Interphase FISH 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.

The Prevalence of Cytogenetic Abnormalities Detected by Interphase FISH Method in Chronic Lymphocytic Leukemia

Abstract Background Chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia. Identification of genomic aberration provides prognostic/predictive information that is helpful in the precision medicine management of these patients. The aim of this study was to determine prevalence of the most common cytogenetic abnormalities of CLL patients in the southwest region (Shiraz) of Iran and correlate with clinical prognostic parameters to clarify their prognostic value. Materials and Methods In this cross-sectional study, 100 patients with CLL were recruited from April 2019 to October 2021. Four milliliters of anticoagulated peripheral blood was collected from each participant. The sample was used for complete blood count (CBC) test and fluorescence in situ hybridization (FISH) test. Interphase FISH (I-FISH) was performed for most common cytogenetic abnormalities, including trisomy 12, 13q14 deletion, 11q deletion, and 17p deletion on interphasic cell nuclei. Results Among 100 patients with CLL, 33 (33%) were females and 67 (67%) were males. The mean age (mean ± standard error [SE]) of the patients was 59.00 ± 1.14 years, with a ranged of 25 to 79 years. Our analysis demonstrated that 86 (86%) patients had at least one chromosomal aberration. The most commonly detected abnormality was 13q deletion (61, 61%), followed by 17q deletion (50 cases, 50%). Trisomy 12 was detected in 14 (14%) cases and 10 cases (10%) had 11q deletion. Conclusion The higher frequency of 13q14 and 17p anomalies in our study may be attributed to delayed medical consultations, leading to the emergence of secondary abnormalities. More studies are recommended for verifying the results.

Complex genomic rearrangements of the Y chromosome in a premature infant

BackgroundChromoanagenesis is an umbrella term used to describe catastrophic “all at once” cellular events leading to the chaotic reconstruction of chromosomes. It is characterized by numerous rearrangements involving a small number of chromosomes/loci, copy number gains in combination with deletions, reconstruction of chromosomal fragments with improper order/orientation, and preserved heterozygosity in copy number neutral regions. Chromoanagesis is frequently described in association with cancer; however, it has also been described in the germline. The clinical features associated with constitutional chromoanagenesis are typically due to copy number changes and/or disruption of genes or regulatory regions.Case presentationWe present an 8-year-old male patient with complex rearrangements of the Y chromosome including a ring Y chromosome, a derivative Y;21 chromosome, and a complex rearranged Y chromosome. These chromosomes were characterized by G-banded chromosome analysis, SNP microarray, interphase FISH, and metaphase FISH. The mechanism(s) by which these rearrangements occurred is unclear; however, it is evocative of chromoanagenesis.ConclusionThis case is a novel example of suspected germline chromoanagenesis leading to large copy number changes that are well-tolerated, possibly because only the sex chromosomes are affected.

Prenatal diagnosis of a trisomy 7 mosaic case: CMA, CNV-seq, karyotyping, interphase FISH, and MS-MLPA, which technique to choose?

ObjectiveThis study aims to perform a prenatal genetic diagnosis of a high-risk fetus with trisomy 7 identified by noninvasive prenatal testing (NIPT) and to evaluate the efficacy of different genetic testing techniques for prenatal diagnosis of trisomy mosaicism.MethodsFor prenatal diagnosis of a pregnant woman with a high risk of trisomy 7 suggested by NIPT, karyotyping and chromosomal microarray analysis (CMA) were performed on an amniotic fluid sample. Low-depth whole-genome copy number variation sequencing (CNV-seq) and fluorescence in situ hybridization (FISH) were used to clarify the results further. In addition, methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was performed to analyze the possibility of uniparental disomy(UPD).ResultsAmniotic fluid karyotype analysis revealed a 46, XX result. Approximately 20% mosaic trisomy 7 was detected according to the CMA result. About 16% and 4% of mosaicism was detected by CNV-seq and FISH, respectively. MS-MLPA showed no methylation abnormalities. The fetal ultrasound did not show any detectable abnormalities except for mild intrauterine growth retardation seen at 39 weeks of gestation. After receiving genetic counseling, the expectant mother decided to continue the pregnancy, and follow-up within three months of delivery was normal.ConclusionIn high-risk NIPT diagnosis, a combination of cytogenetic and molecular genetic techniques proves fruitful in detecting low-level mosaicism. Furthermore, the exclusion of UPD on chromosome 7 remains crucial when NIPT indicates a positive prenatal diagnosis of trisomy 7.

Abstract 4360: Integrative analysis of multiplex interphase FISH copy number with whole exome sequencing identifies tumor evolution in Stage II colon cancer

