Dual-fusion Fluorescence Research Articles

High-grade B-cell lymphoma with a quadruple-hit genetic profile including concurrent MYC, BCL2, BCL6, and CCND1 gene rearrangements.

Several reports of concurrent MYC, BCL2, BCL6, and CCND1 rearrangements in high-grade B-cell lymphoma (HGBL) have been recently described. Herein, we aimed to delineate the scope of this entity through a review of HGBL with a "quadruple-hit" genetic profile identified at our institution. We performed a retrospective review (2015-2023) at our institution of B-cell lymphoma (BCL) cases that were evaluated with concurrent MYC, BCL2, and BCL6 break-apart and IGH::MYC and IGH::CCND1 dual-color dual-fusion fluorescence in situ hybridization studies. Of 203 cases meeting inclusion criteria, 2 (1%) with a quadruple-hit genetic profile were identified. Case 1 represented a 59-year-old female with widespread lymphadenopathy and a diagnosis of HGBL who exhibited primary refractoriness to dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (DA-EPOCH-R) chemotherapy. Case 2 represented a 58-year-old male with mediastinal and abdominal lymphadenopathy and a diagnosis of large BCL who died from disease after 1 cycle of DA-EPOCH-R chemotherapy. Similarly, a literature review of 7 previously reported cases of HGBL with a quadruple-hit profile also demonstrated aggressive disease behavior. Our study adds 2 new cases to the rarely encountered quadruple-hit HGBL, and a brief meta-analysis of the 9 available cases indicates aggressive disease behavior conferred by this constellation of genetic events.

Typical, atypical and cryptic t(15;17)(q24;q21) (PML::RARA) observed in acute promyelocytic leukemia: A retrospective review of 831 patients with concurrent chromosome and PML::RARA dual-color dual-fusion FISH studies.

The diagnosis of acute promyelocytic leukemia (APL) relies on the identification of PML::RARA fusion. While the majority of APL cases harbor a typical t(15;17)(q24;q21), atypical genetic mechanisms leading to the oncogenic PML::RARA fusion have been reported yet their frequency and scope remain poorly characterized. We assessed the genetic findings of 831 cases with APL investigated with concurrent chromosome banding analysis and dual-color dual-fusion fluorescence in situ hybridization (D-FISH) analysis at our institution over an 18.5-year timeframe. Seven hundred twenty-three (87%) cases had a typical balanced t(15;17) with both testing modalities. Atypical karyotypic results including complex translocations, unbalanced rearrangements and insertional events occurred in 50 (6%) cases, while 6 (0.7%) cases were cryptic by conventional chromosome studies despite PML::RARA fusion by D-FISH evaluation. Atypical FISH patterns were observed in 48 (6%) cases despite apparently balanced t(15;17) on chromosome banding analysis. Two hundred fifty (30%) cases displayed additional chromosome abnormalities of which trisomy/tetrasomy 8 (37%), del(7q)/add(7q) (12%), and del(9q) (7%) were most frequent. Complex and very complex karyotypes were observed in 81 (10%) and 34 (4%) cases, respectively. In addition, 4 (0.5%) cases presented as an apparently doubled, near-tetraploid stemline clone. This report provides the largest appraisal of cytogenetic findings in APL with conventional chromosome and PML::RARA D-FISH analysis. By characterizing the frequency and breadth of typical and atypical results through the lens of these cytogenetic testing modalities, this study serves as a pragmatic source of information for those involved in the investigation of APL in both the clinical and research laboratory settings.

The suitability of NONO-TFE3 dual-fusion FISH assay as a diagnostic tool for NONO-TFE3 renal cell carcinoma

