Fluorescence In Situ Hybridization Analysis Research Articles

Recurrent and novel fusions detected by targeted RNA sequencing as part of the diagnostic workflow of soft tissue and bone tumours.

The identification of gene fusions has become an integral part of soft tissue and bone tumour diagnosis. We investigated the added value of targeted RNA-based sequencing (targeted RNA-seq, Archer FusionPlex) to our current molecular diagnostic workflow of these tumours, which is based on fluorescence in situ hybridisation (FISH) for the detection of gene fusions using 25 probes. In a series of 131 diagnostic samples targeted RNA-seq identified a gene fusion, BCOR internal tandem duplication or ALK deletion in 47 cases (35.9%). For 74 cases, encompassing 137 FISH analyses, concordance between FISH and targeted RNA-seq was evaluated. A positive or negative FISH result was confirmed by targeted RNA-seq in 27 out of 49 (55.1%) and 81 out of 88 (92.0%) analyses, respectively. While negative concordance was high, targeted RNA-seq identified a canonical gene fusion in seven cases despite a negative FISH result. The 22 discordant FISH-positive analyses showed a lower percentage of rearrangement-positive nuclei (range 15-41%) compared to the concordant FISH-positive analyses (>41% of nuclei in 88.9% of cases). Six FISH analyses (in four cases) were finally considered false positive based on histological and targeted RNA-seq findings. For the EWSR1 FISH probe, we observed a gene-dependent disparity (p = 0.0020), with 8 out of 35 cases showing a discordance between FISH and targeted RNA-seq (22.9%). This study demonstrates an added value of targeted RNA-seq to our current diagnostic workflow of soft tissue and bone tumours in 19 out of 131 cases (14.5%), which we categorised as altered diagnosis (3 cases), added precision (6 cases), or augmented spectrum (10 cases). In the latter subgroup, four novel fusion transcripts were found for which the clinical relevance remains unclear: NAB2::NCOA2, YAP1::NUTM2B, HSPA8::BRAF, and PDE2A::PLAG1. Overall, targeted RNA-seq has proven extremely valuable in the diagnostic workflow of soft tissue and bone tumours.

The Application of Fluorescence In Situ Hybridization in the Prescreening of Veronica Hybrids.

Fluorescence in situ hybridization (FISH), a molecular cytogenetic technique that enables the visualization and identification of specific DNA sequences within chromosomes, has emerged as a pivotal tool in plant breeding programs, particularly in the case of Veronica species. Veronica, a genus with a complex reproductive system, often poses challenges in accurately identifying hybrids because of its tendency to hybridize, which leads to intricate genetic variation. This study focused on the use of FISH as a prescreening method to identify true hybrids in Veronica breeding programs. FISH analysis was first performed on the parents to identify their 45S and 5S rDNA signals, along with their respective chromosome numbers. The signals were then compared with those of the twenty progenies with reference to their supposed parents. Five true hybrids, seven self-pollinated progenies, and eight false hybrids were identified through FISH. The findings highlight the significance of FISH as a screening method that contributes significantly to the efficiency of Veronica breeding programs by ensuring the preservation of desired genetic traits and minimizing the inadvertent inclusion of misidentified hybrids. To conclude, this study underscores the vital role of FISH in enhancing the precision and success of breeding programs and opens new avenues for improved breeding strategies and crop development.

Performance of HER2 DAKO HercepTest and Ventana 4B5 Immunohistochemical Assays on Detecting HER2 Gene-Amplification in Uterine Serous Carcinomas

We compared the performance of two commonly-used HER2 immunohistochemical (IHC) assays in uterine serous carcinomas (USC), correlating with HER2 gene amplification by fluorescence in-situ hybridization (FISH). Sixty-five USCs were stained by both HercepTest™ and PATHWAY 4B5 assays. FISH was performed by HER2 IQFISH pharmDx. Consensus HER2 IHC scoring was performed, and HER2 testing results were evaluated using USC-specific criteria. Complete concordance between HercepTest and 4B5 assays was achieved in 44/65 tumors (68%). The overall HER2 IHC/FISH concordance was 94% (45/48) by HercepTest and 91% (42/46) by 4B5. All HER2 IHC 3+ cases with HercepTest (n=6) and 4B5 (n=4) were gene-amplified, corresponding to specificities of 100%. For cases with IHC 2+, 41% (7/17) by HercepTest and 42% (8/19) by 4B5 had HER2 gene amplification. The sensitivity for HercepTest and 4B5 were 38% and 25%, respectively, at a cut-off of IHC 3+ (P=0.50), and were 81% and 75%, respectively, at a cut-off of IHC 2+ (P>0.99). Among HER2 IHC 0-1+ cases, 3/42 cases by HercepTest and 4/42 cases by 4B5 showed amplified FISH results, corresponding to overall false negative rates of 19% for HercepTest and 25% for 4B5. By using USC-specific IHC scoring criteria, both HercepTest and 4B5 assays showed high specificities (100%) for HER2 gene amplification in IHC 3+ cases, high IHC/FISH concordance, and comparable sensitivity for detecting HER2 gene amplification. The notable false negative rates using IHC 2+ as a cut-off for reflexing FISH analysis may warrant consideration for performing FISH in IHC 1+ cases until more data become available.