Abstract Colon cancer ranks as the second leading cause of cancer-related death worldwide. Although standard therapies are highly effective in stage II lymph-node negative colon cancers, about 10-15% of these patients show disease relapse within the next 5-year period after intended curative surgery. Extensive prior research has highlighted the role of intratumor heterogeneity, arising from genomic instability, as a leading contributor to metastasis in colon cancer. In the current study, we pioneered the integration of Multiplex Interphase FISH (miFISH) and Whole Exome Sequencing (WES) data aiming to depict intratumor heterogeneity and delineate the landscape of genetic aberrations that underlie the early metastatic potential in stage II colon cancer patients. To this aim, we have showcased the genome-wide copy-number alterations and single nucleotide mutations derived from WES analysis of samples obtained from primary stage II colon tumors and their patient-matched liver metastases of nine patients using multi-region sampling. miFISH was employed for the concurrent quantification of copy-numbers for nine genes relevant to colon cancer and a centromeric control probe in intact tumor nuclei derived from archival patient material allowing us to establish ploidy baselines and gain and loss patterns for the genes analyzed. Our study was revealing of several copy number alterations, such as gains of chromosomes 1q, 7, 8q, 13q and 20, and losses affecting chromosomes 17p and 18, which were confirmed by miFISH. Based on our SNV data, most frequently mutated genes were APC, TP53, KRAS. We were able to show several mutations in genes belonging to specific pathways most commonly altered in colorectal cancer, such as APC, TCF7L2, AXIN2, FBXW7 in WNT signaling, SMAD4 in TGF-ß signaling, PIK3CA and PTEN in PI3K signaling and KRAS in RTK-RAS signaling pathways. In nearly all cases, subclones that led to metastasis were already present in the primary cancer, indicating a direct progression from the primary to metastatic disease. However, the divergence from the primary tumor to metastasis enabled us to uncover several potential genetic aberrations present in the metastasis but not observed in the primary tumor. Moreover, in one of our cases, despite harboring APC and TP53 mutations in both the primary and metastatic tumor, unique mutations for these genes along with several distinct mutations in the metastasis were identified. These observations suggest that the metastasis might not be related to the primary tumor analyzed and may have arisen from an independent lesion. In conclusion, applying miFISH and WES to decipher the mutational and copy number landscape in metastatic stage II colon cancer provided comprehensive insights into the complex and heterogenous nature of primary colon cancer and the clonal evolution leading to subsequent liver metastases. Citation Format: Sanam Yaghoubi, Kerstin Heselmeyer-Haddad, Ivan Archilla, Carolina Parra, Darawalee Wangsa, Giancarlo Castellano, Jack Zhu, Diba Yaghoubi, Sara Lahoz, Veronica Pablo-Fontecha3, Daniela Hirsch, Wei-Dong Chen, Thomas Ried, Miriam Cuatrecasas, Paul S. Meltzer, Jordi Camps. Integrative analysis of multiplex interphase FISH copy number with whole exome sequencing identifies tumor evolution in Stage II colon cancer [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 4360.

Differential effect of estrogen and progesterone on <i>in vitro</i> growth of uterine leiomyoma cells with chromosome 7 deletions

BACKGROUND: The studies on how sex steroid hormones affect growth of uterine leiomyoma cells with chromosomal abnormalities is highly relevant for development of personalized tumor therapy.
 AIM: To study in vitro the isolated and combined effects of estrogen and progesterone on uterine leiomyoma cells with chromosomal aberrations — deletions in 7q.
 MATERIALS AND METHODS: The study was performed on 15 uterine leiomyomas, excised from 15 women of 26–44 years of age who were not treated with hormones. Uterine leiomyoma cells were cultured in hormone-free medium, in the medium supplemented with estrogen, progesterone or both hormones. The chromosome preparations were made and stained with QFH/AcD to perform conventional karyotyping and fluorescence in situ hybridization (FISH) to accurately describe chromosomal rearrangements. The frequency of uterine leiomyoma cells with chromosomal aberrations was assessed by interphase FISH.
 RESULTS: Deletions in 7q were identified in 6 out of 15 karyotyped uterine leiomyomas; four of them had one clone with deletion in 7q whereas two others comprised two clones with 7q deletions of different length. The frequency of cells carrying deletions in 7q greatly varied in uterine leiomyoma samples cultured in hormone-free medium: from 3.5% to 93.6%. Exposure of cell cultures to estrogen and progesterone resulted in a fold change frequency increase in some of the uterine leiomyomas and decrease in the others. The most significant changes in the frequency of cells with deletions in 7q were registered in response to the isolated estrogen and, to a lesser extent, to progesterone exposure; less significant changes were observed after combined hormonal effect.
 CONCLUSIONS: In uterine leiomyomas with deletions in 7q, the frequency of abnormal cells may either increase or decrease in response to estrogen and progesterone in vitro supplementation. The isolated effect of estrogen or progesterone on the frequency of uterine leiomyoma cells with deletion in 7q is more pronounced compared to the combined one.

Abstract B032: Comprehensive analysis of clonality, segregation errors, chromosomal instability and heterogeneity in cancer cell lines and cancer samples reveals that cancer evolution is often promoted by highly aberrant cellular divisions

