Conventional Fluorescence In Situ Hybridization Research Articles

Beyond the Surface: Uncovering the PML::RARA Fusion in Cytogenetically Cryptic and FISH-Negative Acute Promyelocytic Leukemia- A Case Report and Review of the Literature

Abstract Introduction/Objective Chromosomal t(15;17)(q22;q21), resulting in a fusion between the PML gene on chromosome 15q22 and the RARA gene on chromosome 17q21, is the cytogenetic hallmark of acute promyelocytic leukemia (APL). Conventional karyotyping, along with fluorescence in situ hybridization (FISH), can detect the PML/RARA rearrangements in up to 98% of APL cases. Reverse transcriptase-polymerase chain reaction (RT-PCR) is often used to confirm the initial diagnosis. APL with PML::RARA fusion, which is negative with FISH and conventional karyotyping approaches but positive with RT-PCR, is extremely rare. Herein, we report a de novo APL case that was positive by RT-PCR but negative by cytogenetics and FISH studies. A comprehensive review of the literature was also performed. Methods/Case Report A 23-year-old male presented to the ED for left upper extremity weakness 2 days after post- dental cleaning. Upon admission, he developed intracranial hemorrhage, necessitating urgent decompressive hemicraniectomy. Blood smear revealed promyelocytes; flow cytometric analysis showed a CD13+, CD33+, MPO+, CD117+, CD34-, and HLA-DR- abnormal cell population. STAT interphase FISH analyses for PML/RARA fusion and RARA break-apart were negative. A diagnosis of APL was made based on abnormal promyelocytes and characteristic flow cytometry findings. ATRA was immediately administrated. However, the post-operative course was complicated by significant coagulopathy, pancytopenia, and multi-organ failure, and the patient expired 7 days later. RT-PCR returned positive for PML::RARA long fusion transcript; the conventional cytogenetics study was uninformative. The clinical, pathological, and genetic features of 31 cases of FISH/Cytogenetics-negative APL harboring cryptic rearrangements of PML-RARA, detected by RT-PCR in the literature, have been reviewed. Various detection methods were also discussed. Results (if a Case Study enter NA) N/A Conclusion Conventional cytogenetics, FISH and RT-PCR analysis are complementary studies for all clinical suspected APL cases. The clinicopathological characteristics also play critical roles in APL diagnosis when genetic confirmation of the PML::RARA fusion is unavailable.

Multicolour and lineage-specific interphase chromosome Flow-FISH: method development and clinical validation

Flow cytometry can be applied in the detection of fluorescence in situ hybridisation (FISH) signals to efficiently analyse chromosomal aberrations. However, such interphase chromosome (IC) Flow-FISH protocols are currently limited to detecting a single colour. Furthermore, combining IC Flow-FISH with conventional multicolour flow cytometry is difficult because the DNA-denaturation step in FISH assay also disrupts cellular integrity and protein structures, precluding subsequent antigen-antibody binding and hindering concurrent labeling of surface antigens and FISH signals. We developed a working protocol for concurrent multicolour flow cytometry detection of nuclear IC FISH signals and cell surface markers. The protocol was validated by assaying sex chromosome content of blood cells, which was indicative of chimerism status in patients who had received sex-mismatched allogeneic haematopoietic stem cell transplants (allo-HSCT). The method was also adapted to detect trisomy 12 in chronic lymphocytic leukaemia (CLL) subjects. We first demonstrated the feasibility of this protocol in detecting multiple colours and concurrent nuclear and surface signals with high agreement. In clinical validation experiments, chimerism status was identified in clinical samples (n=56) using the optimised IC Flow-FISH method; the results tightly corresponded to those of conventional slide-based FISH (R2=0.9649 for XX cells and 0.9786 for XY cells). In samples from patients who received sex-mismatched allo-HSCT, individual chimeric statuses in different lineages could be clearly distinguished with high flexibility in gating strategies. Furthermore, in CLL samples with trisomy 12, this method could demonstrate that enriched trisomy 12 FISH signal was present in B cells rather than in T cells. Finally, by performing combined labelling of chromosome 12, X chromosome, and surface markers, we could detect rare residual recipient CLL cells with trisomy 12 after allo-HSCT. This adaptable protocol for multicolour and lineage-specific IC Flow-FISH advances the technique to allow for its potential application in various clinical contexts where conventional FISH assays are currently being utilised.

Prognostic Value of Copy Number Aberrations and Copy Neutral Loss of Heterozygosity in Acute Myeloid Leukemia

