Knudson, Ryan A.

We searched for oncogenes activated by copy number increases using whole-genome sequencing data of 535 pancreatic ductal adenocarcinomas (PDACs). We found that gains of 1q were the second most common gain, occurring in 213 (39.8%) of PDACs. Single-cell analysis via fluorescence in situ hybridization on 33 cancers confirmed these results. A portion of 1q, rather than the entire 1q arm, was gained in 75 (14.0%) PDACs, allowing us to pinpoint two ~3-megabase regions of 1q that were nearly always gained. These two regions contained NCSTN and PSEN2 , genes that code two subunits of the γ-secretase complex. Evaluation of 267 precancerous lesions revealed that extra copies of NCSTN and PSEN2 were common (49%) in noninvasive neoplasms (high-grade pancreatic intraepithelial neoplasms), which are at relatively high risk for progression to PDACs, but uncommon (6%) in low-grade pancreatic intraepithelial neoplasia lesions, which have low malignant potential. We hypothesize that γ-secretase genes are genetically activated oncogenes in the early phases of pancreatic neoplasia.

Abstract The development of targeted therapy for patients with multiple myeloma (MM) is hampered by the low frequency of actionable genetic abnormalities. Gain or amplification of chromosome 1q (1q+) is the most frequent arm-level copy number gain in patients with MM and is associated with higher risk of progression and death despite recent therapeutic advances. Thus, developing targeted therapy for patients with MM with 1q+ stands to benefit a large portion of patients in need of more effective management. Here, we used large-scale dependency screens and drug screens to systematically characterize the therapeutic vulnerabilities of MM with 1q+ and displayed increased sensitivity to myeloid cell leukemia-1 (MCL1) and phosphatidyl inositol 3-kinase (PI3K) inhibitors. Using single-cell RNA sequencing, we compared subclones with and without 1q+ within the same patient tumors and demonstrated that 1q+ is associated with higher levels of MCL1 and the PI3K pathway. Furthermore, by isolating isogenic clones with different copy number profiles for part of the chromosome 1q arm, we observed increased sensitivity to MCL1 and PI3K inhibitors with arm-level gain. Lastly, we demonstrated synergy between MCL1 and PI3K inhibitors and dissected their mechanism of action in MM with 1q+, uncovering a cytostatic effect. In conclusion, this study highlights that MM with 1q+ may present enhanced sensitivity to MCL1 and PI3K inhibitors, enabling their use at lower doses without sacrificing efficacy, and may thus accelerate the development of targeted therapy for patients with MM and 1q+.

Abstract Given the heterogeneous clinical behavior of pancreatic neuroendocrine tumors (pNETs), improved prognostic markers are needed to guide management and post‐resection surveillance. Patients who underwent resection of large (≥3 cm) sporadic well‐differentiated pNETs from 2000 to 2019 were identified. The Ki‐67 proliferation index was determined using immunohistochemistry, and alternative lengthening of telomeres (ALT) status was assessed using fluorescence in situ hybridization. Recurrence‐free and overall survival were estimated using Kaplan–Meier analysis. Multivariable Cox regression analysis evaluated factors associated with recurrence‐free survival. A total of 106 patients were identified. ALT was positive in 57 (54%) and negative in 49 (46%). Ki‐67 was ≥3% in 74 (70%) and <3% in 32 (30%). Tumors with Ki‐67 ≥3% were more likely to be ALT positive (61% vs. 38%, p = .046). Stratifying by ALT status and Ki‐67 proliferation index, median recurrence‐free survival was 4.6 years for patients with ALT‐positive/Ki‐67 ≥3% tumors, 3.1 years for patients with ALT‐positive/Ki‐67 <3% tumors, 12.4 years for patients with ALT‐negative/Ki‐67 ≥3% tumors, and 20.2 years for patients with ALT‐negative/Ki‐67 <3% tumors ( p < .001). Initial recurrence was distant in 82% and locoregional in 18%. Across all groups, overall survival was similar ( p = .19). In multivariable analysis, advanced age, ALT positivity, perineural invasion, and lymph node metastases were associated with increased recurrence risk (all p < .05). ALT and Ki‐67 provide complementary information on post‐resection recurrence risk, which can guide subsequent surveillance and management strategies. These data support the incorporation of ALT testing into routine clinical practice.