Abstract Chromosomal instability (CIN) is the most common form of genomic instability in cancer. CIN leads to structural and numerical chromosomal aberrations and to heterogeneity of such aberrations inside the same tumor. Together with other forms of genomic and epigenomic instability, CIN underlies the most dangerous feature of cancer cells, namely, ability to evolve, allowing cancer cells to evade immunologic surveillance, to metastasize, to become resistant to drug treatments. Studies of chromosomal instability are significantly impeded by the lack of comprehensive approaches for simultaneous detection of different forms of ongoing chromosomal instability and their outcomes. We developed an approach that allows to analyze the clonal composition and chromosomal heterogeneity in cell lines and cancer samples, as well as rates of different forms of ongoing chromosomal instability in cancer cell lines. At the heart our analysis is a multiplex interphase FISH (miFISH) with seven sequential hybridizations of color probe panels resulting in the assessment of a total of 35 probes per single cell. Each probe panel is designed to include the centromeric probe and probes on the p- and q-arm for a given chromosome that allows for the simultaneous assessment of centromeric probes and gene-specific arm probes. This approach was applied to interphase nuclei, metaphase spreads and bi-nucleated/dividing cancer cells in colorectal and cervical cell lines and cancer samples. miFISH allows for automated acquisition of nuclei and metaphase spreads on a BioView spotcounting system. A gallery provides detailed views for semi-automatic as well as manual analysis of the obtained images. A total of 10000-25000 cell images were collected from each slide. The simultaneous evaluation of our strategically chosen centromeric and gene-specific probes within the same cells allows unprecedented insights in the patterns of distribution of genetic material among cancer cells in cancer cell population, and, especially, in dividing cells. We evaluated clonality, clonal and non-clonal chromosomal heterogeneity, ploidy distributions, and occurrence of endoreplication. Assessment of dividing cells based on equal or unequal distribution of miFISH centromeric and gene-specific signals allowed to estimate and compare the rates and outcomes of different forms of ongoing chromosomal instability such as structural rearrangements and amplifications, loss of chromosomes or chromosomal fragments in micronuclei, non-disjunctions, grossly asymmetrical cell divisions (including multipolar mitoses). Conclusions: Gradual changes in chromosomal/genomic content of cancer cells occur due to structural rearrangements, losses of chromosomes and chromosomal fragments in micronuclei, and by relatively rare single chromosome non-disjunctions. Grossly abnormal cell divisions (massive non-disjunctions and structural rearrangements) is the main source for the high level of chromosomal abnormalities and aneuploidy in evolved cancer cell populations. Citation Format: Anna Roschke. Comprehensive analysis of clonality, segregation errors, chromosomal instability and heterogeneity in cancer cell lines and cancer samples reveals that cancer evolution is often promoted by highly aberrant cellular divisions [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B032.

Use of the ETV6/RUNX1 probe to verify the performance of the fluorescence in situ hybridization probe before clinical detection

Objective: To explore the standardized performance of a FISH probe before clinical detection. Methods: The probe sensitivity and specificity of ETV6/RUNX1 were analyzed via interphase and metaphase FISH in 20 discarded healthy bone marrow samples. The threshold system of the probe was established using an inverse beta distribution, and an interpretation standard was established. Finally, a parallel-controlled polymerase chain reaction detection study was conducted on 286 bone marrow samples from patients at our hospital. The clinical sensitivity, specificity, and diagnostic coincidence rate of ETV6/RUNX1 FISH detection were analyzed, and the diagnostic consistency of the two methods was analyzed by the kappa test. Results: The probe sensitivity and specificity of the ETV6/RUNX1 probe were 98.47% and 100%, respectively. When 50, 100, and 200 cells were counted, the typical positive signal pattern cutoffs were 5.81%, 2.95%, and 1.49%, respectively, and the atypical positive signal pattern cutoffs were 13.98%, 9.75%, and 6.26%, respectively. The clinical sensitivity of FISH was 96.1%, clinical specificity was 99.6%, diagnostic coincidence rate was 99.00%, diagnostic consistency test kappa value was 0.964, and P value was <0.001. Conclusion: For FISH probes without a national medical device registration certificate, standardized performance verification and methodology performance verification can be performed using laboratory developed test verification standards to ensure a reliable and accurate reference basis for clinical diagnosis and treatment.

JAK2 Kinase Fusion and Myeloid Involvement in Acute Lymphoblastic Leukemia

Introduction JAK2 fusion genes (FGs) are rare but can be found in various phenotypes of myeloid or lymphoid malignancies. Both in the 5th edition of the World Health Organization Classification of Haematolymphoid Tumors (WHO-HAEM5, PMID: 35732831) and the International Consensus Classification (ICC, PMID: 35767897), JAK2-FGs were included in the diagnostic entity of myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions (MLN-TK or MLN-eo), and both suggest MLN-TK/eo as a priority diagnostic entity. It is highlighted in ICC that cases presenting as acute lymphoblastic leukemia (ALL) with M/LN-eo differ from de novo B-ALL and T-ALL in the involvement of myeloid cells. There is still a lack of research reports about how many JAK2-FG-positive ALL cases developed from M/LN-eo or had myeloid involvement. Methods A total of 21 ALL cases (Table 1) were enrolled in this study. The positive for JAK2-FGs were determined by G-banding karyotype analysis, transcriptome sequencing (RNA-seq; PMID: 34135310), reverse transcription PCR (RT-PCR), and interphase fluorescence in situ hybridization (I-FISH) with the JAK2 break-apart probe. Gene mutation analysis (PMID: 29932212) was also performed. According to whether there were FISH-JAK2 split signals in rod and lobed nuclear cells (Figure 1A), the cases were divided into My+ group (with myeloid involved) and My- group (without myeloid involved), respectively. Results Among these cases, there were 14 males and 7 females (M:F = 2:1); 15 B-ALL and 6 T-ALL cases; the median age was 22 years old (range 5-55). All cases were positive for JAK2-FGs in RNA-seq analysis, and all FGs were verified by RT-PCR and Sanger sequencing. A total of 16 JAK2 partner genes were detected (Table 1, Figure 1B/C), of which FNBP1, CDK5RAP2, GTF2I, and C14orf 93 have not been reported previously. All cases retained the complete kinase domain of the JAK2 gene, and 13 cases retained part or all of the JAK2 pseudo-kinase domain in their JAK2-FGs. Karyotype abnormalities were detected in 17 cases (81%), with 9p24 involved in 10 cases (48%), abnormalities other than 9p24 were observed in 7 cases, and karyotype was normal in 4 patients. FISH-JAK2 inspection showed split signals in 20 cases and showed negative results in case #21 who with a complex karyotype abnormality. Twelve cases (12/20=60%) showed JAK2 split signals in rotundum, rod, and lobed nuclei (Table 1, My+ group), suggesting both lymphoid and myeloid lineages were involved. We further investigated samples of 4 cases at the time of morphological complete remission after induction chemotherapy, of which the immunophenotype analysis was also negative for lymphoid blast cells. FISH-JAK2 inspection showed split signals accounting for 13%~79% of the nucleus, with split signals in the rod/lobed nucleus, and RT-PCR also confirmed abundant JAK2-FG transcripts in these samples. There were 8 B-ALL and 4 T-ALL cases in the My+ group, of which 2 T-ALL cases were also positive for STIL::TAL1 fusion, and 3 cases experienced eosinophilia during the course of the disease (Table 1). The 5 cases whose JAK2 partner genes were lymphoid development-related transcription factors (ETV6, PAX5) were all in the My- group. All My- cases and #21 had no documented eosinophilia. Among the 21 cases in this cohort, 13 had white blood cell (WBC) counts &amp;gt;50×10 9/L at diagnosis, and no significant hepatosplenomegaly. The total follow-up time was 2-89m, with a median overall survival (OS) time of 17m. Fourteen cases underwent allo-HSCT, and the median OS was 29.5m in the HSCT group and 9m in the non-HSCT group, respectively. The median OS was 15.5m in the My+ group and 29m in the My- group. The leukemia cells in these cases are prone to be in a high and rapid proliferation state, and they generally have a short OS and dismal prognosis. Conclusions Myeloid involvement accounted for 60% of JAK2-FG positive ALL and My+ cases had a worse prognosis and shorter OS than My- cases in this study. I-FISH inspection can be used as a method to determine whether there was myeloid involvement and provide valuable information for accurate analysis. The incidence of JAK2-FGs is low in ALL, but the fusion partners are diverse. This group of cases is more likely to have cryptic JAK2 translocations and with dismal prognosis. The phenotype of JAK2-FG malignancies is jointly determined by multiple factors under the influence of partner genes, accompanying fusion genes, and accompanying mutations.