Background: Chromosomal abnormalities are key to the diagnosis and prognosis of patients with acute myeloid leukemia (AML). Chromosomal genomic array testing (CGAT) can identify copy neutral loss of heterozygosity (cnLOH) and genomic copy number alterations (CNAs) that are undetected by conventional karyotype or fluorescence in situ hybridization (FISH). Previous studies have shown that cnLOH is correlated with a shorter duration of complete remission (CR) and worse overall survival (OS) in AML (Grosneth et. al, Cancer, 2015). The primary objective of this exploratory study is to examine the predictive power of CGAT in a cohort of AML patients with majority normal karyotype. Methods: CGAT analyses were collected from 187 subjects with AML or other high grade myeloid neoplasm (myeloid blasts >10% in peripheral blood or bone marrow) between 2012 and 2022 at Fred Hutchinson Cancer Center. CGAT utilized CytoScan™ HD Array (ThermoFisher, MA), targeting genome-wide region with 2.4 million markers for copy number and approximately 750,000 genotype-able SNPs. The data were analyzed using the Nexus Copy Number software with manual curation. Based on the assay validation in our CLIA-certified diagnostic lab, the cutoff is 100 Kb for copy number aberrations and 10 Mb for cnLOH. Survival predictive power analysis (SPP), a log-rank based test using Nexus, was applied to identify aberrations significant for event free survival (EFS) and overall survival (OS). SPP analysis tabulated genomic segments based on p-values with the alpha value cutoff at 0.05. Using frequency as the filtering criteria (present in >5 patients), 132 SPP for EFS, and 73 SPP for OS were identified. SPP were then manually curated to (1) remove those representing constitutional absence of heterozygosity (AOH) and those overlapping with benign copy number variance and (2) connect adjacent SPP into contiguous regions. The presence and absence of the identified SPP were then tabulated, cleaned up, and used to generate Kaplan-Meier curves for EFS and OS. This process identified 5 SPP that are significant for EFS and 2 SPP for OS. A multivariate analysis was performed to determine the hazard ratio (HR) of these 5 genetic aberrations for EFS and OS, adjusted for patient age, secondary/treatment related AML, ELN 2022 risk, and receipt of allogeneic hematopoietic cell transplantation (HCT) as a time-varying covariate. Results: In our 187 subject cohort, the median age was 63 (range 20-87). 76 (41%) had secondary or treatment related disease. 97 (52%) went on to receive a hematopoietic stem cell transplant. 3 had favorable risk disease, 14 had adverse risk, and 170 had intermediate risk by karyotype and FISH using ELN 2022 criteria. 120 (64%) had normal karyotype. 5 subjects had 4q cnLOH, 6 had 5q ( EGR1) loss, 7 had 9p cnLOH, 10 had mixed-lineage leukemia gene partial tandem duplication ( MLL-PTD), and 4 had 7q deletion. Univariate analysis identified five SPP that are significant for EFS: 4q cnLOH (p=0.002), 5q ( EGR1) loss (p<0.001), 9p cnLOH (p<0.001), MLL-PTD (p<0.001), and 7q deletion (p<0.001). 2 SPP were significant for OS: 9p cnLOH (p=0.001) and MLL-PTD (p=0.036). Multivariate analysis for EFS adjusted for age, secondary/treatment related disease, ELN 2022 risk, and receipt of HCT as a time-varying covariate demonstrated a hazard ratio (HR) of 3.5 for 4q cnLOH (p=0.008), 2.3 for 5q ( EGR1) loss (p=0.16), 4.5 for 7q deletion (p=0.007), 5.7 for 9p cnLOH (p<0.001), and 2.9 for MLL-PTD (p=0.002). Multivariate analysis for OS adjusted for age, secondary/treatment related disease, ELN 2022 risk, and receipt of HCT as a time-varying covariate demonstrated a hazard ratio (HR) of 2.0 for 4q cnLOH (p=0.23), 0.5 for 5q ( EGR1) loss (p=0.36), 1.3 for 7q deletion (p=0.66), 4.9 for 9p cnLOH (p=0.001), and 2.3 for MLL-PTD (p=0.04). Discussion: This study has found 5 key genetic changes identifiable via CGAT that are significant for shorter EFS and/or OS in AML patients. Our next step is to validate these findings in a large independent cohort of AML patients. CGAT is a powerful adjunct to conventional karyotype and FISH for the detection of cnLOH and can be used to identify CNAs below the level of detection of karyotype. For these reasons, CGAT may be most useful in intermediate risk patients. Further research is warranted to fully understand the prognostic power of CGAT in AML.

Optical Genome Mapping Allows Detection and Characterization of Cytogenetically Cryptic Oncogenic Fusions in Pediatric Acute Myeloid Leukemia

