Fluorescence In Situ Hybridization Abnormalities Research Articles

Prognostic value of clonal evolution identified by sequential FISH in untreated chronic lymphocytic leukaemia

Aim: The aim of the current study was to evaluate the potential clinical impact of clonal evolution detected by fluorescence in situ hybridization (FISH) in untreated chronic lymphocytic leukaemia (CLL) patients managed with a watch-and-wait strategy. Methods: We performed both overall survival (OS) and time to first treatment (TTFT) analysis. For the first one, we exploited a real-life cohort of 123 consecutive CLL patients followed at our institution, for which at least a second FISH evaluation during watch and wait was available. For TTFT analysis, we considered only patients treated after the second FISH sample (n = 69). Results: Considering the original cohort, patients who acquired a FISH abnormality displayed a worse outcome with a median OS of 91.9 months compared to 147.3 months for patients who did not acquire any FISH abnormalities (P = 0.007). Unmutated immunoglobulin heavy chain gene (IGHV) genes were associated with a higher probability of acquiring a FISH abnormality (P = 0.04). Turning to TTFT analysis, patients who gained at least one FISH abnormality (n = 7, 10%) were characterised by an earlier treatment requirement with a median TTFT of 1.1 months, compared to 2.7 months in patients who did not acquire any FISH abnormalities (n = 62, 90%) (P = 0.025). Conclusions: The dynamic acquisition of karyotypic abnormalities by FISH predicts poor outcomes and early treatment requirement in CLL patients. Our results suggest that FISH analysis could be integrated with other clinical and biological features to obtain dynamic scores that are able to predict outcomes at different phases of disease history.

“Double Hit” Myeloma: Detection By FISH and Flow Cytometry in Circulating Myeloma Plasma Cells

“Double hit” myeloma is defined as the presence of 2 of the known high-risk genetic abnormalities in myeloma. This includes amp(1q), del(17p) and IGH translocations involving chromosomes 4, 16 and 20. Given the prognostic significance of these abnormalities, with poor overall survival, identification is crucial. Testing is traditionally by fluorescence in situ hybridization (FISH) on a single-site bone marrow sample at baseline or at specific disease timepoints. We explored whether amp(1q) and del(17p) “double hit” abnormalities could be detected in circulating myeloma plasma cells (CMPC). Aims: We assessed our novel imaging flow cytometry method that simultaneously integrates immunophenotyping and FISH. Called “immuno-flowFISH” this single cell method combines antigenic detection of neoplastic plasma cells, FISH for DNA loci of interest and imaging flow cytometry for automated assessment. Methods: Blood from 16 myeloma patients, at diagnosis or on therapy was studied. Following density gradient centrifugation, cells were incubated with CD38/CD138/CD319-AF647-conjugated antibodies to identify plasma cells, CD19-BV480 (to differentiate normal from neoplastic plasma cells) and CD3-BV510 (T-lymphocyte control). After fixation and cell membrane permeabilization, DNA was denatured (78 oC for 5 minutes). The cells were then incubated with FISH probes to 1q21 ( CKS1B) and 17p13 loci, and to the centromere of chromosome 17 (C17), and hybridized (24 hours for 37 oC). The nuclei were stained with SYTOX AADvanced and 50,000 to 500,000 cells acquired on the Amnis ® ImageStream ®X Mk II imaging flow cytometer (Cytek Biosciences). Digital images and quantitative data derived from computational algorithms (IDEAS software) were used to assess FISH signals overlying the counterstained nuclei. Results: A mean of 264,937 cells were acquired and 110,696 analysed. Cells with a neoplastic plasma cell phenotype (CD38/CD138/CD319-positive, CD19-negative) were present in all samples. The number of these CMPC ranged from 7 to 797, amounting to 0.011- 0.546% of all cells. FISH abnormalities were present in CMPC of 10 of the 16 cases (see Table). Isolated amp(1q) (three CKS1B FISH signals) was present in 4 cases, and isolated del(17p), (one 17p13 and two C17 FISH spots) in CMPC in 3 cases. “Double hit” abnormalities, with both amp(1q) and del(17p), were present in another 3 cases, with the following cytogenetic patterns (illustrated in the Figure): 1. amp(1q) and del(17p) in different cells (Case 8); 2. amp(1q) and del(17p) in the same cell (35.7% of CMPC), in addition to isolated amp(1q) (28.6% of CMPC) (Case 9); 3. amp(1q) and del(17p) in the same cells only (Case 10). All circulating myeloma plasma cells had diploid signals for the chromosome C17 centromeric probe and the CD3-positive T-cells diploid signals for all probes. Conclusion: Incorporating antigenic identification and FISH, with imaging flow cytometric analysis, can detect “double hit” amp(1q) and del(17p) in circulating myeloma plasma cells at high sensitivity (10 -5). This novel approach allowed sub-clonal interrogation, with amp(1q) and del(17p) “double hits” detected in the same (Cases 9, 10) and separate CMPC (Case 8). It could also identify sub-clonal acquisition of secondary del(17p) (Case 9), which may be a consequence of disease evolution or intra-tumoral clonal heterogeneity. FISH analysis of circulating plasma cells using a flow cytometric platform is achievable to low levels of disease through assessment of thousands of cells. It holds significant promise for sensitive holistic real-time blood surveillance to detect high-risk and sub-clonal “double hit” alterations at the single cell level. Blood monitoring for these genetic aberrations overcomes the limitations of single site marrow analysis and may provide insights into the disease trajectory, clonal evolution and prognosis.