Unraveling the Evolutionary Paths in Relapsed Early-Stage Follicular Lymphoma Showing Complete Remission

Background: Follicular lymphoma (FL) develops through a stepwise acquisition of cooperative genetic changes with t(14;18)(q32;q21)/ IGH:: BCL2 occurring early at the pre-B stage of B-cell development. Patients with FL typically show an indolent clinical course, remitting and relapsing, and eventually developing resistance to treatments. Interestingly, the majority of transformed FL do not progress directly from the preceding FL but originate from their clonally related lymphoma precursor cells (CLP). In light of this finding, we wanted to address whether such divergent tumor evolution also occurs in the setting of relapses in early-stage FL in patients who achieved complete remission following initial treatment. Methods: We identified 13 cases of FL with an early clinical stage (stage I = 9; stage II = 4) at the initial diagnosis, who showed complete remission (mean: 5 years; range: 1-11.5 years) following local radiotherapy but subsequently relapsed (≥ 2 in 5). Paired formalin-fixed paraffin-embedded diagnostic and relapsed lymphoma biopsies were obtained in each case. BCL2 translocation status was investigated using interphase fluorescence in-situ hybridization (FISH). Genomic DNA was extracted from the tumor-rich areas in each tissue specimen and targeted next-generation sequencing was performed with a customized gene panel comprising 191 genes/ regions that are implicated in FL, diffuse large B-cell lymphoma (DLBCL), and B-cell biology using the TWIST target enrichment kits and Illumina NextSeq platform. Clonality analysis of IG gene rearrangements was performed by combining the BIOMED-2 PCR assays and Illumina MiSeq sequencing. Sequencing data analyses, variant calling, and filtering were performed using a well-established data analysis pipeline from a previous study (Cucco et al., 2018). Results: Interphase FISH showed BCL2 translocation in 6 cases, BCL2 amplification (up to 8 copies) in 1 case, and BCL2 copy number gain (3-4 copies) in 3 cases. Pathogenic variants were identified in each of the lymphoma biopsies and were typical of those frequently seen in FL and DLBCL. There was no significant difference in the mutational burden between the paired diagnostic (mean: 7.8/sample; range: 1-13) and relapsed FL (7.9/sample; range: 2-13), excluding tFL. Both the diagnostic and relapsed lesions showed similar, frequent mutations in BCL2 (33.3% vs 41.67%), CREBBP (58.3% vs 58.3%), EP300 (41.7% vs 41.7%), KMT2D (50% vs 58.3%), TNFRSF14 (66.7% vs 58.3%), and STAT6 (50% vs 50%). The clonal relationship between the diagnostic FL and relapses was confirmed in 11 cases. In 6 cases, common and distinct variants were seen between the paired diagnostic and relapsed lymphomas, indicating their divergent evolution from a CLP. In 2 cases, distinct B-cell clones were involved in the diagnostic and the relapsed lymphomas, including 1 case involving 2 different BCL2 translocations, indicating independent evolution of the new lymphomas. In the remaining 5 cases, the relapsed lymphomas developed via a linear progression from the preceding FL (n = 5). Conclusion: Both divergent evolution, either from clonally related or unrelated precursors, and linear evolution processes are involved in the development of relapses in patients with an early-stage FL following complete remission, with the former being more frequent. The above findings, together with the evidence of divergent evolution of tFL in the majority of cases, challenge the current therapeutic strategy of early-stage FL, which does not target the premalignant cell population that underpins lymphoma recurrence in the majority of cases. Additionally, the routine diagnostic workup, which includes IG gene clonality and BCL2 translocation analyses, cannot address the evolutionary path in clonally related lymphomas. Our results indicate the significance of mutational profiling at diagnosis as well as at relapse, and the need to comprehensively characterize and target the evasive CLP population to prevent recurrence.

