Normal Fluorescence In Situ Hybridization Research Articles

Single-Cell RNA Sequencing of Circulating Tumor Cells in Precursor Myeloma Patients Reveals Mechanisms of Disease Dissemination

Introduction Multiple Myeloma (MM) is preceded by the precursors Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM), where some patients with precursor disease progress to active MM faster than others. BM biopsies are useful to monitor disease progression, however, they cannot be repeated often for continuous monitoring of tumor burden especially in the precursor disease management setting. Numerous studies have identified circulating tumor cell (CTC) levels in peripheral blood (PB) are a powerful biomarker of disease aggressiveness; CTC levels have associated with disease stage, PFS and OS in various trials and even outperformed BM to assess tumor burden in the SMM setting. Genomic profiling also demonstrated CTCs harbor the same copy number abnormalities, translocations and mutations as BM tumor cells, and CTCs can be serially monitored to detect the emergence of high-risk subclones in the blood. Further investigations of the molecular profile of both normal plasma cells (NPCs) and CTCs are needed to improve our understanding of myeloma pathogenesis and mechanisms of CTC dissemination across the MM disease continuum. Methods A cohort of 309 samples including CD138-enriched paired PB and BM aspirates and matched CD138- BM aspirates were collected from 103 individuals, including patients with MGUS (n=11), SMM (n=46), NDMM (n=17) and healthy donors (n=29). Malignant PCs from BM and PB underwent 5' single-cell RNA sequencing (scRNA-seq) and single-cell B-cell receptor sequencing (scBCR-seq) (10x Genomics), and CD138- BM immune cells underwent 5' scRNA-seq and single-cell T-cell receptor sequencing (scTCR-seq) to study immune alterations related the tumors' ability to circulate. To differentiate malignant from normal PCs, we used clonal V(D)J rearrangements. Differential expression (DE) and composition analyses were conducted using Wilcoxon's rank-sum tests. Results We successfully captured and profiled 774,647 BM tumor cells and 93,878 CTCs from the PB. The percent malignant plasma cells within the PB increased with disease stage, where NDMM patients had significantly more CTCs compared to MGUS patients (p=0.017) and low-risk SMM patients (p=0.0052). A median of 13.51%, 15.53%, and 18.34% CTCs were present from low (n=20), intermediate (n=10), and high-risk (n=16) SMM patients as defined by the International Myeloma Working Group's 2/20/20 criteria, suggesting sequencing-based CTC enumeration captures prognostically relevant differences in tumor burden. High expression of driver genes upregulated in patients with translocations, including CCND1, NSD2, and MAF, were detected in both BM tumor cells and CTCs in patients with t(11;14), t(4;14), and t(14;16) as identified by fluorescence in situ hybridization (FISH). In 5 patients with normal or inconclusive FISH results, we observed high levels of CCND2 and MAF in both BM and CTCs, indicating scRNA-seq can detect missed prognostically relevant cytogenetic abnormalities. A cytogenetic classifier model was developed to determine the accuracy of translocation calling using minimal numbers of CTCs, where the classifier's performance was able to consistently identify translocations downsampled to 50 CTCs. DE analysis of CTCs vs. BM tumor cells highlighted transcriptional similarity between BM and CTCs, validating their utility as a surrogate for analyzing BM tumor cells. DE analysis also revealed 8 genes significantly upregulated and 3 genes significantly downregulated in CTCs compared to BM tumor cells, providing novel insights into genes involved in PC circulatory potential. Pathway enrichment analysis revealed genes upregulated in CTCs were associated with epithelial mesenchymal transition, interferon response, and inflammation, consistent with CTC studies on MM patients, suggesting these pathways are dysregulated earlier in the disease continuum. Conclusions In the largest scRNA-seq study on CTCs to date, we demonstrate the utility of CTC-based molecular profiling for prognostication of patients with early-stage disease and provide novel insights into PC circulatory potential. Additional analyses are ongoing to gain further insight into intra-patient CTC heterogeneity and define high-risk disease CTC signatures that emerge throughout the MM disease continuum.

How to Obtain High-Quality Metaphase Spreads for Molecular Cytogenetics.

Interphase or metaphase nuclei can be accessed in molecular cytogenetic analyses. Metaphase spreads are routinely studied by fluorescence in situ hybridization (FISH) to answer clinical genetic questions. Even though metaphase quality is essential for FISH studies, there is limited ability in clinical cases to improve the quality of cytogenetic preparations. However, the quality of preps is important for the exact localization of FISH signals, which is necessary to identify individual chromosomes and chromosomal sub-regions using inverted DAPI banding. Here we present an efficient and easy-to-perform variant of standard slide pretreatment before a normal FISH procedure. This method reproducibly leads to solid, "steel," nonfuzzy, and well-DAPI-banded metaphases. This protocol works in blood lymphocyte and amniotic fluid-derived fibroblasts. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Slide pretreatment for high-quality metaphases for molecular cytogenetics.

