Normal Fluorescence In Situ Hybridization Research Articles

Chromosome aberrations detected by conventional karyotyping using novel mitogens in chronic lymphocytic leukemia with “normal” FISH: correlations with clinicobiologic parameters

AbstractIt is unclear whether karyotype aberrations that occur in regions uncovered by the standard fluorescence in situ hybridization (FISH) panel have prognostic relevance in chronic lymphocytic leukemia (CLL). We evaluated the significance of karyotypic aberrations in a learning cohort (LC; n = 64) and a validation cohort (VC; n = 84) of patients with chronic lymphocytic leukemia with “normal” FISH. An abnormal karyotype was found in 21.5% and 35.7% of cases in the LC and VC, respectively, and was associated with a lower immunophenotypic score (P = .030 in the LC, P = .035 in the VC), advanced stage (P = .040 in the VC), and need for treatment (P = .002 in the LC, P = < .0001 in the VC). The abnormal karyotype correlated with shorter time to first treatment and shorter survival in both the LC and the VC, representing the strongest prognostic parameter. In patients with chronic lymphocytic leukemia with normal FISH, karyotypic aberrations by conventional cytogenetics with novel mitogens identify a subset of cases with adverse prognostic features.

Monoclonal B‐cell lymphocytosis is closely related to chronic lymphocytic leukaemia and may be better classified as early‐stage CLL

The World Health Organization classification uses a cut-off point of 5·0 × 10(9)/l cells with a chronic lymphocytic leukaemia (CLL)-phenotype in peripheral blood to discriminate between monoclonal B-lymphocytosis (MBL) and B-CLL. This study analysed 298 MBL patients by multi-parameter flow cytometry, chromosome banding analysis (CBA)/fluorescence in situ hybridization (FISH), and IGHV mutation status and compared them with 356 CLL patients. In MBL, CBA more frequently revealed a normal karyotype and FISH identified less frequently del(6q), del(13q) (as sole alterations), and del(17)(p13). Within the MBL cohort, a shorter time to treatment (TTT) was found for ZAP-70-positivity, 14q32/IGH-translocations (CBA), del(11)(q22·3) (FISH) and unmutated IGHV status. Higher CD38 and ZAP-70 expression, del(11)(q22·3) (FISH), trisomy 12 (FISH), and 14q32/IGH-translocations (CBA) were correlated with a shorter TTT in the combined cohort (MBL + CLL); a sole del(13)(q14) (FISH) correlated with longer TTT. Regarding overall survival, unmutated IGHV status and 'other' alterations (CBA) had an adverse impact. There was no correlation between the concentration of CLL-cells and TTT or overall survival. Multivariate analysis confirmed a negative impact on TTT for del(11)(q22·3)/ATM, trisomy 12 (both by FISH), and 14q32/IGH-translocations by CBA. These data emphasize a close relationship between MBL and CLL regarding clinically relevant parameters and provide no evidence to strictly separate these entities by a distinct threshold of clonal B-cells.

TP53 Mutation Profile and New Mutations in Chronic Lymphocytic Leukemia in Normandy