Background: Abnormal gene fusions are predominant oncogenic drivers in pediatric acute myeloid leukemia (AML) and are important diagnostic and prognostic markers. However, several oncogenic fusions specific for or enriched in pediatric AML (like CBFA2T3::GLIS2, NUP98::NSD1, ETV6::MNX1 and others) are not detectable by conventional cytogenetic analysis (cytogenetically cryptic) and may not be included in fluorescence in situ hybridization (FISH) or molecular fusion panels designed for adult patients. Optical genome mapping (OGM) is a powerful new method for genome-wide, high-resolution detection of copy number abnormalities and balanced chromosome rearrangements. This study determined the frequency of cryptic oncogenic fusions in an institutional cohort of pediatric AML cases and evaluated OGM as a method for their detection and characterization. Methods: Retrospective review of genetic testing results was performed for 93 consecutive de novo or relapsed pediatric AML cases tested at the Children's Hospital Los Angeles (CHLA) Center for Personalized Medicine between March 2016 and July 2023. Genetic testing was performed as part of clinical care following standard procedures and consisted of karyotype analysis, FISH testing, chromosomal microarray (CMA) analysis, CHLA custom sequencing panel (OncoKids®) (June 2017 onwards) and RNA-Seq fusion assay (March 2022 onwards). Cases with normal or inconclusive karyotype in which an oncogenic fusion was detected by another clinical assay were selected for OGM testing. High molecular weight DNA from peripheral blood or bone marrow samples was labeled, processed and imaged in a Saphyr System and analyzed in the Bionano Access software following the manufacturer's recommendations (BionanoGenomics, San Diego CA). Results: Out of 93 pediatric AML cases included in the study, a gene fusion was identified as the primary oncogenic driver in 54 (58%) and a DNA sequence variant in 11 (12%). In 28 cases (30%), the primary genetic abnormality remained undetermined after comprehensive genetic testing. In 17/54 (31%) fusion-driven cases [17/93 (18%) overall cases], the oncogenic fusion remained undetected by karyotype analysis (cryptic). OGM was completed in 13/17 fusion positive cases with non-informative karyotype; it could not be performed in 4 cases due to lack of samples (n=2) or poor DNA quality (n=2), and is still in progress for a subset of 28 unknown cases with available material. OGM analysis successfully revealed all cryptic fusions previously detected by FISH, CMA, OncoKids® and/or RNA-Seq, including NUP98::NSD1 (n=3), KMT2A::MLLT10 (n=3), KMT2A::MLLT3 (n=2)and one case each of CBFA2T3::GLIS2, NUP98::KDM5A, DEK::NUP214, MYB::GATA1 and ETV6::MNX1. Chromosomal mechanism and structure of cryptic fusions were resolved by OGM and included: intrinsically cryptic fusions due to chromosome location of partner genes ( NUP98::NSD1, n=4; CBFA2T3::GLIS2, NUP98::KDM5A and ETV6::MNX1, n=1 each); complex rearrangements due to incompatible gene orientation ( KMT2A::MLLT10 n=2; MYB::GATA1, n=1); three-way rearrangements involving an additional chromosome ( KMT2A::MLLT3 n=2) and small insertions ( KMT2A::MLLT10 and DEK::NUP214, n=1 each). OGM also matched the performance of chromosomal microarray (CMA) analysis in detection of copy number variants. Conclusions: We confirm the significant contribution of cryptic chromosome rearrangements to pathogenesis of pediatric AML, and highlight the importance of tailored testing approaches that effectively detect highly heterogeneous fusion drivers. Our preliminary results show potential of OGM to identify clinically significant cryptic fusions in pediatric AML, and to thus markedly increase diagnostic yield compared to karyotype analysis. As a single assay that detects abnormalities currently interrogated by conventional cytogenetics, FISH and CMA, OGM may allow streamlined and more cost-effective genetic evaluation for pediatric AML.

Utility of Next-Generation Sequencing in the Detection of RNA Fusion Genes in Myeloid Malignancies in Singapore

Background Oncogenic fusion genes play an instrumental role in the pathogenesis of haematological malignancies and the identification of these genes is crucial for diagnosis, risk stratification and treatment decisions. Conventional diagnostic methods such as karyotyping and fluorescence in situ hybridization (FISH) are the current standard of care but are limited in the sensitivity and availability of different FISH probes for detection of fusion genes. In our institution, we have routinely integrated a targeted next-generation sequencing (NGS) panel in our diagnostic workflow for patients with suspected myeloid malignancies. Here we aim to review the cases with RNA fusion genes detected via NGS and compare these results with that of conventional diagnostic methods. Methods We analysed bone marrow samples from patients with suspected myeloid malignancies at the time of diagnosis in Singapore General Hospital (SGH). Extracted genomic DNA and RNA were amplified, and library preparation was performed with the Oncomine TM Myeloid Research assay (Thermo Fisher Scientific) to interrogate 40 DNA genes and 29 fusion driver genes. NGS libraries were sequenced on the Ion GeneStudio S5 System (Thermo Fisher Scientific) and analyzed using in-house bioinformatics pipelines and the Ion Reporter software. Limit of detection was determined as 3% allele frequency for DNA variants and 10 -2 for RNA fusions. RNA fusions detected by the NGS panel were consolidated and compared with cytogenetics (karyotyping and FISH) results. Results A total of 436 samples from adult patients with suspected myeloid malignancies were received for NGS testing from October 2021 to March 2023 in SGH, the largest tertiary hospital in Singapore. 306 had paired cytogenetics and NGS testing performed. 27 of the 306 cases (9%) had RNA fusions detected by NGS as summarized in Table 1 and Figure 1; their disease subgroups comprised of 16 acute myeloid leukaemia (AML), 5 chronic myeloid leukaemia (CML), 3 BCR-ABL1-negative myeloproliferative neoplasm (MPN), 2 acute promyelocytic leukaemia (APL) and 1 myelodysplastic syndrome (MDS). Eight AML cases had RUNX1-RUNX1T1.R3R3 fusions, with 6/8 (75%) harbouring additional DNA single nucleotide variants or indel mutations. Two AML cases had CBFB-MYH11 fusions, with 1 associated with KIT D419 deletion, and the other with U2AF1, NF1, and ASXL1 mutations. We also identified 2 adult AML cases (aged 49 and 65 years respectively) with DEK-NUP214.D9N18 fusions, both of which had associated FLT3-ITD mutations. There were 5 cases with KMT2A fusions, with a variety of partners including MLLT3.K9M6 (n=2), ELL.K9E2 (n=1), MLLT1.K8M4 (n=1), and MLLT6.K8M9 (n=1). Two patients with APL had PML-RARA detected for different variants: P3R3 and P6R3. Amongst the 8 MPN cases, 5 had BCR-ABL1 fusions consistent with diagnosis of CML. There was 1 case of MPN-Eo with FLP1L1-PDGFRA fusion, and the remaining 2 with ETV6-ABL1 and KMT2A-ELL.K9E2 rare fusions. The detected RNA fusions had clinical impact in establishing diagnosis (93%), prognosis (96%), and guidance of treatment decisions (89%). Out of the 27 cases with RNA fusions detected by NGS, 23 (85%) had concordant karyotype and FISH results. Four RNA fusions, namely KMT2A-ELL, CBFB-MYH11, FIP1L1-PDGFRA and ETV6-ABL1, were not detected by karyotyping or conventional FISH probes performed in our institution. It likely reflects the sensitivity of the NGS panel in detecting RNA fusions in cases of cryptic translocations. Conclusion We have demonstrated that NGS has a high sensitivity for identification of RNA fusion genes, is complementary to conventional cytogenetics testing, and has vast clinical impact in terms of diagnosis, prognostication and clinical management. We advocate for the integration of NGS with DNA and RNA sequencing into routine investigation of suspected myeloid malignancies for a more precise and comprehensive diagnostic approach.