Normal FISH CLL Represents a Heterogeneous Subgroup Where Prognosis Can be Refined with IGHV Mutational Status

Normal FISH CLL Represents a Heterogeneous Subgroup Where Prognosis Can be Refined with IGHV Mutational Status

Advanced Imaging and Targeted Myeloma Lesion Biopsies to Enhance Global Response Assessment and Evaluate Spatial Heterogeneity in Multiple Myeloma

Advanced Imaging and Targeted Myeloma Lesion Biopsies to Enhance Global Response Assessment and Evaluate Spatial Heterogeneity in Multiple Myeloma

A case of germline mosaicism for a 7q32.1q33 deletion in a sperm donor: consequences on pregnancy follow-up and recommendations

BackgroundGermline mosaicism is considered to be a rare event. However, its occurrence is underestimated due to the limited availability of germ cells. The genomic variations that underlie this phenomenon comprise single nucleotide polymorphism (SNPs), copy number variations (CNVs) and aneuploidies. In the case of CNVs, deletions are more frequent in the paternal germline while duplications are more commonly maternal in origin. Germline mosaicism increases with paternal age as the risk of SNPs increase with the number of germ cell divisions. We here report a case of germline mosaicism in the spermatozoa of a donor that resulted in one pathological pregnancy.ResultsStraws from the same sperm donor were provided to seven recipient couples, resulting in four pregnancies. Second trimester ultrasound analysis revealed bilateral talipes equinovarus associated with growth retardation in one of these pregnancies. Array-comparative genomic hybridization (CGH) carried out after amniocentesis revealed a 4 Mb deletion in the 7q32.1q33 region. The blood karyotypes and array-CGHs were normal in the mother, as well as in the donor. However, the microsatellite profile indicated a paternal origin. Fluorescent in situ hybridization (FISH) analysis of the donor’s spermatozoa revealed the same chromosomal rearrangements in 12% of the spermatozoa population. Due to the documented risk of mental retardation associated with genomic rearrangements in the same region, the couple decided to terminate the pregnancy. Amniocentesis was performed in the other couples, which yielded normal FISH analysis results.ConclusionsSeveral cases of germline mosaicism have been reported to date, but their frequency is probably underestimated. Moreover, it is important to note that germline mosaicism cannot be ruled out by conventional cytogenetic screening of blood cells. This case highlights the need for close follow-up of every pregnancy obtained through gamete donation, given that the occurrence of germline mosaicism may have major consequences when multiple pregnancies are obtained concomitantly.

Chronic lymphocytic leukemia patients with IGH translocations are characterized by a distinct genetic landscape with prognostic implications.

Chromosome 14q32 rearrangements/translocations involving the immunoglobulin heavy chain (IGH) are rarely detected in chronic lymphocytic leukemia (CLL). The prognostic significance of the IGH translocation is controversial and its mutational profile remains unknown. Here, we present for the first time a comprehensive next-generation sequencing (NGS) analysis of 46 CLL patients with IGH rearrangement (IGHR-CLLs) and we demonstrate that IGHR-CLLs have a distinct mutational profile with recurrent mutations in NOTCH1, IGLL5, POT1, BCL2, FBXW7, ZMYM3, MGA, BRAF and HIST1H1E genes. Interestingly, BCL2 and FBXW7 mutations were significantly associated with this subgroup and almost half of BCL2, IGLL5 and HISTH1E mutations reported were previously identified in non-Hodgkin lymphomas. Notably, IGH/BCL2 rearrangements were associated with a lower mutation frequency and carried BCL2 and IGLL5 mutations, while the other IGHR-CLLs had mutations in genes related to poor prognosis (NOTCH1, SF3B1 and TP53) and shorter time to first treatment (TFT). Moreover, IGHR-CLLs patients showed a shorter TFT than CLL patients carrying 13q-, normal fluorescence in situ hybridization (FISH) and +12 CLL, being this prognosis particularly poor when NOTCH1, SF3B1, TP53, BIRC3 and BRAF were also mutated. The presence of these mutations not only was an independent risk factor within IGHR-CLLs, but also refined the prognosis of low-risk cytogenetic patients (13q-/normal FISH). Hence, our study demonstrates that IGHR-CLLs have a distinct mutational profile from the majority of CLLs and highlights the relevance of incorporating NGS and the status of IGH by FISH analysis to refine the risk-stratification CLL model.

Outcome of Multiple Myeloma with Chromosome 1q Gain and 1p Deletion after Autologous Hematopoietic Stem Cell Transplantation: Propensity Score Matched Analysis

The gain/amplification CKS1B gene at chromosome region 1q21 (1q+) is one of the most common genetic aberrations in multiple myeloma (MM). Amplification of CKS1B is frequently associated with the deletion of the CDKN2C gene at chromosome region 1p32 (1p-), which is also associated with inferior outcomes. In this retrospective study, we evaluated the outcomes of patients with 1q+ and/or 1p- after high-dose therapy and autologous hematopoietic cell transplantation (auto-HCT). From January 2006 to December 2015, 1491 newly diagnosed patients with MM underwent upfront high-dose therapy and auto-HCT at our institution. Of those, 899 had the fluorescent in situ hybridization (FISH) data available. FISH was performed at diagnosis and before the start of induction in 686 (76%) patients and after the initiation of induction therapy in 213 (24%) patients. We identified 100 patients with 1q+ and/or 1p- by FISH from the cohort of 899 patients. A control group (n=287) with diploid cytogenetics and normal FISH panel was selected from the same cohort. From the above 2 cohorts, using a propensity score matched analysis, we identified matched controls for 85 of the 100 patients with 1q+/1p-. Patients were matched for age at auto-HCT, sex, International Staging System stage, induction regimen, creatinine level, disease status at auto-HCT, conditioning regimen, and maintenance therapy. Sixty-seven (79%), 4 (5%), and 14 (16%) patients had 1q+, 1p-, or both 1q+ and 1p-, respectively. There was no significant difference in induction therapy, preparative regimen, or maintenance therapy between the 1q+/1p- and the control group. The median follow-up time for all patients was 29.2 months (range, 0.29 to 84.96). The cumulative incidence of 100-day nonrelapse mortality was 1.2% and 0% for the 1q+/1p- and the control group, respectively. Forty-two patients (50%) in the 1q+/1p- group achieved complete response compared with 40 patients (47%) in the control group. The estimated 3-year progression-free survival (PFS) and overall survival (OS) rates were 41% and 79% for the 1q+/1p- group and 56% and 86% for the control group. Patients in the 1q+/1p- group were at significantly increased risk of progression or death compared to the control group (hazard ratio [HR], 2.21; confidence interval [CI], 1.18 to 4.16; P=.014). No significant association between OS in the 2 groups was observed. The outcome of the 1q+/1p- alone (with no additional high-risk cytogenetics) and the propensity score matched control groups was also compared. Median PFS for the 1q+/1p- alone subgroup was 26.6 months, compared with 38.8 months for the control group (HR, 1.9; CI, 0.9 to 4.08; P=.09). The median OS had not been reached for the 1q+/1p- alone subgroup and was 81.1 months for the control group (HR, 1.25; CI, 0.3 to 4.6; P= .73). 1q+/1p- abnormalities with amplification of CKS1B and deletion ofCDKN2Cgenes were associated with shorter PFS compared with a propensity score matched group of patients with diploid cytogenetics and normal a FISH panel. The outcomes of 1q+/1p- patients with MM have improved with the use of more effective induction, conditioning, and maintenance therapy compared with historical controls, but we still need more effective therapeutic approaches to fully overcome the negative impact of 1q+/1p-.