Clinical Outcomes Associated with Del(17p) in Newly Diagnosed Multiple Myeloma Treated with Triplet and Daratumumab-Based Quadruplet Induction Regimens

Background: del(17p) is an independent prognostic marker associated with poor clinical outcome identified in a number of studies and has been incorporated into risk stratification models for multiple myeloma (Palumbo A et al J Clin Oncol 2015). Recent studies have reported that the percentage of myeloma cells with del(17p) by fluorescent in situ hybridization (FISH) can have a significant impact on outcomes (Corre J et al. Blood 2021; Thakurta A et al. Blood 2019; Thanendrarajan S et al. Haematologica 2017). Different collaborative groups have recommended different cut-points for the presence of del(17p) with clinical significance ranging from ≥20% or &amp;gt;55% of affected cells. Herein, we examined clinical outcomes of newly diagnosed multiple myeloma (NDMM) patients with del(17p) treated with novel combination triplet and quadruplet induction regimens, including bortezomib, lenalidomide, dexamethasone (VRd), carfilzomib, lenalidomide, dexamethasone (KRd), +/- daratumumab (DVRd and DKRd). Methods: We conducted a chart review study of 578 NDMM patients treated with VRd, KRd, DVRd and DKRd at Memorial Sloan Kettering (MSK) from 1/1/2015 to 5/1/23. Cutoff date for analysis was 7/19/23. Only patients with available cytogenetic results (FISH and/or SNP-array) were included. Patients who received ≤1 prior cycle of another regimen for MM were included. We analyzed the percentage of cells with del(17p) by inter-phase FISH analyses and/or SNP-array with CD138 enrichment when FISH was not available. A cut-point of 20% for del(17p) was used (del(17p) &amp;lt;20% vs del(17p) ≥20%) and compared with patients with standard-risk disease. Discrete patient characteristics were summarized by frequency (percentage) and continuous characteristics were summarized by median (Interquartile Range, IQR). PFS and OS were evaluated by Kaplan-Meier method. Results: There were 391 patients treated with triplet combination, of which 46 patients had del(17p) on baseline bone marrow biopsy. Among these patients, the presence of del(17p) in &amp;lt;20% of cells was detected in 13 patients, and the presence of del(17p) in ≥20% of affected cells was detected in 30 patients. Three patients did not have measurements. There were 187 patients treated with quadruplet combination, of which 20 had del(17p). Among these patients, 9 had del(17p) &amp;lt;20% of cells, 10 had del(17p) in ≥20% of cells, and one patient did not have measurements. There were 212 standard-risk NDMM patients treated with triplet induction and 99 patients treated with quadruplet regimens. The median age was 65 (IQR 54-73) for patients with del(17p) &amp;lt;20%, 61 (IQR 56-68) for del(17p) ≥20%, and 64 (IQR 56-69) for standard-risk patients (P=0.53). Other patient's baseline characteristics are summarized in Table 1. Best ORR among the three groups after triplet or quadruplet induction regimens were high: 95% for patients with del(17p) &amp;lt;20%, 93% for patients with del(17p) ≥20%, and 90% for standard-risk patients (P=0.53). After a median follow-up of 56.9 months, 2-year estimated PFS was 76% (95%CI, 59%-97%), 69% (95%CI, 55%-86%), and 85% (95%CI, 81%-90%) for del(17p) &amp;lt;20%, del(17p) ≥20%, and standard-risk groups, respectively. Median PFS was 48.9 months (95%CI, 26.9-not reached [NR]) for patients with del(17p) &amp;lt;20%, 34.3 months (29.8-66.7) for del(17p) ≥20%, and not reached (72.7-NR) for patients with standard-risk NDMM (P&amp;lt;0.0001). Median PFS was not significantly different between patients with del(17p) &amp;lt;20% and del(17p) ≥20% treated with triplet and quadruplet induction regimens (P=0.33). 2-year OS was 89% (95%CI 76%-100%), 85% (74%-98%), and 95% (93%-98%) for patients with del(17p) &amp;lt;20%, del(17p) ≥20%, and standard-risk (P=0.043). Conclusion: In the MSK experience, the presence of del(17p) was associated with poorer outcomes compared to standard-risk disease in patients with NDMM treated with both triplet and dara-based quadruplet induction regimens. We did not detect a significant difference in PFS and OS amongst del(17p) patients in our cohort using a cut-point of ≥20% or &amp;lt;20% of cells. These findings suggest that the presence of del(17p) alone portend poor prognosis, regardless of FISH cutoff threshold utilized. Data on TP53 mutation status and longer follow-up outcomes will be presented at the meeting.

