Analytical evaluation of the clonoSEQ Assay for establishing measurable (minimal) residual disease in acute lymphoblastic leukemia, chronic lymphocytic leukemia, and multiple myeloma.

BACKGROUND: The clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) identifies and tracks unique disease-associated immunoglobulin (Ig) sequences by next-generation sequencing of IgH, IgK, and IgL rearrangements and IgH-BCL1/2 translocations in malignant B cells. Here, we describe studies to validate the analytical performance of the assay using patient samples and cell lines. METHODS: Sensitivity and specificity were established by defining the limit of detection (LoD), limit of quantitation (LoQ) and limit of blank (LoB) in genomic DNA (gDNA) from 66 patients with multiple myeloma (MM), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL), and three cell lines. Healthy donor gDNA was used as a diluent to contrive samples with specific DNA masses and malignant-cell frequencies. Precision was validated using a range of samples contrived from patient gDNA, healthy donor gDNA, and 9 cell lines to generate measurable residual disease (MRD) frequencies spanning clinically relevant thresholds. Linearity was determined using samples contrived from cell line gDNA spiked into healthy gDNA to generate 11 MRD frequencies for each DNA input, then confirmed using clinical samples. Quantitation accuracy was assessed by (1) comparing clonoSEQ and multiparametric flow cytometry (mpFC) measurements of ALL and MM cell lines diluted in healthy mononuclear cells, and (2) analyzing precision study data for bias between clonoSEQ MRD results in diluted gDNA and those expected from mpFC based on original, undiluted samples. Repeatability of nucleotide base calls was assessed via the assay's ability to recover malignant clonotype sequences across several replicates, process features, and MRD levels. RESULTS: LoD and LoQ were estimated at 1.903 cells and 2.390 malignant cells, respectively. LoB was zero in healthy donor gDNA. Precision ranged from 18% CV (coefficient of variation) at higher DNA inputs to 68% CV near the LoD. Variance component analysis showed MRD results were robust, with expected laboratory process variations contributing ≤3% CV. Linearity and accuracy were demonstrated for each disease across orders of magnitude of clonal frequencies. Nucleotide sequence error rates were extremely low. CONCLUSIONS: These studies validate the analytical performance of the clonoSEQ Assay and demonstrate its potential as a highly sensitive diagnostic tool for selected lymphoid malignancies.

Topical resiquimod can induce disease regression and enhance T-cell effector functions in cutaneous T-cell lymphoma.

Early-stage cutaneous T-cell lymphoma (CTCL) is a skin-limited lymphoma with no cure aside from stem cell transplantation. Twelve patients with stage IA-IIA CTCL were treated in a phase 1 trial of 0.03% and 0.06% topical resiquimod gel, a Toll-like receptor 7/8 agonist. Treated lesions significantly improved in 75% of patients and 30% had clearing of all treated lesions. Resiquimod also induced regression of untreated lesions. Ninety-two percent of patients had more than a 50% improvement in body surface area involvement by the modified Severity-Weighted Assessment Tool analysis and 2 patients experienced complete clearing of disease. Four of 5 patients with folliculotropic disease also improved significantly. Adverse effects were minor and largely skin limited. T-cell receptor sequencing and flow cytometry studies of T cells from treated lesions demonstrated decreased clonal malignant T cells in 90% of patients and complete eradication of malignant T cells in 30%. High responses were associated with recruitment and expansion of benign T-cell clones in treated skin, increased skin T-cell effector functions, and a trend toward increased natural killer cell functions. In patients with complete or near eradication of malignant T cells, residual clinical inflammation was associated with cytokine production by benign T cells. Fifty percent of patients had increased activation of circulating dendritic cells, consistent with a systemic response to therapy. In summary, topical resiquimod is safe and effective in early-stage CTCL and the first topical therapy to our knowledge that can induce clearance of untreated lesions and complete remissions in some patients. This trial was registered at www.clinicaltrials.gov as #NCT813320.

Eosinophil-Rich Acute Febrile Neutrophilic Dermatosis in a Patient With Enteropathy-Associated T-cell Lymphoma, Type 1.

