CAR T cells and time-limited ibrutinib as treatment for relapsed/refractory mantle cell lymphoma: the phase 2 TARMAC study.

ABSTRACT: CD19-directed chimeric antigen receptor T cells (CAR-T) achieve high response rates in patients with relapsed/refractory mantle cell lymphoma (MCL). However, their use is associated with significant toxicity, relapse concern, and unclear broad tractability. Preclinical and clinical data support a beneficial synergistic effect of ibrutinib on apheresis product fitness, CAR-T expansion, and toxicity. We evaluated the combination of time-limited ibrutinib and CTL019 CAR-T in 20 patients with MCL in the phase 2 TARMAC study. Ibrutinib commenced before leukapheresis and continued through CAR-T manufacture for a minimum of 6 months after CAR-T administration. The median prior lines of therapy was 2; 50% of patients were previously exposed to a Bruton tyrosine kinase inhibitor (BTKi). The primary end point was 4-month postinfusion complete response (CR) rate, and secondary end points included safety and subgroup analysis based on TP53 aberrancy. The primary end point was met; 80% of patients demonstrated CR, with 70% and 40% demonstrating measurable residual disease negativity by flow cytometry and molecular methods, respectively. At 13-month median follow-up, the estimated 12-month progression-free survival was 75% and overall survival 100%. Fifteen patients (75%) developed cytokine release syndrome; 12 (55%) with grade 1 to 2 and 3 (20%) with grade 3. Reversible grade 1 to 2 neurotoxicity was observed in 2 patients (10%). Efficacy was preserved irrespective of prior BTKi exposure or TP53 mutation. Deep responses correlated with robust CAR-T expansion and a less exhausted baseline T-cell phenotype. Overall, the safety and efficacy of the combination of BTKi and T-cell redirecting immunotherapy appears promising and merits further exploration. This trial was registered at www.ClinicalTrials.gov as #NCT04234061.

Persistence of UBTF tandem duplications in remission in acute myeloid leukaemia.

UBTF tandem duplications are recurrent in adult and paediatric acute myeloid leukaemia and have been reported to be associated with a poor prognosis. Co-mutations in WT1 and FLT3 are common while morphological dysplasia is frequent. The role of UBTF-TDs in leukemogenesis is yet to be elucidated; however they have been proposed as early initiating events, making them attractive for assessment of MRD and a potential therapeutic target. We present two cases where the UBTF-TD was observed in remission and discuss the implications of these findings in the clinicobiological understanding of this emerging entity.

Enhancer retargeting of CDX2 and UBTF::ATXN7L3 define a subtype of high-risk B-progenitor acute lymphoblastic leukemia.

Transcriptome sequencing has identified multiple subtypes of B-progenitor acute lymphoblastic leukemia (B-ALL) of prognostic significance, but a minority of cases lack a known genetic driver. Here, we used integrated whole-genome (WGS) and -transcriptome sequencing (RNA-seq), enhancer mapping, and chromatin topology analysis to identify previously unrecognized genomic drivers in B-ALL. Newly diagnosed (n = 3221) and relapsed (n = 177) B-ALL cases with tumor RNA-seq were studied. WGS was performed to detect mutations, structural variants, and copy number alterations. Integrated analysis of histone 3 lysine 27 acetylation and chromatin looping was performed using HiChIP. We identified a subset of 17 newly diagnosed and 5 relapsed B-ALL cases with a distinct gene expression profile and 2 universal and unique genomic alterations resulting from aberrant recombination-activating gene activation: a focal deletion downstream of PAN3 at 13q12.2 resulting in CDX2 deregulation by the PAN3 enhancer and a focal deletion of exons 18-21 of UBTF at 17q21.31 resulting in a chimeric fusion, UBTF::ATXN7L3. A subset of cases also had rearrangement and increased expression of the PAX5 gene, which is otherwise uncommon in B-ALL. Patients were more commonly female and young adult with median age 35 (range,12-70 years). The immunophenotype was characterized by CD10 negativity and immunoglobulin M positivity. Among 16 patients with known clinical response, 9 (56.3%) had high-risk features including relapse (n = 4) or minimal residual disease >1% at the end of remission induction (n = 5). CDX2-deregulated, UBTF::ATXN7L3 rearranged (CDX2/UBTF) B-ALL is a high-risk subtype of leukemia in young adults for which novel therapeutic approaches are required.