Adolescents and Young Adults with Multiple Myeloma, Kuwait Experience

Background Multiple myeloma MM is an incurable plasma cell malignancy with a median age of 65 years at diagnosis worldwide, a small number of publications were focused on young adult Multiple myeloma MM with special characteristics and clinical outcomes. Objectives To evaluate the incidence of MM among adolescent and young adults (AYA) in Kuwait aged 40 and below. Also, to describe the clinical characteristics, high risk features and treatment outcomes at the Kuwait Cancer Control Centre (KCCC). Materials &amp; Methods We collected data retrospectively from the hematological malignancy registry at KCCC and looked at AYA patients (pts) with a confirmed diagnosis of Multiple myeloma at our center from 2015 till 2022, we looked at age of onset, gender, nationality, year of myeloma diagnosis, presence of extramedullary disease, International Staging System score (ISS score), cytogenetic aberrations, induction therapy, receipts of auto-transplant and maintenance therapy. Results From 2015 till 2022, 340 patients were diagnosed with MM in Kuwait, eleven patients were found 40 years old or younger, only one was female patients, the median age was 35 years old, around a third were Kuwaiti, 73% were non-Kuwaiti of middle eastern background, 4 patients were found to have extramedullary disease at diagnosis, with advanced stage ISS III found in 3/9 pts. One patient was found with 1Q gain and the rest of the 10 patients were with negative del17p, t (4,14), t(14,16), gain1q and t(11,14). Of note, interphase FISH cytogenetic is not done with CD138 selection. Six patients were treated with RVD induction (Lenalidomide, Bortizomib, Dexamethasone), two patients treated by CTD (Cyclophosphamide, Thalidomide, Dexamethasone), one pt treated by VCD (Bortizomib,Cyclophosphamide,Dexamethasone), one patient treated by Daratumumab RVD, and one patient treated by VTD. Overall response rate (ORR) was 90.9%, (10/11) responded patients proceeded to HDCT/ASCT and all these patients went on maintenance post-transplant, two patients were lost to follow up while 9 patients still under follow up, median follow up 33 months, one pt had a relapse after transplant. Conclusion MM is an uncommon disease among AYA in Kuwait (3.2%) of all myeloma patients, with favorable durable responses in this group of patients.

Role of Cytogenetics and Fluorescence In Situ Hybridization in the Laboratory Workup of Acute Myeloid Leukemias

AbstractA new understanding of acute myeloid leukemia as a varied group of unique biologic entity has emerged, as a result of the identification of various chromosomal aberrations and their association with clinical prognosis and diagnosis. Following induction treatment, cytogenetic examination can establish the presence of any residual malignant cells, it's recurrence, clonal evolution if any, or the formation of novel abnormalities. The G-banded karyotype has been the gold standard method for detecting all of these aberrations for years. The capacity to examine the entire genome through karyotype analysis quickly enabled the detection of deletions, duplications, and structural rearrangements across every chromosome, and the more frequent ones were associated with particular aberrant clinical symptoms. Fluorescence in situ hybridization (FISH) is a sensitive technology that aids in differential diagnosis or therapeutic planning and provides rapid results. Furthermore, the combination of cytogenetic and molecular profiling enables a more precise evaluation of disease prognosis, diagnosis, classification, risk stratification, and patient treatment. Interphase FISH analysis, in conjunction with G-banded chromosomal analysis, can be used as a major testing tool for the evaluation of hematological neoplasms. For accurate and consistent descriptions of genomic changes identified by karyotyping and FISH, a specified terminology is necessary. The International System for Human Cytogenomic Nomenclature is the main source and provides instructions for documenting cytogenetic and molecular findings in laboratory reports. This review discusses the two methods, karyotyping and FISH, their advantages and limitations, sample requirements, various FISH probes that are used, nomenclature for results reporting, and the necessary quality control measures.

Impact of 1q21 Gain and Amplification on Daratumumab-Treated Multiple Myeloma Patients: Real-World Data from the Australia-New Zealand and Asia-Pacific Myeloma and Related Diseases Registries