NONO-TFE3 RCC is a subtype of Xp11.2 translocation renal cell carcinoma (RCC). So far, only a small amount of NONO-TFE3 RCC have been reported owing to lack of effective diagnosis methods. Utilizing the novel dual-fusion fluorescence in situ hybridization (FISH) probe reported here, 5 cases of NONO-TFE3 RCC were identified and were ultimately confirmed by RT-PCR. Histopathology, all 5 cases were consisted by sheets of epithelial cells and papillary architecture. The cytoplasm was abundantly clear, and nucleoli was not prominent. Besides, the nuclear palisading, subnuclear vacuoles and psammoma bodies were identified. The most distinctive features were strong positive TFE3 staining but equivocal split signals of the TFE3 probe, which might lead to the misdiagnosis of Xp11.2 translocation RCC. The median age and median tumor size of the five patients were 41.2 years and 3.6 cm, respectively. A median following follow-up of 27 months showed moderate disease progression and prognosis in NONO-TFE3 RCC patients. In conclusion, the present study demonstrates the effectiveness and reliability of the NONO-TFE3 dual-fusion FISH probe for diagnosing NONO-TFE3 RCC. Suspected cases of Xp11.2 translocation RCC showing biphasic pattern, strong positive TFE3 staining, and equivocal split signals in the TFE3 FISH assay indicated a possibility of NONO-TFE3 RCC.

Characterization of TCF3 rearrangements in pediatric B-lymphoblastic leukemia/lymphoma by mate-pair sequencing (MPseq) identifies complex genomic rearrangements and a novel TCF3/TEF gene fusion

The TCF3/PBX1 gene fusion is a recurrent genetic abnormality in pediatric B-lymphoblastic leukemia/lymphoma (B-ALL/LBL). While dual-color, dual-fusion fluorescence in situ hybridization (D-FISH) probes can detect TCF3/PBX1 fusions, further characterization of atypical TCF3 FISH patterns as indicated by additional or diminished TCF3 signals is currently limited. Herein we describe the use of a next-generation sequencing assay, mate-pair sequencing (MPseq), to characterize typical and cryptic TCF3/PBX1 fusions and to identify TCF3 translocation partners based on results obtained from our laboratory-developed TCF3/PBX1 D-FISH probe set. MPseq was performed on 21 cases of pediatric B-ALL/LBL with either TCF3/PBX1 fusion, or no TCF3/PBX1 fusion but with additional or diminished TCF3 signals obtained by our PBX1/TCF3 D-FISH probe set. In addition, MPseq was performed on one pediatric B-ALL/LBL case with an apparently normal karyotype and abnormal TCF3 break-apart probe results. Of 22 specimens successfully evaluated by MPseq, 13 cases (59%) demonstrated TCF3/PBX1 fusion, including three cases with previously undescribed insertional rearrangements. The remaining nine cases (41%) harbored various TCF3 partners, including six cases with TCF3/ZNF384, and one case each with TCF3/HLF, TCF3/FLI1 and TCF3/TEF. Our results illustrate the power of MPseq to characterize TCF3 rearrangements with increased precision and accuracy over traditional cytogenetic methodologies.

Acute leukemias harboring KMT2A/MLLT10 fusion: a 10-year experience from a single genomics laboratory.

The MLLT10 (formerly AF10) gene is the fourth most common KMT2A fusion partner across all acute leukemias and requires at least 3 breaks to form an in-frame KMT2A/MLLT10 fusion due to the opposite orientation of each gene. A 10-year retrospective review was performed to identify individuals from all age groups that harbor KMT2A/MLLT10 fusion obtained by our KMT2A/MLLT10 dual-color dual-fusion fluorescence in situ hybridization (D-FISH) assay. Of the 60 unique individuals identified, 31 were male and 29 were female (M:F ratio, 1.1:1) with ages ranging from 3 days to 86 years (mean 21.5 years, median 5.5 years). The diagnoses included acute myeloid leukemia (AML) (49 patients, 82%), B- or T-lymphoblastic leukemia/lymphoma (7 patients, 12%), myeloid sarcoma (3 patients, 5%), and a single case (2%) of undifferentiated leukemia. Twenty-seven of 49 patients (55%) with AML were in the infant or pediatric age group. Fifty-three of 60 patients (88%) had KMT2A/MLLT10 D-FISH signal patterns mostly consisting of single fusions. In addition, 10 (26%) of 38 patients with conventional chromosome studies had "normal" (5 patients) or abnormal (5 patients) chromosome studies that lacked structural or numeric abnormalities involving chromosomes 10 or 11, implying cryptic cytogenetic mechanisms for KMT2A/MLLT10 fusion. Lastly, mate-pair sequencing was performed on 4 AML cases, 2 of which had "normal" chromosome studies and cryptic KMT2A/MLLT10 fusion as detected by KMT2A/MLLT10 D-FISH studies, and verified the multiple breaks required to generate KMT2A/MLLT10 fusion.