Role of Conventional Cytogenetics and FISH in the Laboratory Work Up of Plasma Cell Dyscrasias

AbstractPlasma cell dyscrasias are a heterogeneous group of neoplasms characterized by abnormal proliferation of plasma cells with or without over production of monoclonal immunoglobulins. Chromosomal abnormalities are acquired either early in the course of the disease or during disease progression. Plasma cell dyscrasias are categorized into multiple cytogenetic subtypes that form an integral component of risk-stratified treatment protocols. The primary genetic events are IgH gene translocations and non-random gains of chromosomes 3/5/7/9/11/15/19 and or 21. The secondary genetic events consist of chromosome 1 abnormalities (1p deletion and 1q gain or amplification), deletion 17p/TP53, deletion 13q, and MYC gene rearrangements. Plasma cells being at the end of differentiation spectrum of B cells, have low proliferative potential precluding the use of karyotyping in identification of chromosomal abnormalities. Analysis of enriched plasma cells using interphase fluorescent in situ hybridization (FISH) is the technique of choice for identifying these abnormalities. It is essential to enrich plasma cells before the FISH analysis, and numerous plasma cell enrichment techniques have been described. In the paper, we review the cytogenetic approach to identify clinically significant genetic aberrations including the effective use of FISH panels and plasma cell enrichment techniques.

Practical Applications of Chromosomal Microarray in Prenatal Diagnosis

AbstractG-banded karyotyping is the most common approach for the detection of genomic alterations. However, this is unable to detect genomic changes of less than 5 Mb. The ability of fluorescence in situ hybridization (FISH) to detect cryptic chromosomal rearrangements exceeds the resolution of routine karyotype. However, conventional FISH is for targeted regions only, whereas the chromosomal microarray is a whole-genome copy number evaluation technique with a resolution of 10 to 20 kb. In this article, we discuss the application of chromosomal microarray 750 K to 384 consecutive prenatal diagnosis cases. Overall diagnostic yield is 15.36%, and chromosomal microarray accounts for a 3.6% additional detection rate. We suggest applying this technique in routine prenatal diagnosis as a first-tier test in prenatal diagnosis along with a backup culture in all cases.

Frequency and pattern of chromosomal abnormalities in acute myeloid leukemia from Western India: A retrospective study

Chromosomal abnormalities play an important role in diagnosis and prognosis of hematological diseases. The aim of the present study was to study the pattern and frequency of chromosomal aberrations in acute myeloid leukemia (AML) subgroups from western India. A retrospective study was conducted through evaluating laboratory proforma which were filled during 2005 to 2014 for diagnosis and treatment of AML subjects. We have studied chromosomal aberrations in 282 subjects with AML from western India. AML patients were sub-grouped according to FAB classification. Cytogenetic study using conventional cytogenetics (GTG-banding) and Fluorescence in situ hybridization (FISH) was carried out using FISH probes (AML1/ETO, PML/RARA, CBFB). Student's t test for continuous variables and Pearson's Chi-squared test for categorical variables were used to identify the relationship between variables. Cytomorphological study revealed AML- M3 as most frequent (32.3%) group followed by AML-M2 (25.2%) and AML-M4 (19.9%). Chromosomal abnormalities were identified in 145 (51.42%) of the total AML cases. A high frequency (38.6%) of chromosomal abnormalities was identified in AML-M3 subgroup as compared to AML-M2 (31%) and AML-M4 (20.6%). Cytogenetic study is important for the diagnosis and management of the AML patients. Our study identified chromosomal abnormalities in AML subgroups with varied frequencies. It is important in diagnosis and monitoring of the disease. As younger AML patients were more affected in our study, etiological factors such as environmental factors need to be studied. Combination of conventional cytogenetics and FISH has an advantage of identifying high frequency of chromosomal aberrations in AML patients.

Longitudinal Plasma Proteomics-Derived Biomarkers Predict Response to MET Inhibitors for MET-Dysregulated NSCLC.