JAFFAL: detecting fusion genes with long-read transcriptome sequencing.

In cancer, fusions are important diagnostic markers and targets for therapy. Long-read transcriptome sequencing allows the discovery of fusions with their full-length isoform structure. However, due to higher sequencing error rates, fusion finding algorithms designed for short reads do not work. Here we present JAFFAL, to identify fusions from long-read transcriptome sequencing. We validate JAFFAL using simulations, cell lines, and patient data from Nanopore and PacBio. We apply JAFFAL to single-cell data and find fusions spanning three genes demonstrating transcripts detected from complex rearrangements. JAFFAL is available at https://github.com/Oshlack/JAFFA/wiki .

T cell receptor beta locus sequencing early post-allogeneic stem cell transplant identifies patients at risk of initial and recurrent cytomegalovirus infection.

Identification of patients at risk of initial & recurrent cytomegalovirus (CMV) reactivation following allogeneic stem cell transplant (alloSCT) may help guide prophylactic strategies. T-cell receptor beta (TRB) deep sequencing was used to identify and enumerate the T-cell repertoire harbouring TRB sequences with annotated specificity to CMV (pubCMVrep), as well as the overall T-cell receptor (TCR) repertoire diversity at day +30 & day +60 post-alloSCT for 65 patients. T-cells harbouring TRB sequences with annotated specificity for CMV were identifiable in all patients. 56% of patients required CMV treatment and 23% of the cohort developed recurrent CMV. PubCMVrep size at day +30 was not associated with reactivation, however amongst patients with antecedent CMV viremia a low day +60 pubCMVrep was associated with a greater incidence of recurrent CMV (75% vs. 21%, HR 6.16, 95% CI 1.29-29.40, P = 0.0008). Moreover, patients with high pubCMVrep only developed recurrent CMV in the setting of GVHD. Low TCR diversity at day +30 was associated with a greater incidence of initial CMV reactivation (71% vs. 22%, HR 5.39, 95% CI 1.70-17.09, p = 0.0002). pubCMVrep and TCR diversity are promising biomarkers to identify patients at risk of initial & recurrent CMV who may benefit from novel prophylactic strategies.

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.

High dose-rate brachytherapy of localized prostate cancer converts tumors from cold to hot.

BACKGROUND: Prostate cancer (PCa) has a profoundly immunosuppressive microenvironment and is commonly immune excluded with few infiltrative lymphocytes and low levels of immune activation. High-dose radiation has been demonstrated to stimulate the immune system in various human solid tumors. We hypothesized that localized radiation therapy, in the form of high dose-rate brachytherapy (HDRBT), would overcome immune suppression in PCa. METHODS: To investigate whether HDRBT altered prostate immune context, we analyzed preradiation versus postradiation human tissue from a cohort of 24 patients with localized PCa that received HDRBT as primary treatment (RadBank cohort). We performed Nanostring immune gene expression profiling, digital spatial profiling, and high-throughput immune cell multiplex immunohistochemistry analysis. We also resolved tumor and nontumor zones in spatial and bioinformatic analyses to explore the immunological response. RESULTS: Nanostring immune profiling revealed numerous immune checkpoint molecules (eg, B7-H3, CTLA4, PDL1, and PDL2) and TGFß levels were increased in response to HDRBT. We used a published 16-gene tumor inflammation signature (TIS) to divide tumors into distinct immune activation states (high:hot, intermediate and low:cold) and showed that most localized PCa are cold tumors pre-HDRBT. Crucially, HDRBT converted 80% of these 'cold'-phenotype tumors into an 'intermediate' or 'hot' class. We used digital spatial profiling to show these HDRBT-induced changes in prostate TIS scores were derived from the nontumor regions. Furthermore, these changes in TIS were also associated with pervasive changes in immune cell density and spatial relationships-in particular, between T cell subsets and antigen presenting cells. We identified an increased density of CD4+ FOXP3+ T cells, CD68+ macrophages and CD68+ CD11c+ dendritic cells in response to HDRBT. The only subset change specific to tumor zones was PDL1- macrophages. While these immune responses were heterogeneous, HDRBT induced significant changes in immune cell associations, including a gained T cell and HMWCK+ PDL1+ interaction in tumor zones. CONCLUSION: In conclusion, we showed HDRBT converted "cold" prostate tumors into more immunologically activated "hot" tissues, with accompanying spatially organized immune infiltrates and signaling changes. Understanding and potentially harnessing these changes will have widespread implications for the future treatment of localized PCa, including rational use of combination radio-immunotherapy.