7022 Background: Philadelphia-like Acute Lymphoblastic Leukemia (Ph-like ALL) is a high-risk subset of adult ALL. Until recently, there has not been a systematic platform to recognize this entity in clinical practice. Furthermore, data regarding the role of allogeneic hematopoietic cell transplantation (allo-HCT) is lacking. We conducted this study to identify patients with Ph-like ALL and describe their outcomes in comparison to Ph+ and Ph- ALL with emphasis on the role of allo-HCT. Methods: To identify cases of Ph-like ALL, available diagnostic cytogenetic pellets for patients in the Mayo Clinic ALL cohort (N=365) were tested using a targeted fluorescence in situ hybridization (FISH) panel developed by the Mayo Clinic Genomics Laboratory and includes probes to detect Ph-like-specific rearrangements (i.e., ABL1, ABL2, PDGFRB, JAK2 and CRLF2). Results: Thirty-three (9%) patients with Ph-like ALL were identified, the remaining patients were classified as Ph+ (N=132, 36%) or Ph- ALL (N=200, 55%). Patients with Ph-like ALL were younger (Median: 39 vs. 50 vs. 49 years, P=.01), had higher WBC (Median: 27.9 vs. 21.5 vs. 4.5 x109/L, P<.001), were less likely to achieve CR (91% vs. 99% vs. 96%, P=.02), more likely to be MRD+ (64% vs. 34% vs. 36%, P=.03), had a higher relapse rate (5-year: 39% vs. 24% vs. 38%, P=.01) and lower OS (5-year: 41% vs. 64% vs. 49%, P=.02), see Table. Patients who achieved MRD negativity had better OS (MRD+ vs MRD-, P=.01). Importantly, no statistically significant difference in OS, relapse or non-relapse mortality were noted between the 3 groups in patients who underwent allo-HCT in CR1. Conclusions: Ph-like ALL is a high risk subgroup with increased prevalence in younger adults. Allo-HCT appears to offset the poor prognosis associated with this entity. A targeted FISH panel offers timely recognition of this entity in a clinical setting.[Table: see text]

Abstract Background: Chronic myeloid leukemia (CML) is invariably associated with the reciprocal translocation of BCR and ABL to form the Philadelphia chromosome (Ph), t(9;22)(q34;q11). At diagnosis, a small proportion of patients display additional cytogenetic abnormalities and/or a variable proportion of cytogenetically normal metaphases that coexist with Ph-positive metaphases. The objective of the current study was to examine the prognostic relevance of these two scenarios. Methods: The study population consisted of 65 (47.7% female) consecutive, newly diagnosed CML patients seen at the Mayo Clinic with a median age of 59 years. 44.5 percent were initially treated with interferon alpha (IFN) resulting in complete cytogenetic remission in 10.7 percent for a median length of 5.83 years, partial cytogenetic remission in 3.57 percent for a median length of 2 years. 55.5 percent were treated with imatinib (Gleevec) resulting in complete cytogenetic remission in 54.1 percent for a median length of 3.08 years, partial cytogenetic remission in 13.5% for a median length of 1.08 years. Results: Survival at five years through Kaplan-Meier analysis was approximately 92 percent in patients demonstrating only the Ph chromosome (n = 53), versus 60 percent in patients with additional chromosomal abnormalities (n = 12; p &lt; 0.0001 by both Logrank and Breslow-Gehan-Wilcox analysis). Furthermore, patients with additional chromosomal abnormalities demonstrated lower rates of either complete or partial cytogenetic remission with imatinib therapy. Similarly, five year survival of patients with less than 90% Ph-positive metaphases at diagnosis (n = 4) was approximately 60% compared to 92% in patients with greater than 90% Ph-positive metaphases (n = 61; p = 0.01 by Logrank and p = 0.001 by Breslow-Gehan-Wilcox analysis). Additionally, the former group of patients was significantly less responsive to imatinib. Finally, there was significant correlation between Ph-positive metaphase mosaicism and the presence of additional chromosomal abnormalities (p = 0.05). Conclusion: Although it is tempting to speculate the possibility that Ph-chromosome mosaicism and/or additional cytogenetic abnormalities at presentation of CML is a surrogate for the presence of Ph-negative imatinib-resistant clones, the preliminary results from the current study require validation from a larger study. Figure Figure Figure Figure