Abstract 4593 Introduction:As deletion of 17p13 (del 17p13) in Chronic lymphocytic leukemia (CLL) patients is associated with poor outcome and chemorefractoriness to alkylating agents and purine analogues, current diagnostic guidelines recommend its screening at diagnosis and prior to treatment initiation. TP53 is a tumor suppressor gene, maps to 17p13 and is inactivated through somatic mutations or by 17p13 deletion. Recent studies show that TP53 mutation is of clinical relevance in CLL patients and has practical implication for therapeutic stratification. The purpose of this study is to characterize the TP53 mutation pattern and its incorporation into treatment algorithms in CLL patients treated in our center. Materials and methods:A retrospective study was performed on a cohort of 42 patients with CLL for whom clinical, biological and fluorescent in situ hybridization (FISH) data were available. Ages ranged from 31 to 88 (median, 63 years), 17 patients were in Binet stage A, 12 in B and 13 in stage C. Treatment was not in controlled trials, and standard clinical criteria were used for the initiation of therapy. DNA was extracted from 52 available samples and TP53 exons 2 to 11 were screened for mutations by high resolution melting (HRM) (Roche LC480). Mutations were confirmed by sequencing both strands using different primer sets (Beckman, Ceq 8000) then validated by the IARC and UMD TP53 Mutation Databases. Results:Out of 42 patients, del 17p13 was observed in 11 (26%), del 11q22 in 8 (19%), del 13q14 in 13 (31%), trisomy 12 in 2 (5%) and normal FISH in 20 (47.6%). We found an overall incidence of TP53 mutations of 19% (8 of 42 patients), 1 new mutation was identified and 5 mutations were not previously reported in CLL but in other cancers according to the IARC and UMD TP53 mutation Databases (table 1). A total of 65 polymorphisms were detected with a prevalence for the g.11446C>G polymorphism of 0.9 compared to 0.49 in published data. Amongst the 8 patients with TP53 mutations, 7 (87%) presented a 17p deletion and mutations were mainly located in the DNA binding domain (62%). 7 of 11 patients (63%) with 17p13 deletion harbored a TP53 mutation. In the absence of del 17p13 only 3% of patients (1 of 31) had a TP53 mutation (table 2). 5 of 8 patients with mutations (62%) presented with an advanced clinical stage and a median age of 56 years. Clonal evolution leading to acquisition of the TP53 mutation was not necessary related to therapy; 3 sequential samples were available for a patient with no abnormalities at diagnosis and who developed a TP53 mutation after 2 years and before therapy initiation. Another patient who was initially devoid of TP53 mutations and responded to treatment with standard chemotherapy, developed chemorefractoriness concomitant with a TP53 mutation acquisition. An interesting case was that of a patient who had been in complete remission after treatment with rituximab, fludarabin and cyclophosphamid (RFC) despite the presence of a TP53 mutation without del 17p13 at diagnosis. Sequencing demonstrated that this patient harbored a silent mutation in exon 4 g.12441C>G.Table 1TP53 mutation profile in CLLExonsIntronsMutation DNA levelCodonMutation protein levelMutation type3g.11327G>A*Splice Ac5g.12447delC153 *Frame shiftDeletiong.12411C>G141*p.C141WPMg.12441C>G151*p.P151PPM6g.12653T>C195 *,*p.I195TPM7g.13348G>A273 *,*p.M237IPM8g.13828G>C283*p.R283PPM9g.16884G>A*Splice AcSplice Ac: Splice acceptor site*: not previously reported, **: previosly reported, PM: point mutationTable 2Mutations in relation to del17p13MutatedTP53TotalWild type+7411del 17p13—13031Total83442 In Conclusion:This study shows similar findings to published data except for a higher incidence of patients with TP53 mutations probably due to bias selection and a low cohort of patients. We identified a new mutation and 5 other mutations not previously detected in CLL patients. As TP53 is associated with poor outcome and chemorefractoriness, and its acquisition might be a result of positive pressure selection following treatment, we think it is of utmost importance to look for TP53 mutations in parallel to FISH at diagnosis and in case of chemorefractoriness using the HRM a cheap and sensitive method followed by sequencing positive cases. Disclosures:No relevant conflicts of interest to declare.