Therapeutic options for mucinous ovarian carcinoma.

OBJECTIVE: Mucinous ovarian carcinoma (MOC) is an uncommon ovarian cancer histotype that responds poorly to conventional chemotherapy regimens. Although long overall survival outcomes can occur with early detection and optimal surgical resection, recurrent and advanced disease are associated with extremely poor survival. There are no current guidelines specifically for the systemic management of recurrent MOC. We analyzed data from a large cohort of women with MOC to evaluate the potential for clinical utility from a range of systemic agents. METHODS: We analyzed gene copy number (n = 191) and DNA sequencing data (n = 184) from primary MOC to evaluate signatures of mismatch repair deficiency and homologous recombination deficiency, and other genetic events. Immunohistochemistry data were collated for ER, CK7, CK20, CDX2, HER2, PAX8 and p16 (n = 117-166). RESULTS: Molecular aberrations noted in MOC that suggest a match with current targeted therapies include amplification of ERBB2 (26.7%) and BRAF mutation (9%). Observed genetic events that suggest potential efficacy for agents currently in clinical trials include: KRAS/NRAS mutations (66%), TP53 missense mutation (49%), RNF43 mutation (11%), ARID1A mutation (10%), and PIK3CA/PTEN mutation (9%). Therapies exploiting homologous recombination deficiency (HRD) may not be effective in MOC, as only 1/191 had a high HRD score. Mismatch repair deficiency was similarly rare (1/184). CONCLUSIONS: Although genetically diverse, MOC has several potential therapeutic targets. Importantly, the lack of response to platinum-based therapy observed clinically corresponds to the lack of a genomic signature associated with HRD, and MOC are thus also unlikely to respond to PARP inhibition.

The molecular origin and taxonomy of mucinous ovarian carcinoma.

Mucinous ovarian carcinoma (MOC) is a unique subtype of ovarian cancer with an uncertain etiology, including whether it genuinely arises at the ovary or is metastatic disease from other organs. In addition, the molecular drivers of invasive progression, high-grade and metastatic disease are poorly defined. We perform genetic analysis of MOC across all histological grades, including benign and borderline mucinous ovarian tumors, and compare these to tumors from other potential extra-ovarian sites of origin. Here we show that MOC is distinct from tumors from other sites and supports a progressive model of evolution from borderline precursors to high-grade invasive MOC. Key drivers of progression identified are TP53 mutation and copy number aberrations, including a notable amplicon on 9p13. High copy number aberration burden is associated with worse prognosis in MOC. Our data conclusively demonstrate that MOC arise from benign and borderline precursors at the ovary and are not extra-ovarian metastases.