Diagnosing Mds; Do We Really Need to FISH?

Objectives Making a definitive diagnosis of myelodysplastic syndrome (MDS) currently entails review of morphology on peripheral blood and bone marrow as well as cytogenetics by standard karyotyping. Fluorescence in situ hybridization (FISH) panel is performed to identify specific abnormalities in addition to karyotyping. Methods We reviewed 186 patients who had undergone bone marrow biopsy for suspected MDS from 2016 to 2018 across our health system and subsequently had karyotyping and FISH testing performed. Results We identified 186 patients in our study cohort, 164 of these patients had karyotype and FISH testing available. There was excellent concordance rate of 93% (152 of 164) between karyotype and FISH. Among the 164 bone marrow biopsy samples reviewed, 40(24.4%) eventually had a confirmed diagnosis of MDS. Concordance rate of karyotyping and FISH results in the confirmed cases of MDS was 75% (30 of 40). Overall, 9 of the 40 cases (22.5%) had discordant findings, of these 20% (8 of 40) demonstrated abnormal karyotype analysis with normal FISH while only 1 of 40 (2.5%) had normal karyotype with abnormal FISH diagnostic of MDS. The abnormal FISH finding was a cryptic RUN X1 deletion. Conclusion Our study indicated excellent concordance between karyotype and MDS-FISH. FISH rarely added additional value to karyotype. Disclosures Jamshed: Takeda Pharmaceutical: Honoraria.

The Effect of Cytogenetic Abnormalities on Organ Involvement and Survival in Patients with AL Amyloidosis

Introduction: Systemic light chain amyloidosis (AL) is a clonal plasma-cell neoplasm that carries a poor prognosis. Efforts are being made at recognizing this disease earlier and developing better prognostic tools as AL is frequently diagnosed at an advanced stage. Cytogenetic analysis and its prognostic relevance have been well studied in multiple myeloma (MM), a related disorder, but remain relatively unknown and less widely reported in AL literature. Previous studies have demonstrated that AL amyloidosis exhibits an increased incidence of translocation t(11;14). However, with an extensive array of fluorescence-in-situ hybridization (FISH) probes now available, multiple other cytogenetic abnormalities are being identified in AL at diagnosis. In addition, it is well known that cardiac involvement in AL is an independent negative prognostic factor for these patients; however, the implications of cytogenetics for these high-risk patients remains largely unexplored. Finally, with the advent of novel non-transplant therapy options for AL patients, we look to evaluate the relevance of daratumumab in this setting. As such, the present study aims to a) determine the most relevant FISH abnormalities in AL patients and establish their importance as independent prognostic factors for response to therapy and in survival, b) assess the impact of cytogenetics on the survival of cardiac AL patients, and c) determine the effect of daratumumab on the survival of patients with AL. Methods: A retrospective chart review was performed on 140 consecutive AL patients treated at The Ohio State University. This cohort included 20 patients who received daratumumab. 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), and Kaplan Meier curves were used to calculate PFS and OS. Results: The median age at diagnosis was 62 years (range: 33-88) and 55% were male. Median number of organs involved was 2, and 49% and 65% had cardiac and kidney involvement, respectively. Chromosomal abnormalities were detected in 86 (61%) patients. Translocation t(11;14) was the most prevalent (44%) aberration followed by hyperdiploidy (43%). We observed a statistically significant relationship between several FISH abnormalities and increased plasma cell burden (PC) (≥10%), including gain (+) 5p/5q (p=0.025), del13q (0.009), +11q (p<0.001), and hyperdiploidy (p<0.001). In addition, hyperdiploidy was associated with worsening of PFS (p=0.019) and OS (p=0.032) (Figure 1A). In multivariable analysis, hyperdiploidy was confirmed to be a poor prognostic marker after adjusting for other confounding variables. In patients with cardiac involvement, hyperdiploidy was also associated with worsening of PFS (p=0.0497) and OS (p=0.006) (Figure 1B). Del 13q was found to be associated with cardiac involvement (p=0.01) but showed no prognostic impact on survival. Conversely, survival benefit was seen among these patients with cardiac involvement who had no FISH abnormalities at diagnosis, both in terms of OS and PFS (p=0.019 and p=0.042, respectively). In addition, the overall presence of t(11;14) did not have any prognostic impact on OS (p=0.76) or PFS (p=0.41). However, on further stratification, we did observe a marginal difference in PFS (p=0.09) among four groups (Figure 1C), and more specifically, patients with presence of only t(11;14) did worse compared to those patients with normal FISH in terms of PFS (p=0.021). This potentially suggests that patients with t(11;14) only are at risk for earlier progression. Finally, we evaluated the efficacy of daratumumab, and observed a median OS of 6.1 years and median PFS 2.6 years. Of note, presence of gain 1q was associated with a trend toward better response to daratumumab. 100% of patients (5/5) with gain 1q achieved a hematologic partial response (PR) or better versus only 60% of patients without +1q. Conclusion: Our findings reveal the effect of hyperdiploidy on PC tumor burden, overall survival, and its importance within the high-risk cardiac AL patient population. The results with daratumumab in our patient subset with gain 1q is intriguing in its own right but identification of the mechanism by which the effect of this mutation is abrogated merits further exploration as its use only continues to grow. Disclosures Rosko: Vyxeos: Other: Travel support. Efebera:Takeda: Honoraria; Akcea: Other: Advisory board, Speakers Bureau; Janssen: Speakers Bureau.

