludarabine-alkylator combination therapy with a cumulative 5-year incidence of 10.4% after venetoclax initiation, consistent with rates reported for patients exposed to fludarabine-alkylator combination therapy without venetoclax. To determine whether the altered myelopoiesis reflected the acquisition of mutations, we analyzed samples from patients with no or minimal bone marrow CLL burden (n = 41). Mutations in the apoptosis effector BAX were identified in 32% (13/41). In cellular assays, C-terminal BAX mutants abrogated outer mitochondrial membrane localization of BAX and engendered resistance to venetoclax killing. BAX-mutated clonal hematopoiesis occurred independently of prior fludarabine-alkylator combination therapy exposure and was not associated with t-MNs. Single-cell sequencing revealed clonal co-occurrence of mutations in BAX with DNMT3A or ASXL1. We also observed simultaneous BCL2 mutations within CLL cells and BAX mutations in the myeloid compartment of the same patients, indicating lineage-specific adaptation to venetoclax therapy.

Outcomes of patients with CLL sequentially resistant to both BCL2 and BTK inhibition.

Covalent Bruton tyrosine kinase inhibitors (BTKi's) and the B-cell lymphoma 2 (BCL2) inhibitor venetoclax have significantly improved outcomes for patients with chronic lymphocytic leukemia (CLL), especially those with biologically adverse disease. Patients with CLL resistant to their first targeted agent (TA) can be effectively treated with the alternative class. However, relapses are expected with second-line TA therapy, and the clinical challenge of double class-resistant disease is now emerging with increasing frequency. To define the characteristics and outcomes of patients with double class-resistant disease, we retrospectively analyzed 17 patients who developed progressive disease (PD) on both TA classes for CLL (venetoclax, then BTKi, n=12; BTKi, then venetoclax, n = 5). The cohort was heavily pretreated (median lines of prior therapy, 4) and enriched for adverse disease genetics (complex karyotype, 12 of 12 tested [100%]; del(17p)/TP53 mutations, 15 of 17 [88%]). The median time to progression on prior venetoclax was 24 months (range, 6-94 months) and was 25 months (range, 1-55 months) on prior BTKi. Progression on second-line TA was manifest as progressive CLL in 11 patients and as Richter transformation in 6. The median overall survival after progression on second-line TA was 3.6 months (95% confidence interval, 2-11 months). Patients with double class-resistant CLL have a dismal prognosis, representing a group of high unmet need.

Transient, flexible gene editing in zebrafish neutrophils and macrophages for determination of cell-autonomous functions.

Zebrafish are an important model for studying phagocyte function, but rigorous experimental systems to distinguish whether phagocyte-dependent effects are neutrophil or macrophage specific have been lacking. We have developed and validated transgenic lines that enable superior demonstration of cell-autonomous neutrophil and macrophage genetic requirements. We coupled well-characterized neutrophil- and macrophage-specific Gal4 driver lines with UAS:Cas9 transgenes for selective expression of Cas9 in either neutrophils or macrophages. Efficient gene editing, confirmed by both Sanger and next-generation sequencing, occurred in both lineages following microinjection of efficacious synthetic guide RNAs into zebrafish embryos. In proof-of-principle experiments, we demonstrated molecular and/or functional evidence of on-target gene editing for several genes (mCherry, lamin B receptor, trim33) in either neutrophils or macrophages as intended. These new UAS:Cas9 tools provide an improved resource for assessing individual contributions of neutrophil- and macrophage-expressed genes to the many physiological processes and diseases modelled in zebrafish. Furthermore, this gene-editing functionality can be exploited in any cell lineage for which a lineage-specific Gal4 driver is available. This article has an associated First Person interview with the first author of the paper.

Investigation of monogenic causes of familial breast cancer: data from the BEACCON case-control study.

Breast cancer (BC) has a significant heritable component but the genetic contribution remains unresolved in the majority of high-risk BC families. This study aims to investigate the monogenic causes underlying the familial aggregation of BC beyond BRCA1 and BRCA2, including the identification of new predisposing genes. A total of 11,511 non-BRCA familial BC cases and population-matched cancer-free female controls in the BEACCON study were investigated in two sequencing phases: 1303 candidate genes in up to 3892 cases and controls, followed by validation of 145 shortlisted genes in an additional 7619 subjects. The coding regions and exon-intron boundaries of all candidate genes and 14 previously proposed BC genes were sequenced using custom designed sequencing panels. Pedigree and pathology data were analysed to identify genotype-specific associations. The contribution of ATM, PALB2 and CHEK2 to BC predisposition was confirmed, but not RAD50 and NBN. An overall excess of loss-of-function (LoF) (OR 1.27, p = 9.05 × 10-9) and missense (OR 1.27, p = 3.96 × 10-73) variants was observed in the cases for the 145 candidate genes. Leading candidates harbored LoF variants with observed ORs of 2-4 and individually accounted for no more than 0.79% of the cases. New genes proposed by this study include NTHL1, WRN, PARP2, CTH and CDK9. The new candidate BC predisposition genes identified in BEACCON indicate that much of the remaining genetic causes of high-risk BC families are due to genes in which pathogenic variants are both very rare and convey only low to moderate risk.

Utility of clinical comprehensive genomic characterization for diagnostic categorization in patients presenting with hypocellular bone marrow failure syndromes.

Bone marrow failure (BMF) related to hypoplasia of hematopoietic elements in the bone marrow is a heterogeneous clinical entity with a broad differential diagnosis including both inherited and acquired causes. Accurate diagnostic categorization is critical to optimal patient care and detection of genomic variants in these patients may provide this important diagnostic and prognostic information. We performed real-time, accredited (ISO15189) comprehensive genomic characterization including targeted sequencing and whole exome sequencing in 115 patients with BMF syndrome (median age 24 years, range 3 months - 81 years). In patients with clinical diagnoses of inherited BMF syndromes, acquired BMF syndromes or clinically unclassifiable BMF we detected variants in 52% (12/23), 53% (25/47) and 56% (25/45) respectively. Genomic characterization resulted in a change of diagnosis in 30/115 (26%) including the identification of germline causes for 3/47 and 16/45 cases with pre-test diagnoses of acquired and clinically unclassifiable BMF respectively. The observed clinical impact of accurate diagnostic categorization included choice to perform allogeneic stem cell transplantation, disease-specific targeted treatments, identification of at-risk family members and influence of sibling allogeneic stem cell donor choice. Multiple novel pathogenic variants and copy number changes were identified in our cohort including in TERT, FANCA, RPS7 and SAMD9. Whole exome sequence analysis facilitated the identification of variants in two genes not typically associated with a primary clinical manifestation of BMF but also demonstrated reduced sensitivity for detecting low level acquired variants. In conclusion, genomic characterization can improve diagnostic categorization of patients presenting with hypoplastic BMF syndromes and should be routinely performed in this group of patients.

Publisher Correction: CNspector: a web-based tool for visualisation and clinical diagnosis of copy number variation from next generation sequencing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.