Impact of cytogenetic abnormalities on the risk of disease progression in solitary bone plasmacytomas

AbstractMost patients with solitary bone plasmacytomas (SBP) progress to multiple myeloma (MM) after definitive radiation therapy as their primary treatment. Whether the presence of high-risk (HR) cytogenetic abnormalities by fluorescence in situ hybridization (FISH) in the clonal plasma cells, obtained either directly from the diagnostic SBP tissue or the corresponding bone marrow examination at the time of diagnosis, is associated with a shorter time to progression (TTP) to MM is unknown. This study evaluated all patients diagnosed with SBP at the Mayo Clinic from January 2012 to July 2022. The presence of del(17p), t(14;16), t(4;14), or +1q (gain or amplification) by FISH in clonal plasma cells was defined as HR. A total of 114 patients were included in this cohort, and baseline FISH was available for 55 patients (48%), of which 22 were classified as HR (40%). The median TTP to MM for patients with SBP and HR FISH was 8 months (95% confidence interval [CI], 6.3-26) compared with 42 months (95% CI, 25-not reached [NR]) in patients with SBP without HR FISH (P < .001). In a multivariate analysis, only HR FISH was a significant predictor for shorter TTP to MM, independent of minimal marrow involvement and an abnormal serum free light chain ratio at diagnosis. Deletion (17p) and gain 1q abnormalities were the most common FISH abnormalities responsible for the short TTP to MM. Thus, assessing for HR FISH abnormalities in clonal plasma cells derived from either the diagnostic SBP tissue or the staging bone marrow examination of patients with newly diagnosed SBP is feasible and prognostic for a shorter TTP to MM.

Measurable Residual Disease Conversion Rate with Consolidation Chemotherapy in Acute Myeloid Leukemia

Background Patients with acute myeloid leukemia (AML) who achieve complete remission (CR) following induction therapy but continue to have measurable residual disease (MRD) have inferior outcomes following allogeneic hematopoietic cell transplant (alloHCT). Determining the historical rate of MRD clearance in response to standard consolidative therapy is needed as an initial step in developing a new therapeutic strategy to clear MRD prior to alloHCT. Methods A multi-institution retrospective analysis was performed on consecutively treated AML patients in morphologic CR with detectable MRD post-induction therapy who received standard chemotherapy consolidation from January 2016 to December 2021. MRD was measured by multiparametric flow cytometry (MPFC, sensitivity 0.01-0.1%), next generation sequencing (NGS, sensitivity 1-5%), real-time quantitative PCR (qPCR, sensitivity 1:100,000-0.5%), and/or fluorescence in situ hybridization (FISH)/karyotype (sensitivity 1-5%). Any detectable disease-related abnormalities were considered to be evidence of MRD for the purpose of this study. Results Eighty-three patients met inclusion criteria for this analysis, with a median age at diagnosis of 51 years (IQR 43-63). Adverse-risk disease by ELN 2017 criteria was observed in 36 patients (43%). The majority received induction therapy with "7+3" (cytarabine + idarubicin or daunorubicin, 77 patients, 93%), and most (71 patients, 86%) required only 1 line of therapy to achieve CR. At the time of CR following induction therapy, 35 patients (42%) had MRD detected by MPFC, 38 patients (46%) had MRD detected by NGS, 10 patients (12%) had MRD detected by qPCR, and 32 patients (39%) were found to have an abnormal FISH or karyotype. Twenty-eight patients had MRD detected by more than 1 method. Consolidation therapy consisted of intermediate-dose cytarabine (defined as <3 g/m2, 41 patients, 49%) or high-dose cytarabine (defined as 3 g/m2, 39 patients, 47%) in all patients, except for 3 who received daunorubicin and cytarabine (liposomal) consolidation. The number of cycles of cytarabine consolidation were 1 cycle (36 patients, 43%), 2 cycles (23 patients, 28%), 3 cycles (15 patients, 18%), and 4 cycles (9 patients, 11%), respectively. Sixteen patients received cytarabine consolidation in combination with another agent including sorafenib (1 patient), midostaurin (10 patients), gilteritinib (1 patient), enasidenib (1 patient), and gemtuzumab (3 patients). In response to consolidation therapy, 27 of the 83 patients with measurable disease converted to MRD-negative (33%) after all given cycles of consolidation cytarabine. Of the 35 patients whose AML had MRD detected by MPFC prior to consolidation, 17 converted to MRD-negative (49%). Of the 38 patients whose AML had MRD detected by NGS prior to consolidation, 6 converted to MRD-negative (16%). Of the 10 patients whose AML had MRD detected by qPCR prior to consolidation, 6 converted to MRD-negative (60%). Of the 32 patients whose AML had MRD detected by FISH or karyotype prior to consolidation, 13 converted to MRD-negative (41%). Of note, 22 patients developed progressive disease following consolidation therapy (and therefore also did not achieve MRD-negativity). Multivariable logistic regression was performed to assess potential associations with MRD conversion during consolidation chemotherapy. Older age decreased the odds of conversion of AML to MRD-negative (OR = 0.96, 95% CI 0.92-1.00, p = 0.04). Higher ELN risk category, higher dose of cytarabine, and more cycles of consolidation did not affect the likelihood of conversion of AML to MRD-negative. Fifty-five patients received alloHCT following consolidation therapy. Thirty-one of these patients had AML with MRD detected post-consolidation, of whom 14 had MRD measured by MPFC. With a median follow-up time of 437 days post-transplant (IQR 151-914), two-year progression-free survival post-transplant was 57% in AML patients with MRD detected post-consolidation compared to 71% in AML patients with no MRD (p = 0.28). Conclusions The rate of MRD eradication with standard consolidation chemotherapy in AML is suboptimal. Combined with the known poor outcomes after alloHCT in patients with AML and detectable MRD pre-transplant, this demonstrates an unmet need to develop a new therapeutic strategy for eradicating MRD after induction therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Circulating Tumor Plasma Cells in the Screened Istopmm Smoldering Multiple Myeloma Cohort