Multilineage involvement in KMT2A-rearranged B acute lymphoblastic leukaemia: cell-of-origin, biology, and clinical implications.

B lymphoblastic leukaemia/lymphoma (B-ALL) is thought to originate from Pro/Pre-B cells and the genetic aberrations largely reside in lymphoid-committed cells. A recent study demonstrated that a proportion of paediatric B-ALL patients have BCR::ABL1 fusion in myeloid cells, suggesting a chronic myeloid leukaemia (CML)-like biology in this peculiar subset of B-ALL, although it is not entirely clear if the CD19-negative precursor compartment is a source of the myeloid cells. Moreover, the observation has not yet been extended to other fusion-driven B-ALLs. In this study we investigated a cohort of KMT2A-rearranged B-ALL patients with a comparison to BCR::ABL1-rearranged B-ALL by performing cell sorting via flow cytometry followed by FISH (fluorescence in situ hybridization) analysis on each of the sorted populations. In addition, RNA sequencing was performed on one of the sorted populations. These analyses showed that (1) multilineage involvement was present in 53% of BCR::ABL1 and 36% of KMT2A-rearranged B-ALL regardless of age, (2) multilineage involvement created pitfalls for residual disease monitoring, and (3) HSPC transcriptome signatures were upregulated in KMT2A-rearranged B-ALL with multilineage involvement. In summary, multilineage involvement is common in both BCR::ABL1-rearranged and KMT2A-rearranged B-ALL, which should be taken into consideration when interpreting the disease burden during the clinical course.

Diagnosing liposarcoma on (peri)-renal mass biopsy: A clinicopathological study of 30 cases.

Classification of renal neoplasms on small tissue biopsies is in increasing demand, and maintaining broad differential diagnostic considerations in this setting is necessary. When evaluating a renal or perirenal tumour biopsy with sarcomatoid morphology, together with sarcomatoid renal cell carcinoma and sarcomatoid urothelial carcinoma as top diagnostic considerations, it is vital to additionally consider the possibility of well-differentiated and de-differentiated liposarcoma. This study reports a series of 30 biopsy samples from sites in or around the kidney collected from four institutions in which the correct diagnosis was either well-differentiated or de-differentiated liposarcoma. The majority (26 of 30, 87%) of lesions were accurately diagnosed on biopsy sampling, all of which incorporated testing for MDM2 by immunohistochemistry (IHC), fluorescence in-situ hybridisation (FISH) or a combination of the two as part of the diagnostic work-up. Tumour expression of MDM2 by IHC without confirmatory FISH analysis was sometimes (30%) sufficient to reach a diagnosis, but demonstration of MDM2 amplification by FISH was ascertained in the majority (57%) of biopsy samples. A diagnosis of de-differentiated liposarcoma was not definitively established until resection in four (13%) patients, as no MDM2 testing was performed on the corresponding pre-operative biopsies. When a retroperitoneal tumour is not clinically suspected, histological consideration of a liposarcoma diagnosis may be overlooked. Implementation of ancillary immunohistochemical and cytogenetic testing can ultimately lead to a definitive diagnosis in this potentially misleading anatomical location.

Abstract 562: Frequent copy number gain of MCL1 is a therapeutic target for osteosarcoma