Abstract Deletion 5q in Acute Myeloid Leukemia (AML): a Clinicopathological Description of 79 Cases From a Single Institution Rafael Santana-Davila, Shernan G. Holtan, Rhett P. Ketterling, Ryan A. Knudson, David P. Steensma, Dong Chen, James D. Hoyer, Curtis A. Hanson, Ayalew Tefferi. Background: Deletion 5q (del(5q)) is a lenalidomide-sensitive cytogenetic abnormality commonly seen in patients with myeloid disorders. In this study we analyze the clinicopathological characteristics of patients with AML and del(5q). Methods: Patients with del(5q) and AML were identified through a database query of our institutional cytogenetic database from May, 1992 through December, 2005. Both peripheral blood and bone marrow specimens from each patient were morphologically examined by a hematopathologist (DC, JDH and/or CAH). A morphologic diagnosis was given based on current WHO criteria. A critical review of the patient history was performed for each patient. Results: A total 79 patients were identified. Median age was 71 (range 39–87) years, 56% (n=43)were female. The most common subtype according to the FAB classification was M2 (n=23, 29%). In 35 (44%) patients AML developed a median of 9.4 (range 1–294) months since the diagnosis of a pre-existing myeloid malignancy, of which myelodysplastic syndrome accounted for 24 (9 cases of MDS NOS, 6 of RA, 6 of RAEB1, 2 of RAEB-2, and a single RARS), a myeloproliferative neoplasm in 10 (4 cases of a CMPD NOS, 4 cases of PV and a single case of ET and PMF each) and 1 case of a prior history of AML with a t(8;21). The del(5q) was documented in 7 of this 24 patients in the antecedent hematologic disorder. Neither a history of prior exposure to cytotoxic agents nor a prior myeloid disorder was found in 44% (n=35) of the patients. Del(5q) was commonly associated with a complex karyotype (n=67), however in 9 patients it was found to be the sole abnormality. The most frequent breakpoint was q13q33 (n=47) followed by q15q33 (n=12). Initial pathological description did not identify any morphological abnormalities in 6 cases, in contrast myelodysplasia involving at least two cell lines was appreciated in 58% (n=46) of the patients. The median survival was 2.8 months. The majority of the subjects (n=51, 65%) received palliative treatment, 7 patients were lost to follow up and no treatment information was available for review and the remaining 21 received induction chemotherapy with a curative intent, in this group median survival was 8.8 months with 52% (n=11) of patients achieving a complete response. Surprisingly the only factor that appeared to alter survival in the whole group was the presence of the breakpoint in chromosome 5 being distal to q15 (median 2.8 vs 4.1 months, p=0.025) Conclusions: This study constitutes the largest review of del(5q) in AML, its presence is currently associated with advanced age and a poor survival. Close to half of the patients in our series were classified as de novo AML. Although more commonly encountered as part of a complex karyotype it can also be present as a single abnormality. The different cytogenetic characteristics of the del(5q) may impact survival and this deserves further investigation. Figure. Survival of patients with AML and del(5q) in which the breakpoint was found to be distal to q15 (straight line) compared with patients in which the breakpoint was proximal to q13 (dotted line). Figure. Survival of patients with AML and del(5q) in which the breakpoint was found to be distal to q15 (straight line) compared with patients in which the breakpoint was proximal to q13 (dotted line).

