Hematologists' barriers and enablers to screening and recruiting patients to a chimeric antigen receptor (CAR) T cell therapy trial: a theory-informed interview study.

BACKGROUND: Novel therapies often fail to reach the bedside due to low trial recruitment rates. Prior to conducting one of the first chimeric antigen receptor (CAR) T cell therapy trials in Canada, we used the Theoretical Domains Framework, a novel tool for identifying barriers and enablers to behavior change, to identify physician-related barriers and enablers to screening and recruiting patients for an early phase immunotherapy trial. METHODS: We conducted interviews with hematologists across Canada and used a directed content analysis to identify relevant domains reflecting the key factors that may affect screening and recruitment. RESULTS: In total, we interviewed 15 hematologists. Physicians expressed "cautious hope"; while expressing safety, feasibility, and screening criteria concerns, 14 out of 15 hematologists intended to screen for the trial (domains: knowledge, goals, beliefs about consequences, intentions). Physicians underscored the "challenging contexts," identifying resources, workload, forgetting, and patient wait times to receive CAR T cells as key practical barriers to screening (domains: environmental context and resources, memory, attention and decision-making, behavioral regulation). They also highlighted "variability in roles and procedures" that may lead to missed trial candidates (domain: social and professional role). Left unaddressed, these barriers may undermine trial recruitment. CONCLUSIONS: This study is among the first to use the Theoretical Domains Framework from the physician perspective to identify recruitment challenges to early phase trials and demonstrates the value of this approach for identifying barriers to screening and recruitment that may not otherwise have been elicited. This approach can optimize trial procedures and may serve to inform future promising early phase cancer therapy trials. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03765177 . Registered on December 5, 2018.

Navigating choice in the face of uncertainty: using a theory informed qualitative approach to identifying potential patient barriers and enablers to participating in an early phase chimeric antigen receptor T (CAR-T) cell therapy trial.

OBJECTIVES: Bench to bedside translation of groundbreaking treatments like chimeric antigen receptor T (CAR-T) cell therapy depends on patient participation in early phase trials. Unfortunately, many novel therapies fail to be adequately evaluated due to low recruitment rates, which slows patient access to emerging treatments. Using the Theoretical Domains Framework (TDF), we sought to identify potential patient barriers and enablers to participating in an early phase CAR-T cell therapy trial. DESIGN: We used qualitative semistructured interviews to identify potential barriers and enablers to patients' hypothetical participation in an early phase CAR-T cell therapy trial. We used the TDF and directed content analysis to identify relevant domains based on frequency, relevance and the presence of conflicting beliefs. PARTICIPANTS: Canadian adult patients diagnosed with haematological malignancies. RESULTS: In total, we interviewed 13 participants (8 women, 5 men). Participants ranged in age from 18 to 73 (median=56) and had been living with haematological cancer from a few months to several years. We found participants were unfamiliar with CAR-T cell therapy but wished to know more about treatment safety, efficacy and trial logistics (domains: knowledge, beliefs about consequences). They were motivated by altruistic considerations, though many prioritised personal health benefits despite recognising the goals (ie, establishing safety) of early phase clinical trials (domains: goals, intentions). Every participant valued receiving medical advice from their haematologists and oncologists, though some preferred impartial medical experts to inform their decision making (domain: social influences). Finally, participants indicated that improving access to financial and social supports would improve their trial participation experience (domain: environmental context and resources). CONCLUSION: Using the TDF allowed us to identify factors that might undermine participation to a CAR-T cell therapy trial and to optimise recruitment processes by considering patient perspectives to taking part in early phase trials.Trial regestration: NCT03765177; Pre-results.

Partnering with patients to get better outcomes with chimeric antigen receptor T-cell therapy: towards engagement of patients in early phase trials.