PRCC-TFE3 dual-fusion FISH assay: A new method for identifying PRCC-TFE3 renal cell carcinoma in paraffin-embedded tissue.

PRCC-TFE3 renal cell carcinoma (RCC) is one of the most common types of Xp11.2 translocation renal cell carcinoma (tRCC), of which the diagnosis mainly relies on reverse transcription-polymerase chain reaction (RT-PCR) or chromosomal analysis in fresh frozen samples. Herein, we developed a new dual-fusion fluorescence in situ hybridization (FISH) probe to succinctly identify PRCC-TFE3 RCC in paraffin-embedded tissue. We immunohistochemically analyzed TFE3 and cathepsin K expression in 23 cases of Xp11.2 tRCC which had been confirmed by break-apart TFE3 FISH probe. Next, the dual-fusion FISH assay was performed on these selected cases. Twenty typical cases of clear renal cell carcinoma and 20 cases of papillary renal cell carcinoma were collected as control groups. Seven cases were finally confirmed as PRCC-TFE3 RCC by FISH detection, emerging dual-fusion signals, of which 2 cases were identified as PRCC-TFE3 RCC by RT-PCR previously. All remaining cases were negative for the PRCC-TFE3 rearrangement by FISH. The TFE3 immunohistochemistry was positive in 22/23 cases and the cathepsin K was positive in 16/23 cases. All 7 PRCC-TFE3 RCCs showed positive cathepsin K immunoreactivity. Our results reveal that PRCC-TFE3 dual-fusion FISH probe is an efficient and concise technique for diagnosing PRCC-TFE3 RCC in paraffin-embedded tissue.

Signal Patterns of Dual Color Dual Fusion Fluorescence in Situ Hybridization for Detection of Genetic Abnormality in Adult Patients with ALL and Their Clinical Application

To study the signal patterns of dual color dual fusion fluorescence in situ hybridization (DCDF-FISH) for detection of genetic abnormality in adult acute lymphoblastic leukemia (ALL) patients and their diagnostic value and clinical application. The clinical data of 68 ALL patients confirmed in our hospital were analyzed retrospectively; The bone marrow samples were detected by DCDF-FISH, flow cytometry, conventional cytogenetics (CCG), reverse transcriptase polymerase chain reaction (RT-PCR), and the correlation of these results was compared. And the reaction of patients to treatment was dynamically observed by DCDF-FISH. Sixteen signal patterns were found in DCDF-FISH, including 14 kinds of atypical signal patterns (signal patterns of 1R2G, 2R3G, 2R4G and 3R3G as abnormal signal patterns without BCR/ABL fusion gene. Signal patterns of 1R1G1F, 1R1G3F, 1R1G4F, 1R2G1F, 1R2G2F, 1R2G3F, 1RnG2F (n ≥ 3), 2R2G1F, 1G4F, 1R4F corresponded to t (9;22) karyotype). Ph(+) ALL patients accounted for 17. All cases with Ph chromosome or BCR/ABL positive were B-ALL or My(+)-B-ALL. The Ph chromosome was detected in 12 cases (positive rate was 18%) by CCG. The positive rate was 25% (17/68) by DCDF-FISH and RT-PCR. The DCDF-FISH fluorescence pattern change before and after chemotherapy of the patients showed that the quantity and form of the signal pattern was changed after chemotherapy, and the common characteristics was the Ph chromosome in patients. The DCDF-FISH is a sensitive and reliable method for the detection of BCR/ABL rearrangement. Analyzing the dynamical change of DCDF-FISH signal patterns has been comfirmed to have a important guiding significance in the diagnosis, and anlysis of response to therapy, drug resistance and the prognosis of ALL patients.