Abstract Malignant plasma cell (PC) disorders are clonal diseases based on their immunoglobulin variable region sequence signature. However, intratumor phenotypic, genetic, and morphologic heterogeneity is often observed, fueling the notion that a subpopulation of the malignant PC clone may have extensive self-renewal properties whereas others may not. Little information exists regarding the relationship between these putative tumor subpopulations that is instrumental to our understanding of the growth, progression and therapeutic targeting of PC disorders. We recently described establishment of two novel cell lines from one patient initially diagnosed with primary amyloidosis (ALMC-1), who eventually progressed to a combination of AL and symptomatic multiple myeloma (MM; ALMC-2). Although both cell lines are clonally related, they exhibit a number of intriguing differences, including a marked variability in their makeup of mononuclear (MoN) and multinuclear cells (2 − &gt;10 nuclei; MuN). The ALMC-1 cell line consistently displays a much larger fraction of MuN cells (~50%) than the ALMC-2 cell line (~20%). By contrast, the growth rate of the ALMC-2 cells is significantly higher than ALMC-1 cells suggesting an inverse relationship between growth rate and fraction of MuN cells. It has been demonstrated that MoN plasmablastic morphology is a predictor of poor survival in MM, however the clinical significance of MuN PCs, which are frequently observed in bone marrow biopsies, has yet to be determined. We therefore used the ALMC-1 and ALMC-2 cell lines to begin to test the hypothesis that the MoN fraction in both cell lines contains cells with extensive self-renewal properties whereas the MuN cells are no longer capable of cell division. The central questions become by what mechanism are MuN PCs generated and how are MoN PCs maintained? Two hypotheses that can explain the dual persistence of both types of cells are: random cell fusion; regulated or sporadic cell division of MoN cells into 2 daughter cells, one of which divides normally and the other one fails to undergo cytokinesis. To address the first possibility, each cell line was transfected with GFP or YFP. Single positive (GFP or YFP) cells were then mixed and cocultured for assessing the frequency of dual positive cells by flow cytometry at various time points. However, studies to date suggest this mechanism does not occur. To begin to address the second possibility and determine whether extrinsic factors, e.g., cytokines, may regulate the rate of generation of MuN PCs, we cultured both cell lines +/− IL-6 and IGF-I and assessed numbers of MoN and MuN PCs. Initial analyses suggest that IL-6, but not IGF-I, increases the proportion of MoN PCs in both cell lines. To address the hypothesis that MuN PCs are no longer capable of growth, cells were labeled with bromodeoxyuridine (BrDU) to assess the capability of MuN cells to synthesize DNA. We observed that MuN PCs incorporating BrDU did so uniformly in each nucleus present in the cell. We then assessed the ability of cell division of MuN cells by quantitating normal vs abnormal metaphases in both cell lines and observed that &gt;90% of metaphases were present in MoN PCs, suggesting that MoN PCs largely give rise to MuN PCs and the latter are likely compromised in cytokinesis. As centrosomes play a critical role in regulating cell division and centrosome abnormalities have been noted in MM by others, we next used gamma-tubulin immunofluorescence to detect centrosomes. MoN PCs exhibited uniformly normal centrosome numbers and positioning while MuN PCs showed clear centrosomal abnormalities including accumulation of centrosomes, centrosome clustering, and multiple directional centrosome polarization, suggesting a mechanism by which the MuN cells may be generated. In summary, our data suggest that MuN PCs lack growth potential and that intrinsic and/or extrinsic factors may drive accumulation of these cells. These observations support the possibility that patients with a larger proportion of MuN PCs may have differences in clinical outcome from those patients with largely MoN PCs. To test this, we have begun hematopathological analysis of a large series of patients diagnosed with various plasma cell disorders. Knowledge of the signals that regulate the balance between MoN and MuN cells could lend itself toward identification of therapeutic modalities that may maximize differentiation of tumor cells into end-stage cells.

