Human epidermal growth factor receptor 2 (HER2) positive breast carcinomas due to HER2 amplification are associated with aggressive behavior and a poor prognosis. Anti-HER2-targeted therapies are widely used to treat HER2-positive breast carcinomas with excellent outcomes. Accurate identification of HER2 amplification status in breast carcinomas is of important diagnostic and treatment value. Currently, HER2 amplification status is routinely determined by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH) testing. This study will review our past HER2 data to determine and characterize discordant results between HER2 IHC and FISH. It will also determine a potential impact of HER2 amplification status by next-generation sequencing (NGS) on these patients.

Familial cases of myeloproliferative neoplasms (MPN) are relatively common, yet few inherited risk factors have been identified. Exome sequencing of a kindred with a familial cancer syndrome characterized by both MPN and melanoma produced a germline variant in the ERBB2/HER2 gene that co-segregates with disease. To further investigate whether germline ERBB2 variants contribute to MPN predisposition, the frequency of ERBB2 variants was analyzed in 1604 cases that underwent evaluation for hematologic malignancy, including 236 cases of MPN. MPN cases had a higher frequency of rare germline ERBB2 coding variants compared to non-MPN hematologic malignancies (8.9% vs. 4.1%, OR 2.4, 95% CI: 1.4 to 4.0, p = 0.0028) as well as cases without a blood cancer diagnosis that served as an internal control (8.9% vs. 2.7%, OR 3.5, 95% CI: 1.4 to 8.3, p = 0.0053). This finding was validated via comparison to an independent control cohort of 1587 cases without selection for hematologic malignancy (8.9% in MPN cases vs. 5.2% in controls, p = 0.040). The most frequent variant identified, ERBB2 c.1960A > G; p.I654V, was present in MPN cases at more than twice its expected frequency. These data indicate that rare germline coding variants in ERBB2 are associated with an increased risk for development of MPN. The ERBB2 gene is a novel susceptibility locus which likely contributes to cancer risk in combination with additional risk alleles.

The diffuse variant of follicular lymphoma (dFL) is a rare variant of FL lacking t(14;18) that was first described in 2009. In this study, we use a comprehensive approach to define unifying pathologic and genetic features through gold-standard pathologic review, FISH, SNP-microarray, and next-generation sequencing of 16 cases of dFL. We found unique morphologic features, including interstitial sclerosis, microfollicle formation, and rounded nuclear cytology, confirmed absence of t(14;18) and recurrent deletion of 1p36, and showed a novel association with deletion/CN-LOH of 16p13 (inclusive of CREBBP, CIITA, and SOCS1). Mutational profiling demonstrated near-uniform mutations in CREBBP and STAT6, with clonal dominance of CREBBP, among other mutations typical of germinal-center B-cell lymphomas. Frequent CREBBP and CIITA codeletion/mutation suggested a mechanism for immune evasion, while subclonal STAT6 activating mutations with concurrent SOCS1 loss suggested a mechanism of BCL-xL/BCL2L1 upregulation in the absence of BCL2 rearrangements. A review of the literature showed significant enrichment for 16p13 and 1p36 loss/CN-LOH, STAT6 mutation, and CREBBP and STAT6 comutation in dFL, as compared with conventional FL. With this comprehensive approach, our study demonstrates confirmatory and novel genetic associations that can aid in the diagnosis and subclassification of this rare type of lymphoma.

The measurable residual disease (MRD) assessment provides an attractive predictor of allogeneic hematopoietic cell transplnat (alloHCT) outcomes. Cell-free DNA (cfDNA) has been applied to diagnosis, early detection, and disease burden monitoring in various tumors, but its utility as an MRD test in myeloid malignancies has not been systematically evaluated. We sought to determine the differential sensitivity between bone marrow (BM) and cfDNA MRD and to assess the effect of cfDNA MRD on alloHCT outcomes. The technical and clinical validation cohorts, including 82 patients participating in clinical trials (Bone Marrow Transplant Clinical Trials Network-0201 and 0402), were used. Ultradeep error-corrected targeted sequencing was performed on plasma and BM-derived DNA. We demonstrated that 94.6% (range, 93.9-95.3) of cfDNA was derived from hematopoietic tissue. The mutant allele fraction was congruent between BM and cfDNA (rho = 0.8; P < .0001); however, cfDNA seemed to be more sensitive in detecting clones with a variant allele frequency (VAF) of <0.26%. cfDNA-MRD clearance by day 90 after alloHCT (D90) was associated with improved relapse-free survival (RFS, median survival not reached vs 5.5 months; P < .0001) and overall survival (OS, median survival not reached vs 7.3 months; P < .0001) when compared with patients with persistent MRD. Irrespective of pre-alloHCT MRD, D90 cfDNA MRD was associated with inferior 2-year OS (16.7% vs 84.8%; P < .0001) and RFS (16.7% vs 80.7%; P < .0001). cfDNA seems to be an accurate, minimally invasive alternative to BM aspirates in MRD assessment and confers important prognostic implications in patients with myeloid malignancies undergoing alloHCT.