MET inhibitors have shown promising efficacy for MET-dysregulated non-small cell lung cancer (NSCLC). However, quite a few patients cannot benefit from it due to the lack of powerful biomarkers. This study aims to explore the potential role of plasma proteomics-derived biomarkers for patients treated with MET inhibitors using mass spectrometry. We analyzed the plasma proteomics from patients with MET dysregulation (including MET amplification and MET overexpression) treated with MET inhibitors. Thirty-three MET-dysregulated NSCLC patients with longitudinal 89 plasma samples were included. We classified patients into the PD group and non-PD group based on clinical response. The baseline proteomic profiles of patients in the PD group were distinct from those in the non-PD group. Through protein screening, we found that a four-protein signature (MYH9, GNB1, ALOX12B, HSD17B4) could predict the efficacy of patients treated with MET inhibitors, with an area under the curve (AUC) of 0.93, better than conventional fluorescence in situ hybridization (FISH) or immunohistochemistry (IHC) tests. In addition, combining the four-protein signature with FISH or IHC test could also reach higher predictive performance. Further, the combined signature could predict progression-free survival for MET-dysregulated NSCLC (p < 0.001). We also validated the performance of the four-protein signature in another cohort of plasma using an enzyme-linked immunosorbent assay. In conclusion, the four plasma protein signature (MYH9, GNB1, ALOX12B, and HSD17B4 proteins) might play a substitutable or complementary role to conventional MET FISH or IHC tests. This exploration will help select patients who may benefit from MET inhibitors.

Development and application of universal ND-FISH probes for detecting P-genome chromosomes based on Agropyron cristatum transposable elements.

Fluorescence in situ hybridization (FISH) is a basic tool that is widely used in cytogenetic research. The detection efficiency of conventional FISH is limited due to its time-consuming nature. Oligonucleotide (oligo) probes with fluorescent labels have been applied in non-denaturing FISH (ND-FISH) assays, which greatly streamline experimental processes and save costs and time. Agropyron cristatum, which contains one basic genome, "P," is a vital wild relative for wheat improvement. However, oligo probes for detecting P-genome chromosomes based on ND-FISH assays have not been reported. In this study, according to the distribution of transposable elements (TEs) in Triticeae genomes, 94 oligo probes were designed based on three types of A. cristatum sequences. ND-FISH validation showed that 12 single oligo probes generated a stable and obvious hybridization signal on whole P chromosomes in the wheat background. To improve signal intensity, mixed probes (Oligo-pAc) were prepared by using the 12 successful probes and validated in the diploid accession A. cristatum Z1842, a small segmental translocation line and six allopolyploid wild relatives containing the P genome. The signals of Oligo-pAc covered the entire chromosomes of A. cristatum and were more intense than those of single probes. The results indicate that Oligo-pAc can replace conventional genomic in situ hybridization (GISH) probes to identify P chromosomes or segments in non-P-genome backgrounds. Finally, we provide a rapid and efficient method specifically for detecting P chromosomes in wheat backgrounds by combining the Oligo-pAc probe with the Oligo-pSc119.2-1 and Oligo-pTa535-1 probes, which can replace conventional sequential GISH/FISH assays. Altogether, we developed a set of oligo probes based on the ND-FISH assays to identify P-genome chromosomes, which can promote utilization of A. cristatum in wheat improvement programs.

Genome oligopaint via local denaturation fluorescence in situ hybridization (gold fish) and single-nucleotide variation detection

Cas9 in complex with a programmable guide RNA targets specific double-stranded DNA for cleavage. By harnessing Cas9 as a programmable loader of superhelicase to genomic DNA, we report a physiological-temperature DNA fluorescence in situ hybridization (FISH) method termed genome oligopaint via local denaturation (GOLD) FISH. Instead of global denaturation as in conventional DNA FISH, loading a superhelicase at a Cas9-generated nick allows for local DNA denaturation, reducing nonspecific binding of probes and avoiding harsh treatments such as heat denaturation.

Single HER2-positive tumor cells are detected in initially HER2-negative breast carcinomas using the DEPArray\u2122\u2013HER2-FISH workflow

BackgroundIn breast cancer (BC), overexpression of HER2 on the primary tumor (PT) is determined by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) to stratify samples as negative, equivocal and positive to identify patients (pts) for anti-HER2 therapy. CAP/ASCO guidelines recommend FISH for analyzing HER2/neu (ERBB2) gene amplification and for resolving equivocal HER2 IHC results. However, pre-analytical and analytical aspects are often confounded by sample related limitations and tumor heterogeneity and HER2 expression may differ between the PT and circulating tumor cells (CTCs), the precursors of metastasis. We used a validation cohort of BC patients to establish a new DEPArray™-PT-HER2-FISH workflow for further application in a development cohort, characterized as PT-HER2-negative but CTC-HER2/neu-positive, to identify patients with PT-HER2 amplified cells not detected by routine pathology.Methods50 µm FFPE tumor curls from the validation cohort (n = 49) and the development cohort (n = 25) underwent cutting, deparaffinization and antigen retrieval followed by dissociation into a single-cell suspension. After staining for cytokeratin, vimentin, DAPI and separation via DEPArray™, single cells were processed for HER2-FISH analysis to assess the number of chromosome 17 and HER2 loci signals for comparison, either with available IHC or conventional tissue section FISH. CTC-HER2/neu status was determined using the AdnaTest BreastCancer (QIAGEN, Hilden, Germany).ResultsApplying CAP/ASCO guidelines for HER2 evaluation of single PT cells, the comparison of routine pathology and DEPArray™-HER2-FISH analysis resulted in a concordance rate of 81.6% (40/49 pts) in the validation cohort and 84% (21/25 pts) in the development cohort, respectively. In the latter one, 4/25 patients had single HER2-positive tumor cells with 2/25 BC patients proven to be HER2-positive, despite being HER2-negative in routine pathology. The two other patients showed an equivocal HER2 status in the DEPArray™-HER2-FISH workflow but a negative result in routine pathology. Whereas all four patients with discordant HER2 results had already died, 17/21 patients with concordant HER2 results are still alive.ConclusionsThe DEPArray™ system allows pure tumor cell recovery for subsequent HER2/neu FISH analysis and is highly concordant with conventional pathology. For PT-HER2-negative patients, harboring HER2/neu-positive CTCs, this approach might allow caregivers to more effectively offer anti-HER2 treatment.

