Meyer, Reid G.

Abstract MYC amplification (amp) is a marker of poor prognosis in many non-hematologic malignancies. While MYC translocations in B cell lymphoma (BCL) have been extensively studied, little is known about the significance of MYC amp. Recent studies describe increased MYC copy numbers (3-10 copies/cell) to be associated with more aggressive BCL. The WHO 2017 does not include MYC amp in the definition of high-grade BCL (HGBCL) with MYC and BCL2 and/or BCL6 rearrangement ("double-hit lymphoma", DHL). However, it also states that high-level MYC amp occurring together with a MYC rearrangement, and concurrent with BCL2 rearrangement likely has a similar clinical impact as classic DHL. Although increased MYC copy number is commonly identified by routine fluorescence in situ hybridization (FISH) testing in BCL, the experience of our large cytogenetics reference laboratory suggests that high-level MYC amp, defined as uncountable MYC signals, is far less common. Therefore, we sought to characterize the clinical, pathologic and cytogenetic features of patients with BCL showing high-level MYC amp. The Mayo Clinic cytogenetic database was retrospectively reviewed for all cases of BCL with high-level MYC amp seen by FISH from January 2010 - February 2018. All FISH studies reported as MYC amp were re-reviewed by a cytogeneticist to verify the level of amp and the MYC probes involved. Pathology was reviewed by two independent hematopathologists. Clinical information was collected through chart review. Survival analysis was performed using Kaplan-Meier curves and the Wilcoxon rank-sum test. FISH analysis for MYC aberrations identified 44/9715 (0.45%) cases with high-level MYC amp. Of cases with available H&E, the most common morphology was diffuse large BCL (DLBCL) (82%; 28/34), followed by HGBCL (15%; 5/34) and plasmablastic BCL (3%; 1/34). Hans cell of origin (COO) algorithm immunohistochemistry (IHC) identified 21/25 (84%) germinal center B-cell-like (GCB), and 4/25 (16%) non-GCB cases. 21/27 (78%) cases were BCL2+ by IHC. MYC+ by IHC was ≥40% in 21/28 (75%) and <40% in 7/28 (25%) cases. 9/17 (53%) were "double expressers" (DEL) by IHC. MYC amp probe signals appeared in a cloud-like distribution (CLD) in 31 (70%) or in a single homogenous staining region (HSR) in 13 (30%) [Figure 1A]. Among 38 cases with amp in a MYC break-apart probe, 21 (55%) had amp of 5' alone, 15 (40%) of intact and 2 (5%) of the 3' probe alone. 7/44 (16%) had MYC translocations (5 IGH; 2 non-IGH). BCL2 rearrangement was seen in 15/39 (38%) cases, and BCL6 rearrangement in 3/36 (8%). Only 2/44 (4%) cases met the current WHO 2017 definition of DHL. Clinical data was available for 20 cases. Median age at diagnosis was 64.5 (range 25-88) years with M:F of 1.5:1. Only 1/14 (7%) had bone marrow while 16/18 (88.8%) had other extranodal sites of involvement (8 gastrointestinal). The clinical presentation was heterogeneous: 13 de novo, 3 post-transplant, 3 transformed from low grade and 1 mediastinal BCL. 9/14 had an elevated LDH, median 387 (225 - 2063) U/L. R-CHOP was the most common first line therapy in 12/17 (70%). At median follow-up of 18.2 (range 0.4 - 88.9) months, 9 patients had died, 3 were in relapse and 8 remained in first complete remission. Lymphoma relapse/progression was the most common cause of death in 7/9 (78%). The median overall survival (OS) was 29.3 months [Figure 1B]. There was no statistically significant difference in OS by morphologic classification (DLBCL vs HGBCL, p=0.6), double expresser (Yes/No, p=0.19), COO (GCB vs non-GCB, p=0.08), MYC amp pattern (HSR vs CLD, p=0.48), MYC amp probe (3' vs 5' vs intact, p=0.31), MYC rearrangement (Yes/No, p=0.1), or MYC amp with concurrent BCL2/BCL6 rearrangement (Yes/No ,p=0.2). To our knowledge, this is the first study to characterize the clinical, pathologic and cytogenetic features of BCL with strictly-defined high level MYC amp identified by FISH. The 5' signal alone, or the intact MYC probe are most frequently amplified, and two distinct patterns of amp can be seen. Predominant extra nodal involvement is an important clinical observation. These cases are usually DLBCL, GCB type, and infrequently have concurrent MYC/BCL2/BCL6 rearrangement. Our study suggests that BCL with high-level MYC amp may have an aggressive disease course regardless of MYC, BCL2 and BCL6 gene rearrangement status. A larger series is necessary to further understand the clinical significance of high-level MYC amp in BCL. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