Background The presence and number of circulating tumor plasma cells (CTPCs) in peripheral blood (PB) as detected by flow cytometry has previously been associated with increased risk of progression from smoldering multiple myeloma (SMM) to multiple myeloma (MM). CTPCs may therefore be a marker for tumor burden and/or tumor aggressiveness in SMM that can, if the correlation to progression can be verified, potentially spare patients from bone marrow biopsies. However, the rate of detectable CTPCs in the overall SMM population is not known and the dynamics of CTPC numbers between diseases stages and risk categories in a screened cohort of individuals with SMM have not been studied. Aim To investigate the detection and quantification of CTPCs in individuals with SMM diagnosed in the screened population-based Iceland Screens, Treats, or Prevents Multiple Myeloma study (iStopMM), and to associate the findings to known clinical risk models of progression from SMM to MM. Methods Individuals diagnosed with SMM, monoclonal gammopathy of undetermined significance (MGUS) and MM in the iStopMM study were included. Next-generation flow (NGF) cytometry was used to assess CTPC numbers in PB and the limit of detection (LOD) was set at ≥20 tumor PC. An optimal cutoff value of the percentage of CTPC to discriminate between SMM and MM was found using receiver operating characteristic (ROC) curve analysis of individuals with CTPCs detected. In a subgroup of individuals with SMM, interphase fluorescent in situ hybridization (FISH) analysis was performed. The difference in frequency of CTPC was tested between risk groups according to the PETHEMA and Mayo 2/20/20 models, the groups with, and without high-risk FISH abnormalities and between clinical entities (MGUS, SMM and MM). The Mann-Whitney U test or the Kruskal-Wallis tests were used to assess differences observed between two or more than two groups, respectively. Results A total of 93 individuals with SMM, 29 with MM, and 103 with MGUS were included. CTPCs were detected in 64 (69%) of individuals with SMM, with a median frequency of 0.0009% (range 0.0002-0.078%), or 0.054 CTPC/μL (range 0.011-3.99 CTPC/μL). The median sensitivity reached for samples with undetectable CTPCs was 2.3x10-6 (range 4.8-1.7x10-6). The detection rate and concentration of CTPCs increased from MGUS to SMM and MM (p<0.001 for both, figure) and from low to higher risk categories according to both risk models (table and figure). The increase in frequency of CTPCs according to risk score was only significant for the PETHEMA model (p<0.001), but not the Mayo 2/20/20 model (p=0.19). However, when low-risk individuals were compared to the intermediate/high risk groups according to the Mayo 2/20/20 model, the difference reached statistical significance (p=0.05). An optimal cutoff of 0.0017% CTPCs for SMM vs. MM was found with a sensitivity of 0.70, specificity of 0.69, and area under the curve 0.76 (figure). In total, 20 (22%) individuals with SMM had CTPC over the cutoff. This proportion increased with higher Mayo 2/20/20 risk categories (20% in low-risk, 23% in intermediate-risk and 25% in high-risk SMM) and PETHEMA risk categories (18% in low-risk, 23% in intermediate-risk and 30% in high-risk SMM). In the 40 individuals with SMM who underwent FISH, 12 (30%) had high-risk FISH abnormalities (del(17p), t(4;14) or t(14;16)) present, and of those 75% had CTPCs detected vs. 71% in those without high-risk FISH abnormalities, no difference in CTPC frequency was found between the groups (p=0.11) Conclusion CTPCs are a potential marker of tumor burden or tumor aggressiveness in SMM and are present in 69% of the overall population with SMM. Both the detection and numbers of CTPCs increase progressively from MGUS, to SMM, and MM. A similar trend was observed with increasing risk score in SMM, according to two established risk stratification models. These results indicate that NGF of PB to detect CTPCs might have a role in the management of SMM. Future studies with serial monitoring of CTPCs will help to further define the clinical value of CTPCs in relation to the risk of progression, and to define how CTPC monitoring impacts the role of established markers, especially to identify individuals with low risk of progression where bone marrow sampling might be omitted. This would focus clinical care and treatments in SMM and MM to those who benefit the most from them while limiting interventions in those who do not need them. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Enhanced Global Disease Assessment with Advanced Imaging and Targeted Myeloma Lesion Biopsy Highlights Spatial Heterogeneity and Detects Residual Disease in Multiple Myeloma