AIM: Though patient engagement in clinical research is growing, recent reports suggest few clinical trials report on such activities. To address this gap, we describe our approach to patient engagement in the development of a clinical trial protocol to assess a new immunotherapy for blood cancer (chimeric antigen receptor T-cell therapy, CAR-T cell therapy). METHODS: Our team developed a clinical trial protocol by working with patient partners from inception. Two patient partners with lived blood cancer experience were identified through referrals from our team's professional network and patient organization contacts. Our patient partners were onboarded to the team and engaged in several studies conducted to develop the clinical trial protocol, including a systematic review of the existing literature on the therapy, patient interviews and a survey to obtain perspectives on barriers and enablers to participating in the trial, an early economic analysis, and a retrospective cohort study. RESULTS: Engaging patient partners enhanced our research in ways that would not have otherwise occurred. By selecting patient important outcomes for data collection, our partners helped flag that quality of life and health utility measures have not been reported in previous CAR-T cell therapy trials for blood cancer. Our partners also co-developed a non-technical summary of the systematic review that summarized results in an accessible manner. Our patient partners reviewed interview and survey questions, to improve the language and appropriateness; provided recruitment suggestions; and provided a patient perspective on the results, thereby confirming the importance of findings. Input was also obtained on costs for the early economic analysis. Our patient partners identified costs that may be a burden to both patients and caregivers during a trial and helped to confirm that the overall structure of the economic model reflected the patient care pathway. Our patient partners also shared their diagnosis and treatment stories, which helped to provide the research team with insight into this experience. CONCLUSIONS: Contributions by our patient partners were invaluable to each component study, as well as the overall development of the trial protocol. We plan to use this approach in the future in order to meaningfully engage patients in the development of other clinical trials; we also hope that by reporting our methods this will help other research teams to do the same. TRIAL REGISTRATION: Affiliated with the development of NCT03765177.

Near haploidization is a genomic hallmark which defines a molecular subgroup of giant cell glioblastoma.

BACKGROUND: Giant cell glioblastoma (gcGBM) is a rare histologic subtype of glioblastoma characterized by numerous bizarre multinucleate giant cells and increased reticulin deposition. Compared with conventional isocitrate dehydrogenase (IDH)-wildtype glioblastomas, gcGBMs typically occur in younger patients and are generally associated with an improved prognosis. Although prior studies of gcGBMs have shown enrichment of genetic events, such as TP53 alterations, no defining aberrations have been identified. The aim of this study was to evaluate the genomic profile of gcGBMs to facilitate more accurate diagnosis and prognostication for this entity. METHODS: Through a multi-institutional collaborative effort, we characterized 10 gcGBMs by chromosome studies, single nucleotide polymorphism microarray analysis, and targeted next-generation sequencing. These tumors were subsequently compared to the genomic and epigenomic profile of glioblastomas described in The Cancer Genome Atlas (TCGA) dataset. RESULTS: Our analysis identified a specific pattern of genome-wide massive loss of heterozygosity (LOH) driven by near haploidization in a subset of glioblastomas with giant cell histology. We compared the genomic signature of these tumors against that of all glioblastomas in the TCGA dataset (n = 367) and confirmed that our cohort of gcGBMs demonstrated a significantly different genomic profile. Integrated genomic and histologic review of the TCGA cohort identified 3 additional gcGBMs with a near haploid genomic profile. CONCLUSIONS: Massive LOH driven by haploidization represents a defining molecular hallmark of a subtype of gcGBM. This unusual mechanism of tumorigenesis provides a diagnostic genomic hallmark to evaluate in future cases, may explain reported differences in survival, and suggests new therapeutic vulnerabilities.

Multidisciplinary analysis of pediatric T-ALL: 9q34 gene fusions.

T-cell acute lymphoblastic leukemia (T-ALL) is not as frequently reported as the B-cell counterpart (B-ALL), only occurring in about 15% of pediatric cases with a typically heterogeneous etiology. Approximately 8% of childhood T-ALL cases have rearrangements involving the ABL1 tyrosine kinase gene at 9q34.12; although a t(9;22), resulting in a fusion of ABL1 with the BCR gene at 22q11.23 is a common occurrence in B-ALL, it is not a typical finding in T-ALL. A subset of 10 of 40 documented cases of T-ALL analyzed over a 5-year period is presented, each having gene rearrangements within band 9q34 that resulted in fusions other than BCR/ABL1. These cases included fusions involving ABL1, SET (9q34.11), NUP214 (9q34.13), SPTAN1 (9p34.11), and TNRC6B (22q13.1). Among the 10 cases are: six SET/NUP214 fusions, two ABL1/NUP214 fusions (one of which was associated with episomal amplification) and novel SPTAN1/ABL1 and TNRC6B/ABL1 fusions. The evaluations of these clones were each significantly aided by FISH analysis, which directed subsequent microarray and anchored multiplex PCR testing for fusion confirmations.

