BRAF Mutations in Patients with Myeloid Neoplasms: A Cancer Center Multigene Next-Generation Sequencing Analysis Experience.

BRAF mutations are rare in myeloid neoplasms and are reported to be associated with poor treatment outcomes. The purpose of our study is to characterize BRAF mutations in myeloid neoplasms using a next-generation sequencing (NGS) panel based on the experiences of a single cancer center. We conducted a retrospective review of patients with myeloid neoplasms who underwent the HopeSeq studies between January 2018 and September 2023. A total of 14 patients with myeloid neoplasms carrying BRAF mutations were included in our cohort. The clinical, pathological, and molecular features of these patients were investigated. Our study indicates that BRAF mutations are rare in myeloid neoplasms, constituting only 0.53% (14/2632) of all myeloid neoplasm cases, with the most common BRAF mutation being BRAF V600E (4/14; 28.6%). Interestingly, we observed that six out of seven patients with acute myeloid leukemia (AML) exhibited AML with monocytic differentiation, and all the patients with AML exhibited an extremely poor prognosis compared to those without BRAF mutations. TET2 (5/14; 35.7%), ASXL1 (4/14; 28.6%), and JAK2 (4/14; 28.6%) were the three most frequently co-mutated genes in these patients. Moreover, we noted concurrent KMT2A gene rearrangement with BRAF mutations in three patients with AML (3/7; 42.9%). Our study suggests that although BRAF mutations are rare in myeloid neoplasms, they play a crucial role in the pathogenesis of specific AML subtypes. Furthermore, RAS pathway alterations, including BRAF mutations, are associated with KMT2A gene rearrangement in AML. However, these findings warrant further validation in larger studies.

High response rates and transition to transplant after novel targeted and cellular therapies in adults with relapsed/refractory acute lymphoblastic leukemia with Philadelphia-like fusions.

Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) is associated with a poor response to standard chemotherapy. However, outcomes with novel antibody and cellular therapies in relapsed/refractory (r/r) Ph-like ALL are largely unknown. We conducted a single-center retrospective analysis of adult patients (n = 96) with r/r B-ALL and fusions associated with Ph-like who received novel salvage therapies. Patients were treated with 149 individual novel regimens (blinatumomab = 83, inotuzumab ozogamicin [InO] = 36, and CD19CAR T cells = 30). The median age at first novel salvage therapy was 36 years (range; 18-71). Ph-like fusions were IGH::CRLF2 (n = 48), P2RY8::CRLF2 (n = 26), JAK2 (n = 9), ABL-class (n = 8), EPOR::IGH (n = 4) and ETV6::NTRK2 (n = 1). CD19CAR T cells were administered later in the course of therapy compared to blinatumomab and InO (p < .001) and more frequently in recipients who relapsed after allogeneic hematopoietic cell transplantation (alloHCT) (p = .002). Blinatumomab was administered at an older age compared to InO and CAR T-cells (p = .004). The complete remission (CR)/CR with incomplete hematologic recovery (CRi) rates were 63%, 72%, and 90% following blinatumomab, InO and CD19CAR, respectively, among which 50%, 50%, and 44% of responders underwent consolidation with alloHCT, respectively. In multivariable analysis, the type of novel therapy (p = .044) and pretreatment marrow blasts (p = .006) predicted the CR/CRi rate, while the Ph-like fusion subtype (p = .016), pretreatment marrow blasts (p = .022) and post-response consolidation with alloHCT (p < .001) influenced event-free survival. In conclusion, novel therapies are effective in inducing high remission rates in patients with r/r Ph-like ALL and successfully transitioning the responders to alloHCT.

Favorable impact of allogeneic stem cell transplantation in patients with therapy-related myelodysplasia regardless of TP53 mutational status.