Background: Multiple myeloma (MM) is a biologically complex disease with marked heterogeneity; limitations remain in our standard myeloma disease assessment at diagnosis and in response to therapy. In this study, we aimed to prospectively investigate the utilization of advanced imaging and targeted focal lesion (FL) biopsies to augment global disease evaluation, detect residual tumor burden, and explore the diverse histologic and molecular landscape of this multifocal neoplasm. Herein, we present our final analysis of this clinical trial. Methods: Adult patients with newly diagnosed clinical MM (NDMM) and bone or extramedullary (EM) FL were eligible to participate in this prospective study. Advanced imaging with positron emission tomography/computed tomography (PET/CT) or whole body magnetic resonance imaging (WB-MRI), standard bone marrow (BM) biopsy and aspiration, and targeted FL biopsy were performed at enrollment and after four 28-day cycles of induction therapy with carfilzomib (36 mg/m2 intravenously twice weekly), lenalidomide (25 mg orally days 1-21), and dexamethasone (40 mg orally weekly) (KRd), with dose modifications as needed. Conventional clinical response, using IMWG Response Criteria (Kumar S et al, 2016), was also measured after each cycle of treatment. Patients were followed for 12 months after end of treatment or until data cutoff on February 23rd, 2022. Results: 27 patients were enrolled in this study between June 2018 and August 2021, with 25 evaluable for global response assessment. Median age was 61 years (range, 42-76 years) and 14.8% of patients were of Black or African American race. 85.2% of patients had Revised International Staging System (R-ISS) stage II or III disease. 51.9% of patients were identified as having EM disease arising out of bone or soft tissue and 44.4% of patients had high risk cytogenetics inclusive of chromosome 1 abnormalities. In sum, 70.4% of our cohort had at least 1 high risk feature at diagnosis (Table 1). 80% of patients had myeloma FL identified on advanced imaging that were missed on conventional skeletal survey. Of the 7 patients with cytogenetic and fluorescence in situ hybridization (FISH) testing performed on initial FL biopsy, 6 (85.7%) had findings discrepant from those seen on cytogenetic testing from standard BM samples. 9 patients had cytogenetic and FISH studies on repeat myeloma FL biopsy after KRd induction with 2 showing residual FISH abnormalities that were not detected on standard BM obtained for response assessment. Clinical response rates after 4 cycles of KRd were high in our study population with 88% of patients achieving a very good partial response (VGPR) or better and 20% with a stringent complete response (sCR) with minimal residual disease (MRD) negativity by flow cytometry. However, of the 22 patients with ≥VGPR by IMWG response assessment, 18 had residual disease on advanced imaging with WB-MRI (10 patients), PET/CT (7 patients), or MRI total spine (1 patient), including 3 of the 5 patients with sCR (Figure 1). 2 patients experienced disease progression during the study period with median progression free survival not reached by the patient cohort. Both patients with progression had a VGPR by conventional response assessment, though with residual FL on advanced imaging post-KRd induction. Conclusions: While tumor spatial differences in MM have been observed retrospectively, prospective data on this important component of myelomagenesis is limited. In our cohort of high risk NDMM, we demonstrated spatial heterogeneity both at diagnosis and after KRd induction. Despite the deep clinical responses observed by IMWG response assessment, 81.8% of patients with ≥VGPR had ongoing abnormalities on advanced imaging which could potentially serve as disease reservoirs for future progression and development of treatment resistance. A novel assessment method in MM, inclusive of advanced imaging and targeted FL biopsies, may overcome the limitations of our standard response assessment with resultant improved knowledge of the varied intra-tumor disease biology as well as development of more refined treatment approaches. A potential limitation of our study is the smaller sample size with underrepresentation of minority populations. Data on whole exome sequencing of paired BM and FL biopsies will be reported separately. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Combined utility of p16 and BRAF V600E in the evaluation of spitzoid tumors: Superiority to PRAME and correlation with FISH.

Spitzoid melanocytic neoplasms are diagnostically challenging; criteria for malignancy continue to evolve. The ability to predict chromosomal abnormalities with immunohistochemistry (IHC) could help select cases requiring chromosomal evaluation. Fluorescence in situ hybridization (FISH)-tested spitzoid neoplasms at our institution (2013-2021) were reviewed. p16, BRAF V600E, and preferentially expressed antigen in melanoma (PRAME) IHC results were correlated with FISH. A total of 174 cases (1.9F:1M, median age 28 years; range, 5 months-74 years) were included; final diagnoses: Spitz nevus (11%), atypical Spitz tumor (47%), spitzoid dysplastic nevus (9%), and spitzoid melanoma (32%). Sixty (34%) were FISH positive, most commonly with absolute 6p25 gain (RREB1> 2). Dermal mitotic count was the only clinicopathologic predictor of FISH. Among IHC-stained cases, p16 was lost in 55 of 134 cases (41%); loss correlated with FISH positive (p< 0.001, Fisher exact test). BRAF V600E (14/88, 16%) and PRAME (15/56, 27%) expression did not correlate with FISH alone (p= 0.242 and p= 0.359, respectively, Fisher exact test). When examined together, however, p16-retained/BRAF V600E-negative lesions had low FISH-positive rates (5/37, 14%; 4/37, 11% not counting isolated MYB loss); all other marker combinations had high rates (56%-75% of cases; p< 0.001). p16/BRAF V600E IHC predicts FISH results. "Low-risk" lesions (p16+ /BRAF V600E- ) uncommonly have meaningful FISH abnormalities (11%). PRAME may have limited utility in this setting.

Conventional Karyotyping and Fluorescence In Situ Hybridization for Detection of Chromosomal Abnormalities in Multiple Myeloma.

BackgroundMultiple myeloma (MM) is a genetically heterogeneous disease, with cytogenetic findings that determine disease behavior. Genetic abnormalities can be assessed by fluorescence in situ hybridization (FISH) analysis and/or G-banded karyotyping. The two methods produce unique and overlapping information, and the clinical utility of using both is investigated here.MethodsSeventy patients diagnosed with MM at a hospital in Southern California were retrospectively reviewed for the FISH and G-banded karyotyping results obtained from bone marrow specimens.ResultsKaryotype was normal in 71% (50/70), abnormal in 27% (19/70), and inadequate in 1% (1/70). Among patients with abnormal karyotype, FISH provided additional information about genetic aberrations in 95% of cases (18/19). Among cases with abnormal FISH, karyotype provided additional information about genetic aberrations in 27% of cases (18/66). When numerical abnormalities were present (detected by FISH and/or karyotype), FISH detected them in 95% (54/57), of which karyotype missed 70% (38/54) of the time. Karyotyping detected numerical abnormalities in 33% (19/57), which FISH missed 16% (3/19) of the time.ConclusionsKaryotyping and FISH analysis in MM each provide unique information. For most patients, performing both tests together will provide more information than either test alone.

Amp 1q21 is more predictable with dismal survival than gain 1q21 of newly diagnosed multiple myeloma in real-world analysis.