Development of an NPM1/MLF1 D-FISH Probe Set for the Detection of t(3;5)(q25;q35) Identified in Patients with Acute Myeloid Leukemia

The t(3;5)(q25;q35) NPM1/MLF1 fusion has an incidence of approximately 0.5% in acute myeloid leukemia (AML) and has an intermediate prognosis at diagnosis. We have developed a dual-color, dual-fusion fluorescence in situ hybridization (D-FISH) assay to detect fusion of the MLF1 and NPM1 genes. A blinded investigation was performed using 25 normal bone marrow specimens and 26 bone marrow samples from patients with one or more metaphases with a t(3;5)(q21-q25;q31-q35) or a der(5)t(3;5)(q21-q25;q31-q35) previously identified by chromosome analysis. Once unblinded, the results indicate our D-FISH method identified NPM1/MLF1 fusion in 15 of the 26 fully evaluated patient samples. Excluding three samples with a single abnormal t(3;5) metaphase, 15 of 17 (88%) patient samples with a balanced t(3;5) demonstrated NPM1/MLF1 fusion, and 0 of 6 patient samples with a der(5)t(3;5) demonstrated NPM1/MLF1 fusion, suggesting only the balanced form of this 3;5 translocation as observed by karyotype is associated with NPM1/MLF1 fusion. Overall, the FISH results demonstrated five different outcomes (NPM1/MLF1 fusion, MLF1 disruption, MLF1 duplication, NPM1 deletion, and normal), indicating significant molecular heterogeneity when the 3;5 translocation is identified. The development of this sensitive D-FISH strategy for the detection of NPM1/MLF1 fusion adds to the AML FISH testing repertoire and is effective in the detection of this translocation at diagnosis as well as monitoring residual disease in AML patients.

Complex/variant translocations in chronic myelogenous leukemia (CML): genesis and prognosis with 4 new cases

In 5–10% of cases with CML, variant or complex translocations (CT) are seen that may result in atypical fluorescence in situ hybridization signal patterns. Dual color, dual fusion fluorescence in situ hybridization (D-FISH) patterns are instrumental in identifying the genesis of these CT, but their prognostic implications remain controversial. The most common mechanism is a two-step process in which a standard two-way translocation (9;22) is followed by subsequent rearrangements involving other chromosomes. The second common mechanism is the one-step process wherein breakage occurs simultaneously on different chromosomes leading to CT. The typical D-FISH pattern seen with the one-step mechanism is 1F2G2R, while the pattern for the two-step mechanism can be variable (2F1G1R, 1F1G1R, 1F1G2R, 1F2G1R, etc.). We have studied 4 cases of CT using metaphase FISH with triple color, dual fusion ASS1, ABL1 and BCR probes to understand the genesis of these CT. All the patients were treated with imatinib, but only patients 3 and 4 showed remission. Our results indicate that the CT in cases 1, 3 and 4 arose from a one-step mechanism and case 2 from a multi-step mechanism. Response to imatinib varied from full remission to no response. Long term follow-up is necessary to evaluate the prognostic implications of these CT.

Early monitoring drug-resistance of patients with BCR/ABL(+) ALL by DCDF-FISH

This study was aimed to investigate the application value of the dual color dual fusion fluorescence in situ hybridization (DCDF-FISH) in BCR/ABL (+) acute lymphoblastic leukemia patients with complex chromosomal translocation. The clinical presentations of a patient with ALL were monitored regularly by bone marrow cell morphology test, chromosome analysis, flow cytometry and DCDF-FISH technique, and the reaction of patients to treatment and disease progression were dynamically observed by DCDF-FISH. The results indicated that the patient showed the typical presentation of B lineage acute lymphoblastic leukemia (B-ALL) with expression of CD10, CD19 and CD34; the chromosome analysis showed 46,XY, i(8), ider(9)t (9; 22) [23]/47, idem, +der(22) t (9;22) [7] karyotype in the bone marrow cells, FISH showed that 83% cells contained BCR/ABL fusion gene in the patient's bone marrow, among which 5% cells showed 1R1G2F signalling model, 14% cells showed 1R1G3F, and 64% cells showed 1R1G4F. The patient got complete remission when the imatinib chemotherapy combined with VTLP was carried out, and the tumor cells decreased to 19%, but the cells with 1R1G2F signal model increased to 18%. The 1R1G2F cell signal model increased up to 38% when patient relapsed. The patient died of the drug-resistance. It is concluded that the BCR/ABL (+) leukemia patient with complex translocation has multiple tumor cell subsets, and the responses of different cell subsets to the treatment are different, therefore the response to therapy and drug resistance of patient can be monitored early by the signal model of DCDF-FISH and the observation of dynamical changes of different cell subset.