AimsThe aims of this study were to review our 5‐year experience with clinical FISH testing for TP63 rearrangements using both TP63 break‐apart (BAP) and TBL1XR1/TP63 dual‐fusion (D‐FISH) probes to evaluate the frequency of TP63 rearrangements and the distribution of TBL1XR1 vs. alternate partner loci, and to assess whether both probe sets are necessary in all cases undergoing FISH testing.Methods and resultsA retrospective review of the Mayo Clinic cytogenetic database identified 470 patients evaluated by FISH testing for TP63 rearrangements in formalin‐fixed paraffin‐embedded (FFPE) tissue using both BAP and D‐FISH probes. Of these, 25 (5.3%) had TP63 rearrangements. All samples were being investigated for anaplastic large‐cell lymphoma or other T cell lymphoma subtypes. A TBL1XR1 partner was identified by D‐FISH in 12 (48%) of 25 cases. All cases positive by TBL1XR1/TP63 D‐FISH were also positive by TP63 BAP FISH.ConclusionThis is the largest series of TP63 rearrangements to date. The frequency of positive results among cases referred to a large reference laboratory for TP63 FISH testing was 5.3%. Approximately half of TP63 rearrangements have a TBL1XR1 partner. TP63 BAP FISH testing is sufficient for up‐front testing of FFPE tissue samples. However, because of the genomic proximity of the TP63 and TBL1XR1 loci, we recommend reflex TBL1XR1/TP63 D‐FISH testing in positive and equivocal cases.

AbstractObjectivesMyeloid sarcoma with CBFB-MYH11 fusion may be enriched in abdominal sites. The clinicopathologic features of 11 cases are reported.MethodsWe collected clinical features, morphology, immunophenotype, and bone marrow (BM) involvement of myeloid sarcoma cases with CBFB-MYH11 fusion.ResultsEleven of 29 total myeloid sarcoma cases were CBFB-MYH11 positive and all 11 involved abdominal sites. The blastic infiltrate was associated with eosinophils in four of 11 cases and plasmacytoid dendritic cell (pDC) nodules in four of six cases. CD34, CD117, and myeloperoxidase were expressed in eight of nine, 10 of 10, and 10 of 10 cases, respectively. Ten of 10 cases showed no BM involvement.ConclusionsOur current series, combined with a literature review, identifies a compelling series of 31 (94%) of 33 cases of myeloid sarcoma with CBFB-MYH11 fusion showing a marked predilection for abdominal sites. In addition, the lack of obvious associated eosinophils, presence of pDC nodules, and lack of concurrent BM involvement suggest that “myeloid sarcoma with CBFB-MYH11 fusion” may represent a unique phenomenon.

3144 Background: Updated ASCO/CAP Guidelines for HER2 testing in breast cancer have been most impactful on the resolution of certain challenging groups of FISH results. We review the change in assignment of HER2 status in a large series of breast cancers referred to a large national reference laboratory for FISH testing since the introduction of the 2018 updated guidelines. Methods: Patient samples submitted to the Mayo Clinic Cytogenetics Laboratory (N = 2208) were analyzed by FISH. Samples with Group 2, Group 3, or Group 4 FISH results were reflexed to immunohistochemistry (IHC) in our central laboratory; FISH slides for those cases with equivocal 2+ IHC results were re-scored in the regions of invasive cancer showing more intense membranous staining. A subset of 202 samples with Group 4 FISH results were also reflexed to the previously employed reflex FISH assay (HER2/D17S122), and these were also re-analyzed according to the new reflex IHC/FISH process. Results: 382 of 2208 breast cancer samples tested (17.3%) had FISH results categorized as Group 2 (N = 17, 0.8%), Group 3 (N = 34, 1.5%), or Group 4 (N = 331, 15%) and required reflex IHC testing, and of those, 75% were 2+ equivocal and required targeted re-analysis of the FISH slide according to the 2018 updated guidelines. Re-analysis of the FISH slide resulted in switching between Groups 1-5 in 19.4% of cases, but HER2 status was changed by FISH re-scoring in only 7.7% of cases re-scored (1.0% of all samples), generally due to only minor shifts in HER2 copy number and HER2/control ratios between the initial and IHC-guided reflex FISH scores. In the subset of 202 cases tested by both reflex methods, the previously employed HER2/D17S122 reflex probe set was positive in 123 cases (60.9%), whereas reflex IHC/FISH was positive in only 10 cases (7.9%). Including positive reflex IHC (0.4%) and positive reflex FISH results (2.1%), the overall assignment of positive HER2 status on our series of 2208 cases was 11.5%. Conclusions: Overall rates of HER2 positive FISH results have declined under the most recent ASCO/CAP guideline update as a consequence of new recommendations for reflex testing for Groups 2-4. This change is largely due to reassignment of Group 2 and Group 4 results as negative in the absence of positive IHC.