Introduction: Gain/amplification of 1q21 (1q21+) is found in 40% of patients (pts) with newly diagnosed Multiple Myeloma (MM). There is increasing evidence that presence of gain of 1q21 [gain(1q21), 3 copies] or amplification of 1q21 [amp(1q21), ≥4 copies] are independent poor prognostic factors. A recent post-hoc analysis of the ICARIA-MM and IKEMA studies showed that the addition of monoclonal anti-CD38 antibody isatuximab in combination with pomalidomide-dexamethasone or carfilzomib-dexamethasone abrogated the negative impact of 1q21 on progression-free survival (PFS). However, the impact of 1q21+ has not been consistently assessed in daratumumab (Dara) exposed pts. Methods: We identified MM pts treated with Dara between Jan 2020 and Jan 2023 from the Australia-New Zealand and Asia-Pacific Myeloma and Related Diseases Registries. Pts who had interphase fluorescence in situ hybridization (FISH) testing were included. High-risk cytogenetics (HRCyto) was defined as the presence of t(4;14), t(14;16) or del(17p) by FISH, and standard risk cytogenetics (SRCyto) as pts without 1q21+ and HRCyto. Pt groups analysed were: (1) without 1q21+ vs with 1q21+ (2) SRCyto vs isolated 1q21+ vs HRCyto without 1q21+ vs HRCyto with 1q21+. Subgroup analyses performed include: (3) gain(1q21) vs amp(1q21). Overall survival (OS) and PFS from commencement of Dara were estimated using the Kaplan-Meier method. Comparisons of PFS and OS were performed using the log-rank test. Categorical data were compared using a chi-square test and continuous variables with a rank-sum test. Results: Of 654 pts who received Dara, 451 had interphase FISH testing performed and were included. There were 132 pts with 1q21+ [gain(1q): 23, amp(1q):14, gain/amp status unknown: 95] and 319 without. Median follow up was 15 months (95% CI 14-18). Median prior lines of therapy was 1 (IQR 1-2). Dara-based regimens used were: Dara monotherapy (17.7%), Dara-bortezomib-dex (54.6%), Dara-lenalidomide-dex (11.3%), Dara-pomalidomide-dex (DPd) (9.3%), Dara-carfilzomib-dex (DKd) (2.5%) and Dara-bortezomib-lenalidomide-dex (4.6%). There were no differences in age, gender, ECOG, line of therapy in which Dara was used, or rate of autologous stem cell transplantation between pts with 1q+ vs pts without (Table 1). Pts with 1q21+ had higher rates of other HRCyto (37.1% vs 20.4%; p=0.005) and ISS Stage 3 disease (44.1% vs 29.3%; p=0.005) vs those without 1q21+. There was no significant difference in overall response rate (65.9% vs 71.6%; p=0.32). Through univariate analyses, PFS for 1q21+ pts was significantly shorter than those without 1q21+ [median PFS 8.2 vs 18.4 months (HR 1.7, 95% CI 1.3-2.3; p&amp;lt;0.001)] (Figure 1). This difference was still significant when adjusted for ISS, the presence of other HRCyto and country of treatment (HR 1.61, 95% CI 1.17-2.22; p=0.004). Pts with 1q21+ with or without HRCyto, and those with HRCyto and no 1q21+ had inferior PFS vs SRCtyo (median PFS 7.7 vs 8.9 vs 8.5 vs 30.3 months; p&amp;lt;0.001) (Figure 2). Of the pts where 1q21+ copy number was available, OS was significantly poorer in pts with amp(1q) vs those with gain(1q) (median OS 22.6 vs 46.9 months; p=0.035). There was also a trend towards poorer PFS in pts with amp(1q) (median PFS 4.9 vs 10.5 months; p=0.096) Conclusions: 1q21+ often co-exists with other HRCyto abnormalities. However, it remains an independent negative prognostic marker. The use of Dara does not appear to abrogate the negative impact of 1q21+ on PFS. Amp(1q21) potentially confers an even poorer outcome. This finding differs from an earlier retrospective study (Parrondo et al, Blood 2022), although &amp;lt;10% of patients in our study received DKd and DPd combinations. As such, more data on Dara in combination with carfilzomib and pomalidomide is needed.

Role of Interphase FISH Assay on Air-Dried Smears in Identifying Specific Structural Chromosomal Abnormalities among Pediatric Patients with Acute Leukemias.

The online version contains supplementary material available at 10.1007/s12288-023-01699-2.

Fluorescence in situ hybridisation for interphase chromosomal aberration-based biological dosimetry.

Metaphase spreads stained with Giemsa or painted with chromosome-specific probes by fluorescence in situ hybridisation (FISH) have been in use since long for retrospective dose assessment (biological dosimetry). However, in cases of accidental exposure to ionising radiation, the culturing of lymphocytes to obtain metaphase chromosomes and analysis of chromosomal aberrations is time-consuming and problematic after high radiation doses. Similarly, analysing chromosomal damage in G0/G1 cells or nondividing cells by premature chromosome condensation is laborious. Following large-scale radiological emergencies, the time required for analysis is more important than precision of dose estimate. Painting of whole chromosomes using chromosome-specific probes in interphase nuclei by the FISH technique will eliminate the time required for cell culture and allow a fast dose estimate, provided that a meaningful dose-response can be obtained by scoring the number of chromosomal domains visible in interphase nuclei. In order to test the applicability of interphase FISH for quick biological dosimetry, whole blood from a healthy donor was irradiated with 8Gy of gamma radiation. Irradiated whole blood was kept for 2h at 37°C to allow DNA repair and thereafter processed for FISH with probes specific for Chromosomes-1 and 2. Damaged chromosomal fragments, distinguished by extra color domains, were observed in interphase nuclei of lymphocytes irradiated with 8Gy. These fragments were efficiently detected and quantified by the FISH technique utilising both confocal and single plane fluorescence microscopy. Furthermore, a clear dose-response curve for interphase fragments was achieved following exposure to 0, 1, 2, 4 and 8Gy of gamma radiation. These results demonstrate interphase FISH as a promising test for biodosimetry and for studying cytogenetic effects of radiation in nondividing cells.

17p Deletion in Patients with De Novo Acute Myeloid Leukemia and their Clinico-Haematologic Features

Objective: To determine the frequency of 17p deletion in patients with Acute Myeloid Leukemia and their clinical-hematologic features using fluorescence in situ hybridization.&#x0D; Study Design: Cross-sectional study.&#x0D; Place &amp; Duration of Study: Department of Hematology, Armed Forces Institute of Pathology, Rawalpindi Pakistan, from Dec 2018 to Dec 2019.&#x0D; Methodology: All diagnosed cases of Acute Myeloid Leukemia of all ages and genders were included. Interphase FISH testing was performed using blood or bone marrow specimens using 10μL of the Meta systems XL p53 probe, and a total of 500 nuclei per assay were analyzed using a fluorescent microscope. Del 17p positivity and negativity were noted. Clinico-haematologic features of the patients with and without del 17p were also noted.&#x0D; Results: In our targeted population of 84 patients, there were 25(29.8%) females and 59(70.2%) males. The mean age of presentation was 36.3±1.6 years. The mean total leucocyte count(TLC)was 31.2±4.2×109/L, and the mean platelet count was 69.3±4.5×109/L. Del 17p was detected in a total of 8(9.5%) patients. The median age of patients with del 17p at diagnosis was 33 years, the mean TLC was 12.32±10.36x109/L, and the mean platelet count was 80±26.73*109/L. Prominent clinical features among patients with del 17p included fever and pallor.&#x0D; Conclusion: Our study suggests a relatively low frequency of del 17p in AML (9.5%), which is consistent with international data.