The use of fluorescence in situ hybridization analysis on sperm: indications to perform and assisted reproduction technology outcomes.

To determine the consequences of an altered sperm fluorescence in situ hybridization (FISH) result for ART outcomes and the indications for a sperm FISH analysis. Data from 439 infertile men were collected. Bivariate analyses were performed to determine the association of men's age, seminal alterations, and sperm FISH indication, with the incidence of X, Y, 13, 18, and 21 sperm chromosomal abnormalities. A multivariate logistic regression analysis was performed to establish the most predictive variables for altered sperm FISH. Results from the IVF/ICSI cycles were collected for 248 out of 439 patients. Two distinct groups were established: 151 couples that used their own oocytes and 97 couples involved in egg donation programs. In both groups, ART outcomes were compared between normal and altered sperm FISH. Teratozoospermia and oligozoospermia were associated with sperm chromosome anomalies (p < 0.05). Indications for sperm FISH analysis with the highest predictability were teratozoospermia, male age, oligozoospermia, and implantation failure (AUC = 0.702). Embryo quality (p = 0.096), pregnancy rate (p = 0.054), and implantation rate (p = 0.089) were higher in own-oocytes couples with normal sperm FISH than in altered sperm FISH couples, although differences were not statistically significant. In donor-oocytes couples, in which high-quality embryos were transferred later than in own-oocytes couples (3.8 vs. 3.0 days), we did not identify differences in the ART outcome between normal and altered sperm FISH couples. In both groups, the possible interference of woman age was negligible. Sperm FISH is indicated in middle-aged oligoteratozoospermic patients with implantation failures in previous IVF/ICSI cycles. Sperm chromosome anomalies have a moderate detrimental impact on embryo quality, implantation, and pregnancy rates.

Sperm chromosomal abnormalities and their contribution to human embryo aneuploidy.

In this work we reviewed 18years of experience using fluorescence in situ hybridization (FISH) for sperm aneuploidy testing. We evaluated parameters associated with increased numerical sperm chromosome abnormalities and determined the male contribution to embryo aneploidies in terms of reproductive outcome by increased sperm aneuploidy. This retrospective study analyzed data from 2008 sperm samples of infertile males undergoing FISH analysis because of clinical history of repetitive implantation failure, recurrent miscarriage, impaired sperm parameters, or mixed causes. Sperm concentration was the only sperm parameter associated with FISH results-we observed a gradual increase of abnormal sperm FISH results in males with decreasing sperm concentration. However, a great proportion of normozoospermic males also showed increased sperm aneuploidies, suggesting that sperm parameters alone do not enable identification of a substantial proportion of infertile males at risk of sperm aneuploidies. Regarding reproductive outcomes, couples with normal sperm FISH results for the male had similar outcomes regardless of conventional in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), or preimplantation genetic testing for aneuploidies (PGT-A). However, couples with abnormal sperm FISH results for the male showed better clinical outcomes after PGT-A, suggesting a potential contribution of sperm to embryo aneuploidy. Moreover, PGT-A cycles showed better clinical outcomes when 24 chromosomes were analyzed by array comparative genome hybridization (aCGH) or next-generation sequencing (NGS) instead of only nine chromosomes analyzed by FISH. In conclusion, sperm FISH analysis offers clinical prognostic value to evaluate reproductive possibilities in infertile couples. Therefore, couples with abnormal sperm FISH results should be offered genetic counseling and presented with clinical options such as PGT-A.

Impact of t(11;14) on the Outcome of Autologous Transplantation in Multiple Myeloma: A Matched-Pair Analysis