Therapy-related myelodysplastic syndrome is a long-term complication of cancer treatment in patients receiving cytotoxic therapy, characterized by high-risk genetics and poor outcomes. Allogeneic hematopoietic cell transplantation is the only potential cure for this disease, but the prognostic impact of pre-transplant genetics and clinical features has not yet been fully characterized. We report here the genetic and clinical characteristics and outcomes of a relatively large cohort of patients with therapy-related myelodysplastic syndrome (n=67) who underwent allogeneic transplantation, comparing these patients to similarly treated patients with de novo disease (n=199). The 5-year overall survival was not different between patients with therapy-related and de novo disease (49.9% versus 53.9%; P=0.61) despite a higher proportion of individuals with an Intermediate-2/High International Prognostic Scoring System classification (59.7% versus 43.7%; P=0.003) and high-risk karyotypes (61.2% versus 30.7%; P<0.01) among the patients with therapy-related disease. In mutational analysis, TP53 alteration was the most common abnormality in patients with therapy-related disease (n=18: 30%). Interestingly, the presence of mutations in TP53 or in any other of the high-risk genes (EZH2, ETV6, RUNX1, ASXL1: n=29: 48%) did not significantly affect either overall survival or relapse-free survival. Allogeneic stem-cell transplantation is, therefore, a curative treatment for patients with therapy-related myelodysplastic syndrome, conferring a similar long-term survival to that of patients with de novo disease despite higher-risk features. While TP53 alteration was the most common mutation in therapy-related myelodysplastic syndrome, the finding was not detrimental in our case-series.

Outcomes of allogeneic hematopoietic cell transplantation in adults with fusions associated with Ph-like ALL.

Allogenic hematopoietic cell transplantation (alloHCT) is a well-established curative modality for acute lymphoblastic leukemia (ALL), yet large amounts of data describing alloHCT outcomes in Philadelphia (Ph)-like ALL are lacking. We retrospectively analyzed archived DNA samples from consecutive adults with B-cell Ph-negative ALL who underwent alloHCT in complete remission (CR) (n = 127) at our center between 2006 and 2020. Identification of fusions associated with Ph-like ALL was performed using cumulative results from RNA-seq, conventional cytogenetics, fluorescence in situ hybridization, and whole genome array studies. Fusions associated with Ph-like ALL were detected in 56 (44%) patients, of whom 38 were carrying CRLF2r. Compared with other non-Ph-like ALL (n = 71), patients with fusions associated with Ph-like ALL were more frequently Hispanic (P = .008), were less likely to carry high-risk cytogenetics (P < .001), and were more likely to receive blinatumomab prior to HCT (P = .019). With the median followup of 3.5 years, patients with Ph-like ALL fusions had comparable posttransplant outcomes compared with other B-cell ALL: 3-year relapse-free survival (RFS) (41% vs 44%; P = .36), overall survival (OS) (51% vs 50%; P = .59), and relapse (37% vs 31%; P = .47). In multivariable analysis, age (P = .023), disease status at the time of transplant (P < .001), and donor type (P = .015) influenced OS. RFS (primary endpoint) was significantly influenced by disease status (P < .001) and conditioning regimen intensity (P = .014). In conclusion, our data suggest that alloHCT consolidation results in similarly favorable survival outcomes in adult patients with Ph-like fusions and other high-risk B-cell ALL.

Papillary Thyroid Carcinoma with High-Grade Features Versus Poorly Differentiated Thyroid Carcinoma: An Analysis of Clinicopathologic and Molecular Features and Outcome.

Background: Similar to poorly differentiated thyroid carcinoma (PDTC), papillary thyroid carcinoma with high-grade features (PTC HGF) demonstrates increased mitotic activity and/or necrosis; however, PTC HGF is excluded from the World Health Organization (WHO) definition of PDTC based on maintained nuclear features of PTC. Methods: Consecutive tumors that met criteria for PTC HGF, defined as tumors with maintained nuclear features of PTC and mitoses numbering 5 or more per 10 contiguous high-power fields and/or tumor necrosis, and PDTC (defined as per the WHO criteria) were identified. Clinicopathologic characteristics, follow-up data, and targeted next-generation sequencing results were compared between groups. Results: There were 15 PTC HGF and 47 PDTC. PTC HGF was associated with a higher rate of pT4 disease (53% vs. 13%, p = 0.0027) and lymph node metastases (73% vs. 38%, p = 0.049). The disease-specific survival was worse for patients with PTC HGF compared with those with PDTC using Kaplan-Meier estimation (p < 0.001) and was worse in subgroup analysis evaluating patients with widely invasive PDTC (i.e., those with a similar rate of pT4 disease) and PTC HGF (p = 0.040). PTC HGF had a higher BRAFV600E mutation rate (42% vs. 3%; p = 0.003), a trend toward more gene fusions (25% vs. 3%; p = 0.052), and a higher rate of relative gain of 1q (67% vs. 15%; p = 0.002) than PDTC. Conclusions: Our results demonstrate that PTC HGF are important to recognize based on their aggressive behavior. The molecular differences between PTC HGF and PDTC suggest that PTC HGF should be considered a distinct group from PDTC.