ABSTRACTThe MLLT10 (formerly AF10) gene is the fourth most common KMT2A fusion partner across all acute leukemias and requires at least 3 breaks to form an in‐frame KMT2A/MLLT10 fusion due to the opposite orientation of each gene. A 10‐year retrospective review was performed to identify individuals from all age groups that harbor KMT2A/MLLT10 fusion obtained by our KMT2A/MLLT10 dual‐color dual‐fusion fluorescence in situ hybridization (D‐FISH) assay. Of the 60 unique individuals identified, 31 were male and 29 were female (M:F ratio, 1.1:1) with ages ranging from 3 days to 86 years (mean 21.5 years, median 5.5 years). The diagnoses included acute myeloid leukemia (AML) (49 patients, 82%), B‐ or T‐lymphoblastic leukemia/lymphoma (7 patients, 12%), myeloid sarcoma (3 patients, 5%), and a single case (2%) of undifferentiated leukemia. Twenty‐seven of 49 patients (55%) with AML were in the infant or pediatric age group. Fifty‐three of 60 patients (88%) had KMT2A/MLLT10 D‐FISH signal patterns mostly consisting of single fusions. In addition, 10 (26%) of 38 patients with conventional chromosome studies had “normal” (5 patients) or abnormal (5 patients) chromosome studies that lacked structural or numeric abnormalities involving chromosomes 10 or 11, implying cryptic cytogenetic mechanisms for KMT2A/MLLT10 fusion. Lastly, mate‐pair sequencing was performed on 4 AML cases, 2 of which had “normal” chromosome studies and cryptic KMT2A/MLLT10 fusion as detected by KMT2A/MLLT10 D‐FISH studies, and verified the multiple breaks required to generate KMT2A/MLLT10 fusion.

AbstractT‐lymphoblastic leukemia/lymphoma (T‐ALL/LBL) accounts for approximately 15% of pediatric and 25% of adult ALL. While the underlying frequency of KMT2A (MLL) gene rearrangements has been identified in approximately 4‐8% of T‐ALL/LBL cases, a paucity of literature is available to characterize further the KMT2A rearrangements in pediatric/young adult T‐ALL/LBL. A 10‐year retrospective review was performed to identify KMT2A rearrangements in specimens sent for T‐ALL/LBL fluorescence in situ hybridization studies in patients under the age of 30 years. Of 806 T‐ALL/LBL FISH studies performed on unique individuals, 27 (3.3%) harbored KMT2A rearrangements. Nineteen patients were male and eight were female (M:F ratio, 2.4:1) with ages ranging from 1 to 20 years (mean 12, median 12). Of the 27 cases, nine (33%) had KMT2A/MLLT1 fusions, eight (30%) had KMT2A/AFDN fusions, two (7%) had KMT2A/ELL fusions, and one (4%) had a KMT2A/MLLT10 fusion. In addition, five (19%) had KMT2A rearrangements with unidentified gene fusion partners and two (7%) had 3’KMT2A deletions. Our results indicate that MLLT1 and AFDN account for the majority (63%) of KMT2A gene partners in pediatric/young adult T‐ALL/LBL, while no KMT2A/AFF1 or KMT2A/MLLT3 fusions were observed despite their common identification in B‐ALL and acute myeloid leukemia, respectively. In addition to diagnostic and prognostic value, detecting specific KMT2A fusions may also be of clinical importance in the era of targeted therapies.