Abstract:Background:The translocation t(11;14)(q13;q32) is the most common chromosomal translocation in multiple myeloma (MM) with a reported frequency of 15-20%. Most, but not all, reports in the literature have identified this translocation with a relatively favorable outcome. In a smaller cohort, we had previously reported the impact of t(11;14) on the outcomes of MM patients who underwent autologous hematopoietic transplantation (auto-HCT) at our institution1. In this study, we present an updated analysis with a larger cohort of t(11;14) patients, and compared their outcomes to a propensity-matched control group of MM patients with normal diploid cytogenetics and no high-risk abnormalities detected by Fluorescence in Situ Hybridization (FISH).Methods and patients:A total of 1365 MM patients underwent auto-HCT at our institution from 2007 to 2015. We identified 95 patients with t(11;14) by FISH before auto-HCT. Out of these 95 patients, 44 had t(11;14) alone, 26 had co-existent high-risk abnormalities and remaining 25 had standard-risk abnormalities. The control group included 287 MM patients with normal diploid cytogenetics and no abnormalities detected by FISH. From the above two cohorts, using a 1:1 propensity score-matched analysis without replacement, we identified matched controls for 79 patients with t(11;14). Clinical response, relapse, and progression were defined by the International Myeloma Working group criteria. The FISH technique was performed using a LSI IGH/CCND1 XT dual color, dual fusion translocation probe from Abbott Molecular, Inc. The normal cut off for IGH/MYEOV/CCND1 rearrangement using LSI IGH/CCND1 XT probe established at 95 (P<0.05) confidence level in the our Cytogenetics Laboratory is 0.4% for 1R1G2F; 1.1% for 2R2G1F; 3.4% for 3R2G; 7.4% for 2R3G and 0% for 1R3F signal patterns.Results:Table 1 includes the baseline characteristics of the matched doublets. Patients in both groups were well matched for age, ISS stage, serum creatinine, response to induction therapy, induction and preparative regimens, and maintenance therapy. The median follow-up time for the cohort was 48.1 months (range: 0.8-124.6). The overall response rate (CR+VGPR+PR) after auto-HCT was 77/79 (97%) and 78/79 (99%) patients in the t(11;14) and the control group, respectively (P = 1.00). Twenty eight (35%) and 37 (47%) patients achieved a CR in the t(11;14) and the control group, respectively. VGPR rate in the t(11;14) and the control group was 35 (44%) and 31 (39%), respectively. The median PFS for the t(11;14) and the control groups were 33.3 (95%CI: 25.8-not reached) and 39.7 (95%CI: 36.6-not reached) months, respectively (p = 0.24, stratified log-rank test). The median OS has not been reached for both groups (p=0.35). The 4-year PFS rates in the t(11;14) and the control groups were 42% (95%CI: 31%-57%) and 50% (95%CI: 38%-65%), respectively (Fig. A). The 4-year OS rates in the t(11;14) and the control groups were 76% (95%CI: 65%-90%) and 87% (95%CI: 79%-97%), respectively (Fig. B).Conclusions:On a propensity score matching analysis, multiple myeloma patients with translocation t(11;14) had similar response rates, PFS and OS to auto-HCT as standard-risk patients with normal cytogenetics or FISH studies.

Outcome of Multiple Myeloma with Chromosome 1q Gain and 1p Deletion after High-Dose Therapy and Autologous Hematopoietic Stem Cell Transplantation

Background and AimThe gain/amplification CKS1B gene at chromosome region 1q21 (1q+) is one of the most common genetic aberrations in multiple myeloma (MM). CKS1Bamplification and overexpression contribute to increased p27Kip1 degradation, cell cycle upregulation and poor outcomes in MM patients. Amplification of CKS1B is frequently associated with the deletion of CDKN2C gene at chromosome region 1p32 (1p-). Deletion of CDKN2C, a tumor suppressor gene, leads to an increased proliferative rate of plasma cells in MM patients and is also associated with inferior outcomes. There are limited data on the impact of 1q+/1p- on the outcomes after high-dose therapy and autologous stem cell transplantation (auto-HCT).MethodsIn this retrospective study, we evaluated outcomes of patients with 1q+ and/or 1p- after high-dose therapy and auto-HCT. From 2007 to 2015, 1365 MM patients underwent high-dose therapy and auto-HCT at our institution. We identified 100 patients with 1q+ and/or 1p- by fluorescent in situ hybridization (FISH) from that cohort. The cut off value used for gain/amplification of a 1q21/CKS1B was 7.9%; gain/amplification of two 1q21/CKS1B was 4.4%; and deletion of a 1p32.3/CDKN2C was 6.8%. A control group (N=287) with diploid cytogenetics and normal FISH panel with no high risk abnormalities was selected from the same cohort. From the above two cohorts, using a 1:1 propensity score-matched analysis without replacement, we were able to identify a matched control for 85 patients with 1q+/1p-.ResultsBaseline characteristics of the 1q+/1p- and control groups are summarized in table 1. Sixty-seven (79%), 4 (5%) and 14 (16%) patients had 1q+, 1p- or both 1q+ and 1p-, respectively. There was no significant difference in induction therapy, preparative regimen, or maintenance therapy between the two groups. The median follow-up time for all patients was 29.2 months (range: 0.29 -84.96). The cumulative incidence of 100-day nonrelapse mortality was 1.1% and 0% for the 1q+/1p- and the control groups, respectively. Forty-two patients (51%) in the 1q+/1p- group achieved a CR compared to 40 patients (49%) in the control group (P=0.6). VGPR rates in the 1q+/1p- and controls groups were 47% and 53% (P=0.5) respectively. The overall response rate for the 1q+/1p-and controls group was 96% and 100%, respectively (P=0.3). Median PFS for the 1q+/1p- and the controls groups were 26.5 months and 38.8 months, respectively. The estimated 3-year PFS for the 1q+/1p- and the control groups were 41% and 56%, respectively (HR 2.2, CI 1.18-4.16, P=0.01) (Figure 1A). The 3-year OS for the 1q+/1p- and the control groups were 79% and 86%, respectively (HR 1.5, CI 0.61-4.05, P=0.34) (Figure 1B).Conclusion1q+/1p- abnormalities with amplification CKS1B and deletion of CDKN2C genes were seen in approximately 7% of MM patients undergoing auto-HCT between 2007 and 2015. They were associated with a shorter PFS when compared to a propensity matched group of patients with diploid cytogenetics and normal FISH panel. Patients with 1q+/1p- may need more aggressive therapeutic approaches. DisclosuresThomas:Amgen Inc: Research Funding; Bristol Myers Squibb Inc.: Research Funding; Array Pharma: Research Funding; Acerta Pharma: Research Funding; Celgene: Research Funding. Lee:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies Corporation: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Chugai Biopharmaceuticals: Consultancy; Takeda Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees. Patel:Poseida Therapeutics, Inc.: Research Funding; Takeda: Research Funding; Abbvie: Research Funding; Celgene: Research Funding. Shpall:Affirmed GmbH: Research Funding. Orlowski:Bristol-Myers Squibb: Consultancy; Kite Pharma: Consultancy; Celgene: Consultancy; Amgen: Consultancy, Research Funding; Sanofi-Aventis: Consultancy; Janssen: Consultancy; Spectrum Pharma: Research Funding; BioTheryX: Research Funding; Takeda: Consultancy. Champlin:Otsuka: Research Funding; Sanofi: Research Funding.