Abstract Background: For most patients with MDS, the disease-associated molecular abnormalities are unknown, which contributes to diagnostic uncertainty and has limited development of effective therapies. Chromosomal deletions such as del(5q) or monosomy 7 are more common than translocations in MDS, but balanced translocations are more likely to be pathobiologically informative. Here we report cloning of an MDS-associated translocation that revealed a novel recurrent molecular abnormality. Methods: We first studied a 71-year old man who presented with MDS and t(6;9)(p21.3;q34) as an isolated chromosomal abnormality in 18 of 20 metaphases. FISH assays for DEK/CAN and ABL rearrangements were unrevealing. We generated somatic hybrid murine/human cell lines from a buffy coat; microdissected and amplified derivative chromosomes; and hybridized to 6p/9q custom comparative genomic hybridization (CGH) arrays. The CGH results considerably narrowed the 6p and 9q breakpoints, which were then amplified by long-range PCR and sequenced to define the rearrangement. Results: The IER3 gene (Immediate Early Response 3; also known as IEX-1) at 6p21.3 was found to be separated from its upstream regulatory elements in this rearrangement and translocated to a transcript-poor region of 9q. RT-PCR confirmed marked downregulation of IER3 expression in the patient compared to healthy controls. IER3 is a plausible candidate for involvement in MDS because of its known role in regulating death receptor-induced apoptosis, interaction with NF-κb pathways, and its importance in response to genotoxic stresses such as ionizing irradiation. We then identified archival bone marrow cell pellets from 204 additional patients with various clonal hematological disorders and chromosomal rearrangements involving 6p21 or 6p22 (i.e., translocations, inversions, deletions, or additions), and designed a splitsignal FISH probe set to assay IER3. FISH results were abnormal in 8 additional patients (9/205 pts total abnormal, 4.4%): 3 split signals and 6 amplifications (one patient had both abnormalities); all 8 patients had MDS and 6p rearrangements as part of a more complex karyotype. FISH studies in MDS patients without 6p rearrangements are ongoing. RTPCR in MDS patients without 6p rearrangements demonstrated down-regulation of IER3 by &gt;2-fold in 38% of patients (67% lower-risk) and up-regulation in 12% of patients (50% higher-risk) when compared to the mean for healthy controls. Direct sequencing of the IER3 coding region and the promoter region in 30 patients without 6p rearrangements revealed no point mutations, but the prevalence of non-synonymous single nucleotide polymorphism rs3094124 (p.A127P) was higher than expected in patients (variant in 28.3 % of MDS alleles vs. 8.6% of 116 HAP-CEU controls, p=0.0015). IER3−/− mice are known to have hypertension, cardiac hypertrophy, and epithelial proliferation, but are not anemic (Sommer SL et al J Appl Physiol 2006). We performed methylcellulose hematopoietic colony growth assays using bone marrow mononuclear cells from IER3−/− mice and wildtype controls, and noted no differences in BFU-E, CFU-E, and CFU-GM growth under unstressed conditions. Studies of IER3 expression induction under stressed conditions in primary MDS cells are ongoing. Conclusion: IER3 rearrangements represent a novel clonally-restricted recurrent genetic abnormality in MDS. Dysregulation of IER3 expression is common in MDS, even in patients without 6p rearrangements. This is the first time IER3 has been linked to human disease. Clarification of the role of IER3 in MDS is likely to yield new insights into MDS pathobiology.

Abstract BACKGROUND: DNA rearrangements that result in the inappropriate activation of the PDGFRA gene at 4q12 and the PDGFRB gene at 5q31-q33 occur rarely in patients with chronic myeloproliferative disorders. Approximately 10% of patients with systemic mast cell disease/hypereosinophilic syndrome have a unique mutational mechanism resulting in PDGFRA overexpression due to a novel microdeletion of the CHIC2 region resulting in the juxtaposition of FIP1L1 and PDGFRA. PDGFRB activation has been observed in patients with chronic myelomonocytic leukemia/atypical chronic myeloid leukemia and has been associated with 11 translocation partners. Since patients with demonstrable breakpoints in the PDGFRA and PDGFRB genes often have a dramatic disease response to the tyrosine kinase inhibitor imatinib, we searched the Mayo Clinic Cytogenetic database to identify additional translocation partners involving these regions. METHODS: Homebrew dual-color FISH probes were created which flank the PDGFRA gene at 4q12 and the PDGFRB gene at 5q31-q33. Archived bone marrow karyotypes analyzed in the Mayo Clinic Cytogenetics laboratory from a 15 year period (1989-2004) were reviewed to determine the frequency of specimens with breakpoints at 4q12 and 5q31-33. Of