Detection of cryptic and variant IGH-MYC rearrangements in high-grade non-Hodgkin's lymphoma by fluorescence in situ hybridization: implications for cytogenetic testing

In recent years it has become increasingly evident that MYC rearrangements are not confined to classical Burkitt lymphoma (BL), but also occur in diffuse large B-cell lymphoma (DLBCL) and in the new subtype, “B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL” (BCLU), which was recently described in the 2008 revision of the World Health Organization classification. The accurate identification of MYC rearrangements in these three subtypes of high-grade lymphoma is becoming increasingly critical both in terms of diagnosis of classical BL and in light of the prognostic implications in cases of DLBCL and BCLU. We describe three cases of high-grade lymphoma in which cryptic insertion events, resulting in clinically significant IGH-MYC rearrangements, were detectable using an IGH/MYC three-color, dual-fusion fluorescence in situ hybridization (FISH) probe set, but were not detected using break-apart MYC FISH probes, thus highlighting the limitations of using break-apart probes as a stand-alone test, particularly with the increased use of interphase FISH analysis of formalin-fixed, paraffin-embedded tissue sections in the diagnostic work-up of these patients.

Evaluation of the PAX8/PPARG Translocation in Follicular Thyroid Cancer with a 4-Color Reverse-Transcription PCR Assay and Automated High-Resolution Fragment Analysis

Molecular testing of thyroid malignancies, in combination with cytologic and histologic examination, is becoming increasingly attractive as a tool for refining traditional morphologic diagnosis. The molecular changes associated with follicular thyroid carcinoma (FTC) are point mutations in RAS oncogenes or the presence of PAX8/PPARG (paired box 8/peroxisome proliferator-activated receptor gamma) rearrangement. We developed and validated a clinical assay for the detection of PAX8/PPARG rearrangements that uses a 4-color reverse-transcription PCR (RT-PCR) assay and high-resolution fragment analysis. The RT-PCR assay is applicable for detecting the various described fusion transcripts of PAX8/PPARG in formalin-fixed, paraffin-embedded thyroid tissue and in fine-needle aspirate biopsy washes from thyroid nodules. The analytical sensitivity of the assay is 1 abnormal cell in a background of 100-10 000 translocation-negative cells. A comparison of the RT-PCR assay with dual-fusion fluorescence in situ hybridization showed an overall concordance of 95%. With this assay, we obtained a prevalence for the PAX8/PPARG rearrangement in FTC of 62% (13 of 21 cases), compared with a 5% prevalence (3 of 55) for other follicular cell-derived neoplasms. The introduction of this assay into clinical practice could provide useful information for the diagnosis and possibly for the prognosis and treatment of thyroid cancer in the future.

Application of tri-colour, dual fusion fluorescence in situ hybridization (FISH) system for the characterization of BCR-ABL1 fusion in chronic myelogenous leukaemia (CML) and residual disease monitoring

BackgroundWe studied the application of the BCR-ABL1 + 9q34 tri-colour dual fusion fluorescence in situ hybridization (FISH) system in the characterization of fusion signal pattern and the monitoring of residual disease in chronic myelogenous leukaemia (CML). The signal constellation on metaphases with the tri-colour dual fusion system was defined. The knowledge of various signal patterns obtained from the different genetic rearrangements was further applied to the analysis of hybridization signals on interphase nuclei.MethodsBCR-ABL1 dual colour, dual fusion FISH (D-FISH) was performed on diagnostic samples of 22 CML patients. The tri-colour FISH system was performed on cases that showed aberrant signal patterns other than the classical 1 green (G) 1 orange (O) 2 fusions (F). Using the aqua band-pass filter, random signal overlap in interphase nuclei would be indicated by the presence of an aqua signal (ASS1), while genuine fusion was represented by the absence of the ASS1 signal.ResultsUsing the D-FISH system, the signal patterns could be categorized into 4 groups: group 1 (n = 17) showed the classical 1G1O2F; group 2 (n = 2) showed 2G1O1F indicating ABL1 deletion; group 3 (n = 1) showed 1G2O1F indicating BCR deletion; group 4 (n = 2) with 1G1O1F indicating reciprocal ABL1-BCR deletion. The tri-colour dual fusion system correlated with the D-FISH system for cases with der(9) deletion. The added aqua-labelled ASS1 probe was useful in differentiating random signal overlap from genuine BCR-ABL1 fusion in the interphase cells (group 4).ConclusionAlthough the D-FISH probe was valuable in establishing the different patterns of aberrant signals and monitoring patients with the classic 2-fusion signals in CML, the tri-colour dual fusion probe should be used for patients with der(9) deletion to monitor response to treatment.