Prognostic Value of Specific High-Risk Cytogenetic Abnormalities and Ethnicity in Outcomes after Autologous Stem Cell Transplant in Multiple Myeloma

Background:Chromosomal abnormalities in multiple myeloma (MM) patients, identified by either conventional metaphase cytogenetics or fluorescence in situ hybridization (FISH), stratify those at high risk of relapse and poorer survival after treatment. However the prognostic value of high-risk (HR) features in regard to survival after autologous hematopoietic stem cell transplantation is unclear. In addition, recent epidemiologic studies describe difference between Hispanic and non-Hispanic whites (NHW) in incidence, age at presentation, time to initial treatment, and rate of auto-HSCT within one year of diagnosis, but the presence of cytogenetic abnormalities as a prognostic factor in Hispanic patients has not yet been described.Methods:We conducted a retrospective analysis of 367 MM patients at the University of Miami Sylvester Comprehensive Cancer center who underwent auto-HSCT between January 2014 and December 2020. Patients were classified as HR if either conventional cytogenetics or FISH demonstrated at least one of the following: 1q+, 1p-, 17p-, 13q-, t(4;14), or t(14;16). All other patients with normal chromosomal studies or with trisomies and/or hyperdiploidy were considered standard risk (SR). Survival analysis were performed using the log-rank test, with significance at p-value < 0.05.Results:Male patient comprised 58% of our patient population with 43% of patients of Hispanic ethnicity. Of the 367 patients who underwent auto-HSCT, 183 (50%) had at least one HR cytogenetic abnormality. Overall, HR patients exhibited inferior PFS (32.9m vs 50.6m, p=0.017) and OS (median not reached/NR for both, p=0.0086) compared to SR patients.When evaluating outcomes with specific HR cytogenetic abnormalities, we identified cohorts that did not exhibit survival benefit, either in overall survival (OS) or progression-free survival (PFS) after transplant compared to SR patients, in particular patients with 17p-, 13q-, or 1q+ (Table 1). Patients with 1p-, t(14;16) or t(4;14) derived partial benefit from transplant in terms of PFS but not OS. Notably, patients with 1p- exhibited significantly worse OS compared to other HR patients (38.3m vs NR, p=0.007).We next evaluated differences in outcome when stratifying across ethnicity. Of the 183 patients with HR cytogenetic abnormalities, 75 (41%) were of Hispanic ethnicity, while 58 (32%) were NHW and 46 (25%) were of African-American (AA) ethnicity. The proportion of patients with SR or each individual HR cytogenetic abnormality was mostly equivalent amongst each ethnicity with the exception of 13q- (35% of Hispanic patients, vs. 44% of NHW and 26% of AA patients). Nevertheless, we observed that Hispanic patients with either t(4;14) or 13q- had significantly worse OS and PFS than their NHW and AA counterparts, and similarly observed inferior OS in Hispanic patients with 1q+ (Table 2).Conclusion:In our single-center retrospective analysis of HR MM patients undergoing auto-HSCT, we have identified specific patient populations that derive some survival benefit from transplant as well as populations that did not derive any benefit. Additionally, we demonstrate that despite similar incidence of certain HR abnormalities when comparing across ethnicities, Hispanic patients with particular chromosomal abnormalities have inferior overall survival outcomes after transplant. Further investigation is warranted to identify patient-specific and treatment-related factors leading to inferior outcomes in this patient population. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comparison of Next-Generation Sequencing and Fluorescence In Situ Hybridization for Detection of Segmental Chromosomal Aberrations in Neuroblastoma.

The aim of this study was to compare next-generation sequencing (NGS) with the traditional fluorescence in situ hybridization (FISH) for detecting segmental chromosomal aberrations (SCAs) such as 1p deletion, 11q deletion and 17q gain, which are well-known predictive markers for adverse outcome in neuroblastoma. The tumor tissue obtained from 35 patients with neuroblastoma was tested by FISH and targeted NGS, which is specially designed to detect copy number alterations across the entire chromosomal region in addition to mutations in 353 cancer-related genes. All chromosomal copy number alterations were analyzed using the copy number variation plot derived from targeted NGS. FISH was performed to detect 1p deletion, 11q deletion and 17q gain. The copy numbers of 1p, 11q, and 17q obtained via NGS were correlated with those acquired via FISH. The SCAs determined by NGS were matched with those by FISH. Most 17q gain of mismatched cases detected by NGS alone showed a subsegmental gain of 17q. FISH revealed 11q deletion and 17q gain in a few tumor cells of two cases, which were not detected by NGS. NGS can be a sensitive complementary and alternative method to the conventional FISH for detecting SCAs.