Features of Feingold syndrome 1 dominate in subjects with 2p deletions including MYCN.

Interstitial deletions of the distal short arm of chromosome 2 including MYCN have only been reported for a small number of individuals. Germline deletions and mutations of MYCN cause Feingold syndrome 1 (FS1), a rare disorder characterized by microcephaly, digit anomalies, gastrointestinal atresias, short stature, dysmorphic features, and intellectual disability. We present a series of six individuals referred for SNP microarray with overlapping deletions of 2p ranging from 3.4 to 16.8 Mb in size, with a common overlapping region of 1.53 Mb spanning (14,614,477-16,148,021) [hg19] and including five genes: NBAS, DDX1, MYCNUT, MYCNOS, and MYCN. Clinical information was available for five individuals. Clinical features included core features of FS1 such as microcephaly, digit anomalies, and gastrointestinal atresias as well as structural cardiac defects, hearing loss, and renal anomalies, which are features less consistently associated with FS1. Other features observed in several individuals, that have not specifically been associated with FS1 were motor delay, structural brain abnormalities, genital abnormalities, and radioulnar synostosis. These results indicate that while individuals with deletions of 2p spanning several megabases and including MYCN can present with features not typically associated with FS1, the common core features are usually present.

Double-hit mantle cell lymphoma with MYC gene rearrangement or amplification: a report of four cases and review of the literature.

Mature B-cell lymphomas with both BCL2 and MYC translocations are known as "double hit" lymphomas. These lymphomas are aggressive and show high proliferation rate due to the growth advantages provided by MYC and BCL2 translocation and overexpression. Mantle cell lymphoma (MCL) is a neoplasm of mature B-lymphocytes with characteristic t(11;14) and subsequent Cyclin D1 overexpression. Secondary cytogenetic changes are frequent in MCL, but MYC translocation has only been rarely reported. In this study, we report four cases of MCL with MYC translocation or MYC gene amplification detected by conventional cytogenetics, fluorescence in situ hybridization and whole genome single nucleotide polymorphism (SNP) array, and determined the clinicopathologic features. Our study provides further evidence supporting the concept of "double hit" MCL with co-involvement of MYC gene rearrangement and/or amplification and CCND1 gene rearrangement.

Fatal Henoch-Schönlein purpura in an adult with Dieulafoy lesions.

Henoch-Schönlein purpura (HSP) is considered a benign disease of children. We report a severe case of HSP in an adult causing renal failure and gastrointestinal (GI) hemorrhage. Despite aggressive treatment with corticosteroids, cyclophosphamide, and plasmapheresis, the patient developed massive GI bleeding from 2 Dieulafoy lesions and died weeks after bowel resection. Although uncommon, when massive GI hemorrhage occurs, actively bleeding Dieulafoy lesions, although uncommon, should be suspected and evaluated early.

Clinical utility of single nucleotide polymorphism arrays.

Detection of chromosomal abnormalities has evolved over the past 30 years. Microarray analysis allows for the detection of abnormalities at a level of resolution 100 times greater than chromosomal analysis. In this article, one specific array, a single nucleotide polymorphism array, is reviewed. This array not only allows for increased resolution to detect copy number changes, but the genotyping aspect of the array allows copy neutral detection (for both uniparental disomy and consanguinity). Additionally, use of this array in constitutional postnatal, prenatal, and products of conception studies is reviewed along with the use of the array in oncology studies.

Inverted duplications on acentric markers: mechanism of formation.