Abstract Osteosarcoma (OS) is the most common primary malignant bone tumor that predominantly occurs in children, adolescents, and young adults. The treatment for OS that combines surgery with chemotherapy, which consists of a four-drug combination of adriamycin (DOX), cisplatin (CDDP), high-dose methotrexate (MTX), and ifosfamide, was established in 1970s, and it is still used as a standard therapy. Oncogenic driver mutations and fusion genes common to OS have not been identified, which is one of the reasons for the lack of success in drug development for OS. Herein, to explore the characteristic vulnerability in OS, molecular targeted drugs were screened against OS cell lines and patient-derived cells; obatoclax, a pan-BH3 mimetic, and a concomitant drug that enhances obatoclax-induced apoptosis, OSI906, a dual inhibitor of IGF1R and IR, were found. Fluorescence in situ hybridization (FISH) analysis revealed that copy number gains of MCL1, an antiapoptotic Bcl-2 family protein, were observed in 46.3% of formalin-fixed paraffin-embedded (FFPE) specimens from patients with OS (N = 41) and in 45.5% of OS cell lines (N = 11), thereby causing vulnerability to BH3 mimetics. IHC staining also revealed high expression of Mcl-1 protein in specimens with MCL1-amplification in FISH analysis. Pharmacological or genetic inhibition of Mcl-1 induced potent apoptosis in MCL1-amplified OS cells, which was enhanced by the co-inhibition with Bcl-xL. Moreover, chromosome 1q21.2-3, where MCL1 is located, contains several IGF1R/PI3K pathway-related genes, including PIP5K1A, TARS2, OUTD7B, and ENSA, whose copy numbers were also increased in MCL1-amplified OS cells, suggesting that 1q21.2-3-amplified OS cells readily activate downstream pathways via IGF1R. IGF signaling activation in 1q21.2-3-amplified OS cells negated the obatoclax-induced apoptosis, which was overcome by OSI906 treatment. Combined obatoclax and OSI906 administration remarkably suppressed MCL1-amplified OS tumor growth in the xenograft mouse model and resulted in tumor regression; however, none of the treatments reduced tumor growth of OS without MCL1 amplification. These results suggest that combining obatoclax with OSI906 could be a potential therapeutic intervention for 1q21.2-3-amplified OS, and that prior stratification of OS based on the 1q21.2-3 amplification status would be important for the success of this combination therapy. Thus, we elucidated the mechanism of action of drugs and their potential as a novel therapeutic strategy for approximately 50% of patients with OS. Moreover, a FISH analysis that can detect copy number gains of MCL1 in FFPE specimens from patients with OS was established to stratify patients eligible for this therapy. Citation Format: Satoshi Takagi, Ryohei Katayama. Frequent copy number gain of MCL1 is a therapeutic target for osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 562.

Mixed Gonadal Dysgenesis with 45,X/46,X,idic(Y)/46,XY Karyotype: A Case Report

Background: The purpose of the current study was to report a case with 45,X/ 46,XY/46,X,idic(Yp) mosaicism showing the male phenotype with mixed gonadal dysgenesis.
 Case Presentation: A 27 year-old individual, phenotypically male, presented with azoospermia and a micropenis. Both testes were not visualized in the scrotal sac. Due to the presence of a small-sized uterus, the individual was referred to the KSHEMA Center for Genetic Services for chromosomal analysis. Karyotyping revealed a mosaic karyotype of 45,X[44]/46,XY[5]/46,X,idic(Yp)[1]. This finding was further confirmed through fluorescent in situ hybridization (FISH) analysis. The individual's mosaic karyotype consisted of three cell lines, with a higher proportion of the 45,X cell line and lower proportions of the idic(Yp) and 46,XY cell lines. It is worth noting that this mosaic condition in postnatal peripheral blood has not been reported in the literature thus far.
 Conclusion: The case report demonstrated the importance of performing karyotype and FISH analysis in understanding genetic defects including mosaicism and other chromosomal aberrations, which can influence not only growth and puberty but also sexual development and maturation. Hence, performing cytogenetic and molecular cytogenetic analysis will help clinicians to take a further step in understanding and managing the condition.

High-resolution genomic profiling and locus-specific FISH in subcutaneous and visceral adipose tissue of obese patients.

Obesity is known as a heterogeneous and multifactorial disease. The distribution of body fat is crucial for the development of metabolic complications. Comprehensive genetic analyses on different fat tissues are rare but necessary to provide more detailed information. Therefore, we performed genetic analyses of three patients with obesity using high resolution genome wide SNP array (blood, visceral fat tissue) and fluorescence in situ hybridization (FISH) analyses (visceral and subcutaneous fat tissue). Altogether, we identified 31 small Copy Number Variations (losses: 1p31.1, 1p22.2, 1q21.3, 2q34, 2q37.1, 3q28, 6p25.3, 7q31.33, 7q33, 8p23.3, 10q22.3, 11p15.4, 11p15.1, 11p14.2, 11p12, 13q12.3, 15q11.2-q13.1, 15q13.3, 20q13.2, 22q11.21; gains: 2q22.1-q22.2, 3p14.3, 4p16.3, 4q32.2, 6q27, 7p14.3, 7q34, 11p12, 12p11.21, 16p11.2-p11.1, 17q21.31) and 289 small copy-neutral Loss of Heterozygosity (cn-LOH). For the chromosomal region 15q11.2-q13.1, we detected a microdeletion (Prader-Willi-Syndrome) in one patient. Interestingly, we identified chromosomal SNP differences between EDTA-blood and visceral fat tissue (deletion and gain). Small losses of 7q31.33, 7q33, 11p14.2, 11p12, 13q12.3 as well as small gain of 7q34 were detected only in fat tissue and not in blood. Furthermore, FISH analyses on 7q31.33, 7q33 and 11p12 revealed differences between subcutaneous and visceral fat tissue. Generally, the deletions were detected more frequent in visceral fat tissue. Predominantly detected cn-LOH vs. CNV suggests a meaning of these cn-LOH for the pathogenesis of obesity. We conclude that the SNP array and FISH analyses used is applicable to generate more information for basic research on difficult cell subpopulations (e.g., visceral adipose tissue) and could opens up new diagnostic aspects in the field of obesity. Altogether, the significance of these mostly not yet described genetic aberrations in different fat tissues needs to confirmed in a larger series.