Abstract LB190: DNAscopeTM: A novel chromogenic in-situ hybridization technology for high-resolution detection of DNA copy number and structural variations

Abstract Genomic DNA anomalies such as copy number variations (gene duplication, amplification, deletion) and gene rearrangements are important biomarkers and drug targets in many cancer types. DNA in-situ hybridization (ISH) is the gold standard method to directly visualize these molecular alterations in formalin-fixed paraffin-embedded (FFPE) tumor tissues at single-cell resolution within a histological section. However, currently available fluorescent ISH (FISH) assays provide limited morphological detail due to the use of fluorescent nuclear staining compared to chromogenic staining. Furthermore, FISH techniques rely on expensive fluorescence microscopes, risk loss of fluorescent signal over time and involve tedious imaging at high magnifications (100X). There is thus an unmet need for a sensitive and robust chromogenic DNA-ISH assay that can enable high-resolution detection of genomic DNA targets with the ease of bright-field microscopy. We present here DNAscope - a novel chromogenic DNA-ISH assay - for detecting and visualizing genomic DNA targets under a standard light microscope. DNAscope is based on the widely used RNAscope® double-Z probe design and signal amplification technology and provides unparalleled sensitivity and specificity with large signal dots readily visualized at 40X magnification and with full morphological context. Furthermore, DNAscope ensures specific DNA detection without interference from RNA due to the use of a novel RNA removal method. Using a duplex chromogenic detection assay in red and blue, we demonstrate highly specific and efficient detection of gene rearrangements (ALK, ROS1, RET and NTRK1), gene amplification (ERBB2, EGFR, MET) and deletion (TP53 and CDKN2A). The DNAscope assay has been carefully optimized for probe signal size and color contrast to enable easy interpretation of signal patterns under conventional light microscopy or digital pathology. Compared to conventional FISH assays, DNAscope probes are standard oligos that are designed in silico to be free of any repetitive sequences and can be rapidly synthesized for any DNA target. In conclusion, the DNAscope assay provides a powerful and convenient alternative to commonly used FISH assays in many cancer research applications. Citation Format: Li-Chong Wang, Farzaneh Tondnevis, Courtney Todorov, Jayson Gaspar, Aparna Sahajan, Vasudha Murlidhar, Bingqing Zhang, Xiao-Jun Ma. DNAscopeTM: A novel chromogenic in-situ hybridization technology for high-resolution detection of DNA copy number and structural variations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB190.

Optimizing the Diagnostic Strategy to Identify Genetic Abnormalities in Miscarriage.

IntroductionThe single most common cause of miscarriage is genetic abnormality.ObjectiveWe conducted a prospective cohort study to compare the performance of conventional karyotyping and chromosomal microarray analysis (CMA) using array comparative genomic hybridization (array-CGH) and single nucleotide polymorphism array (SNP-array) to identify genetic abnormalities in miscarriage specimens.MethodsA total of 63 miscarriage specimens were included. Conventional karyotyping, array-CGH, and SNP-array were performed and the results compared.ResultsGenetic abnormalities were detected in 31 cases (49.2%) by at least one testing modality. Single autosomal trisomy was the most common defect (71.0%), followed by polyploidy (16.1%), multiple aneuploidy (9.7%), and monosomy X (3.2%). Mosaicisms were identified in four cases and confirmed by fluorescence in situ hybridization (FISH) using appropriate probes. SNP-array had a higher detection rate of genetic abnormalities than array-CGH (93.5 vs. 77.4%), and conventional karyotyping had the lowest detection rate (76.0%). SNP-array enabled the detection of all types of genetic abnormalities, including polyploidy.ConclusionsAlthough conventional karyotyping and FISH are still needed, SNP-array represents the first choice for miscarriage because the technique showed excellent performance in the detection of genetic abnormalities and minimized the probability of testing failure as well as time, costs, and labor.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40291-021-00523-9.

Genome oligopaint via local denaturation fluorescence in situ hybridization

Cas9 in complex with a programmable guide RNA targets specific double-stranded DNA for cleavage. By harnessing Cas9 as a programmable loader of superhelicase to genomic DNA, we report a physiological-temperature DNA fluorescence in situ hybridization (FISH) method termed genome oligopaint via local denaturation (GOLD) FISH. Instead of global denaturation as in conventional DNA FISH, loading a superhelicase at a Cas9-generated nick allows for local DNA denaturation, reducing nonspecific binding of probes and avoiding harsh treatments such as heat denaturation. GOLD FISH relies on Cas9 cleaving target DNA sequences and avoids the high nuclear background associated with other genome labeling methods that rely on Cas9 binding. The excellent signal brightness and specificity enable us to image nonrepetitive genomic DNA loci and analyze the conformational differences between active and inactive X chromosomes. Finally, GOLD FISH could be used for rapid identification of HER2 gene amplification in patient tissue.

Is There an Unequal Benefit of Autologous Stem Cell Transplant in Different Cytogenetic Groups of High Risk Patients with Multiple Myeloma: The University of Miami Experience