Acentric inverted duplication (inv dup) markers, the largest group of chromosomal abnormalities with neocentromere formation, are found in patients both with idiopathic mental retardation and with cancer. The mechanism of their formation has been investigated by analyzing the breakpoints and the genotypes of 12 inv dup marker cases (three trisomic, six tetrasomic, two polysomic and one X chromosome derived marker) using a combination of fluorescence in situ hybridization, quantitative SNP array and microsatellite analysis. Inv dup markers were found to form either symmetrically with one breakpoint or asymmetrically with two distinct breakpoints. Genotype analyses revealed that all inv dup markers formed from one single chromatid end. This observation is incompatible with the previously suggested model by which the acentric inv dup markers form through inter-chromosomal U-type exchange. On the basis of the identification of DNA sequence motifs with inverted homologies within all observed breakpoint regions, a new general mechanism is proposed for the acentric inv dup marker formation: following a double-strand break an acentric fragment forms, during either meiosis or mitosis. The open DNA end of the acentric fragment is stabilized by the formation of an intra-chromosomal loop promoted by the presence of sequences with inverted homologies. Likely coinciding with the neocentromere formation, this stabilized fragment is duplicated during an early mitotic event, insuring the marker's survival during cell division and its presence in all cells.

Paxillin is a target for somatic mutations in lung cancer: implications for cell growth and invasion.

Lung cancer is characterized by abnormal cell growth and invasion, and the actin cytoskeleton plays a major role in these processes. The focal adhesion protein paxillin is a target of a number of oncogenes involved in key signal transduction and important in cell motility and migration. In lung cancer tissues, we have found that paxillin was highly expressed (compared with normal lung), amplified (12.1%, 8 of 66) and correlated with increased MET and epidermal growth factor receptor (EGFR) gene copy numbers, or mutated (somatic mutation rate of 9.4%, 18 of 191). Paxillin mutations (19 of 21) were clustered between LD motifs 1 and 2 and the LIM domains. The most frequent point mutation (A127T) enhanced lung cancer cell growth, colony formation, focal adhesion formation, and colocalized with Bcl-2 in vitro. Gene silencing from RNA interference of mutant paxillin led to reduction of cell viability. A murine in vivo xenograft model of A127T paxillin showed an increase in tumor growth, cell proliferation, and invasion. These results establish an important role for paxillin in lung cancer.

Synaptic defects at meiosis I and non-obstructive azoospermia.

BACKGROUND: Recent advances in immunofluorescence methodology have made it possible to directly monitor protein localization patterns in germ cells undergoing meiosis. We used this technology to examine the early stages of meiosis in testicular material obtained from men presenting for evaluation at infertility clinics. METHODS: Specifically, we compared meiotic progression, synapsis and recombination in 34 individuals with obstructive azoospermia ('controls') to 26 individuals with non-obstructive azoospermia (NOA) ('cases'). RESULTS: In 9 of the 26 cases, no germ cells were identified, but in the remaining 17, there was at least some progression through meiosis. Most of these individuals appeared to have normal levels of spermatogenic activity, with little evidence of meiotic impairment. However, in three individuals, we observed either complete or partial meiotic arrest associated with abnormalities in synapsis. CONCLUSIONS: This suggests that >10% of cases of unexplained NOA may be attributable to severe meiotic defects. The characterization of these meiotic arrest phenotypes may guide further research into the molecular basis of unexplained infertility.

Identification and molecular characterization of a de novo supernumerary ring chromosome 18 in a patient with Klippel-Trenaunay syndrome.

Klippel-Trenaunay syndrome (KTS) is a congenital vascular disorder comprised of capillary, venous and lymphatic malformations associated with overgrowth of the affected tissues. In this study, we report the identification of a de novo supernumerary ring chromosome in a patient with mild mental retardation, long tapering fingers, elongated, thin feet and Klippel-Trenaunay syndrome (KTS). The ring marker chromosome was found to be mosaic, present in 24% of cells, and was later shown to be derived from chromosome 18, r(18). Fluorescence in situ hybridization (FISH) was used to define the breakpoints involved in the formation of r(18). The chromosome 18p breakpoint was localized between the markers WI-9619 and D18S1150, which is less than 10 cM to the centromere. The 18q breakpoint was localized between the centromere and BAC clone 666n19, which is a region of less than 40 kb. These data suggest that the r(18) mostly originated from 18p, with an estimated size of less than 10 cM. These studies identify and characterize a new marker chromosome 18, provide insights into the understanding of the relationships between the clinical phenotypes and marker chromosomes, and establish a framework for finding a potential vascular and/or overgrowth gene located on chromosome 18.