IntroductionThe gain/amplification (amp) of 1q21 is one of the most common high‐risk chromosome abnormality (HRCA) in multiple myeloma (MM). The prognostic value of 1q21+ remains to be controversial on the status of gain or amp and the combination of other HRCAs.MethodsIn this retrospective study, we included 318 newly diagnosed MM (NDMM) patients who had fluorescence in situ hybridization (FISH) data and treated with novel agents in our department.ResultsOur study noted MM patients with amp 1q21 were more likely accompanied with t(4;14), t(14;16), and t(14;20). Patients with amp 1q21 presented with elder age, advanced Revised International Staging System (R‐ISS) stages, anemia, and more plasma cells in bone marrow compared to patients with gain 1q21 alone and no 1q21+. Moreover, amp 1q21 alone correlated with shorter progression‐free survival (PFS) (22.8m vs. 40.5m vs. 39.6m) and overall survival (OS) (45.2m vs. NA vs. 83.5m) compared with gain 1q21 alone and no FISH abnormalities. Although the high ratio of proteasome inhibitor and immunomodulatory drugs used in patients with amp 1q21, the overall response (ORR) was the lowest compared with no 1q21+ and gain 1q21. Multivariate analysis defined amp 1q21 as an independent prognostic marker for NDMM patients, rather than gain 1q21.ConclusionThe amp 1q21 predict inferior treatment response and survival, especially coexisted with high‐risk IgH translocation.

Variation in Use of Immunoglobulin and Impact on Survival in Multiple Myeloma: A Report from the Australian and New Zealand Myeloma and Related Diseases Registry (MRDR)

Variation in Use of Immunoglobulin and Impact on Survival in Multiple Myeloma: A Report from the Australian and New Zealand Myeloma and Related Diseases Registry (MRDR)

Recurrent Chromosomal Abnormalities in Tissues Involved by Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma.

Prognostically relevant chromosomal abnormalities in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) are routinely identified by fluorescence in situ hybridization (FISH) on peripheral blood or bone marrow specimens. We studied the prevalence of chromosomal abnormalities on extramedullary tissues involved by CLL/SLL and evaluated their association with prominent proliferation centers (PPCs). FISH for recurrent abnormalities in CLL/SLL was performed on formalin-fixed, paraffin-embedded biopsy sections. PPCs were identified on H&E-stained sections. Available FISH results on peripheral blood or bone marrow specimens were also reviewed. Recurrent FISH abnormalities were detected in 69% of 320 CLL/SLL biopsy specimens studied, including +12 (35%), 13q- (24%), 11q- (15%), 17p- (6%), 6q- (2%), and IGH/BCL2 (0.9%). Forty-three patients had abnormal blood or bone marrow FISH analyses, of whom 7 (16%) had discordant +12 and/or 13q-, and 3 (7%) had discordant 17p- or 11q-. Morphology was positive (17%), negative (78%), or equivocal (6%) for PPCs on 247 evaluable biopsy specimens, a finding not significantly associated with FISH results (P = .7). Trisomy 12 is overrepresented in tumoral CLL/SLL involvement, compared with the known predominance of 13q- in blood. Discrepancies between leukemic and tissue FISH findings are occasionally encountered. FISH results do not correlate with the presence of PPCs.

The Cytogenetic Aberrations and Their Prognostic Value in 181 Untreated Patients with Lymphoplasmacytic Lymphoma/ Waldenström Macroglobulinemia in China

Background: lymphoplasmacytic lymphoma/ Waldenström macroglobulinemia (LPL/WM) is a rare B cell proliferative malignancy characterized by immunoglobulin M monoclonal gammopathy and /or bone marrow infiltration by lymphoplasmacytic cells. Cytogenetic aberrations are now the main prognostic predicators in some B-NHLs such as CLL. However, their prognostic valve in LPL/WM remains controversial and recurrently chromosomal abnormalities have rarely been reported. Furthermore, the genetic basis of LPL/WM in China is also unclear.Methods: A panel of DNA probes was used to detect cytogenetic aberrations by fluorescence in situ hybridization (FISH), including RB1/D13S25 at 13q14, ATM at 11q22, TP 53 at 17p13, CEP12 and IGH translocation at 14q32, MYB at 6q23. Chromosomes were identified by G-banding and karyotypes were described according to the International System for Human Cytogenetics Nomenclature (ISCN, 1995). Totally, 181 untreated LPL/WM patients were enrolled in this study.Results: The median age of 181 patients was 61 years (range 32-87). The male to female ratio was 2.75 (133/48). 90% and 15.7% patients showed an elevated serumβ2 microglobulin and LDH. The percentage of hepatomegaly, splenomegaly, lymphadenopathy and B symptom were 15.3%, 35.2%, 50% and 27.3%, respectively. Almost half patients were in the high risk ISSWM group. The chromosomal karyotypes were successful in 153/181 (84.5%) cases, of which 15 patients showed clonal abnormalities. 8 of the 15 patients with abnormal chromosome were complex. The main aberrations of chromosomal karyotypes were 6q deletion (3/15, 20%), and loss of sex chromosomes (3/15, 20%). The other abnormalities include trisomy 12, trisomy 4, trisomy 3, 20q deletion, 17p deletion and so on. All the 6q deletions patients were in the complex karyotype group. Moreover, the trisomy 12 detected by FISH were always accompanied with chromosomal karyotypes abnormalities. Of 181 patients, 139 patients had FISH detection, of which 25 patients (18%) had FISH abnormalities. The most common aberrations were 6q deletion (6/28, 21.4%), trisomy 12 (5/59, 8.5%), 17p deletion (8/131, 6.1%). 13q deletion, gain 1q21and 11qdeletion were 4.6% (6/130) and 2.8% (3/36) and 2% (2/99), respectively. About 5.4% (7/130) patients had IGH gene translocation. Although it was higher in our study, this aberration always accompanied with other clones. Besides, 17p deletion was also more frequent in complex clone group, while trisomy 12 was more common in the single clone group. In LPL/WM, the chromosomal abnormities was an adverse predictor for both progress-free survival (PFS) and overall survival (OS). The median PFS and OS for patients with this abnormity was 62 months (62m vs 69m, p=0.022) and 75 months (75m vs 109m, p=0.033), which was significantly higher than normal patients. In different FISH groups, only TP 53 deletion had a poor effect on both PFS and OS in LPL/WM patients. The median PFS and OS were significantly shorter than those without the abnormality (median PFS 34m vs 77m, p=0.008; median OS 34m vs 135m, p=0.006). However, the 6q deletion and trisomy 12 did not significantly influence both PFS and OS. In addition, we found that the number of different clones had an effect on the survival of patients. The complex clone group showed shorter PFS and OS than the single clone group (p<0.05).Conclusion: Although the detection of cytogenetic aberration is rare in Chinese patients, it plays an essential role in diagnosis and prognostication. DisclosuresNo relevant conflicts of interest to declare.