The presence of eosinophils within the neutrophilic infiltrates of acute febrile neutrophilic dermatosis (Sweet syndrome) is documented in the literature. Here, the authors describe a case of eosinophil-rich acute febrile neutrophilic dermatosis in the setting of new onset enteropathy-associated T-cell lymphoma (EATL), type 1. Histopathologic evaluation of the skin biopsies demonstrated a mixed superficial perivascular and inflammatory infiltrate composed of neutrophils, lymphocytes, and abundant eosinophils. EATL, type 1 is an aggressive although rare primary intestinal lymphoma that may be associated with celiac disease. This lymphoma is associated with a poor prognosis due to treatment resistance or bowel perforation. To the authors' knowledge, Sweet syndrome has not been reported in a patient with EATL.

Long-term Remissions Following CD20-Directed Chimeric Antigen Receptor-Adoptive T-cell Therapy.

Chimeric antigen receptor (CAR) T-cell therapy produces high response rates in refractory B-cell non-Hodgkin lymphoma, but long-term data are minimal to date. In this study, we present long-term follow-up of a pilot trial testing a CD20-targeting third-generation CAR in patients with relapsed B-cell lymphomas following cyclophosphamide-only lymphodepletion. Two of the three patients in the trial, with mantle cell lymphoma and follicular lymphoma, had remissions lasting more than 7 years, though they ultimately relapsed. The absence of B-cell aplasia in both patients suggested a lack of functional CAR T-cell persistence, leading to the hypothesis that endogenous immune responses were responsible for these long-term remissions. Correlative immunologic analyses supported this hypothesis, with evidence of new humoral and cellular antitumor immune responses proximal to clinical response time points. Collectively, our results suggest that CAR T-cell therapy may facilitate epitope spreading and endogenous immune response formation in lymphomas. Significance: Two of three patients treated with CD20-targeted CAR T-cell therapy had long-term remissions, with evidence of endogenous antitumor immune response formation. Further investigation is warranted to develop conditions that promote epitope spreading in lymphomas.

The Stil protein regulates centrosome integrity and mitosis through suppression of Chfr.

Stil (Sil, SCL/TAL1 interrupting locus) is a cytosolic and centrosomal protein expressed in proliferating cells that is required for mouse and zebrafish neural development and is mutated in familial microcephaly. Recently the Drosophila melanogaster ortholog of Stil was found to be important for centriole duplication. Consistent with this finding, we report here that mouse embryonic fibroblasts lacking Stil are characterized by slow growth, low mitotic index and absence of clear centrosomes. We hypothesized that Stil regulates mitosis through the tumor suppressor Chfr, an E3 ligase that blocks mitotic entry in response to mitotic stress. Mouse fibroblasts lacking Stil by genomic or RNA interference approaches, as well as E9.5 Stil(-/-) embryos, express high levels of the Chfr protein and reduced levels of the Chfr substrate Plk1. Exogenous expression of Stil, knockdown of Chfr or overexpression of Plk1 reverse the abnormal mitotic phenotypes of fibroblasts lacking Stil. We further demonstrate that Stil increases Chfr auto-ubiquitination and reduces its protein stability. Thus, Stil is required for centrosome organization, entry into mitosis and cell proliferation, and these functions are at least partially mediated by Chfr and its targets. This is the first identification of a negative regulator of the Chfr mitotic checkpoint.

t(11;19)(q21;p13) translocation in mucoepidermoid carcinoma creates a novel fusion product that disrupts a Notch signaling pathway.

Truncation of Notch1 has been shown to cause a subtype of acute leukemia, and activation of Notch4 has been associated with mammary and salivary gland carcinomas of mice. Here we identify a new mechanism for disrupting Notch signaling in human tumorigenesis, characterized by altered function of a new ortholog of the Drosophila melanogaster Notch co-activator molecule Mastermind. We cloned the t(11;19) translocation that underlies the most common type of human malignant salivary gland tumor. This rearrangement fuses exon 1 from a novel gene of unknown function at 19p13, termed mucoepidermoid carcinoma translocated 1 (MECT1), with exons 2-5 of a novel member of the Mastermind-like gene family (MAML2) at 11q21 (ref. 3). Similar to D. melanogaster Mastermind and MAML1 (refs. 4,5), full-length MAML2 functioned as a CSL (CBF-1, suppressor of hairless and Lag-1)-dependent transcriptional co-activator for ligand-stimulated Notch. In contrast, MECT1-MAML2 activated transcription of the Notch target gene HES1 independently of both Notch ligand and CSL binding sites. MECT1-MAML2 induced foci formation in RK3E epithelial cells, confirming a biological effect for the fusion product. These data suggest a new mechanism to disrupt the function of a Notch co-activator in a common type of malignant salivary gland tumor.