Detection of the Philadelphia chromosome in adult B-lineage acute lymphoblastic leukemia using dual-color dual-fusion interphase fluorescence in situ hybridization

To investigate the incidence of Philadelphia chromosome (Ph) in adult B-lineage acute lymphoblastic leukemia (B-ALL). One hundred and twelve adult patients with previously untreated B-ALL were prospectively investigated by interphase dual-color dual-fusion fluorescence in situ hybridization (DD-FISH) with two-color break apart probe BCR-ABL and the results were compared with that of conventional cytogenetics (CC). The incidence of Ph chromosome was 17.98% (16/89) and 31.25% (35/112) by CC and DD-FISH, respectively. The mean positive rate of Ph+cells by FISH was 66.23% (ranging 18.5%-99%). Of the 35 Ph+ALL patients by FISH, 25 were successfully karyotyped by CC which included 5 normal karyotypes, 20 abnormal karyotypes including 16 Ph chromosome and 13 complex abnormalities. The incidence of Ph chromosome was 31.25% in adult with B-ALL. DD-FISH with BCR-ABL probe provides a powerful technique for the diagnosis of Ph+B-ALL. It is an important supplement to the CC analysis. DD-FISH technique should be used as a routine method for the diagnosis for adult acute B-ALL.

A dual colour dual fusion fluorescence in situ hybridisation study on the genesis of complex variant translocations in chronic myelogenous leukaemia

Complex variant 9;22 translocations occur in a significant minority of chronic myelogenous leukaemia (CML) patients. Different mechanisms of their formation have been described. We report dual colour dual fusion fluorescence in situ hybridisation data in 12 Chinese CML patients with complex translocations. Three previously reported breakpoint hotspots in a third partner chromosome (14q32, 17q25, 1q21) were observed. In 10/12 (83.3%) patients, the abnormality occurred as a single step 3-break event. Only a single abnormal clone harbouring the complex translocation was seen in this group. The remaining 2 cases in the chronic phase showed a 4-break mechanism (2/12, 16.7%). Deletion of 5' ABL at der(9) was not observed in any of the 12 patients, however, the loss of 3' BCR was observed in 1 patient (1/12, 8.3%). Together with previous findings, these data suggest that these variant translocations occur more often as a 3-break single-step process with no reciprocal ABL-BCR fusion. On the other hand, a 4-break event is also regularly seen during the initial stages of leukaemogenesis, which likely predisposes to der(9) deletion. The observed difference in rates of der(9) deletion reported in a series of CML patients with variant translocations may be related to a difference in rates of a 4-break event.

Detection of FOXO1 (FKHR) Gene Break-apart by Fluorescence In Situ Hybridization in Formalin-fixed, Paraffin-embedded Alveolar Rhabdomyosarcomas and Its Clinicopathologic Correlation

Chromosomal translocations of t(2;13)(q35;q14) and t(1;13)(p36;q14), resulting in PAX3-FOXO1 (FKHR) and PAX7-FOXO1 (FKHR) gene fusions, have been found to be specific molecular markers for alveolar rhabdomyosarcomas (ARMS) and can be identified in approximately 80% cases. As the prognosis of ARMS is worse than that of embryonal rhabdomyosarcomas (ERMS), it is important to accurately distinguish between these 2 subtypes. This distinction may be difficult on the basis of morphology alone. To detect the genetic alterations, reverse transcriptase polymerase chain reaction (RT-PCR) or dual-color dual-fusion fluorescence in situ hybridization (FISH) have been used in most studies so far. In this study, we used FOXO1 (FKHR) gene break-apart FISH probe, which can detect both of the translocations involving the FOXO1 gene, and tested 20 cases of rhabdomyosarcoma (RMS) including 6 cases of ARMS, 8 ERMS, 1 pleomorphic type, 5 not otherwise specified (RMS-NOS), and 10 non-RMS sarcomas. A home-brew RT-PCR that could detect both PAX3-FOXO1 and PAX7-FOXO1 was also performed. Four pathologists independently reviewed all RMS and a consensus diagnosis was also reached in discrepant cases. Histologic and molecular findings were correlated with clinical outcomes with an average of a 49-month follow-up. FOXO1 break-apart by FISH was positive in 4 of 6 (66%) ARMS and 2 of 5 (40%) RMS-NOS cases. All other cases, including all ERMS, were negative. RT-PCR assay confirmed all FISH results. While 2 of 6 (33%) RMS patients with a FOXO1 break-apart died of the disease, there were no deaths among the patients with negative result. The FOXO1 gene break-apart FISH probe is a simple and accurate tool to detect the translocations associated with ARMS. As characteristic genetic alterations of ARMS can be identified in 40% of RMS-NOS cases in our study, the FISH assay would provide an additional useful tool in the diagnosis and prognosis of ARMS, and an alternative to RT-PCR.

