Smadbeck, James B.

Abstract Introduction/Objective High-grade endometrial cancer (EC) recurs in 50% of patients following surgery and first-line chemotherapy. There are no established blood-based biomarkers with sufficient specificity and sensitivity to monitor EC patients for residual disease and recurrence. The ability to detect and monitor EC currently relies on serial imaging based approaches. Liquid biopsies via blood-based tests for cell-free circulating tumor DNA (ctDNA) are becoming increasingly popular, but none have been FDA approved for EC and current approaches have not been validated in patients with uncommon tumor mutations. We hypothesized that personalized testing panels based on tumor-specific chromosomal rearrangements would allow ctDNA to be monitored and that ctDNA presence correlates with other clinical indicators of recurrence. Methods/Case Report Twelve patients with FIGO Stage I-IV and biopsy confirmed high-grade EC were prospectively enrolled and underwent primary tumor resection. DNA was isolated from tumor tissue and sequenced on Illumina NGS platforms [San Diego, CA]. BIMA algorithm and SVAtools identified junctions of somatic chromosomal rearrangements and 4-5 junctions were chosen per patient, based primarily on copy number/amplification level. Blood was collected before surgery, and longitudinally between 1 and 7 timepoints. Total cell-free DNA (cfDNA) was extracted from platelet poor plasma with the Qiagen CNA kit [Germany]. Personalized ctDNA qPCR Taqman [IDT, Coralville, Iowa] probe assays were developed with pooled pre-amplification to maximize sensitivity for the multiple junctions and normalized to input. Results (if a Case Study enter NA) ctDNA was detected in 9/12 and 5/12 cases pre- and post-surgery, respectively. The detection or absence of measurable ctDNA following surgery showed high correlation with cancer recurrence as detected by CT or MRI scans. Conclusion The detection of ctDNA in high-grade EC using personalized junction panels shows promise for a blood-based assay that may help physicians better monitor and treat patients.

AbstractAcute lymphoblastic leukemia (B‐ALL) with intrachromosomal amplification of chromosome 21 (iAMP21‐ALL) represents a recurrent high‐risk cytogenetic abnormality and accurate identification is critical for appropriate clinical management. Identification of iAMP21‐ALL has historically relied on fluorescence in situ hybridization (FISH) using a RUNX1 probe. Current classification requires ≥ five copies of RUNX1 per cell and ≥ three additional copies of RUNX1 on a single abnormal iAMP21‐chromosome. We sought to evaluate the performance of the RUNX1 probe in the identification of iAMP21‐ALL. This study was a retrospective evaluation of iAMP21‐ALL in the Mayo Clinic and Children's Oncology Group cohorts. Of 207 cases of iAMP21‐ALL, 188 (91%) were classified as “typical” iAMP21‐ALL, while 19 (9%) cases were classified as “unusual” iAMP21‐ALL. The “unusual” iAMP21 cases did not meet the current definition of iAMP21 by FISH but were confirmed to have iAMP21 by chromosomal microarray. Half of the “unusual” iAMP21‐ALL cases had less than five RUNX1 signals, while the remainder had ≥ five RUNX1 signals with some located apart from the abnormal iAMP21‐chromosome. Nine percent of iAMP21‐ALL cases fail to meet the FISH definition of iAMP21‐ALL demonstrating that laboratories are at risk of misidentification of iAMP21‐ALL when relying only on the RUNX1 FISH probe. Incorporation of chromosomal microarray testing circumvents these risks.

Abstract The World Health Organization category of myeloid/lymphoid neoplasms with eosinophilia and PDGFRA rearrangements is composed of a heterogeneous group of neoplasms that can present as a myeloproliferative neoplasm, acute myeloid leukemia, myeloid sarcoma, or lymphoblastic leukemia/lymphoma. The overall outcome of these neoplasms is favorable with imatinib therapy. Herein, we describe an adult female patient with a myeloid neoplasm accompanied by eosinophilia and a novel USP25::PDGFRA gene fusion.