Integrated analysis of next generation sequencing minimal residual disease (MRD) and PET scan in transplant eligible myeloma patients.

Minimal residual disease (MRD) assays allow response assessment in patients with multiple myeloma (MM), and negativity is associated with improved survival outcomes. The role of highly sensitive next generation sequencing (NGS) MRD in combination with functional imaging remains to be validated. We performed a retrospective analysis on MM patients who underwent frontline autologous stem cell transplant (ASCT). Patients were evaluated at day 100 post-ASCT with NGS-MRD and positron emission tomography (PET-CT). Patients with ≥ 2 MRD measurements were included in a secondary analysis for sequential measurements. 186 patients were included. At day 100, 45 (24.2%) patients achieved MRD negativity at a sensitivity threshold of 10-6. MRD negativity was the most predictive factor for longer time to next treatment (TTNT). Negativity rates did not differ according to MM subtype, R-ISS Stage nor cytogenetic risk. PET-CT and MRD had poor agreement, with high rates of PET-CT negativity in MRD-positive patients. Patients with sustained MRD negativity had longer TTNT, regardless of baseline risk characteristics. Our results show that the ability to measure deeper and sustainable responses distinguishes patients with better outcomes. Achieving MRD negativity was the strongest prognostic marker and could help guide therapy-related decisions and serve as a response marker for clinical trials.

Prospective evaluation of minimal residual disease in the phase II FORTE trial: a head-to-head comparison between multiparameter flow cytometry and next-generation sequencing.

Background: Limited data are available on the concordance between multiparameter flow cytometry (MFC) and next-generation sequencing (NGS) for minimal residual disease (MRD) detection in a large trial for multiple myeloma (MM) patients. Methods: MRD was explored in the FORTE trial for transplant-eligible MM patients randomised to three carfilzomib-based induction-intensification-consolidation treatments and carfilzomib-lenalidomide (KR) vs R maintenance. MRD was assessed by 8-colour 2nd-generation flow cytometry in patients with ≥very good partial response before maintenance. NGS was performed in case of suspected complete response (CR) in a correlative subanalysis. Biological/prognostic concordance between MFC and NGS, conversion to MRD negativity during maintenance, and 1-year/2-year sustained MRD negativity were explored. Findings: Between September 28, 2015 and December 22, 2021, 2020 samples were available for MFC and 728 for the simultaneous MFC/NGS correlation in the "suspected CR population". Median follow-up was 62 months. Biological agreement was 87% at the 10-5 and 83% at the 10-6 cut-offs. A remarkable prognostic concordance was observed: hazard ratios in MFC-MRD and NGS-MRD-negative vs -positive patients were 0.29 and 0.27 for progression-free survival (PFS) and 0.35 and 0.31 for overall survival, respectively (p < 0.05). During maintenance, 4-year PFS was 91% and 97% in 1-year sustained MFC-MRD-negative and NGS-MRD-negative patients (10-5), respectively, and 99% and 97% in 2-year sustained MFC-MRD-negative and NGS-MRD-negative patients, regardless of treatment received. The conversion rate from pre-maintenance MRD positivity to negativity during maintenance was significantly higher with KR vs R both by MFC (46% vs 30%, p = 0.046) and NGS (56% vs 30%, p = 0.046). Interpretation: The significant biological/clinical concordance between MFC and NGS at the same sensitivity suggests their possible use in the evaluation of one of the currently strongest predictors of outcome. Funding: Amgen, Celgene/Bristol Myers Squibb, Multiple Myeloma Research Foundation.

Challenges to Using Big Data in Cancer.