Whole-Genome Mate Pair Sequencing Reflex Test to Characterize Chromosome Rearrangements in Hematologic Neoplasia

Chromosomal alterations such as translocations, inversions and deletions involving one or more breaks are common in hematologic malignancies. Mate pair sequencing (MPseq) is highly informative when a chromosome rearrangement of uncertain significance is identified by conventional chromosome or fluorescence in situ hybridization (FISH) studies and characterization of the genes affected could be helpful, but an alternative clinical assay is not available for this purpose. MPseq utilizes a modified library preparation method to generate paired end sequencing from long fragments of genomic DNA, resulting in higher sensitivity and lower cost compared to conventional Next Generation Sequencing (NGS) for detecting structural variation. Precise molecular characterization by MPseq of clinically significant genes at or near the breakpoints of chromosome rearrangements can provide tremendous diagnostic, prognostic and predictive value, and this assay has already demonstrated its potential to offer targeted therapeutic treatment opportunities for patients with hematologic malignancies.Of the 33 MPseq tests that have been performed following abnormal chromosome or FISH studies of a hematologic malignancy, 15 were for lymphoid and 18 were for myeloid disorders. Each case had a chromosome alteration detected by routine chromosome or by FISH studies. In all cases, multiple partner genes or just one partner gene was known and the MPseq analysis was limited to the region targeted by the rearrangement. Sanger sequencing confirmed each abnormal result identified by MPseq. Of these 33 samples, 26 were found to have direct concordance between MPseq and the targeted rearrangement, 4 demonstrated concordance but the rearrangement was found to be more complex than appreciated from conventional cytogenetic studies and in 3 cases MPseq did not identify the abnormality likely due to the presence of a low level clone and/or as a result of the repetitive nature of the DNA near the rearrangement. Of the 30 concordant cases, MPseq identified in 21 of these a clinically relevant gene at the breakpoint resulting in identification of a fusion gene or deletion of a potentially significant gene within the rearrangement. In 5 cases, the gene at the breakpoint was predicted to be influenced by a position effect such as translocation to an immunoglobulin locus. In all 26 cases where a clinically significant gene was identified, MPseq provided diagnostic, prognostic and/or predictive value which had not been appreciated with conventional cytogenetic studies alone.MPseq was critical for the identification of fusion genes allowing for the classification of Philadelphia Chromosome (Ph)-Like B-ALL. In one example of adult B-ALL, an inv(1)(q25q41) was identified by chromosomes and subsequent MPseq confirmed a chimeric gene comprised of the 5' end of RCSD1 fused to the tyrosine kinase domain of ABL2 resulting in an RCSD1-ABL2 fusion. A second example of relapsed pediatric B-ALL identified an ETV6 rearrangement by FISH and subsequent MPseq confirmed a chimeric gene composed of exons 1-4 of ETV6 and exons 15-20 including the tyrosine kinase domain of NTRK3 . Each fusion identified may be targetable by tyrosine kinase inhibitors. MPseq was also valuable in identification of fusion genes in patients with myeloid malignancies. In one example of infant AML, FISH studies identified a cryptic NUP98 rearrangement and subsequent MPseq confirmed a chimeric gene comprised on exons 1-13 of NUP98 fused to exons 25-28 of the KDM5A gene. This fusion is a known recurrent rearrangement associated with non-Down syndrome acute megakaryocytic leukemia. In summary, by presenting our initial clinical experience along with case examples, we demonstrate that MPseq represents an NGS technology that has the potential to revolutionize the diagnosis of hematologic malignancies and provide an opportunity to advance precision medicine. DisclosuresNo relevant conflicts of interest to declare.

Clinical Utility of Targeted Next-Generation Sequencing Assay to Detect Copy Number Variants Associated with Myelodysplastic Syndrome in Myeloid Malignancies

Copy number variants (CNVs) and gene mutations are important for diagnosis and treatment of myeloid malignancies. In a routine clinical setting, somatic gene mutations are detected by targeted next-generation sequencing (NGS) assay, but CNVs are commonly detected by conventional chromosome analysis and fluorescence in situ hybridization (FISH). The aim of this proof-of-principle study was to investigate the feasibility of using targeted NGS to simultaneously detect both somatic mutations and CNVs. Herein, we sequenced 406 consecutive patients with myeloid malignancies by targeted NGS and performed a head-to-head comparison with the results from a myelodysplastic syndrome (MDS) FISH and conventional chromosome analysis to detect CNVs. Among 91 patients with abnormal MDS FISH results, the targeted NGS revealed all 120 CNVs detected by MDS FISH (including -5/5q-, -7/7q-, +8, and 20q-) and 193 extra CNVs detected by conventional chromosome analysis. The targeted NGS achieved 100% concordance with the MDS FISH. The lower limit of detection of MDS CNVs by the targeted NGS was generally 5% variant allele fraction for DNA, based on the lowest percentages of abnormal cells detected by MDS FISH in this study. This proof-of-principle study demonstrated that the targeted NGS assay can simultaneously detect both MDS CNVs and somatic mutations, which can provide a more comprehensive genetic profiling for patients with myeloid malignancies using a single assay in a clinical setting.

AL amyloidosis: The effect of fluorescent in situ hybridization abnormalities on organ involvement and survival.