Abstract Purpose: Multiple myeloma (MM) is a plasma cell malignancy characterized by a collection of cytogenetic abnormalities that drive clinical presentation, response to treatment, and prognosis. The most common immunoglobulin heavy chain (IGH) translocation found in MM, t(11;14), results in CCND1 overexpression. Although t(11;14) is considered a standard risk cytogenetic abnormality, heterogeneity in patient outcomes and response to therapy within the t(11;14) group has been reported. In contrast to t(11;14) mantle cell lymphoma, where most cases (73%) have a typical, balanced t(11;14) rearrangement, only 47% of t(11;14) MM have a typical, balanced t(11;14) FISH pattern; the remaining 53% of MM are characterized by an atypical t(11;14) FISH result (Dalland, Genes Chromosomes Cancer, 2021). Here, we compared the genetic characteristics of typical vs. atypical t(11;14) MM and the association with other genomic abnormalities, CCND1 expression, IGH breakpoint location and patient outcomes. Methods: We performed a retrospective study of newly diagnosed t(11;14) MM patients seen at Mayo Clinic (N=411) or enrolled in the MMRF CoMMpass trial (N=167). The t(11;14) FISH results of the Mayo Clinic samples were segmented into two groups: typical CCND1/IGH signal pattern (n=179, 3 red, 3 green, 2 fusion signals) and atypical CCND1/IGH signal pattern (n=232, any FISH pattern deviating from typical). The CoMMpass cases had both RNAseq and long-insert WGS data and were analyzed to detect gene expression, structural and copy number variants and IGH breakpoint locations. These data were used to classify the t(11;14) as typical (n=63) or atypical (n=104) and whether there was a single t(11;14) fusion (n=107) or a gain of the t(11;14) fusion (n=60), and whether these cases were associated with changes in gene expression or breakpoint location. Differences between the groups were measured using χ2 and Wilcoxon tests. Survival curves were estimated using Kaplan Meier and compared using the Log-Rank test. Statistical significance was determined at p<0.05. Results: Of the Mayo t(11;14) cohort, 232/411 (56%) had an atypical t(11;14) FISH pattern and 179/411 (44%) had a typical t(11;14) FISH pattern. Although an atypical t(11;14) FISH pattern was not significantly associated with monosomy 17/17p deletion, MYC rearrangements, 1q gain or ISS stage III, it was significantly associated with the presence of any trisomy in comparison to typical t(11;14) cases (40/222=18% vs.12/166=7%, p=0.002). Atypical t(11;14) cases had an increase in CCND1 gene expression compared to typical t(11;14) cases (median 375.0 RPKM vs. 266.4 RPKM, p<0.0001) and cases with a gain of the t(11;14) fusion were associated with even greater CCND1 expression compared to cases with a single t(11;14) fusion (median 459.4 RPKM vs. 263.2 RPKM, p<0.0001). We next evaluated whether the t(11:14) FISH pattern was associated with differences in IGH breakpoint locations. Nearly 80.0% of atypical t(11;14) cases had a breakpoint within the constant region of the IGH locus commonly reported for MM, while only 61.0% of the typical cases had constant region breakpoints (p=0.035); the remaining cases had breakpoints involving the VDJ region. We next evaluated the impact of atypical t(11;14) FISH on OS. In Mayo cases, OS was significantly shorter in patients with atypical FISH patterns compared to typical t(11;14) FISH patterns (6.9 years [95% CI: 5.1-7.8] vs. 9.0 years [95% CI: 7.0-11.6], p=0.019). Although atypical t(11;14) was associated with increased risk of death (RR 1.45, p=0.021), this finding was not retained in a multivariate model including high risk abnormalities such as age, ISS stage, 17p deletion and 1q gain demonstrating atypical t(11;14) is not an independent predictor of poor outcome when treated with standard therapies. Conclusions: Atypical t(11;14) FISH in MM is found in approximately half of t(11;14) cases, is associated with trisomies, higher CCND1 expression and is more likely to have a breakpoint involving the constant region of the IGH locus. The enrichment of VDJ breakpoints within typical t(11;14) MM is consistent with enrichment of VDJ IGH breakpoints in MCL, which are mostly characterized by typical t(11;14) FISH patterns. While not an independent predictor of outcome with standard therapies, it remains to be evaluated whether patients with typical or atypical t(11;14) have a differential response to novel agents, such as venetoclax. Disclosures Fonseca: Janssen: Consultancy; Novartis: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; Merck: Consultancy; Juno: Consultancy; Caris Life Sciences: Membership on an entity's Board of Directors or advisory committees; Kite: Consultancy; Aduro: Consultancy; OncoTracker: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; AbbVie: Consultancy; Patent: Prognosticaton of myeloma via FISH: Patents & Royalties; Mayo Clinic in Arizona: Current Employment; Amgen: Consultancy; BMS: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Bayer: Consultancy; Scientific Advisory Board: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees. Bergsagel: Pfizer: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Oncopeptides: Consultancy, Honoraria; Genetech: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Patents & Royalties: human CRBN mouse. Kumar: Merck: Research Funding; Carsgen: Research Funding; Roche-Genentech: Consultancy, Research Funding; Beigene: Consultancy; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bluebird Bio: Consultancy; Antengene: Consultancy, Honoraria; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Research Funding; Sanofi: Research Funding.