Big data in healthcare can enable unprecedented understanding of diseases and their treatment, particularly in oncology. These data may include electronic health records, medical imaging, genomic sequencing, payor records, and data from pharmaceutical research, wearables, and medical devices. The ability to combine datasets and use data across many analyses is critical to the successful use of big data and is a concern for those who generate and use the data. Interoperability and data quality continue to be major challenges when working with different healthcare datasets. Mapping terminology across datasets, missing and incorrect data, and varying data structures make combining data an onerous and largely manual undertaking. Data privacy is another concern addressed by the Health Insurance Portability and Accountability Act, the Common Rule, and the General Data Protection Regulation. The use of big data is now included in the planning and activities of the FDA and the European Medicines Agency. The willingness of organizations to share data in a precompetitive fashion, agreements on data quality standards, and institution of universal and practical tenets on data privacy will be crucial to fully realizing the potential for big data in medicine.

Case Studies for Overcoming Challenges in Using Big Data in Cancer.

The analysis of big healthcare data has enormous potential as a tool for advancing oncology drug development and patient treatment, particularly in the context of precision medicine. However, there are challenges in organizing, sharing, integrating, and making these data readily accessible to the research community. This review presents five case studies illustrating various successful approaches to addressing such challenges. These efforts are CancerLinQ, the American Association for Cancer Research Project GENIE, Project Data Sphere, the National Cancer Institute Genomic Data Commons, and the Veterans Health Administration Clinical Data Initiative. Critical factors in the development of these systems include attention to the use of robust pipelines for data aggregation, common data models, data deidentification to enable multiple uses, integration of data collection into physician workflows, terminology standardization and attention to interoperability, extensive quality assurance and quality control activity, incorporation of multiple data types, and understanding how data resources can be best applied. By describing some of the emerging resources, we hope to inspire consideration of the secondary use of such data at the earliest possible step to ensure the proper sharing of data in order to generate insights that advance the understanding and the treatment of cancer.

Minimal Residual Disease in Myeloma: Application for Clinical Care and New Drug Registration.

The development of novel agents has transformed the treatment paradigm for multiple myeloma, with minimal residual disease (MRD) negativity now achievable across the entire disease spectrum. Bone marrow-based technologies to assess MRD, including approaches using next-generation flow and next-generation sequencing, have provided real-time clinical tools for the sensitive detection and monitoring of MRD in patients with multiple myeloma. Complementary liquid biopsy-based assays are now quickly progressing with some, such as mass spectrometry methods, being very close to clinical use, while others utilizing nucleic acid-based technologies are still developing and will prove important to further our understanding of the biology of MRD. On the regulatory front, multiple retrospective individual patient and clinical trial level meta-analyses have already shown and will continue to assess the potential of MRD as a surrogate for patient outcome. Given all this progress, it is not surprising that a number of clinicians are now considering using MRD to inform real-world clinical care of patients across the spectrum from smoldering myeloma to relapsed refractory multiple myeloma, with each disease setting presenting key challenges and questions that will need to be addressed through clinical trials. The pace of advances in targeted and immune therapies in multiple myeloma is unprecedented, and novel MRD-driven biomarker strategies are essential to accelerate innovative clinical trials leading to regulatory approval of novel treatments and continued improvement in patient outcomes.

Generation after generation: exploring the psychological impact of providing genetic services through a cascading approach.

PURPOSE: The provision of genetic services often occurs in a cascading fashion within families experiencing inherited diseases. This study examines whether previous family experiences with genetic services influences levels of psychological well-being of family members receiving services later. METHODS: Two hundred ninety-seven persons from 38 families with Lynch syndrome completed questionnaires before receiving genetic services. Baseline levels of test-related distress, depressive symptoms, and cancer worries were assessed in relationship to the (1) amount of time elapsed since services were provided to the index case and (2) generation of the family member relative to the index case. RESULTS: Family members in the same generation as the index case experienced significant increases in test-related distress (P = 0.003) and cancer worry (P = 0.001) with increasing time between receipt of genetic test results by the index case and provision of services to family members. Change in the number of depressive symptoms was not significant (P = 0.17). CONCLUSION: The provision of genetic services through a cascading approach significantly increases distress and worry among family members within the same generation as the index case who receive services at increasingly distant time intervals. Additional research is needed to explore social influences after the introduction of genetic services.

Sil phosphorylation in a Pin1 binding domain affects the duration of the spindle checkpoint.