Clinical Significance of Notch1 Mutation in Chinese B-CLL Patients,

Abstract 3892Chronic lymphocytic leukaemia (CLL) is a heterogeneous disease with variable clinical presentation and evolution, known as the most frequent leukemia in adults in Western countries. The molecular changes leading to the pathogenesis of the disease are not fully understood. Together with Notch 2, 3, 4, Notch 1 constitute a family of heterodimeric transmembrane proteins and encodes a class I transmembrane protein functioning as a ligand-activated transcription factor and playing an important role in cell differentiation, proliferation, and apoptosis. An oncogenic role for Notch signaling has well established in T-acute lymphoblastic leukemia (T-ALL). It has been reported that up to 60% of T-ALL have activated gain-of-function mutations in Notch1 gene. Nevertheless, few Notch1 mutations have been reported in CLL.Recently, using whole-genome sequencing, Puente et al identified 46 somatic mutations that potentially affect gene functions in CLL, further analysis of these mutations identified that Notch 1 is recurrently mutated. Through functional and clinical analyses, it is confirmed that Notch 1 mutation is oncogenic change that contribute to the clinical evolution of CLL. Mauro et al eported that 2/43(4.6%) B-CLL patients had Notch 1 activating mutations and both had unmutated IGHV, ZAP-70 positive and had a very poor prognosis. Giulia et al reported 10/120 (8.3%) patients at CLL diagnosis harbor Notch 1 mutation, and these patients also had higher incidence of unmutated IGHV, ZAP-70 positive and poor prognosis when compared with CLL patients harboring wild-type Notch 1. To date, there is no study on Notch 1 mutation in Chinese patients with CLL, the aim of this study was to investigate Notch 1 mutation in Chinese patients with CLL, to confirm whether it could be a prognostic marker in Chinese CLL cohort. In this study, Exon 26, 27 and 34 of Notch1 were successfully investigated by polymerase chain reaction (PCR) and sequence screening in 100 CLL patients and the results were compared with other factors including clinical stages, IGHV gene status and ZAP-70 expression. PEST domain mutation locating in exon 34 of Notch1 was found in 8/100(8.0%) CLL patients while there were no mutations of other three domains including HD-N, HD-C and TAD. The mutation occurs in 5 males, 3 females and median age at diagnosis was 70 years (range 51–83 years). According to the Binet staging system, 6/7 (85.7%) patients were in stage B+C, 1/7 (14.3%) patients were in stage A (p <0.01) and one patient did not got disease staging at diagnosis. 4/6(66.7%) patients carried unmutated IGHV genes and 3/7 (42.9%) CD38 and 2/7 (28.6%) ZAP-70 were positive. Chromosomal anomalies were assessed by fluorescent in situ hybridization (FISH) on 7 of the 8 Notch1 mutated patients. Of which, two patients presented with del13q14 and one with a normal FISH pattern, moreover, one presented with del17p13 and 14q32 rearrangement, one with trisomy 12 and del17p13, one with del17p13 and del11q22, one with del17p13 and del13q14. Time-to-treatment (TTT) was shorter in patients with Notch1 mutation than patients with wild-type Notch1 (4 vs. 12 months). One patient with Notch1 mutation had an invasive progression and died. Results also show that Patients with Notch1 mutation had higher rates of unmutated IGHV genes (66.7% vs. 29.1%), older ages (70 years vs. 60 years), more progressive stage (Binet B+C 85.7% vs. 45.9%) when compared with those with wild-type Notch1. The study showed that patients with Notch1 mutation indicate poor prognosis and the status of mutation may be a novel prognostic factors in CLL. Disclosures:No relevant conflicts of interest to declare.

Automated Enumeration and Characterization of Circulating Multiple Myeloma Cells in Blood