Abstract Purpose: Multiple myeloma (MM) is a plasma cell (PC) malignancy with an increasing incidence in the United States. Fluorescence in situ hybridization (FISH) is currently the gold-standard diagnostic assay to detect recurrent genomic abnormalities of prognostic and therapeutic significance in MM. Hyperdiploid MM, a standard risk abnormality, is characterized by gains of odd-numbered chromosomes including chromosome 15. While gains of chromosome 15 have been reported in approximately 53% of standard risk MM patients (Chretien, Blood, 2015), losses of chromosome 15 are rarely reported. Enumeration of chromosome 15 centromere is detected using the alpha satellite FISH probe. We previously discovered ~9% of cases had false-negative chromosome 15 FISH results when compared to Mate Pair Sequencing (MPseq) (Smadbeck, BCJ, 2019). To our knowledge, no previous studies have reported a reduced performance of the alpha satellite D15Z4 FISH probe in PC FISH studies. A false-negative result for D15Z4 could result in an underreporting of trisomy 15 and overreporting of monosomy 15. Here, we investigated the incidence of false-negative D15Z4 FISH results by examining the presence of monosomy 15 from patients with a monoclonal gammopathy. Methods: We identified 1231 samples from patients with a monoclonal gammopathy who had undergone uniform testing to identify MM-specific cytogenetic abnormalities. DNA from bone marrow samples was genotyped on the Precision Medicine Research Array and biogeographical ancestry was assessed using the Geographic Population Structure Origins tool (Elhaik, Nat Commun, 2014; Home DNA 2016). FISH of immunoglobulin (cIg)-stained PCs was performed as described (Baughn, BCJ, 2018). Chromosome 15 centromere probe (Vysis CEP 15 D15Z4, Abbott Molecular, Abbott Park, IL) was used for chromosome 15 enumeration. Samples were divided into two groups: Cases with or without evidence of monosomy 15 by FISH. Mann-Whitney U test was applied to compare difference between groups for data deviating from normal distribution. A Chi-squared test evaluating the differences across these two groups was used. Results: We identified monosomy 15 in 31 cases (2.5% of cohort). Monosomy 15 was observed in nearly 100% of both the PC (cIg+) and non-PC (cIg-) populations, by contrast to the other recurrent PC-specific genomic abnormalities which were restricted to only the PC population. All samples with a banded analysis revealed most metaphases had two normal chromosome 15s demonstrating that the D15Z4 FISH probe failed to correctly enumerate the number of chromosome 15 centromere signals. Of the samples with a monosomy 15 result, the median African ancestry was 76.2% (95% percentiles: 77.5%-89.0%), while the median African ancestry of the non-monosomy 15 cohort was 2.3% (95% percentiles: 0%-87.7%) (). Although not all patients with >50% African ancestry had evidence of monosomy 15, 26/31 (83.9%) of patients with monosomy 15 had >50% African ancestry. Accordingly, patients with African ancestry (similarity ≥ 0.70) had 8.02-fold (95% confidence interval: 3.73-17.25, P = 9.92 X10 -8) increased possibility to have monosomy 15. Of cases with monosomy 15, 22 (71.0%) had an IGH rearrangement with the most prevalent IGH partner being CCND1. The incidence of t(11;14) was greater in the monosomy 15 group (13/31:41.9%), in contrast to the non-monosomy 15 group (330/1200=27.5%) (P = 0.12). Conclusions: We report a polymorphic variant of the chromosome 15 centromere resulting in a false-negative FISH result using the D15Z4 FISH probe. Surprisingly, the false-negative FISH result was not uniformly distributed among patients and was highly enriched in individuals of African ancestry. This may be due to a variation of the chromosome 15 alpha satellite in some individuals that prevents the D15Z4 FISH probe from sufficiently binding (O'Keefe, Genome Research, 2000). Although this study focused on identification of monosomy 15, the incidence of a false-negative result is likely greater than we have reported in this study since we did not evaluate cases that had a true trisomy 15 that was reported as normal chromosome 15 enumeration by FISH. This study highlights a critical need to ensure diagnostic reagents have equal performance for all patients independent of their ancestry. Evaluation of genomic events using unbiased approaches such as whole genome sequencing may circumvent some of these limitations. Disclosures Elhaik: DDC: Consultancy. Baird: DDC: Current Employment. Kumar: Antengene: Consultancy, Honoraria; Bluebird Bio: Consultancy; Beigene: Consultancy; BMS: Consultancy, Research Funding; Tenebio: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Novartis: Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche-Genentech: Consultancy, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Consultancy; Carsgen: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding.