SIL is an immediate-early gene that is essential for embryonic development and is implicated in T-cell leukemia-associated translocations. We now show that the Sil protein is hyperphosphorylated during mitosis or in cells blocked at prometaphase by microtubule inhibitors. Cell cycle-dependent phosphorylation of Sil is required for its interaction with Pin1, a regulator of mitosis. Point mutation of the seven (S/T)P sites between amino acids 567 and 760 reduces mitotic phosphorylation of Sil, Pin1 binding, and spindle checkpoint duration. When a phosphorylation site mutant Sil is stably expressed, the duration of the spindle checkpoint is shortened in cells challenged with taxol or nocodazole, and the cells revert to a G2-like state. This event is associated with the downregulation of the kinase activity of the Cdc2/cyclin B1 complex and the dephosphorylation of the threonine 161 on the Cdc2 subunit. Sil downregulation by plasmid-mediated RNA interference limited the ability of cells to activate the spindle checkpoint and correlated with a reduction of Cdc2/cyclin B1 activity and phosphorylation on T161 on the Cdc2 subunit. These data suggest that a critical region of Sil is required to mediate the presentation of Cdc2 activity during spindle checkpoint arrest.

Pubertal impairment in Nhlh2 null mice is associated with hypothalamic and pituitary deficiencies.

Pubertal development is impaired in mice lacking the basic helix-loop-helix transcription factor Nhlh2. The mechanisms underlying changes in reproduction in Nhlh2-deficient mice (Nhlh2(-/-)) are unclear. Here we show that hypothalamic GnRH-1 content is reduced in adult Nhlh2(-/-) mice as is the number of GnRH-1 neurons localized to mid- and caudal hypothalamic regions. This reduction was detected postnatally after normal migration of GnRH-1 neurons within nasal regions had occurred. Phenotype rescue experiments showed that female Nhlh2(-/-) mice were responsive to estrogen treatment. In contrast, puberty could not be primed in female Nhlh2(-/-) mice with a GnRH-1 regimen. The adenohypophysis of Nhlh2(-/-) mice was hypoplastic although it contained a full complement of the five anterior pituitary cell types. GnRH-1 receptors (GnRHRs) were reduced in Nhlh2(-/-) pituitary gonadotropes as compared with wild type. In vitro assays indicated that Nhlh2 expression is regulated in parallel with GnRHR expression. However, direct transcriptional activity of Nhlh2 on the GnRHR promoter was not found. These results indicate that Nhlh2 plays a role in the development and functional maintenance of the hypothalamic-pituitary-gonadal axis at least at two levels: 1) in the hypothalamus by regulating the number and distribution of GnRH-1 neurons and, 2) in the developing and mature adenohypophysis.

Gene expression differences in normal esophageal mucosa associated with regression and progression of mild and moderate squamous dysplasia in a high-risk Chinese population.

A randomized, double-blinded, placebo-controlled 2 x 2 factorial chemoprevention trial was conducted in Linxian, China to assess the effects of selenomethionine and celecoxib on the natural history of esophageal squamous dysplasia. Results from this study indicated that asymptomatic adults with mild dysplasia were more likely to show an improvement when treated with selenomethionine compared with placebo (P = 0.02). Prompted by this finding, we examined the molecular profiles associated with regression and progression of dysplastic lesions in normal mucosa from 29 individuals, a subset of the Linxian cohort, using the Affymetrix U133A chip. Twenty differentially expressed genes were associated with regression and 129 were associated with progression when we compared the change in gene expression over time. Genes associated with immune response (n = 15), cell cycle (n = 15), metabolism (n = 15), calcium transport or calcium ion activity (n = 10), regulation of transcription (n = 9), signal transduction (n = 7), cytoskeleton and microtubules (n = 5), nucleotide processing and biosynthesis (n = 4), G-coupled signaling (n = 4), and apoptosis (n = 3) were present in the list of 149 genes. Using the Expression Analysis Systematic Explorer pathway analysis program, only the immune response pathway was significantly overrepresented among these 149 genes. Individuals whose lesions regressed seemed to have higher expression of genes associated with immune stimulation, such as antigen presentation, survival of T cells, and T-cell activation (HLA-DRA, HLA-DPA1, HLA-DBQ1, CD58, and FCER1A). In contrast, individuals whose lesions progressed had higher expression of genes involved in immune suppression and inflammation (CNR2, NFATC4, NFRKB, MBP, INHBB, CMKLR1, CRP, ORMS, SERPINA7, and SERPINA1). These data suggest that local and systemic immune responses may influence the natural history of esophageal squamous dysplasia.