PD09-03 COST-EFFECTIVENESS OF SPERM FLUORESCENCE IN SITU HYBRIDIZATION TESTING FOR INFERTILE MEN WITH SUSPECTED SPERM ANEUPLOIDY

You have accessJournal of UrologyInfertility: Epidemiology & Evaluation I1 Apr 2018PD09-03 COST-EFFECTIVENESS OF SPERM FLUORESCENCE IN SITU HYBRIDIZATION TESTING FOR INFERTILE MEN WITH SUSPECTED SPERM ANEUPLOIDY Taylor P. Kohn, Alexander W. Pastuszak, Jaden R. Kohn, Katherine M. Rodriguez, Dolores J. Lamb, and Larry I. Lipshultz Taylor P. KohnTaylor P. Kohn More articles by this author , Alexander W. PastuszakAlexander W. Pastuszak More articles by this author , Jaden R. KohnJaden R. Kohn More articles by this author , Katherine M. RodriguezKatherine M. Rodriguez More articles by this author , Dolores J. LambDolores J. Lamb More articles by this author , and Larry I. LipshultzLarry I. Lipshultz More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.600AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Men with recurrent spontaneous abortions (SAB) or implantation failures (IF) may have abnormal sperm aneuploidy as diagnosed by fluorescent in situ hybridization (FISH). In vitro fertilization (IVF) with pre-implantation genetic screening (PGS) is the most effective assisted reproductive technique (ART) for men with sperm aneuploidy, yet the cost-effectiveness of FISH testing in the context of available genetic testing of the embryo remains unclear. Here we examine the cost effectiveness of sperm FISH testing in the context of ART. METHODS Couples with a history of either 2 IVF failures or 2 SABs without known female factor were surveyed via phone call to document subsequent pregnancy attempts. Of 98 couples surveyed, 80 men underwent FISH testing while 18 did not. Men were grouped by normal FISH, abnormal FISH, and not tested for FISH, and outcomes of pregnancies conceived by natural intercourse, IVF, intracytoplasmic sperm injection (ICSI), and IVF/PGS were assessed. The cost-effectiveness for each category and reproductive method, as well as the overall cost of performing or not performing FISH prior to assisted reproduction, were determined. RESULTS Of the 80 men who underwent FISH, 78 had abnormal results, while 2 were normal. Success and failure rates, costs, and cost-effectiveness of each reproductive method in our sample are shown in Table 1a. The costs per pregnancy for men without FISH testing using PGS with IVF were $30,268 and $49,600, respectively. For men with normal and abnormal FISH, PGS after IVF was more cost-effective than either IVF alone or ICSI with IVF (Table 1a). The total costs, associated with FISH testing in men with suspected sperm aneuploidy was $17,351 for FISH testing prior to attempted conception and $16,318 for bypassing FISH and proceeding directly to the reproductive method of choice - more than cost of the FISH testing (Table 1b). CONCLUSIONS PGS after IVF is more cost-effective than IVF alone, or ICSI with IVF for men with possible sperm aneuploidy. Sperm FISH testing adds additional cost especially when the rate of rate of ″abnormal″ is so high. Thus, couples utilizing assisted reproductive techniques may consider utilizing sperm FISH only when a sperm donor would be considered, or proceeding directly to PGS without undergoing sperm FISH testing. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e222-e223 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Taylor P. Kohn More articles by this author Alexander W. Pastuszak More articles by this author Jaden R. Kohn More articles by this author Katherine M. Rodriguez More articles by this author Dolores J. Lamb More articles by this author Larry I. Lipshultz More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The 11q-Gain/Loss Aberration Occurs Recurrently in MYC-Negative Burkitt-like Lymphoma With 11q Aberration, as Well as MYC-Positive Burkitt Lymphoma and MYC-Positive High-Grade B-Cell Lymphoma, NOS.

ObjectivesThe latest revision of lymphoma’s World Health Organization classification describes the new provisional entity “Burkitt-like lymphoma with 11q aberration” (BLL, 11q) as lacking MYC rearrangement, but harboring the specific11q-gain/loss aberration. We report genetic characteristics of 11 lymphoma cases with this aberration.MethodsClassical cytogenetics, fluorescence in situ hybridization (FISH), and single nucleotide polymorphism/array comparative genomic hybridization.ResultsThe 11q aberrations were described as duplication, inversion, and deletion. Array comparative genomic hybridization showed two types of duplication: bigger than 50 megabase pairs (Mbp) and smaller than 20 Mbp, which were associated with bulky tumor larger than 20 cm and amplification of the 11q23.3 region, including KMT2A. Six cases revealed a normal FISH status of MYC and were diagnosed as BLL,11q. Five cases showed MYC rearrangement and were diagnosed as Burkitt lymphoma (BL) or high-grade B-cell lymphoma, not otherwise specified (HGBL, NOS).ConclusionsThe 11q-gain/loss is not specific for BLL, 11q, but occurs recurrently in MYC-positive BL and MYC-positive HGBL.