Abstract P13: Genetic and epigenetic characterization of B-cell chronic lymphocytic leukemia with IG::BCL3-translocation provides evidence for a distinct biological entity

Abstract IG translocations occur in various B-cell neoplasms and result in the deregulation of (onco)genes due to their juxtaposition with regulatory elements in the immunoglobulin loci, predominantly the immunoglobulin heavy chain (IGH) locus (14q32). Some of these IGH translocations are pathognomonic for distinct disease entities, like those involving MYC, BCL2 or CCND1 in Burkitt, Follicular or Mantle Cell Lymphomas, respectively. In Chronic Lymphocytic Leukemia (CLL), the BCL3 gene (19q13), a transcriptional coactivator of the nuclear factor-kappaB (NF-κB) family, is a known partner gene in these translocations. Two subsets of IG::BCL3 translocation-positive B-cell neoplasms have been described that exhibit differences in chromosomal aberrations, IGHV mutation status, and histopathology [Martín-Subero et al. 2007]. In CLL, IG::BCL3 translocation is linked to a younger age at diagnosis, a more aggressive clinical course, and an atypical tumor cell phenotype [Michaux et al. 1997; Au et al. 2002]. A total of 89 B-cell neoplasms, primarily diagnosed as CLL, displaying IG::BCL3 translocation detected by fluorescence in situ hybridization (FISH), were examined. Analysis of DNA methylation and copy number aberrations (CNA) was conducted for 86 IGH::BCL3-translocated B-cell neoplasms using 850K EPIC BeadChip array data. Evaluation of chromosomal aberrations typical for CLL was moreover performed by FISH and the IGHV mutation status was determined. IGH::BCL3 breakpoint junctions were sequenced in 23 samples using targeted capture and whole-genome sequencing approaches. RNA expression of BCL3 was assessed using the HTG mRNA pan B-cell panel. In UMAP analysis of DNA methylation, CLLs with IG::BCL3 translocation exhibited segregation from other CLLs, irrespective of IGH translocations. Supervised analysis revealed global hypomethylation of B-cell neoplasms with IG::BCL3 translocation compared to other CLL. A total of 66/69 CLLs with IG::BCL3 translocation showed an unmutated IGHV status (96%). Copy number determination using BeadChip data (n=86) and FISH analysis (n=85) showed a significantly higher incidence of trisomy 12 in IG::BCL3-translocated cases compared to the general CLL population (p=0.011). Breakpoint sequencing indicated IG::BCL3 junctions to be recurrently associated with aberrant class-switch recombination at the IGH locus, involving IGHA (n=11/23), IGHG (n=5/23) and IGHM segments (n=5/23). Breakpoints at the BCL3 locus displayed two groups. 80 cases (all CLL) were located upstream of BCL3 and two other B-cell neoplasms (NHL, DLBCL) were located downstream of BCL3. BCL3 expression analysis in 15 IG::BCL3-translocated CLLs confirmed its transcriptional upregulation compared to the general CLL population. Our genetic and epigenetic analyses support the view that CLL with IG::BCL3 translocation may constitute a genetically and epigenetically distinct subtype of B-cell neoplasms, diverging from the typical CLL. Citation Format: Cosima Drewes, Cristina López, Nnamdi Okeke, Sina Hillebrecht, Petra Schütz, Anja Fischer, Anja Mottok, Susanne Bens, Christof Schneider, Stephan Stilgenbauer, Eugen Tausch, Reiner Siebert. Genetic and epigenetic characterization of B-cell chronic lymphocytic leukemia with IG::BCL3-translocation provides evidence for a distinct biological entity [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P13.

Evaluation of Combined p57KIP2 Immunohistochemistry and Fluorescent in situ Hybridization Analysis for Hydatidiform Moles Compared with Genotyping Diagnosis.