Clinical implications of der(9q) deletions detected through dual-fusion fluorescence in situ hybridization in patients with chronic myeloid leukemia

Poor outcomes of some chronic myeloid leukemia ((CML) patients have been associated with submicroscopic der(9q) deletions, particularly the 5′ ABL region. Deletion profiles of 120 BCR/ABL+ CML patients were studied using the dual-fusion fluorescence in situ hybridization probe. Poor prognosis was associated with 5′ ABL deletion but not with 3′ BCR deletion. Overall survival (OS) and chronic phase duration (CPD) were significantly shorter for 5′ ABL deletion than for those without deletions (OS time: 27 vs. 61 months, P = 0.02; CPD: 17 vs. 56 months, P = 0.02). In addition, when isolated 5′ ABL deletion patients were compared to those without it, a greater impact on prognosis was detected (OS time: 18 vs. 59 months, P = 0.0008; CPD: 7 vs. 54 months, P = 0.0003). Isolated 5′ ABL deletion seems to have a greater impact on survival than does concomitant 5′ ABL and 3′ BCR deletion, although the difference was not statistically significant in this aspect (OS time: 18 vs. 28 months, P = 0.08).

Automated Duet spot counting system and manual technologist scoring using dual-fusion fluorescence in situ hybridization (D-FISH) strategy: comparison and application to FISH minimal residual disease testing in patients with chronic myeloid leukemia

The automated BioView Duet system was compared with manual technologist scoring (MTS) using a BCR/ABL dual-fusion FISH (D-FISH) probe strategy for chronic myeloid leukemia (CML) specimens. In the first study, 500 nuclei were evaluated for 10 distinct signal patterns in various abnormal cell percentages from each of 89 specimens. The Duet system correctly identified all 27 normal specimens and the abnormal signal pattern of all 63 abnormal specimens. The percentage of abnormal nuclei detected was also concordant, with an average difference between MTS and the Duet system of only 2.7%. However, achievement of accurate quantitative results required reclassification by a technologist for nearly 50% of nuclei per specimen. Next, the Duet system was used to evaluate BCR/ABL D-FISH for FISH minimal residual disease (MRD) detection in CML patients. Up to 6,000 nuclei were evaluated for four signal pattern categories for each of 60 CML MRD samples. Excluding four abnormal specimens with insufficient samples, the Duet system correctly identified all of the abnormal specimens and identified four additional abnormal specimens previously diagnosed as normal by MTS. The technologist time required for evaluation and reclassification of the Duet system data for the FISH MRD samples averaged only 1 minute per case, saving significant technologist effort. We conclude that the Duet system appears to be more sensitive and cost-effective than MTS for CML FISH MRD testing.

Can morphological assessment limit the use of specific genetic testing to exclude chronic myeloid leukaemia?