MP35-09 SPERM ANEUPLOIDY IS ASSOCIATED WITH WORSE GENERAL HEALTH IN INFERTILE MEN

You have accessJournal of UrologyInfertility: Epidemiology & Evaluation II1 Apr 2017MP35-09 SPERM ANEUPLOIDY IS ASSOCIATED WITH WORSE GENERAL HEALTH IN INFERTILE MEN Taylor P. Kohn, Alexander W. Pastuszak, Katherine M. Rodriguez, Zachary J. Solomon, Matthew F. Cherches, Dolores J. Lamb, and Larry I. Lipshultz Taylor P. KohnTaylor P. Kohn More articles by this author , Alexander W. PastuszakAlexander W. Pastuszak More articles by this author , Katherine M. RodriguezKatherine M. Rodriguez More articles by this author , Zachary J. SolomonZachary J. Solomon More articles by this author , Matthew F. CherchesMatthew F. Cherches More articles by this author , Dolores J. LambDolores J. Lamb More articles by this author , and Larry I. LipshultzLarry I. Lipshultz More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1099AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Numerous studies have examined the relationship between male infertility and men′s health but most have not investigated specific causes of infertility and their health impact. Here we examine the relationship between general male health and sperm aneuploidy. METHODS We used the Charlson Co-Morbidity Index (CCI) to assess and compare the health status of men with abnormal sperm aneuploidy to five groups of men who received care at an academic infertility clinic: men with normal sperm ploidy (normal FISH), men with genetic abnormalities that may cause infertility (genetic infertile), men presenting for infertility with varicocele (varicocele), men with idiopathic infertility (idiopathic) and proven-fertile men. Sperm ploidy was assessed using fluorescence in situ hybridization (FISH). Genetic infertile men had proven Y chromosome microdeletions or abnormal karyotype. CCI for men with sperm FISH results was completed via telephone survey and CCI for genetic infertile men was extracted from intake forms. Men who were diagnosed with a varicocele or idiopathic infertility had not conceived after 12 months. Men with varicoceles, men with idiopathic infertility, and fertile controls completed the CCI in clinic. Fertile controls included men who presented for vasectomy and had fathered a child in the past 5 years. RESULTS 402 men were included in this analysis: 92 with an abnormal sperm FISH results, 7 with a normal sperm FISH test, 49 genetic infertile men, 85 men with idiopathic infertility, 86 infertile men with varicocele, and 83 fertile controls. Mean age, semen density, and CCI of all groups are described in Table 1. Of note, malignancy was common in our cohort, exceeding rates in the SEER database for comparable age (Table 1). Controlling for age, we explored infertility groups as a predictor of elevated CCI score, and found that only men with abnormal sperm FISH (p=0.002) had increased risk of a higher CCI score. Age also increased the risk for a higher CCI score, with each additional year raising the CCI by 0.0117 points (p=0.009). CONCLUSIONS Men with elevated sperm aneuploidy have nearly double the prevalence of general health issues, particularly malignancy, when compared with men with infertility of other etiologies. These findings further support the relationship between genetic causes of male infertility and general health. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e460-e461 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Taylor P. Kohn More articles by this author Alexander W. Pastuszak More articles by this author Katherine M. Rodriguez More articles by this author Zachary J. Solomon More articles by this author Matthew F. Cherches More articles by this author Dolores J. Lamb More articles by this author Larry I. Lipshultz More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Target fluorescence in-situ hybridization (Target FISH) for plasma cell enrichment in myeloma.

BackgroundCytogenetic abnormalities are important prognostic markers in plasma cell myeloma (PCM) and detection is routinely performed by interphase fluorescence in-situ hybridization (FISH) with a panel of probes after enrichment of the plasma cells in the bone marrow specimen. Cell sorting by immunomagnetic beads and concurrent labeling of the cytoplasmic immunoglobulin are the usual enrichment methods. We present an alternative method of plasma cell enrichment termed Target FISH, which is an automated system that combines the images of May-Grünwald- Giemsa (MGG) staining and FISH study on the same plasma cell for analysis.ResultsOur experience of Target FISH on 40 PCM patients was described. Briefly, plasma cells were MGG stained, image captured, de-stained, FISH probe hybridized and finally relocated for simultaneous analysis of morphology and FISH signal pattern. The FISH probe panel was TP53/CEP17, t(4;14) IGH/FGFR3, t(14;16) IGH/MAF and CKS1B(1q21)/CDKN2C(P18). Gain of 1q21 was the most common abnormality detected in 18 patients (45 %), to be followed by t(4;14) IGH/FGFR3 detected in 11 patients (27.5 %). Of note, 10 patients showed coexistence of both t(4;14) and 1q21 gain. Two patients showed del(17p)/TP53, one in association with t(4;14) and 1q gain while the other was stand alone. None of this patient cohort showed t(14;16) IGH/MAF. Using the critical binomial function, the normal cutoff FISH positive value for del(17p)/TP53 was 3.4 %, t(4;14) IGH/FGFR3 was 6.8 %, t(14;16) IGH/MAF was 5.6 % and +1q21 was 5.7 %.ConclusionsThe equipment cost notwithstanding, when compared with cell sorting, the total reagent cost was around 10 % lower in Target FISH. The total processing time was longer for Target FISH but manual fluorescence microscopy was no longer necessary. The main advantage of Target FISH was the complete certainty that the cytogenetic abnormality was detected in the cells of interest, and hence a more stringent analytical cutoff value might be considered. Optimization of the cell collection and slide preparation process upfront was required to accrue adequate target cells on each slide for analysis. Our experience suggested that Target FISH was applicable as a routine method of plasma cell enrichment in clinical diagnostic laboratories.