Immunostaining with p57KIP2 is a widely used diagnostic technique to differentiate complete hydatidiform moles (CHMs) from partial hydatidiform moles (PHM) and non-molar hydropic abortion. However, distinguishing between PHMs and non-molar hydropic abortions using histopathology alone is often challenging. This study aimed to evaluate the technical validity and additional benefits of using fluorescence in situ hybridization (FISH) in combination with p57KIP2 immunostaining to diagnose molar and non-molar conceptuses. The study involved 80 specimens, which underwent genetic diagnosis using short tandem repeat analysis, including 44 androgenetic CHMs, 20 diandric monogynic PHMs, 14 biparental non-molar hydropic abortions, 1 monoandric digynic triploid abortion, and 1 vaginal specimen of gestational trophoblastic neoplasia. Two pathologists independently diagnosed the cases based on morphology and p57KIP2 immunostaining while the clinical information was masked. FISH analysis was performed using 3 probes (CEP17, CEPX, and CEPY), which revealed that all androgenetic CHM and biparental diploid non-molar hydropic abortion specimens were diploid. Among the 20 diandric monogynic PHM cases examined by analyzing short tandem repeat polymorphisms, 18 were triploid, and the remaining 2 were diploid. These two specimens were possibly androgenetic/biparental mosaics based on FISH analysis, where the three-signal ratios counting 50 cells were clearly within the diploid ranges. Eight of the 20 genetic PHMs and 2 of the 14 genetically confirmed non-molar hydropic abortions that were falsely diagnosed based on morphology and immunohistochemistry by at least 1 pathologist were correctly diagnosed as PHM and non-molar hydropic abortion, respectively, by FISH analysis. However, 1 monoandric digynic villus was classified as triploid by FISH analysis, leading to a false PHM diagnosis. In conclusion, the combination of FISH analysis with p57KIP2 immunostaining helps in diagnosing molar and non-molar conceptuses in numerous cases; nevertheless, exceptional cases should be considered.

Report and literature review of four cases of EWSR1::NFATC2 round cell sarcoma

BackgroundEWSR1::NFATC2 rearranged sarcomas are a group of rare round, undifferentiated sarcomas with clinicopathological features different from those of Ewing's sarcoma (ES) family and other non-ES sarcomas. We report 4 cases of this rare sarcoma and review their features.Materials and methodsFour cases of EWSR1::NFATC2 rearranged round cell sarcoma of the bone from the Pathology Department of Peking University People's Hospital were retrospectively studied. Clinical and pathological data were summarized, and immunohistochemical staining, fluorescence in situ hybridization (FISH), and Next-generation sequencing (NGS) were performed. Relevant literature reports were also reviewed.ResultsAmong the four cases of EWSR1::NFATC2 rearranged round cell sarcoma, three were male, and one was female, with the age ranged from 14 to 34 years old at diagnosis (mean age: 27.5 years). All tumors were located in the femur and ranged in size from 4 to 8cm (mean 6cm), involving the surrounding soft tissues. All four patients underwent surgical treatment, and three received chemotherapy and radiotherapy postoperatively. Follow-up results showed that all four patients were alive. Histologically, the tumors exhibited small round cell sarcoma phenotype, with the stroma rich in mucin or exhibiting a glassy appearance. The tumor cells diffusely expressed CD99, NKX2.2, NKX3.1 and focal expression of CK and EMA was observed. FISH analysis showed that EWSR1 gene rearrangement was detected in all 4 cases, accompanied by 5' locus amplification. EWSR1::NFATC2 fusion probe demonstrated multi yellow fusion signals. NGS identified EWSR1::NFATC2 breakpoints in exon 9 and exon 3 in all 4 cases. The average follow-up duration of the study group was 88 months (range from 26—180 months). One case experienced both local recurrence and metastasis to the lung and chest wall. One case presented with local recurrence. The remaining two cases did not have the recurrence or metastasis.ConclusionAlthough the disease can locally recur and metastasize to the lungs, its mortality rate is significantly lower than that of Ewing sarcoma and other high-grade small round cell undifferentiated sarcomas. Therefore, it supports to classify this tumor as a separate subtype of small round cell sarcoma.

Syndromic MEN1 parathyroid adenomas consist of both subclonal nodules and clonally independent tumors.

Primary hyperparathyroidism with parathyroid tumors is a typical manifestation of Multiple Endocrine Neoplasia Type 1 (MEN1) and is historically termed "primary hyperplasia". Whether these tumors represent a multi-glandular clonal disease or hyperplasia has not been robustly proven so far. Loss of Menin protein expression is associated with inactivation of both alleles and a good surrogate for a MEN1 gene mutation. The cyclin-dependent kinase inhibitor 1B (CDKN1B) gene is mutated in MEN4 and encodes for protein p27 whose expression is poorly studied in the syndromic MEN1 setting.Here, we analyzed histomorphology and protein expression of Menin and p27 in parathyroid adenomas of 25 patients of two independent, well-characterized MEN1 cohorts. The pattern of loss of heterozygosity (LOH) was assessed by fluorescence in situ hybridization (FISH) in one MEN1-associated parathyroid adenoma. Further, next-generation sequencing (NGS) was performed on eleven nodules of four MEN1 patients.Morphologically, the majority of MEN1 adenomas consisted of multiple distinct nodules, in which Menin expression was mostly lost and p27 protein expression reduced. FISH analysis revealed that most nodules exhibited MEN1 loss, with or without the loss of centromere 11. NGS demonstrated both subclonal evolution and the existence of clonally unrelated tumors.Syndromic MEN1 parathyroid adenomas therefore consist of multiple clones with subclones, which supports the current concept of the novel WHO classification of parathyroid tumors (2022). p27 expression was lost in a large fraction of MEN1 parathyroids and must therefore be used with caution in suggesting MEN4.