the 29,047 abnormal specimens, 64 possessed a 4q12 breakpoint and 164 possessed a 5q31-q33 breakpoint (excluding simple deletions). Of these 228 patients, residual bone marrow specimens were available from 170 patients for FISH analysis. RESULTS: Eleven of 50 patients with a 4q12 breakpoint yielded a break with the PDGFRA FISH probe. Eight patients had the previously described t(4;12)(q12;p13) which results in a reciprocal exchange between the TEL oncogene and has a break near, but not within, the PDGFRA gene. Three patients had breaks within the PDGFRA gene and had novel translocation partners including 1q44, 3q25 and 17q23. Twelve of 120 patients with a 5q31-q33 anomaly had a break detected with the PDGFRB FISH probe. Nine patients had the classic t(5;12)(q33;p13) involving PDGFRB and TEL. Three patients had novel PDGFRB translocation partners, including 1q21, 14q32 and 16p13.1. CONCLUSIONS: Breakpoints involving the PDGFRA and PDGFRB genes appear to be quite uncommon as only 23 patient samples were abnormal in our series of 29,407 abnormal bone marrow samples. With the description of three new translocations involving PDGFRB, at least 14 unique translocation partners have been identified with this gene. With the exception of the recurrent t(5;12) between PDGFRB and TEL, most translocations involving PDGFRB appear to be unique. The microdeletion of CHIC2 at 4q12 resulting in the juxtaposition of FIP1L1 and PDGFRA has been the sole mechanism thus far described resulting in the activation of this gene. The identification of the three translocations involving the PDGFRA gene represent the first classic cytogenetically visible rearrangements involving this novel gene region. While rare, we propose that all chromosome anomalies identified with breakpoints in the 4q12 and 5q31-q33 regions should receive appropriate FISH testing to determine the potential involvement of the PDGFRA and PDGFRB genes.

Abstract Background: Deletion 5q is a lenalidomide-sensitive cytogenetic abnormality seen usually in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In the current study, we looked into the occurrence and clinicopathologic correlates of del(5q) in myeloproliferative neoplasms (MPNs) defined according to the World Health Organization (WHO) criteria. Methods: Patients with del(5q)-positive MPN were identified through database query involving over 2000 patients with myeloproliferative neoplasms (MPD) including no less than 500 patients with primary myelofibrosis (PMF), 600 patients with essential thrombocytosis (ET), 500 patients with polycythemia vera (PV), and the remaining comprised of patients with molecularly-undefined chronic eosinophilic leukemia, hypereosinophilic syndrome, systemic mastocytosis (SM), chronic neutrophilic leukemia, and “MPN, unclassifiable”. Results: A total of 23 patients with del(5q) were identified (12 at time of initial diagnosis), suggesting an overall prevalence rate of 1%. A central pathological review was conducted to confirm the diagnosis in each instance. Specific diagnoses included PMF (n=14; estimated prevalence of 3%), MPN, unclassifiable (n=4), PV (n=2), ET (n=1), post ET-myelofibrosis (n=1) and SM (n=1). Del(5q) was a sole abnormality in 5 patients, accompanied by one other abnormality in 6 patients, and part of a complex karyotype in 12 patients. The most common breakpoint was q13q33 (n=10) followed by q15q33 (n=6). Based on sample size adequacy, we focused on del(5q)-positive PMF patients and compared their bone marrow histological and clinical features with their del(5q)-negative counterparts seen at the Mayo Clinic during the same period of time (n=311). There was no difference in gender or age distributions. Histopathological review did not show the typical tight clusters of megakaryocytes seen in classic PMF. The megakaryocytes were not the predominate feature of the biopsy, in general were less lobulated and smaller in size, and had a higher grade of fibrosis (med = 3+) than classic PMF. Furthermore, del(5q)-positive PMF patients presented with significantly lower hemoglobin level (p=0.0009) and platelet count (p=0.0003). The percentage of patients presenting with a hemoglobin level of &lt; 10 g/dL or a platelet count of &lt; 100 × 109/L were 70% and 92% in del(5q)-positive patients as opposed to 43% and 18% in del(5q)-negative patients. However, the presence of del(5q) did not significantly affect either overall survival (Figure) or leukemic transformation in PMF (14% vs 9%). Conclusions: Del(5q) is generally rare in MPNs but might occur in as much as 3% of patients with PMF. The presence of del(5q) does not appear to affect overall survival in PMF despite its clustering with adverse prognostic features for the disease including anemia and thrombocytopenia. Finally, megakaryocyte morphology and distribution in del(5q)-associated PMF are characteristically different from classic PMF. Figure Figure