Malignant Melanoma of the Nail Apparatus

Background Malignant melanoma of the nail apparatus is exceedingly rare. Increasingly, genetic studies have been employed to aid in distinguishing between malignant melanoma and benign melanocytic nevi. Methods Archived nail apparatus melanomas were analyzed by fluorescence in situ hybridization (FISH) using probes targeting the genes at 6p25 (RREB1), 11q13 (CCND1), 8q24.1 (MYC), 6q23 (MYB), 9p21 (CDKN2A) and the centromeres of chromosomes 8 (D8Z2) and 6 (D6Z1). The results were correlated with clinical and demographic information. Results Mean patient age was 57.8 years (range 23-92 years). In all, 5 of 7 (71%) cases involved the upper extremity digits. RREB1 gain was seen in all cases. CCND1 gain was seen in 6 of 7 (86%) cases, 3 of which were amplified. MYB loss and MYC gain were both seen in 5 of 7 (71%) cases. Homozygous loss of CDKN2A was not observed in any case. Two of 7 (28.6%) patients had lymph node metastasis and died of widely metastatic disease. These 2 patients harbored the most genetic aberrations: gains of RREB1, CCND1, and MYC, and MYB loss. Both benign melanocytic nevi controls showed normal FISH results. Conclusions RREB1 and CCND1 gains are common in nail apparatus melanoma as in most melanomas, and an increased number of genetic aberrations may be associated with a poorer prognosis, though the limited number of cases precludes definitive correlation. FISH appears to be a useful adjunct in the diagnosis of nail apparatus melanomas and improves diagnostic confidence even in the setting of unambiguous histomorphology.

Identification of Combinatorial Genomic Abnormalities Associated with Prostate Cancer Early Recurrence

Multiple biomarkers are needed to distinguish aggressive from indolent prostate cancer. We tested the prognostic utility of a three-marker fluorescent in situ hybridization (FISH) panel (TMPRSS2/ERG rearrangements, AR gain, and PTEN deletion) in a retrospective cohort (n = 210; median follow-up, 5.7 years). PTEN deletion was associated with an increased risk of biochemical recurrence (BcR; hazard ratio, 3.58; 95% CI, 1.39-9.22; P < 0.01) by multivariable Cox regression analyses and earlier BcR (P < 0.02) by Kaplan-Meier analysis. AR gain coexisted with X-chromosome gain and was associated with advanced tumor stage. When this panel was applied, two categories of combinatorial abnormalities proved clinically important. First, PTEN deletion without TMPRSS2/ERG rearrangement was enriched in pT3/4 tumors (70% versus 48%) and tumors with Gleason grades of 8 to 9 (60% versus 17%) compared with the entire cohort. These patients had earlier BcR than patients with normal FISH panel results (P < 0.01). In contrast, patients with PTEN deletion and ERG rearrangement had a BcR rate similar to patients who tested normal for all three markers (P > 0.1). Second, AR gain and concurrent trisomy 10 without TMPRSS2/ERG rearrangement were enriched in pT3/4 tumors and tumors with Gleason grades of 8 to 9. The three-marker FISH panel demonstrated prognostic utility and identified genomic aberrations associated with advanced disease state and early BcR in prostate cancer.

Abstract A33: Utility of multiplexed biomarker analysis in non-small-cell lung cancer patients with discrepancies between FISH and immunohistochemical results of ALK testing

Abstract Background: The clinical implications in Non-small cell lung cancer (NSCLC) patients with discrepancy between fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) assessing ALK status are unclear. Materials and Methods: From 1131 archived tissue samples, we identified 41 NSCLC patients with discrepant results showing positive or borderline ALK FISH and negative IHC using ALK1 antibody. Of these, 21 cases with sufficient tissue remaining for selected reaction monitoring (SRM) and an additional case within normal range FISH split (4%) were sent to confirm ALK IHC using other antibodies (5A4 and D5F3). Target protein levels measured by multiplexed SRM were correlated with records of clinical outcomes after treatment with crizotinib or cytotoxic chemotherapy. Results: Of 22 cases with various ALK FISH results and negative IHC (ALK1), IHC using other antibodies (5A4 and D5F3) identified 4 positive cases, whereas the SRM assay identified 7 cases with tumor-specific ALK protein. IHC showed lower sensitivity in specimen with delayed fixation but SRM showed superior sensitivity in those samples as SRM is independent to modified epitope. Of 8 crizotinib-treated cases, 4 cases with detectable levels of either ALK or cMET proteins had complete or partial responses; cases without either of these proteins (n = 4) did not respond to crizotinib regardless of ALK FISH results. Cases with 3 or more predictive proteins specific to chemotherapeutic agents were found to be more responded to those agents and had a higher probability of progression-free survival. Conclusions: The SRM assay showed superior sensitivity in poorly fixed specimens and can inform therapeutic decision-making in NCSLC patients with ALK screening discordances. Citation Format: TAE-JUNG KIM, HIDEKI TERAI. Utility of multiplexed biomarker analysis in non-small-cell lung cancer patients with discrepancies between FISH and immunohistochemical results of ALK testing. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A33.