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

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

Phaseolus vulgaris mutants reveal variation in the nuclear genome.

Phaseolus vulgaris L. (common bean) is an essential source of proteins in the human diet worldwide. Bean breeding programs to increase genetic diversity based on induced mutagenesis have a long tradition in Bulgaria. Common bean varieties with high productivity, wide environmental adaptability, good nutritional properties, and improved disease resistance have been successfully developed. In this study, we aimed to investigate selected nuclear genome features, such as the genome size, the number and chromosomal distribution of 5S and 35S rDNA loci by using the fluorescence in situ hybridization (FISH), as well as the level of DNA damage in some local Bulgarian accessions and mutants of P. vulgaris. Flow cytometry analyses revealed no significant differences in genome size between analyzed lines except for one of the analyzed mutants, M19. The value of genome size 2C DNA is about 1.37 pg2C -1 for all lines, whereas it is 1.42 pg2C-1 for M19. The chromosome number remains the same (2n=22) for all analyzed lines. Results of FISH analyses showed that the number of 5S rDNA was stable among accessions and mutant lines (four loci), while the number of 35S rDNA loci was shown as highly polymorphic, varying between ten and sixteen, and displaying differences in the size and location of 35S rDNA loci between analyzed genotypes. The cell cycle profile was different for the analyzed genotypes. The results revealed that wide variation in genome organization and size as well as DNA damage characterizes the analyzed genetic resources of the common bean.

Revealing RB1 loss in an emerging entity: report of two cases of PRRX1-rearranged mesenchymal tumours

AimsPRRX1-rearranged mesenchymal tumours are a recently identified and rare subgroup of soft tissue neoplasms with distinct morphological features and genetic alterations. This study aims to further investigate the immunohistochemical profile...

Cytogenetics of Multiple Myeloma

Multiple myeloma is a type of cancer that affects the plasma cells in the bone marrow. In this abstract, we discuss the cytogenetic abnormalities in multiple myeloma patients and their correlation with age and gender. We also describe the techniques used for cytogenetic analysis in the Indian population. Our study included 70 patients with multiple myeloma, and cytogenetic analysis was performed using conventional karyotyping and Fluorescence In-situ Hybridization (FISH) techniques. We observed a higher incidence of cytogenetic abnormalities in patients above 60 years of age, with male predominance. FISH analysis showed a higher detection rate of cytogenetic abnormalities compared to conventional karyotyping. FISH also provided additional information on the extent and complexity of the chromosomal aberrations. It is a cytogenetic technique that uses fluorescently labelled probes to detect genetic abnormalities in the chromosomes of cells obtained from bone marrow. This analysis can provide valuable information about the type and stage of the disease, Translocations t(4;14),t(14;16), t(6;14), and t(14;20) were associated with anaemia, t(4;14) was associated with a higher serum monoclonal protein and plasma cell proliferation. Monosomy 13 is associated with a short survival rate with transition monoclonal gammopathy. In conclusion, our study confirms the age and male predominance in multiple myeloma and highlights the importance of cytogenetic analysis in the diagnosis and prognosis of the disease. By studying the chromosomal abnormalities in MM, we can gain insights into the underlying causes of the disease. In this study, we will discuss the cytogenetics of MM and explore how it can help understand the genetics of cancer. The study highlights the chromosomal abnormalities associated with MM and its implications for diagnosis and prognosis.

[Clinicopathological and molecular features of metaplastic thymoma].

Objective: To investigate the clinicopathological features, and molecular genetic alterations of metaplastic thymoma (MT). Methods: A total of ten MT cases, diagnosed from 2011 to 2021, were selected from the Department of Pathology of Jinling Hospital, Nanjing University Medical School, Nanjing, China for clinicopathological and immunohistochemical (IHC) examination and clinical follow-up. Fluorescence in situ hybridization (FISH), next-generation sequencing (NGS), and YAP1 C-terminus (YAP1-CT) IHC were performed to detect YAP1::MAML2 fusions. Results: There were four males and six females, ranging in age from 29 to 60 years (mean 50 years, median 54 years). Microscopically, all tumors showed a typical biphasic morphology consisting of epithelial components and gradually or abruptly transitioning spindle cell components. The two components were present in varying proportions in different cases. Immunophenotypically, the epithelial cells were diffusely positive for CKpan, CK5/6 and p63. The spindle cells were diffusely positive for vimentin and focally positive for EMA. TdT was negative in the background lymphocytes. Ki-67 proliferation index was less than 5%. YAP1 and MAML2 break-apart FISH analyses showed that all ten cases had narrow split signals with a distance of nearly 2 signal diameters and may be considered false-negative. Using YAP1::MAML2 fusion FISH assays, abnormal fusion signals were observed in all the ten cases. NGS demonstrated YAP1::MAML2 fusions in all eight cases with adequate nucleic acids; in two cases the fusions were detected by DNA sequencing and in eight cases by RNA sequencing. All ten cases of MT demonstrated loss of YAP1 C-terminal expression in epithelioid cells. Conclusions: MT is a rare and low-grade thymic tumor characterized by a biphasic pattern and YAP1::MAML2 fusions. Break-apart FISH assays may sometimes show false-negative results due to the proximity of YAP1 and MAML2, while YAP1 C-terminal IHC is a highly sensitive and specific marker for MT. Loss of YAP1 C-terminal expression can also be used to screen YAP1::MAML2 fusions for possible MT cases.