BACKGROUND: Stratification using cytogenetics (CG), either metaphase karyotyping or fluorescence in situ hybridization (FISH) is used to identify patients (pts) with multiple myeloma (MM) who are at higher risk of relapse and tend to have poorer survival. It is largely unknown if autologous stem cell transplantation (auto-HCT) after high dose melphalan (200 mg/m2) is able to modify the survival of some of these high-risk MM pts and to make it comparable to standard risk. METHODS: Pts were classified as high risk (HR) if either conventional cytogenetics or FISH demonstrated at least one of the following 1q+, 1p-, 17p-, 13q-, t(4;14), or t(14;16), realizing that the inclusion of 13q- by FISH alone and 1p- in the HR MM definition is controversial. Pts with normal chromosomes or those with trisomies and hyperdiploidy were considered standard-risk (SR). We compared progression-free survival (PFS) and overall survival (OS) via a retrospective analysis of pts at the University of Miami Sylvester Comprehensive Cancer Center who underwent auto-HCT between January 2014 and December 2017 for MM. Survival analyses were performed using the log-rank test, with significance at p-value &amp;lt; 0.05. RESULTS: Male pts comprised 56% of the population, and 40% of pts were of Hispanic ethnicity. Of 205 pts undergoing auto-HCT, 108 (53%) had at least one HR cytogenetic abnormality. Interestingly, the depth of response to pre-transplant induction was higher in pts classified as HR, with 71% (77 of 108) achieving at least a very good partial response (VGPR), while 24% (23 of 96) SR pts achieved VGPR. While OS remained largely unaffected in HR pts (34.0 m vs. 35.1 m, p = 0.27); HR pts had an inferior PFS compared to SR pts (21.9 m vs. 25.7 m, p = 0.041). The presence of trisomies did not negate the poorer PFS of HR pts. When we evaluated specific HR CG abnormalities, OS and PFS in patients with 1q+ or t(4;14) were surprisingly comparable to SR pts, indicating a significant benefit from auto-HCT. On the other hand, OS was significantly decreased in pts with 1p- when compared to standard risk (16.5 m vs. 35.1 m, p = 0.004) or other high-risk patients (16.5 m vs. 35.4 m, p = 0.01), implying that 1p- group derive no benefit from auto-HCT. Similarly, OS was shorter in pts with t(14;16) (16.5 m vs. 34.4 m, p = 0.025) and with 17p- (26.6 m vs. 35.1 m, p=0.01), however the PFS was not affected in these populations. In pts with 13q-, PFS was significantly shorter (20.3 m vs. 25.7 m, p=0.023) compared to SR pts without affecting OS. CONCLUSION: At our center in a retrospective analysis of 205 pts: a) patients with HR MM responded better and faster than SR pts to induction, b) those with 1p- did not derive any benefit from transplant and c) pts with 17p- and t(14;16) had some short term benefit (similar PFS to SR group) but at the end their OS remained inferior. Nevertheless, we consider a very important finding the fact that, by having auto-HCT, pts with 1q+ and t(4;14), equalized their PFS and OS to those of SR pts. Based on these findings, patients with 1q+ and t(4;14) should still have a transplant in CR1 even after optimal induction. Whether 17p- and t(14;16) pts can extend PFS benefit after transplant into OS benefit with maintenance regimens stronger than lenalidomide alone remains to be determined. Disclosures Hoffman: Celgene: Honoraria, Speakers Bureau; Loxo: Current equity holder in publicly-traded company; Seattle Genetics: Research Funding.

Single-cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection.

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

The Myoepithelial Cells of Salivary Intercalated Duct-type Intraductal Carcinoma Are Neoplastic: A Study Using Combined Whole-slide Imaging, Immunofluorescence, and RET Fluorescence In Situ Hybridization.

Intraductal carcinoma (IDC) is a salivary gland tumor currently believed to be analogous to breast ductal carcinoma in situ, consisting of a complex neoplastic epithelial proliferation surrounded by a continuous layer of myoepithelial cells presumed to be native and non-neoplastic. Recent molecular insights have shown that there are at least 3 different types of IDC: (1) intercalated duct-like, with frequent NCOA4-RET fusions; (2) apocrine, with multiple mutations similar to salivary duct carcinoma; and (3) mixed intercalated duct-like and apocrine with frequent RET fusions, especially TRIM27-RET. Recent observations (eg, IDC occurring in lymph nodes) have challenged the notion that the myoepithelial cells of IDC are non-neoplastic. Five IDCs with known RET fusions by RNA sequencing were retrieved from the authors' archives, including 4 intercalated duct-like IDCs with NCOA4-RET, and 1 mixed intercalated duct-like/apocrine IDC with TRIM27-RET. A panel of immunohistochemistry antibodies (S100 protein, p63 or p40, mammaglobin, smooth muscle actin, calponin, androgen receptor) was tested. To precisely localize RET split-positive cells, each case was subjected to sequential retrieval of whole-slide imaging data of hematoxylin and eosin (HE) staining, immunofluorescence staining for calponin, and fluorescence in situ hybridization (FISH) for RET. Because NCOA4-RET is an inversion difficult to visualize on conventional RET FISH, a novel 3-color FISH technique was utilized to demonstrate it clearly. In all 5 cases, the proliferative ducts were completely surrounded by a layer of myoepithelial cells that were positive for p63 or p40, smooth muscle actin, and calponin. Using combined HE, calponin immunofluorescence, and RET FISH imaging, the positive signals were unmistakably identified in both calponin-negative ductal cells and peripheral, calponin-positive myoepithelial cells in all 5 cases. Utilizing combined HE, calponin immunofluorescence, and RET FISH imaging, we demonstrated that IDCs with RET fusions harbored this alteration in both the ductal and myoepithelial cells. This is compelling evidence that the myoepithelial cells of IDC are not mere bystanders, but are rather a component of the neoplasm itself, similar to other biphasic salivary gland neoplasms like pleomorphic adenoma and epithelial-myoepithelial carcinoma. This finding raises questions about the appropriate terminology, classification, and staging of IDC.