Mutation and immune profiling of metaplastic breast cancer: Correlation with survival.

The goal of this study is to characterize the genomic and immune profiles of metaplastic breast cancer (MpBC) and identify the association with survival through an analysis of archived tumor tissue. A next-generation sequencing-based mutational assay (Onco-48) was performed for 21 MpBC patients. Clinicopathologic characteristics were captured, including relapse free survival (RFS) and overall survival (OS). Immunohistochemistry (IHC) for CD3, CD4, CD8, and programmed death-ligand 1 (PD-L1) was also performed. Recurrence free survival (RFS) at 5 years was 57% (95% CI 0.34-0.75) and overall survival (OS) at 5 years was 66% (95% CI 0.41-0.82). The most commonly altered genes were TP53 (68.4%, 13/19), PIK3CA (42.1%, 8/19), and PTEN (15.8%, 3/19. For patients with PIK3CA mutations, RFS and OS were significantly worse than for those without (HR 5.6, 95% CI 1.33-23.1 and HR 8.0, 95% CI 1.53-41.7, respectively). Cox regression estimated that PD-L1 expression was associated with worse RFS and OS (HR 1.08, 95% CI 1.01-1.16 and HR 1.05, 95% CI 1.00-1.11, respectively, for an absolute increase in PD-L1 expression of 1%). In conclusion, PIK3CA mutation and PD-L1 expression confer poor prognosis in this cohort of patients with MpBC.

Allogeneic Hematopoietic Cell Transplantation Outcomes in Patients Carrying Isocitrate Dehydrogenase Mutations.

BACKGROUND: Mutations in isocitrate dehydrogenase (IDH)1/2 genes result in nicotinamide adenine dinucleotide phosphate-dependent reduction of α-ketoglutarate and formation of 2-hydroxyglutarate, which blocks normal cellular differentiation and promotes leukemogenesis. Nearly 20% of acute myeloid leukemia (AML) patients carry IDH1/2 mutations. Although multiple investigators have described the prognostic implications of IDH mutations in AML patients receiving chemotherapy, the effect of these mutations on outcomes after allogeneic (allo) hematopoietic cell transplantation (HCT) is unknown. PATIENTS AND METHODS: We report on the clinical outcome of a cohort of AML patients, who were tested for IDH mutations and underwent alloHCT at City of Hope (2015-2017). Of a total of 317 screened patients, 99 (31%) underwent alloHCT, of whom 23 carried and 76 did not carry IDH mutations (control). RESULTS: No statistical significance was detected in patient's overall survival (P = .84). With a median follow-up of 7.8 months, 1-year relapse rate of 29% and 13% was seen in the IDH-mutated and control group, respectively (P = .033). IDH1/2 mutation status remained significantly associated with relapse (hazard ratio, 2.8; P = .046) after inclusion of pre-HCT disease status in a multivariable model. CONCLUSION: Our results, despite low patient numbers, indicate that IDH mutations are associated with higher relapse rate after alloHCT. Further prospective studies on post transplantation IDH inhibition is required to improve outcomes in AML patients who carry IDH mutations.

Infant hearing loss and connexin testing in a diverse population.