Fluorescence In-situ Hybridization (FISH) and Its Application in Oncology

Fluorescence in situ hybridization (FISH) technique was developed in early 1970s for DNA sequence identification. FISH has gained wide application in cytogenetic research, thanks for its high sensitivity and specificity. FISH analysis has also become a powerful tool for cancer research and treatment, through detecting genetic aberrations in cancer cells. Compared to other cytogenetic tests, FISH analysis is fast and compatible with various cell types. It is particularly useful under clinical settings, by providing crucial information of chromosomal abnormalities, such as gene microdeletion, amplification, and translocation, to guide cancer therapy selection, and monitor effectiveness of the treatment. For example, FISH is used as a companion diagnostic tool for non-small-cell lung cancer (NSCLC) patients with ALK or ROS1 gene rearrangement. It’s also a confirmative test for breast cancer patients with HER2 abnormality. Through combination with other novel technologies, newer versions of FISH have been developed to improve the throughput and sensitivity, which will further expand the potential of FISH analysis in fighting cancer.

Residual Mast Cells Can Explain Persistent Molecular Positivity in Difference from Normal Flow Cytometric-Defined MRD Negative Core Binding Factor AML

Introduction: Core binding factor acute myeloid leukemia (CBF-AML) accounts for 30% of pediatric and 15% adult AMLs and is defined by fusions involving RUNX1 or CBFB. RT-PCR is commonly used to monitor measurable residual disease (MRD) in patients with CBF-AML. However, it has been reported that these fusions can be detected in patients during complete remission (CR) or after hematopoietic cell transplant (HCT). This raises the question of if these patients should be considered MRD positive, given they appear otherwise disease free. We utilized flow cytometric cell sorting and fluorescent in situ hybridization (FISH) to determine the source of persistent CBF fusions. Methods: Flow cytometry was used to isolate mast cells (CD117 bright/CD34 negative) from 13 patients with CBF-AML fusions (8 RUNX1:: RUNX1T1 and 5 CBFB:: MYH11) alongside progenitor cells (CD34+/CD117 heterogeneous, 12 patients) and T cells (CD3+, 11 patients) as available for FISH analysis. These patients were fusion positive by RT-PCR but had no evidence of MRD by difference from normal multidimensional flow cytometry (ΔN), the gold standard for determination of residual disease in Children's Oncology Group-sponsored clinical trials for AML. Cells were sorted from 6 additional patients with a history of RUNX1:: RUNX1T1 CBF-AML who were MRD negative by ΔN but without RT-PCR results available. Results: In 11/13 patients, the mast cell fraction was positive for the CBF fusion but all other sorted cell fractions tested were negative. In the remaining two specimens, all cell fractions were negative, likely due to FISH sensitivity compared to RT-PCR. Both CBF fusions exhibited the same pattern, with fusions detected only in the mast cell fractions (7 RUNX1:: RUNX1T1 and 4 CBFB:: MYH11). The additional 6 patients who were MRD negative by ΔN but without RT-PCR data followed the same pattern, with RUNX1:: RUNX1T1 detected by FISH in the mast cells (6/6 patients) and not in other sorted populations (5/6 patients sorted for progenitor and T cells). Two patients had a clinical history of CBF-AML and systemic mastocytosis (SM). ΔN revealed no evidence of residual AML but showed abnormal mast cells with expression of CD2 and CD25, consistent with abnormalities associated with SM. FISH analysis of sorted mast cells revealed the presence of the RUNX1:: RUNX1T1 fusion, while progenitor and T cells were both negative. In all other patients the mast cells showed no phenotypic abnormalities, demonstrating that CBF fusions can be detected in both phenotypically normal and abnormal mast cells in patients who are free of residual AML. Two patients with CBFB:: MYH11 fusions had specimens evaluated by FISH on sorted cells over a month apart where the fusion persisted only in the mast cells. Two patients with RUNX1:: RUNX1T1 fusions had serial specimens analyzed at 4 different timepoints. One patient had fusion positive mast cells for over 8 months after residual disease was last detected by ΔN. The other patient, who had previously undergone HCT, harbored fusion positive mast cells for 13 months. Both patients remained MRD negative by ΔN over the course of testing. Discussion: These data demonstrate that in CBF-AML, fusions can be detected exclusively in the mast cells of otherwise leukemia-free patients, resulting in positive RT-PCR results in the absence of any other disease pathology. These mast cells can be normal or abnormal and can persist after successful HCT. Given that these patients are free of disease by other measures, these fusion positive mast cells do not appear to contribute to AML pathogenesis. Additionally, this post-HCT fusion positive persistence demonstrates a potential clinical implication: if patients such as reported here are assessed pre-transplant using RT-PCR on unsorted specimens alone, they may not be considered candidates for HCT even though the fusion is confined to terminally differentiated cells and successful transplant can be achieved. For these reasons, while RT-PCR is a specific and accurate assay, caution is required for CBF-AML patients. Flow cytometry and cell sorting in combination with genetic testing, in this case FISH, protect against these mast cell driven positive results and provide an accurate assessment of disease status. These data highlight the importance of considering AML biology and combining multiple testing methods to monitor AML patients comprehensively and accurately.