High-throughput sequencing of the T cell receptor ß gene identifies aggressive early-stage mycosis fungoides.

Mycosis fungoides (MF), the most common cutaneous T cell lymphoma (CTCL) is a malignancy of skin-tropic memory T cells. Most MF cases present as early stage (stage I A/B, limited to the skin), and these patients typically have a chronic, indolent clinical course. However, a small subset of early-stage cases develop progressive and fatal disease. Because outcomes can be so different, early identification of this high-risk population is an urgent unmet clinical need. We evaluated the use of next-generation high-throughput DNA sequencing of the T cell receptor ß gene (TCRB) in lesional skin biopsies to predict progression and survival in a discovery cohort of 208 patients with CTCL (177 with MF) from a 15-year longitudinal observational clinical study. We compared these data to the results in an independent validation cohort of 101 CTCL patients (87 with MF). The tumor clone frequency (TCF) in lesional skin, measured by high-throughput sequencing of the TCRB gene, was an independent prognostic factor of both progression-free and overall survival in patients with CTCL and MF in particular. In early-stage patients, a TCF of >25% in the skin was a stronger predictor of progression than any other established prognostic factor (stage IB versus IA, presence of plaques, high blood lactate dehydrogenase concentration, large-cell transformation, or age). The TCF therefore may accurately predict disease progression in early-stage MF. Early identification of patients at high risk for progression could help identify candidates who may benefit from allogeneic hematopoietic stem cell transplantation before their disease becomes treatment-refractory.

Randomized comparison of phone versus in-person BRCA1/2 predisposition genetic test result disclosure counseling.

PURPOSE: This study evaluated whether phone results were equivalent to in-person result disclosure for individuals undergoing BRCA1/2 predisposition genetic testing. METHODS: A total of 111 of 136 subjects undergoing education and counseling for BRCA1/2 predisposition genetic testing agreed to randomization to phone or in-person result disclosure. Content and format for both sessions were standardized. Data from the State-Trait Anxiety Inventory and the Psychological General Well-Being index were collected at baseline and then again at 1 week and 3 months after disclosure of test results. Baseline measures were administered after the following had occurred: counseling/education session had been conducted, informed consent had been obtained, and decision to be tested had been made. Satisfaction and cost assessments were administered after the result session. At 1 week, participants were asked their preferred method of result disclosure. RESULTS: There were no differences in anxiety and general well-being measures between 50 phone and 52 in-person results disclosure. Both groups reported similar rates of satisfaction with services. Among those with a preference, 77% preferred the notification method assigned. There was a statistically significant preference for phone results among the 23% who did not prefer the method assigned. Greater costs were associated with in-person result disclosure. CONCLUSIONS: These data suggest that phone results are a reasonable alternative to traditional in-person BRCA1/2 genetic test disclosure without any negative psychologic outcomes or compromise in knowledge. However, further study is needed in a more clinically representative population to confirm these findings.

Predisposition to arrhythmia and autonomic dysfunction in Nhlh1-deficient mice.

Nhlh1 is a basic helix-loop-helix transcription factor whose expression is restricted to the nervous system and which may play a role in neuronal differentiation. To directly study Nhlh1 function, we generated null mice. Homozygous mutant mice were predisposed to premature, adult-onset, unexpected death. Electrocardiograms revealed decreased total heart rate variability, stress-induced arrhythmia, and impaired baroreceptor sensitivity. This predisposition to arrhythmia is a likely cause of the observed death in the mutant mice. Heterozygosity for the closely related transcription factor Nhlh2 increased the severity of the Nhlh1-null phenotype. No signs of primary cardiac structural or conduction abnormalities could be detected upon necropsy of the null mice. The pattern of altered heart rhythm observed in basal and experimental conditions (stress and pharmacologically induced) suggests that a deficient parasympathetic tone may contribute to the arrhythmia in the Nhlh1-null mouse. The expression of Nhlh1 in the developing brain stem and in the vagal nuclei in the wild-type mouse further supports this hypothesis. The Nhlh1 mutant mouse may thus provide a model to investigate the contribution of the autonomic nervous system to arrhythmogenesis.