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

Next Generation Sequencing is now routinely used in the practice of diagnostic pathology to detect clinically relevant somatic and germline sequence variations in patient samples. However, clinical assessment of copy number variations (CNVs) and large-scale structural variations (SVs) is still challenging. While tools exist to estimate both, their results are typically presented separately in tables or static plots which can be difficult to read and are unable to show the context needed for clinical interpretation and reporting. We have addressed this problem with CNspector, a multi-scale interactive browser that shows CNVs in the context of other relevant genomic features to enable fast and effective clinical reporting. We illustrate the utility of CNspector at different genomic scales across a variety of sample types in a range of case studies. We show how CNspector can be used for diagnosis and reporting of exon-level deletions, focal gene-level amplifications, chromosome and chromosome arm level amplifications/deletions and in complex genomic rearrangements. CNspector is a web-based clinical variant browser tailored to the clinical application of next generation sequencing for CNV assessment. We have demonstrated the utility of this interactive software in typical applications across a range of tissue types and disease contexts encountered in the context of diagnostic pathology. CNspector is written in R and the source code is available for download under the GPL3 Licence from https://github.com/PapenfussLab/CNspector . A server running CNspector loaded with the figures from this paper can be accessed at https://shiny.wehi.edu.au/jmarkham/CNspector/index.html .

Frequent activating STAT3 mutations and novel recurrent genomic abnormalities detected in breast implant-associated anaplastic large cell lymphoma.

Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a rare form of T-cell lymphoma that occurs after implantation of breast prostheses. We performed comprehensive next generation sequencing based genomic characterization of 11 cases of BIA-ALCL including sequence variant detection on 180 genes frequently mutated in haematological malignancy, genome-wide copy number assessment, structural variant detection involving the T-cell receptor loci and TRB deep-sequencing. We observed sequence variants leading to JAK/STAT activation in 10 out of 11 patients. We also observed germline TP53 mutations in two cases. In addition we detected a recurrent copy number loss involving RPL5 as well as copy number amplifications involving TNFRSF11A [RANK] (in 2 cases), MYC, P2RX7, TMEM119 and PDGFRA. In summary, our comprehensive genomic characterisation of 11 cases of BIA-ALCL has provided insight into potential pathobiological mechanisms (JAK/STAT, MYC and TP53) as well as identifying targets for future therapeutic intervention (TNFRSF11A, PDGFRA) in this rare entity.

Molecular Mechanisms of Disease Progression in Primary Cutaneous Diffuse Large B-Cell Lymphoma, Leg Type during Ibrutinib Therapy.

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT) is one of the well-recognized extranodal lymphomas commonly addicted to the B-cell receptor-MYD88 superpathway. We aimed to describe the genomic changes in a patient who progressed through treatment with ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor. An 80-year-old woman presented with multiply relapsed PCDLBCL-LT after multiple lines of chemoimmunotherapy and radiotherapy. Pre-treatment testing of the localized cutaneous tumor lesion on a lymphoid amplicon panel demonstrated an MYD88 p.L265P mutation. Ibrutinib therapy was subsequently commenced, resulting in complete resolution of the skin disease. Despite an ongoing skin response, the patient developed progressive nodal disease at two months. Genomic analysis of the cutaneous tumor sample at baseline was compared to that of the inguinal lymph node upon progression, and revealed the acquisition of multiple genomic changes. These included several aberrations expected to bypass BTK inhibition, including two CARD11-activating mutations, and a deleterious mutation in the nuclear factor kappa B (NF-κB) negative regulator, NFKBIE. In addition, an IgH-IRF8 translocation was detected (which brings the IRF8 transcription factor under control of the immunoglobulin heavy chain locus), representing a third plausible mechanism contributing to ibrutinib resistance. Several copy-number changes occurred in both samples, including an amplification of 18q, which encodes the anti-apoptotic protein BCL2. We describe the first case of novel genomic changes of PCDLBCL-LT that occurred while on ibrutinib, providing important mechanistic insights into both pathogenesis and drug resistance.