BackgroundSystemic light chain (AL) amyloidosis is a clonal plasma‐cell neoplasm that carries a poor prognosis. Although AL amyloidosis and Multiple Myeloma (MM) can co‐exist and share various cytogenetic chromosomal abnormalities, little is known about Fluorescent in situ hybridization (FISH) and its prognostic relevance in AL amyloidosis.Aim:The study aims to evaluate the most prevalent FISH cytogenetic abnormalities in AL patients as independent prognostic factors, and assess the impact of cytogenetics on the survival of high‐risk cardiac AL patients.Materials & MethodsThis retrospective study reviewed 113 consecutive AL patients treated at The Ohio State University (OSU). Patients were divided into subgroups based on FISH data obtained within 90 days of diagnosis. Hyperdiploidy was defined as trisomies of at least 2 chromosomal loci. Primary endpoints were progression free survival (PFS) and overall survival (OS). Kaplan Meier curves were used to calculate PFS and OS. The log‐rank test and Cox proportional hazard models were used to test the equality of survival functions and further evaluate the differences between groups.ResultsFISH abnormalities were detected in 76% of patients. Patients with abnormal FISH trended toward lower overall survival (OS) (p=0.06) and progression free survival (PFS) (p=0.06). The two most prevalent aberrations were translocation t(11;14) (39%) and hyperdiploidy‐overall (38%). Hyperdiploidy‐overall was associated with worsening PFS (p=0.018) and OS (p=0.03), confirmed in multivariable analysis. Patients with del 13q most frequently had cardiac involvement (p=0.006) and was associated with increased bone marrow plasmacytosis (p=0.02). Cardiac AL patients with no FISH abnormalities had much improved OS (p=0.012) and PFS (p=0.018)ConclusionsOur findings ultimately reveal the association of hyperdiploidy on survival in AL amyloidosis patients, including the high‐risk cardiac AL population.

AL Amyloidosis: The Effect of Maintenance Therapy on Autologous Stem Cell Transplantation Outcomes.

Background: Autologous stem cell transplantation (ASCT) remains an effective treatment option for many patients with systemic light chain (AL) amyloidosis. While maintenance post ASCT in multiple myeloma is now standard, the decision to utilize maintenance in AL amyloidosis remains largely unexplored. The present study aims to determine the prognostic significance of utilizing maintenance therapy following ASCT and assess the impact of fluorescent in situ hybridization (FISH) abnormalities, bone marrow plasma cell burden (BMPC), and degree of organ involvement on this decision. Methods and results: This is a retrospective analysis of fifty AL amyloidosis patients who underwent ASCT at The Ohio State University. Twenty-eight patients received maintenance and twenty-two did not. Kaplan–Meier survival analysis was used to compare the effect of maintenance therapy with no significant difference in PFS (p = 0.66) and OS (p = 0.32) between the two groups. There was no difference in survival based on maintenance when further categorized by FISH, PFS (p = 0.15), and OS (p = 0.65); BMPC ≥ 10%, PFS (p = 0.49), and OS (p = 0.32); or with 2 or more organs involved, PFS (p = 0.34) and OS (p = 0.80). Conclusion: Maintenance therapy post ASCT did not impact PFS or OS when categorized by FISH abnormalities, increasing BMPC, or ≥2 organs involved in AL amyloidosis patients.

Redefining the Prognostic Significance of t(11;14) Multiple Myeloma

Background In the era prior to introduction of novel agents, multiple myeloma (MM) harboring t(11;14) was characterized as standard risk. More recently, its unique biology, predictive ability and the prospect of targeted therapeutic agents have renewed interest in t(11;14) MM. Using a large, contemporary real-world database, we investigated the characteristics and outcomes of t(11;14) MM. Methods We used the Flatiron Health Electronic Health Record (EHR)-derived de-identified database to source patients (pts) with newly diagnosed MM from 1/2011 to 2/2020 with available Fluorescence in situ Hybridization (FISH) results documented within 90 days of diagnosis. We compared characteristics of t(11;14)+ patients [without additional high-risk FISH abnormalities: del(17p), Ch1 abnormality (Ch1a), t(4;14), t(14;16) or t(14;20)] vs. t(11;14)- patients (without additional high risk FISH) vs. del(17p) (irrespective of other abnormality) vs. Ch1a (Ch1a without additional high-risk FISH) vs. high-risk translocations [t(4;14), t(14;16) or t(14;20) without del(17p)]. We subsequently compared real-world progression-free survival (PFS) and overall survival (OS) across these five subsets. Additionally, we assessed the impact of t(11;14) as additional FISH abnormality in patients with del(17p) and in patients with Ch1a. We used Kaplan Meier methods with log-rank test and Cox proportional hazard regression model for survival analysis with date of diagnosis as the index date for follow-up. Results 6039 patients in the database met the inclusion criteria. Overall, 83.6% of patients received initial therapy with immunomodulatory agent (IMiD) and/or proteasome inhibitor (PI); of these 40.3% received combination of IMiD and PI. Overall, 27.1% received autologous hematopoietic cell transplantation. Median follow up was 2.1 years (IQR 0.8-4.0). There were 637 pts in t(11;14)+ group, 3173 in t(11;14)- group, 587 in del(17p), 1205 in Ch1a and 437 with high-risk translocations. The t(11;14)+ group had a higher proportion of men, IgM and light-chain isotype, as well as a higher proportion of patients with serum creatinine ³ 2mg/dl (Table). Patients in t(11;14)+ group had worse PFS (mPFS 3.1 vs. 3.3 years, p=0.02) and worse OS (mOS 5.9 vs. 6.5 years , p=0.04) compared to t(11;14)-, but better PFS and OS than the other three high-risk groups (Figure panels A and B). Worse PFS for t(11;14)+ was demonstrable even after adjustment for sex, age, race/ethnicity, immunoglobulin isotype, stage, comorbidities, and treatment received (adjusted HR=0.87, 95% C.I. 0.77-0.98, P=0.027). We subsequently analyzed the impact of presence of t(11;14) in MM with del(17p) or Ch1a.. The presence of t(11;14) in addition to del(17p) resulted in worse OS compared to del 17p without t(11;14) (mOS 2.8y vs. 3.7y; p=0.04). Indeed, the impact of t(11;14) on del(17p) was comparable to the impact of t(4;14) (Figure, Panel C). There was no difference in survival with concomitant presence of t(11;14) with Ch1a (Figure, Panel D). Conclusion MM with t(11;14) has distinct features at presentation and even when treated with modern therapy carries worse prognosis than otherwise standard-risk MM. The concomitant presence of t(11;14) portends a negative prognostic impact to MM with del(17p) but does not appear to impact MM with Ch1a. When present alongside del17p, t(11;14) behaves like a high-risk translocation and identifies a subset of MM in greatest need of newer therapies. Figure 1 Disclosures Costa: Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria; AbbVie: Consultancy; Celgene: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Genentech: Consultancy.