PURPOSE: Previous studies of connexin-related hearing loss have typically reported on mixed age groups or adults. To further address epidemiology and natural history of connexin-related hearing loss, we conducted a longitudinal study in an ethnically diverse cohort of infants and toddlers under 3 years of age. Our study compares infants with and without connexin-related hearing loss to examine differences in the prevalence of connexin and non-connexin-related hearing loss by ethnic origin, detection by newborn hearing screening, phenotype, neonatal risk factors, and family history. This is the first study to differentiate infants with and without connexin-related hearing loss. METHODS: We enrolled 95 infants with hearing loss from whom both exons of Cx26 were sequenced and the Cx30 deletion was assayed. Demographic, family history, newborn hearing screening data, perinatal, and audiologic records were analyzed. RESULTS: Genetic testing identified biallelic Cx26/30 hearing loss-associated variants in 24.7% of infants with a significantly lower prevalence in Hispanic infants (9.1%). Eighty-two infants underwent newborn hearing screening; 12 infants passed, 3 had connexin-related hearing loss. No differences in newborn hearing screening pass rate, neonatal complications, or hearing loss severity were detected between infants with and without connexin-related hearing loss. Family history correlates with connexin-related hearing loss. CONCLUSIONS: Connexin-related hearing loss occurs in one quarter of infants in an ethnically diverse hearing loss population but with a lower prevalence in Hispanic infants. Not all infants with connexin-related hearing loss fail newborn hearing screening. Family history correlates significantly with connexin-related hearing loss. Genetic testing should not be deferred because of newborn complications. These results will have an impact on genetic testing for infant hearing loss.

Impact of RAS and BRAF mutations on carcinoembryonic antigen production and pattern of colorectal metastases.

AIM: To investigate the impact of RAS and BRAF mutations on the pattern of metastatic disease and carcinoembryonic antigen (CEA) production. METHODS: In this retrospective study, we investigated the impact of RAS and BRAF mutational status on pattern of metastatic disease and CEA production. Only patients presenting with a newly diagnosed metastatic colorectal cancer (CRC) were included. Patients' characteristics, primary tumor location, site of metastatic disease and CEA at presentation were compared between those with and without RAS and BRAF mutations. RESULTS: Among 174 patients, mutations in KRAS, NRAS and BRAF were detected in 47%, 3% and 6% respectively. RAS mutations (KRAS and NRAS) were more likely to be found in African American patients (87% vs 13%; P value = 0.0158). RAS mutations were associated with a higher likelihood of a normal CEA (< 5 ng/mL) at presentation. BRAF mutations were more likely to occur in females. We were not able to confirm any association between mutational status and site of metastatic disease at initial diagnosis. CONCLUSION: No association was found between RAS and BRAF mutations and sites of metastatic disease at the time of initial diagnosis in our cohort. Patients with RAS mutations were more likely to present with CEA levels < 5 ng/mL. These findings may have clinical implications on surveillance strategies for RAS mutant patients with earlier stages of CRC.

A novel method for creating artificial mutant samples for performance evaluation and quality control in clinical molecular genetics.

The lack of readily available, patient-derived materials for molecular genetic testing of many heterozygous or rare disorders creates a major impediment for laboratory proficiency and quality control procedures. The paucity of clinically derived mutation-positive samples could be surmounted if it were possible to construct artificial samples containing mutations of interest that would sufficiently resemble natural human samples. Such samples could then function as acceptable and realistic performance evaluation challenges and quality control reagents for recipient laboratories. Using the cystic fibrosis gene (CFTR) as a prototype, we have devised and executed experiments designed to generate unique DNA samples that could be used for these purposes. We used site-directed mutagenesis to generate mutations of interest in plasmid DNA derived from common bacterial artificial chromosome sources containing the cystic fibrosis transmembrane conductance receptor gene. CFTR mutations G85E and 1078delT were chosen to represent mutations in the original American College of Medical Genetics-recommended population-screening panel of 25 mutations. DNA samples containing predetermined concentrations and ratios of wild-type and mutated plasmids, bacterial artificial chromosomes of interest, and nonhuman genomic carrier DNA were characterized and tested in-house and in a group of nine pilot testing laboratories using a variety of technical platforms. The results indicate that these constructs, containing CFTR mutations in heterozygous and homozygous states, can serve as valid and accessible materials for quality assurance, including performance evaluation, proficiency testing, and assay quality control.