Internal tandem duplications of the juxtamembrane domain of FLT3 (FLT3/ITD) are among the most common mutations in Acute Myeloid Leukemia (AML). Resulting in constitutive activation of the kinase, FLT3/ITD portends a particularly poor prognosis, with reduced overall survival and increased rates of relapse. We previously generated a knock-in mouse, harboring an internal tandem duplication at the endogenous Flt3 locus, which develops a fatal myeloproliferative neoplasm (MPN), but fails to develop acute leukemia, suggesting additional mutations are necessary for transformation. To investigate the potential cooperativity of FLT3/ITD and mutant DNMT3A, we bred a conditional Dnmt3a knockout to a substrain of our Flt3/ITD knock-in mice, and found deletion of Dnmt3a significantly reduced median survival of Flt3ITD/+ mice in a dose dependent manner. As expected, pIpC treated Flt3ITD/+ mice solely developed MPN, while Flt3ITD/+;Dnmt3af/f and Flt3ITD/+;Dnmt3af/+ developed a spectrum of neoplasms, including MPN, T-ALL, and AML. Functionally, FLT3/ITD and DNMT3A deletion cooperate to expand LT-HSCs, which exhibit enhanced self-renewal in serial re-plating assays. These results illustrate that DNMT3A loss cooperates with FLT3/ITD to generate hematopoietic neoplasms, including AML. In combination with FLT3/ITD, homozygous Dnmt3a knock-out results in reduced time to disease onset, LT-HSC expansion, and a higher incidence of T-ALL compared with loss of just one allele. The co-occurrence of FLT3 and DNMT3A mutations in AML, as well as subsets of T-ALL, suggests the Flt3ITD/+;Dnmt3af/f model may serve as a valuable resource for delineating effective therapeutic strategies in two clinically relevant contexts.

Somatic genetic alterations including copy number and point mutations in the androgen receptor (AR) are associated with resistance to therapies targeting the androgen/AR axis in patients with metastatic castration resistant prostate cancer (mCRPC). Due to limitations associated with biopsying metastatic lesions, plasma derived cell-free DNA (cfDNA) is increasingly being used as substrate for genetic testing. AR mutations detected by deep next generation sequencing (NGS) of cfDNA from patients with mCRPC have been reported at allelic fractions ranging from over 25% to below 1%. The lower bound threshold for accurate mutation detection by deep sequencing of cfDNA has not been comprehensively determined and may have locus specific variability. Herein, we used NGS for AR mutation discovery in plasma-derived cfDNA from patients with mCRPC and then used droplet digital polymerase chain reaction (ddPCR) for validation. Our findings show the AR (tTC>cTC) F877L hotspot was prone to false positive mutations during NGS. The rate of error at AR (tTC>cTC) F877L during amplification prior to ddPCR was variable among high fidelity polymerases. These results highlight the importance of validating low-abundant mutations detected by NGS and optimizing and controlling for amplification conditions prior to ddPCR.

The sinonasal tract is a common extranodal site for Rosai-Dorfman disease (RDD). Recently, histiocytes with features of RDD were identified in the clinical setting of chronic sinusitis. This study evaluates whether this phenomenon should be considered part of the RDD spectrum or classified separately as RDD-like histiocytes.

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML), classified by a translocation between chromosomes 15 and 17 [t(15;17)], that is considered a true oncologic emergency though appropriate therapy is considered curative. Therapy is often initiated on clinical suspicion, informed by both clinical presentation as well as direct visualization of the peripheral smear. We hypothesized that genomic imprinting of morphologic features learned by deep learning pattern recognition would have greater discriminatory power and consistency compared to humans, thereby facilitating identification of t(15;17) positive APL. By applying both cell-level and patient-level classification linked to t(15;17) PML/RARA ground-truth, we demonstrate that deep learning is capable of distinguishing APL in both discovery and prospective independent cohort of patients. Furthermore, we extract learned information from the trained network to identify previously undescribed morphological features of APL. The deep learning method we describe herein potentially allows a rapid, explainable, and accurate physician-aid for diagnosing APL at the time of presentation in any resource-poor or -rich medical setting given the universally available peripheral smear.

Wilms tumor 1 (WT1) is a zinc finger transcriptional regulator, and has been implicated as both a tumor suppressor and oncogene in various malignancies. Mutations in the DNA-binding domain of the WT1 gene are described in 10-15% of normal-karyotype AML (NK-AML) in pediatric and adult patients. Similar WT1 mutations have been reported in adult patients with myelodysplastic syndrome (MDS). WT1 mutations have been independently associated with treatment failure and poor prognosis in NK-AML. Internal tandem duplication (ITD) mutations of FMS-like tyrosine kinase 3 (FLT3) commonly co-occur with WT1-mutant AML, suggesting a cooperative role in leukemogenesis. The functional role of WT1 mutations in hematologic malignancies appears to be complex and is not yet fully elucidated. Here, we describe the hematologic phenotype of a knock-in mouse model of a Wt1 mutation (R394W), described in cases of human leukemia. We show that Wt1 +/R394W mice develop MDS which becomes 100% penetrant in a transplant model, exhibit an aberrant expansion of myeloid progenitor cells, and demonstrate enhanced self-renewal of hematopoietic progenitor cells in vitro. We crossbred Wt1 +/R394W mice with knock-in Flt3 +/ITD mice, and show that mice with both mutations (Flt3 +/ITD/Wt1 +/R394W) develop a transplantable MDS/MPN, with more aggressive features compared to either single mutant mouse model.

Genomic testing is often limited by the exhaustible nature of human tissue and blood samples. Here we describe biotinylated amplicon sequencing (BAmSeq), a method that allows for the creation of PCR amplicon based next-generation sequencing (NGS) libraries while retaining the original source DNA.