Abstract 1825Detection of circulating Multiple Myeloma cells (CMMC) by flow cytometry is an indicator of active disease. In addition, circulating plasma cells can be detected in earlier stages of disease, including MGUS and Smoldering Multiple Myeloma, and appear to correlate with prognosis. The capture and characterization of these circulating plasma cells from peripheral blood may provide novel biomarkers for the management of Multiple Myeloma patients, particularly in monitoring minimal residual disease and in progression from MGUS or Smoldering Multiple Myeloma to active disease. The enumeration and characterization of circulating tumor cells (CTC) in patients with metastatic breast, prostate or colorectal cancer using the CellSearch® technology, has been shown to provide clinically relevant prognostic and predictive information. Here we describe the development of an automated assay for detecting circulating normal plasma cells (CPC) and multiple myeloma cells (CMMC) in blood using CellSearch. Assay results from Multiple Myeloma, MGUS patients, and from an aged matched control population are presented.The CellTracks® AutoPrep® System and CellTracks Analyzer II® systems were used to capture and enumerate CPC and CMMC. Magnetic particles conjugated to anti-CD138 are used to capture myeloma cells from 4.0mL of blood. Enriched cells are then stained with the nucleic acid dye DAPI and anti-CD38-Phycoerythrin (PE) antibody. Allophycocyanine (APC) conjugated anti-CD45 and anti-CD19 were used to exclude leukocytes and B-cells. In addition, FITC labeled anti-CD56 was added as a biomarker. The enriched and stained cells were transferred to a CellTracks® cartridge and MagNest® for magnetic mounting. The cartridge was scanned using the CellTracks Analyzer II®. Individual images of cells were presented to the operator for review, and scored as CMMCs, based on fluorescence and cell morphology. In a model spike-in system the assay consistently recovered ∼60% of the cells from the Multiple Myeloma cell line H929 spiked into 4.0mL of blood from healthy donors. The assay was linear over the tested range of from 0 to 2000 spiked H929 cells (r2 0.98, slope 0.50, intercept 10). The assay was validated using blood from age matched healthy donors (n=22) and patients with Multiple Myeloma (n=66) and MGUS (n=7). In 4.0mL blood from normal donors, 0 CPC were detected in 12/22 (55%) and low numbers (1–6 CPC) were detected in 10/22 (45%) samples. Interestingly, one CD56 positive CPC (CMMC?) was found in a normal donor. CMMC in Multiple Myeloma patients ranged from 0 – 17,000 /4.0mL blood. One or more CMMC were detected in 91% of the patients, > 5 in 68%, > 10 in 58% and > 100 in 35%. Expression of CD56 was highly variable in the patient population. CMMC in MGUS patients ranged from 0 – 112 /4.0mL blood. One or more CMMC were detected in 6/7 of the patients, > 5 in 4/6, > 10 in 2/6 and > 100 in 1/6.To further characterize CMMC, and differentiate CPC from CMMC, an interphase fluorescent in situ hybridization (FISH) assay was developed to be used with the capture and detection system described above. A four color FISH probe was used to simultaneously detect high risk mutations including two recurrent translocations of the IgH locus (t(4;14)(p16;q32) and t(14;16)(q32;q23)) as well as deletion of the TP53 locus (Δ17p13). The FISH assay was verified on cell lines H929, MM1s, and U266, which showed mutations at t(4;14), t(14;16) and Δ17p13, respectively. The FISH assay was tested on 9 CMMC patient samples and 8 samples yielded evaluable results. Two samples showed t(4;14)fusions, 3 patients showed aberrant FISH signal patterns indicating aneuploidy of chromosome 4 or 14 and the remaining patients showed normal FISH patterns.Well controlled prospective clinical studies are needed to establish the prognostic and predictive value of the presence, and characteristics, of CMMC in multiple myeloma or MGUS. In addition, as with CTC, this automated CMMC assay should prove useful in evaluating the effectiveness of new treatments as well as the assessment of potential treatment targets on CMMC in this difficult disease. Disclosures:Gross:Johnson and Johnson: Employment, Equity Ownership. Foulk:Johnson and Johnson: Employment, Equity Ownership. Patel:Johnson and Johnson: Employment, Equity Ownership. Connelly:Johnson and Johnson: Employment, Equity Ownership. Mata:Johnson and Johnson: Employment, Equity Ownership.

Fluorescence In Situ Hybridization to Visualize Genetic Abnormalities in Interphase Cells of Acinar Cell Carcinoma, Ductal Adenocarcinoma, and Islet Cell Carcinoma of the Pancreas

To use fluorescence in situ hybridization (FISH) to visualize genetic abnormalities in interphase cell nuclei (interphase FISH) of acinar cell carcinoma, ductal adenocarcinoma, and islet cell carcinoma of the pancreas. Between April 4, 2007, and December 4, 2008, interphase FISH was used to study paraffin-embedded preparations of tissue obtained from 18 patients listed in the Mayo Clinic Biospecimen Resource for Pancreas Research with a confirmed diagnosis of acinar cell carcinoma, ductal adenocarcinoma, islet cell carcinoma, or pancreas without evidence of neoplasia. FISH probes were used for chromosome loci of APC (see glossary at end of article for expansion of all gene symbols), BRCA2, CTNNB1, EGFR, ERBB2, CDKN2A, TP53, TYMP, and TYMS. These FISH probes were used with control probes to distinguish among various kinds of chromosome abnormalities of number and structure. FISH abnormalities were observed in 12 (80%) of 15 patients with pancreatic cancer: 5 of 5 patients with acinar cell carcinoma, 5 of 5 patients with ductal adenocarcinoma, and 2 (40%) of 5 patients with islet cell carcinoma. All 3 specimens of pancreatic tissue without neoplasia had normal FISH results. Gains of CTNNB1 due to trisomy 3 occurred in each tumor with acinar cell carcinoma but in none of the other tumors in this study. FISH abnormalities of all other cancer genes studied were observed in all forms of pancreatic tumors in this investigation. FISH abnormalities of CTNNB1 due to trisomy 3 were observed only in acinar cell carcinoma. FISH abnormalities of genes implicated in familial cancer, tumor progression, and the 5-fluorouracil pathway were common but were not associated with specific types of pancreatic cancer.