Between January 2004 and June 2006, a total of 66 unselected peripheral blood or marrow aspirate samples from patients with evidence of myeloproliferation were received by our cytogenetics and molecular laboratory for BCR/ABL detection to exclude chronic myeloid leukaemia (CML) [morphological diagnosis: polycythaemia vera (PV) in 10, essential thrombocythaemia (ET) in 22, chronic idiopathic myelofibrosis (CIMF) in 12, hypereosinophilic syndrome in one, chronic myeloproliferative diseases (CMPD), unspecified in 20 and myelodysplastic/myeloproliferative diseases (MDS/MPD) in one]. Significant peripheral basophilia (absolute basophil count >1 × 109/l) was only seen in four of 57 patients with available white cell differential counts (two with CMPD, one with PV and one with CIMF), and none of them had a count >2 × 109/l. Marrow morphology was examined by haematopathologists before genetic testing. In 60 patients with available marrow aspirate specimen, proliferation of small and hypolobulated megakaryocytes typical of CML were found and documented in only four cases (one each of CMPD, unspecified, PV, ET and MDS/MPD). As none of these 66 patients received a morphological diagnosis of CML from initial peripheral blood and/or marrow examination, according to our protocol we did not perform conventional cytogenetics but employed dual-colour dual-fusion fluorescence in-situ hybridisation (D-FISH; Vysis, Downers Grove, IL, USA) for exclusion of BCR/ABL fusion in this setting (Wan et al, 2003). Three hundred nuclei were scored in each case. Test sensitivity was 0·8%. BCR/ABL fusion was not detected in any of the 66 cases. A further 14 cases with a pretest diagnosis of reactive cytosis were analysed in the same period by D-FISH and all showed a negative result. During this period, 72 cases with a morphological diagnosis of CML were analysed by conventional cytogenetics in our laboratory (plus FISH in nine cases that showed no growth or a normal karyotype). Of these, 51 cases were previously untreated and had blood counts and bone marrow examination results reported before karyotyping. All 51 cases were documented to have proliferation of small and hypolobulated megakaryocytes in bone marrow and significant peripheral basophilia of >1 × 109/l (range 1·15–38·82 × 109/l, median 10·64 × 109/l; basophil count >2 × 109/l in 47 cases). The diagnosis was confirmed in 70 cases by the detection of t(9;22)(q34;q11.2) in 63 out of 72 cases examined or positive FISH fusion signals in seven out of nine cases examined. A review of the records during this period showed that a Ph chromosome was detected in 11 non-CML cases by conventional cytogenetics. None of them had a pretest diagnosis of CMPD (precursor lymphoblastic leukaemia in nine, acute myeloid leukaemia in two). Although one may argue that the present FISH technique with 0·8% test sensitivity may not be able to detect a very small BCR/ABL fusion-positive clone, this should not be relevant in our series as most patients were newly diagnosed and untreated. None of them had received imatinib, which could have reduced the clone size to this level. Our results show that cases lacking characteristic morphological features of CML are highly unlikely to be positive for BCR/ABL fusion. Careful morphological examination can therefore help to decide whether further genetic testing to exclude CML is indicated. The well-accepted, but not well-scrutinised, approach of screening all patients with CMPD for BCR/ABL fusion to exclude CML needs to be re-evaluated. Careful re-examination of those rarely reported cases of ‘PV-like’ or ‘ET-like’ CML for these highly sensitive morphological features is warranted. We propose that in patients with evidence of myeloproliferation but without significant peripheral blood basophilia and lacking characteristic small hypolobulated megakaryocytes on bone marrow examination, specific genetic tests performed solely for the purpose of excluding CML can safely be withheld, unless suggestive morphological features develop during monitoring. With this approach, laboratory resources can be utilised more cost-effectively.

Primary application of spectral karyotyping in leukemia

To establish a spectral karyotyping (SKY) technique and explore the value of SKY in leukemia research. SKY technique was conducted on 2 samples of peripheral blood from 2 healthy volunteers, then on the samples from 8 patients with leukemia, including chronic myelocytic leukemia (CML) and acute myelocytic leukemia (AML) confirmed by R-banding. In addition, four patients underwent dual fusion-fluorescence in situ hybridization (DF-FISH) to detect the mixed lineage leukemia (MLL), PML/RARa, and BCR/ABL fusion genes. By comparing the results of SKY, R-band karyotyping, and DF-FISH, the stability and reliability of SKY was judged. All 10 samples were successfully hybridized and karyotyped. The 2 cases of healthy volunteers showed normal karyotypes, thus, a specific SKY technique was successfully established. In the 8 cases of leukemia patients, SKY identified aberrations including 9q-, t (9; 22), t (15; 17) and the complex karyotype 47, XY, +9?ins (1;5) (q23;q23), t (6;7) (q23?; p13), in addition, the SKY technique detected some number abnormalities. The results of SKY confirmed the results of R-band karyotyping and DF-FISH; moreover, the SKY technique provided more accurate karyotypes. With high stability, accuracy, and sensitivity, the SKY technique established by this study can be applied in leukemia research.