Human artificial chromosomes with alpha satellite-based de novo centromeres show increased frequency of nondisjunction and anaphase lag.

Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fully recapitulate normal centromere function has not been explored. Here, we have used two kinds of alpha satellite DNA, DXZ1 (from the X chromosome) and D17Z1 (from chromosome 17), to generate human artificial chromosomes. Although artificial chromosomes are mitotically stable over many months in culture, when we examined their segregation in individual cell divisions using an anaphase assay, artificial chromosomes exhibited more segregation errors than natural human chromosomes (P < 0.001). Naturally occurring, but abnormal small ring chromosomes derived from chromosome 17 and the X chromosome also missegregate more than normal chromosomes, implicating overall chromosome size and/or structure in the fidelity of chromosome segregation. As different artificial chromosomes missegregate over a fivefold range, the data suggest that variable centromeric DNA content and/or epigenetic assembly can influence the mitotic behavior of artificial chromosomes.

Analysis of microsatellite instability and X-inactivation in ovarian borderline tumors lacking numerical abnormalities by comparative genomic hybridization.

The genetic events underlying development of ovarian borderline tumors (tumors of low malignant potential) are not well understood. In our previous studies of microdissected samples from serous borderline tumors, comparative genomic hybridization (CGH) and/or fluorescence in situ hybridization (FISH) analyses showed that 3 of 13 tumors had detectable numerical abnormalities; the remaining 10 had none. In the present study, we examined microsatellite instability (MSI) and clonality in this same set of tumors. Although absence of chromosomal imbalances has been associated with the presence of MSI in some types of solid tumors, the extent of MSI in borderline tumors, and its role in their pathogenesis, is unclear. In our set of 13 tumors, no MSI was detected despite analysis with microsatellite markers recommended by the National Cancer Institute for assessment of MSI. Quantitative X-inactivation studies were informative at the androgen receptor gene AR in 9 of the 13 tumors and revealed that each of the 9 tumors was clonal. In two patients, bilateral tumors showed identical patterns of skewed X-inactivation. These studies confirm the clonality of borderline tumors and suggest that some borderline tumors may develop through mechanisms other than chromosomal imbalances or microsatellite instability.

Covariation of synaptonemal complex length and mammalian meiotic exchange rates.

Analysis of recombination between loci (linkage analysis) has been a cornerstone of human genetic research, enabling investigators to localize and, ultimately, identify genetic loci. However, despite these efforts little is known about patterns of meiotic exchange in human germ cells or the mechanisms that control these patterns. Using recently developed immunofluorescence methodology to examine exchanges in human spermatocytes, we have identified remarkable variation in the rate of recombination within and among individuals. Subsequent analyses indicate that, in humans and mice, this variation is linked to differences in the length of the synaptonemal complex. Thus, at least in mammals, a physical structure, the synaptonemal complex, reflects genetic rather than physical distance.

Chemotherapy-induced O(6)-benzylguanine-resistant alkyltransferase mutations in mismatch-deficient colon cancer.