2.54 Targeting Refractory CLL Lacking ATM Function with Sapacitabine-Containing Therapy

Cytogenetic abnormalities are common, and have prognostic implications, in chronic lymphocytic leukemia (CLL). In about 7080% CLL cases, chromosomal abnormalities can be identified by fluorescence in-situ hybridization (FISH) using a disease specific probe set. The most common genomic aberrations include deletion 13q, trisomy 12, and deletion in 11q, 17p and 6q. Approximately 10% of previously untreated patients with CLL exhibit a substantial deletion in the q arm of chromosome 11, the site of the ATM gene at (11q22.3). This lesion occurs at increased incidence of about 40% in patients relapsing on fludarabine-cyclophosphamide-rituximab (FCR) chemoimmunotherapy. The residual ATM allele is often mutated, suggesting that the region containing ATM may be important for response to chemoimmunotherapy. Sapacitabine is the orally bioavailable prodrug of the nucleoside analog CNDAC. It is undergoing clinical trials and has presented favorable clinical activity in advanced acute leukemias and myelodysplastic syndromes. CNDAC induces cell death by a unique mechanism of action. Incorporation of CNDAC triphosphate into DNA results in a single-strand break (SSB), which is converted to a one-ended double-strand break (DSB) upon collapse of the DNA replication fork. We have demonstrated that CNDAC-induced DSBs, which are lethal if not repaired, are repaired predominantly through homologous recombination (HR), relative to other repair pathways, such as transcription-coupled nucleotide excision (TC-NER) and non-homologous end-joining (NHEJ). CNDAC greatly sensitizes the viability of cells lacking HR components, such as ATM, Rad51D, Xrcc3 and Brca2. Based on this evidence, we hypothesize that CLL patients with 11q deletion that lack ATM function will be selectively sensitized to sapacitabinecontaining therapy. In order to distinguish ATM non-functional and functional individuals among del(11q22-23) patients characterized by FISH, we have established an assay of ATM activity. ATM is activated in response to irradiation and will further phosphorylate its downstream targets. We have shown that phosphorylation of KAP1 and Smc1 is dependent on the activity of ATM. In this assay, samples from eight patients with normal FISH karyotype were collected. These samples were mock treated or irradiated at dose of 10 Gy and protein lysates were harvested. The lysates were then combined as a positive pool comparator for the 11q deletion CLL samples, and both total and phosphorylated proteins were quantitated in immunoblots. The averaged phosphorylation level of the KAP1 and Smc1 proteins among the CLL patient samples was calculated and compared with those of the positive pool. The value of the samples to the positive pool of 30% was used as indicative of deficient ATM function. Using this cutoff, we identified five ATM non-functional individuals among ten del(11q22-23) CLL samples isolated from archived stocks. A phase II clinical trial of sapacitabine in combination with cyclophosphamide and rituximab (SCR) for previously treated patients with CLL having del(11q22-23) is open for patient recruitment. We will extend clinical investigations of sapacitabine to these CLL patients, focusing on the comparisons of responses of patients defined by the loss of ATM function. The results will test the hypothesis that loss of ATM function will sensitize the disease to sapacitabine therapy and prevent disease progression.

Usefulness of a break-apart FISH assay in the diagnosis of Xp11.2 translocation renal cell carcinoma

Xp11.2 translocation renal cell carcinoma (RCC) is a rare subtype of RCC predominantly reported in young patients. It results from gene fusions between the transcription factor E3 (TFE3) gene, which is located on chromosome Xp11.2, and various fusion partners. Recently, a dual color, break-apart fluorescence in situ hybridization (FISH) assay to detect Xp11.2 translocation was reported. We performed this study to evaluate the usefulness of the FISH assay in the diagnosis of Xp11.2 translocation RCC using a commercially available TFE3 break-apart probe. We immunohistochemically analyzed TFE3 nuclear expression in 809 cases of RCCs using 14 tissue microarray blocks and selected nine cases those showed moderate to strong positive nuclear immunoreactivity for TFE3. The extent of TFE3 nuclear expression was variable. The TFE3 FISH assay was performed in these 9 selected cases and 44 negative control cases. Only four out of nine selected cases showed the TFE3 break-apart signal. TFE3 FISH-positive cases mainly showed diffuse and strong TFE3 immunopositivity, but one case revealed focal and moderate TFE3 staining. On the contrary, TFE3 FISH-negative cases mainly revealed focal and moderate TFE3 immunoreactivity, however, one FISH-negative case revealed diffuse and strong TFE3 nuclear immunopositivity. All negative control cases revealed normal TFE3 FISH results. Our results reveal that TFE3 immunohistochemistry can show false-positive results, and that the TFE3 break-apart FISH assay is a useful complementary method for confirming the diagnosis of Xp11.2 translocation RCC.