Fluorescence in Situ Hybridization (FISH) Utility for Risk Score Assessment in Patients With MDS With Normal Metaphase Karyotype

BackgroundCytogenetic profile is an essential parameter in myelodysplastic syndromes (MDS) risk stratification by both International Prognostic Symptom Score (IPSS) and Revised (R)-IPSS. Almost one-half of patients with MDS have normal cytogenetics by metaphase karyotype. Here we report the yield of MDS fluorescence in situ hybridization (FISH) panel detecting cytogenetic abnormalities in these patients and its impact on risk stratification. Patients and MethodsAmong patients with normal metaphase karyotype, we assessed those patients who had cytogenetic abnormalities detected by an MDS FISH panel, which included probes for del (5), del (7), del (20), trisomy 8, and del (17p). Risk stratification was calculated by both IPSS and R-IPSS. ResultsOf 1600 patients with MDS with normal metaphase karyotype, 53 (3%) patients had cytogenetic abnormality detected by MDS FISH panel. Integrating the MDS FISH panel cytogenetics (IPSS + FISH restaging) resulted in upstaging the score, where 53% of low-risk IPSS were upstaged to intermediate (int)-1, 56% of int-1 were upstaged to int-2, and 78% of int-2 were upstaged to high risk. Based on the R-IPSS, 61% of very low-risk patients, all low-risk patients, 92% of intermediate-risk patients, and 50% of high-risk patients with FISH abnormalities were upstaged, respectively. ConclusionThe yield of MDS FISH panel detecting cytogenetic abnormalities in patients with normal karyotype by G-banding is low and may not warrant ordering the panel in all patients. Among the 3% of patients with normal karyotype who had cytogenetic abnormality detected by FISH, the risk score assignment by IPSS and R-IPSS was upstaged.

Enucleated globes with choroidal melanoma: A retrospective histopathological study and correlation with cytogenetic profile in 2 eye centers

BackgroundUveal melanoma is the commonest intraocular malignant tumor in adults and the choroid is the commonest involved location. It is more prevalent in Caucasians; however, the demographics are widely variable based on ethnicity. Histopathological features have been correlated to the cytogenetic profile, which we intend to report through the study of enucleated eyes with choroidal melanoma (CM). Materials and MethodsA retrospective review of 28 enucleated globes with CM in 2 tertiary eye centers (January 2000-December 2017). The tumors were histopathologically classified based on the 8th edition of the American Joint Committee on Cancer (AJCC). The histopathological risk factors and the AJCC classifications were correlated with Fluorescence in situ hybridization (FISH) for chromosomes 3 and 8 available results in 18/28 eyes. ResultsWe have included 28 patients with a mean age of 56 years, 13 males (46.4%) and 15 females (53.6%). None had lymph node involvement or metastatic disease. The tumor size was categorized as 3 and 4 in 68% of eyes. Half tumors were of spindle cell type and were associated with absent cytogenetic abnormality in chromosomes 3 and 8 (P=0.005). Closed vascular loops presence was significantly associated with abnormal chromosomes 3 and 8 (P=0.027). ConclusionPatients in our area presented late with larger tumor size. The spindle cell CM was the commonest and correlated with negative FISH results, while the presence of closed vascular loops was a risk factor for abnormal FISH results hence expected worse prognosis. AJCC classification did not correlate well with our FISH results.

Metaphase cytogenetics and plasma cell proliferation index for risk stratification in newly diagnosed multiple myeloma

AbstractMetaphase cytogenetic abnormalities, plasma cell proliferation index (PCPro), and gain 1q by fluorescence in situ hybridization (FISH) are associated with inferior survival in newly diagnosed multiple myeloma (MM) treated with novel agents; however, their role in risk stratification is unclear in the era of the revised International Staging System (R-ISS). The objective of this study was to determine if these predictors improve risk stratification in newly diagnosed MM when accounting for R-ISS and age. We studied a retrospective cohort of 483 patients with newly diagnosed MM treated with proteasome inhibitors and/or immunomodulators. On multivariable analysis, R-ISS, age, metaphase cytogenetic abnormalities (both in aggregate and for specific abnormalities), PCPro, and FISH gain 1q were associated with inferior progression-free (PFS) and overall survival (OS). We devised a risk scoring system based on hazard ratios from multivariable analyses and assigned patients to low-, intermediate-, and high-risk groups based on their cumulative scores. The addition of metaphase cytogenetic abnormalities, PCPro, and FISH gain 1q to a risk scoring system accounting for R-ISS and age did not improve risk discrimination of Kaplan-Meier estimates for PFS or OS. Moreover, they did not improve prognostic performance when evaluated by Uno's censoring-adjusted C-statistic. Lastly, we performed a paired analysis of metaphase cytogenetic and interphase FISH abnormalities, which revealed the former to be insensitive for the detection of prognostic chromosomal abnormalities. Ultimately, metaphase cytogenetics lack sensitivity for important chromosomal aberrations and, along with PCPro and FISH gain 1q, do not improve risk stratification in MM when accounting for R-ISS and age.