The ability of O(6)-benzylguanine (BG) to inactivate alkyltransferase (AGT) to potentiate the antitumor efficacy of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is being tested in clinical trials. As of now, there are no examples of acquired resistance to BG+BCNU in the clinical setting. However, we hypothesized that genetically unstable tumors might develop resistance to the combination after repeated drug-exposures to achieve therapeutic efficacy. To evaluate this possibility, we treated three colon cancer cell lines that are either proficient in mismatch repair (MMR) [SW480 (MMR wild type)] or deficient in MMR [HCT116 (hMLH1 mutant) and HCT15 (hMSH6 mutant)] with three cycles of BG+BCNU. After drug-treatments, HCT116 and HCT15 were completely resistant to BG-potentiated cytotoxicity of BCNU. In these two cell lines, the acquired BG resistance resulted from two de novo and different mutations at amino acid 165 in AGT: 165-lysine (K) to glutamic acid (E) (K165E in HCT116), and 165-lysine to asparagine (N) (K165N in HCT15). Both K165-mutated AGTs had markedly decreased enzymatic activity because of unstable AGT protein but were remarkably resistant to BG inactivation. FISH analysis showed that only one copy of MGMT gene exists in HCT116 cells, and the status of promoter methylation of MGMT in HCT15 showed that one allele of the MGMT promoter has an aberrant methylation. Thus, the MGMT gene expressing AGT either from one copy (HCT116) or from unmethylated allele (HCT15) was mutated because of the exposure to BG+BCNU in these two MMR-deficient cell lines. Conversely, MMR-proficient SW480 cells, treated with three cycles of BG+BCNU, maintained wt AGT and the sensitivity to BG-potentiated BCNU-cytotoxicity. To confirm that K165-mutated AGT proteins were responsible for resistance to BG+BCNU, we transfected K165E and K165N MGMT cDNAs into Chinese hampster ovary (CHO) cells. Transfected CHO cells had low AGT activity but increased IC(50) for either BCNU or temozolomide (TMZ), compared with parental CHO cells. BG did not potentiate the cytotoxicity of these two alkylating agents at concentrations up to 200 microM; in contrast, BG, at 25 microM, sensitized CHO-AGT (transfected with wt MGMT cDNA) cells to BCNU or TMZ-cytotoxicity by 3-4 fold. These results suggest that K165 AGT mutants arising in MMR-deficient tumor cells after treatment with chemotherapeutic agents are both resistant to BG-inactivation and are active in the repair of alkylated DNA adducts.

Rapid interphase analysis for prenatal diagnosis of translocation carriers using subtelomeric probes.

Interphase fluorescence in situ hybridization (FISH) has become an accepted laboratory technique for the rapid and preliminary prenatal assessment of chromosome aneuploidy. The introduction of subtelomeric FISH probes now allows for the molecular-cytogenetic analysis of terminal chromosome rearrangements. In a prospective study, we examined the prenatal use of subtelomeric probes on interphase cells to rapidly detect the carrier status of a fetus when a parent carried a known reciprocal or Robertsonian chromosome translocation. Three of the cases were identified as being abnormal. All cases were confirmed by routine cytogenetic analysis. These findings clearly demonstrated the utility of this technique and these probes to rapidly and correctly identify balanced and unbalanced chromosome anomalies of a fetus that could result from parental translocations.

Chromosomal autonomy of hMLH1 methylation in colon cancer.

Silencing of hMLH1 expression by aberrant hMLH1 promoter methylation accounts for the majority of sporadic colon cancers with microsatellite instability. We have previously shown hMLH1 silencing is biallelic and actively maintained. To study the mechanism of aberrant hMLH1 methylation, we assayed whether an hMLH1 methylated cell could transfer methylation and silencing to an exogenous hMLH1 promoter in somatic cell hybrids between hMLH1 methylated-silenced and hMLH1 unmethylated-expressing colon cancer cells. Conversely, we assayed whether these hybrids could reactivate expression of initially methylated and silenced hMLH1 alleles. Compellingly, within the hybrids each hMLH1 allele remained unchanged, retaining the expression status of its parental cell of origin. This chromosomal autonomy may not be simply determined by DNA methylation, as it is reasserted after experimentally forced demethylation of all hMLH1 alleles in the hybrids. Confirming findings included hMLH1 methylated cells being unable to methylate single transferred exogenous hMLH1 expressing chromosomes or transfected hMLH1 reporter constructs. hMLH1 silencing does not conform to either a dominant or recessive model, and is not determined by trans-acting factors differing between hMLH1 expressing or silenced genomes. We posit that hMLH1 methylation is dependent on and maintained by cis chromosomal marks, whose nature remains to be elucidated.