Current guidelines suggest screening all patients with idiopathic pulmonary arterial hypertension for genetic aberrations, particularly mutations in Bone Morphogenic Protein Receptor Type II (BMPR2), the gene most commonly implicated in the pathogenesis of PAH. Herein, we present a novel technique used to identify a pathogenic germline BMPR2 alteration in a 36‐year‐old female and family members with hereditary pulmonary arterial hypertension who each screened negative by standard cytogenetics and molecular genetics testing.

Abstract Purpose: Structural variants (SV) of the MYC gene region are common in multiple myeloma and influence disease progression. However, the prognostic significance of different MYC SVs in multiple myeloma has not been clearly established. Experimental Design: We conducted a retrospective study of multiple myeloma comparing MYC SV subtypes identified by next-generation sequencing (NGS) and FISH to MYC expression and disease survival using 140 cases from Mayo Clinic and 658 cases from the MMRF CoMMpass study. Results: MYC SVs were found in 41% of cases and were classified into nine subtypes. A correlation between the presence of a MYC SV and increased MYC expression was identified. Among the nine MYC subtypes, the non-immunoglobulin (non-Ig) insertion subtype was independently associated with improved outcomes, while the Ig insertion subtype, specifically involving the IgL gene partner, was independently associated with poorer outcomes compared with other MYC SV subtypes. Although the FISH methodology failed to detect approximately 70% of all MYC SVs, those detected by FISH were associated with elevated MYC gene expression and poor outcomes suggesting a different pathogenic role for FISH-detected MYC subtypes compared with other MYC subtypes. Conclusions: Understanding the impact of different MYC SVs on disease outcome is necessary for the reliable interpretation of MYC SVs in multiple myeloma. NGS approaches should be considered as a replacement technique for a more comprehensive evaluation of the multiple myeloma clone.

AbstractPlasma cell neoplasms (PCN) and mantle cell lymphoma (MCL) can both harbor t(11;14)(q13;q32) (CCND1/IGH), usually resulting in cyclin D1 overexpression. In some cases, particularly at low levels of disease, it can be morphologically challenging to distinguish between these entities in the bone marrow (BM) since PCN with t(11;14) are often CD20‐positive with lymphoplasmacytic cytology, while MCL can rarely have plasmacytic differentiation. We compared the difference in CCND1/IGH by fluorescence in situ hybridization (FISH) in PCN and MCL to evaluate for possible differentiating characteristics. We identified 326 cases of MCL with t(11;14) and 279 cases of PCN with t(11;14) from either formalin‐fixed, paraffin‐embedded tissue or fresh BM specimens. The “typical,” balanced CCND1/IGH FISH signal pattern was defined as three total CCND1 signals, three total IGH signals, and two total fusion signals. Any deviation from the “typical” pattern was defined as an “atypical” pattern, which was further stratified into “gain of fusion” vs “complex” patterns. There was a significantly higher proportion of cases that showed an atypical FISH pattern in PCN compared with MCL (53% vs 27%, P < .0001). There was also a significantly higher proportion of cases that showed a complex FISH pattern in PCN compared with MCL (47% vs 17%, P < .0001). We confirmed these findings using mate‐pair sequencing of 25 PCN and MCL samples. PCN more often have a complex CCND1/IGH FISH pattern compared with MCL, suggesting possible differences in the genomic mechanisms underlying these rearrangements in plasma cells compared with B cells.

AbstractA crucial mutational mechanism in malignancy is structural variation, in which chromosomal rearrangements alter gene functions that drive cancer progression. Herein, the presence and pattern of structural variations were investigated in twelve prospectively acquired treatment‐naïve pancreatic cancers specimens obtained via endoscopic ultrasound (EUS). In many patients, this diagnostic biopsy procedure and specimen is the only opportunity to identify somatic clinically relevant actionable alterations that may impact their care and outcome. Specialized mate pair sequencing (MPseq) provided genome‐wide structural variance analysis (SVA) with a view to identifying prognostic markers and possible therapeutic targets. MPseq was successfully performed on all specimens, identifying highly rearranged genomes with complete SVA on all specimens with > 20% tumour content. SVA identified chimeric fusion proteins and potentially immunogenic readthrough transcripts, change of function truncations, gains and losses of key genes linked to tumour progression. Complex localized rearrangements, termed chromoanagenesis, with broad pattern heterogeneity were observed in 10 (83%) specimens, impacting multiple genes with diverse cellular functions that could influence theragnostic evaluation and responsiveness to immunotherapy regimens. This study indicates that genome‐wide MPseq can be successfully performed on very limited clinically EUS obtained specimens for chromosomal rearrangement detection and potential theragnostic targets.