Frequency of PDGFRA, PDGFRB and FGFR1 Gene Rearrangements in Patients with Eosinophilia and Their Clinico-Haematologic Parameters

Objective: To determine the frequency and clinico-haematological features of PDGFRA, PDGFRB and FGFR1 gene rearrangements in patients with persistent Eosinophilia using Fluorescence in situ hybridization.&#x0D; Study Design: Cross-sectional study.&#x0D; Place and Duration of Study: Department of Hematology, Armed Forces Institute of Pathology, Rawalpindi Pakistan, from Dec 2018 to Dec 2019.&#x0D; Methodology: All Patients who presented to AFIP having absolute eosinophil count &gt;1.5x109/L persistent for over six months or with Myeloid or Lymphoid neoplasms with persistent Eosinophilia were studied. Patients having reactive Eosinophilia and those on treatment were excluded. Interphase FISH studies were performed. In addition, 2.5ml of sodium heparin blood was taken. After the denaturation of DNA, slides were set up according to standard protocol. FIP1L1/CHIC2/PDGFRA dual colour probe was applied for PDGFRA, 5q32 PDGFRA break apart probe for PDGFRB and XL FGFR1 break apart probe for FGFR1 gene rearrangement.&#x0D; Results: A total of 60 patients were included in the study. Of these, 50(83.3%) were males, and 10(16.7%) were females, with an average absolute Eosinophilia count of 5.92±7.10x109/L. The only rearrangement detected in patients with Eosinophilia was FIPILI-PDGFRA gene fusion, detected in 20% of the patients. No other rearrangement was found.&#x0D; Conclusion: PDGFRA, PDGFRB and FGFR1 mutations are rare yet most prominent in patients with clonal Eosinophilia. About 80% of eosinophilic patients were found to have idiopathic Eosinophilia, which requires further consideration to address the disease prevalence.

Correlation between conventional karyotype, interphase fish and immunophenotype in risk stratification of newly diagnosed plasma cell myeloma: An observational study

BackgroundPlasma cell myeloma (PCM) is a common adult hematological neoplasm of terminally differentiated B-cells resulting in accumulation of monoclonal plasma cells. PCM is heterogeneous disease with survival time varies from months to years, determined by age, stage, cytogenetics abnormalities, and treatment response. There is conflicting evidence in role of immunophenotype as a prognostic indicator. This study identified frequency of cytogenetics abnormalities and established correlation between cytogenetics and immunophenotyping in risk stratification in newly diagnosed PCM. MethodsPilot study was undertaken on 30 newly diagnosed PCM over 4 years. Apart from extensive myeloma laboratory and imaging profile, multicolor flow cytometry using panel of CD138, CD38, CD45, CD56, CD19, CD20, CD28, CD200, CD117, κ, and λ light-chains. Karyotyping was done using GTG banding and interphase fluorescence in situ hybridization analysis on magnetically sorted plasma cells for t(4;14), t(11;14), del(13)q, and del(17)p probes. ResultsOf 30 myeloma patients, 16(53.3%) were men with mean age 57.23 ± 11.90 years. The del(13)q was commonest cytogenetics abnormality. CD56-positive cases were strongly associated with presence of del(17)p and stage III of disease (p < 0.05). Negative expression of CD117 was seen in advanced stage of disease and high-risk genetic factor, t(4;14) (p = 0.049). Absent CD28 expression was associated with higher albumin levels, lower levels of β2MG, and lack of high-risk genetic abnormalities. Advanced clinical stages were associated with expression of CD56, CD28, and absent expression of CD45 and CD117. ConclusionImmunophenotypic antigens positive and negative expressions are associated with high-risk genetic abnormalities and advanced clinical stages of PCM. Institution ethic committee reference numberIEC/2017/230, AFMC, Pune.

Parallel deletion and duplication at 7q11.23 in a silent carrier for two reciprocal syndromic disorders

Partial deletions at chromosome 7q11.23 are causative for the autosomal-dominant Williams-Beuren syndrome (WBS), whereas the partial duplication of this region leads to the 7q11.23 duplication syndrome. Both syndromes are highly penetrant and occur with a frequency of 1:7500-10,000 (WBS) and 1:13,000-20,000 (7q11.23 duplication syndrome). They are associated with multiple organ defects, intellectual disability, and typical facial dysmorphisms showing broad phenotypic variability. The 7q11.23 region is susceptible to chromosomal rearrangements due to flanking segmental duplications and regions of long repetitive DNA segments. Here, we report on a family with two children affected by WBS and clinically unaffected parents. Interestingly, metaphase fluorescence in situ hybridization (FISH) revealed a deletion on 7q11.23 in the father. Intensive genetic testing, using interphase FISH, whole genome sequencing and optical genome mapping led to the confirmation of a 1.5 Mb deletion at one 7q11.23 allele and the identification of a reciprocal 1.8 Mb duplication at the other allele. This finding is highly important regarding genetic counseling in this family. The father is a silent carrier for two syndromic disorders, thus his risk to transmit a disease-causing allele is 100%. To the best of our knowledge we, here, report on the first case in which the phenotype of a microdeletion/microduplication syndrome was compensated by its reciprocal counterpart.