Fluorescent in Situ Hybridization Analysis in Multiple Myeloma: Enhancing Risk Stratification, Autologous Transplant Utilization and Treatment Decisions in Limited-Resource Settings

Introduction: Multiple myeloma (MM) is a heterogeneous and incurable disease with a high risk of relapse. Recent advancements in MM treatment, such as proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), and monoclonal antibodies, have improved outcomes. Risk stratification plays a crucial role in prognosis, with cytogenetic abnormalities being an important predictor. However, conventional karyotyping is limited in its sensitivity, while interphase Fluorescent In Situ Hybridization (FISH) has emerged as a powerful prognostic marker. In lower- and middle-income countries (LMICs), where MM management faces challenges, identifying high-risk patients through FISH can guide treatment decisions and improve outcomes, even in the absence of novel drugs like Daratumumab. Methods: We conducted a retrospective study on newly diagnosed MM (NDMM) patients from January 2016 to December 2022 in two South Indian centres. Patients receiving at least 16 weeks of induction chemotherapy ± autologous stem cell transplant (ASCT) were included. The cohort was divided into two groups: those with FISH results evaluable (FRE group) and those with FISH results unavailable (FRU group). FISH analysis used 5 probes (Del13q14.3, TP53/ CEN 17, IgH (14q32.3)/ FGFR3 (4p16), IgH (14q32.3)/CCND1 (11q13), IgH (14q32.3)/MAF (16q23)) till March 2020 and 7 probes (XL CDKN2C/CKS1B, XL t(4;14) FGFR3/IGH DF, XL 5p15/9q22/15q22 Hyperdiploidy, XL t(11;14) MYEOV/IGH, XL IGH BA, XL t(14;16) IGH/MAF DF, XL TP53/17cen) from April 2020. Data collected included demographics, International Staging System (ISS), mSMART 3.0-based FISH risk stratification, induction regimen, ASCT, maintenance, relapse and response. Survival analyses employed Kaplan-Meier (KM) and log-rank tests. IBM SPSS Version 26 Software was used for analysis. Results: Of the 427 screened NDMM patients, 333 were included: 192 (57.7%) in FRE and 141(42.3%) in FRU group. Baseline characteristics were comparable, except for hypercalcemia being significantly higher in FRE (p-0.015). In FRE, 128(66.67%) had standard risk, and 64 (33.33%) had high-risk MM. VRd induction was more frequently used in FRE (p-<0.001) with no significant difference in induction response. A higher percentage of FRE patients underwent ASCT (22.9% vs. 9.93%, p<0.002), achieving more complete responses post-transplant (p<0.001). FRE group also showed an increased use of doublet maintenance (p-0.014). No significant difference observed in the mortality (p-0.069). ASCT correlated with better median PFS (64 Vs. 37 months, p-0.013). Conclusions: In this resource-limited setting, the availability of FISH results seemed to have an impact on MM patient management and outcomes. FRE patients received more aggressive induction therapy, resulting in better response rates and increased ASCT utilization. Additionally, dual-agent maintenance was preferred for high-risk patients in FRE. However, it is essential to acknowledge that this was a retrospective analysis, and other factors may have influenced the outcomes, even though both cohorts were mostly matched. Notably, none of the patients had received Daratumumab as induction therapy, suggesting that FISH availability in LMICs may positively impact patient outcomes even in the absence of access to novel drugs. Further validation through multicenter prospective investigations is warranted to strengthen these findings and explore the broader implications of incorporating FISH into risk stratification strategies. By utilizing FISH, LMICs can enhance MM management and form tailored treatment strategies, thereby improving care for NDMM patients.