Mitoxantrone/ifosfamide/etoposide salvage regimen with rituximab for in vivo purging in patients with relapsed lymphoma.

Treatment with the anti-CD20 antibody rituximab prior to stem cell collection may lead to tumor-free stem cell collections in patients with B-cell lymphoma undergoing autologous stem cell transplantation. To test the feasibility of obtaining polymerase chain reaction (PCR)-negative stem cell collection, 30 patients with a variety of B-cell lymphomas were enrolled in a protocol employing a common MINE (mitoxantrone/ifosfamide/etoposide) salvage regimen with rituximab (in vivo purging). Rituximab 400 mg/m2 was administered weekly for 3 weeks on days 1, 6, and 8 in relation to the last MINE cycle, which was followed by growth factor-stimulated peripheral stem cell collection. The median number of CD34(+) cells/kg was 2.5 million cells/kg collected over a median of 5 days. Polymerase chain reaction amplification for the t (14;18) or the heavy-chain gene rearrangement was performed prior to treatment and on the leukapheresis sample. Out of 15 patients who had a positive PCR signal prior to treatment, 10 had PCR-negative stem cell collections, whereas 5 had PCR-positive stem cell collections. After high-dose chemotherapy and stem cell transplant, all patients with a PCR-positive signal pretreatment became PCR negative. We conclude that rituximab may increase the yield of tumor-free stem cells. Higher rates of PCR negativity have been reported when more intense and protracted chemoimmunotherapy regimens have been employed. The magnitude of clinical benefit and the significance of the PCR analysis in stem cells after rituximab requires larger studies.

Characterization of 107 genomic DNA reference materials for CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1: a GeT-RM and Association for Molecular Pathology collaborative project.

Pharmacogenetic testing is becoming more common; however, very few quality control and other reference materials that cover alleles commonly included in such assays are currently available. To address these needs, the Centers for Disease Control and Prevention's Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, have characterized a panel of 107 genomic DNA reference materials for five loci (CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1) that are commonly included in pharmacogenetic testing panels and proficiency testing surveys. Genomic DNA from publicly available cell lines was sent to volunteer laboratories for genotyping. Each sample was tested in three to six laboratories using a variety of commercially available or laboratory-developed platforms. The results were consistent among laboratories, with differences in allele assignments largely related to the manufacturer's assay design and variable nomenclature, especially for CYP2D6. The alleles included in the assay platforms varied, but most were identified in the set of 107 DNA samples. Nine additional pharmacogenetic loci (CYP4F2, EPHX1, ABCB1, HLAB, KIF6, CYP3A4, CYP3A5, TPMT, and DPD) were also tested. These samples are publicly available from Coriell and will be useful for quality assurance, proficiency testing, test development, and research.

Development and characterization of reference materials for MTHFR, SERPINA1, RET, BRCA1, and BRCA2 genetic testing.

Well-characterized reference materials (RMs) are integral in maintaining clinical laboratory quality assurance for genetic testing. These RMs can be used for quality control, monitoring of test performance, test validation, and proficiency testing of DNA-based genetic tests. To address the need for such materials, the Centers for Disease Control and Prevention established the Genetic Testing Reference Material Coordination Program (GeT-RM), which works with the genetics community to improve public availability of characterized RMs for genetic testing. To date, the GeT-RM program has coordinated the characterization of publicly available genomic DNA RMs for a number of disorders, including cystic fibrosis, Huntington disease, fragile X, and several genetic conditions with relatively high prevalence in the Ashkenazi Jewish population. Genotypic information about a number of other cell lines has been collected and is also available. The present study includes the development and commutability/genotype characterization of 10 DNA samples for clinically relevant mutations or sequence variants in the following genes: MTHFR; SERPINA1; RET; BRCA1; and BRCA2. DNA samples were analyzed by 19 clinical genetic laboratories using a variety of assays and technology platforms. Concordance was 100% for all samples, with no differences observed between laboratories using different methods. All DNA samples are available from Coriell Cell Repositories and characterization information can be found on the GeT-RM website.