Diagnostic Yield of Bone Marrow and Peripheral Blood FISH Panel Testing in Clinically Suspected Myelodysplastic Syndromes and/or Acute Myeloid Leukemia

It is unclear how often and in what setting fluorescence in situ hybridization (FISH) panels for myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) provide additional information over metaphase cytogenetics alone. Furthermore, the usefulness of peripheral blood vs bone marrow FISH has also not been directly compared. We prospectively compared metaphase cytogenetics and FISH for -5/5q-, -7/7q-, +8, and 20q- in 433 cases of suspected MDS/AML. FISH testing was abnormal in 6 (14%) of 43 and 10 (19%) of 54 cases with fewer than 20 normal metaphases or no growth, respectively. FISH was only rarely abnormal in cases with 20 normal metaphases obtained (6/222 [2.7%]). Comparison of peripheral blood and bone marrow results in 48 cases showed abnormal peripheral blood FISH results in 18 (69%) of 26 cases with abnormal bone marrow FISH results and in 5 (23%) of 22 cases with normal bone marrow FISH results. These findings, the largest published comparison of FISH vs metaphase cytogenetics in MDS/AML, provide a rational strategy for FISH testing in peripheral blood and bone marrow.

Utility of quantitative fluorescent in situ hybridization (FISH) to predict non-muscle-invasive bladder cancer (NMIBC) recurrence.

286 Background: Urovysion's (Abbot Laboratories, Downers Grove, IL) FISH analysis is used to monitor bladder cancer recurrence in patients with a history of NMIBC and is often reported as a binary variable (normal/abnormal). We investigated whether the percentage of abnormal cells as determined by FISH analysis in patients with a history of NMIBC correlated with risk of recurrence. Methods: At our institution, barbotage FISH analysis is routinely done along with cystoscopy and cytology on both high risk (Ta/T1 high grade or CIS) and low or intermediate risk patients (all others) at every 3-month follow-up for the first year post-resection. We retrospectively reviewed 241 consecutive NMIBC patients and identified 399 FISH analyses for which we had one year follow-up. Normal FISH analyses were defined as 2 or fewer abnormal cells per sample. We calculated the percentage of abnormal cells and correlated that to the number of patients who had a recurrence of NMIBC as defined by positive high grade cytology or tumor on cystoscopy during the first year of follow-up. Results: The sensitivity, specificity, and positive and negative predictive values of FISH analysis if reported as a binary variable was 55, 43, 16 and 89%, respectively. Considering only those patients with abnormal FISH, the average percentage of abnormal cells for patients who were found to have NMIBC recurrence at 1 year was 38% (range 6–100) compared to 21% (range 6–100) for patients who were recurrence-free at 1 year (p&lt;0.0001). High risk patients who recurred within 1 year had a statistically higher percentage of abnormal cells as compared to those who did not recur within 1 year (50% [range 6–100] vs. 25% [range 6– 100], respectively p=0.001). There was no difference in the percentage of abnormal cells for those patients with low or intermediate risk disease based on recurrence within 1 year (22% [range 6–100] vs. 20% [range 6–100], respectively p=0.25). Conclusions: The percentage of abnormal cells in FISH analysis correlates with risk of recurrence for patients with high risk disease and can be used to guide surveillance interval decisions in patients with no other evidence of recurrence. [Table: see text]

New US Myeloma Registry (Connect MM® – The Multiple Myeloma Disease Registry): Features of Newly Diagnosed Patients In 2010

AbstractAbstract 5032 Background:In the past decade, with the availability of novel therapies, the paradigm for myeloma management has changed. In 2010 it is especially important to understand baseline features and initial treatment decisions. The goal of the Connect MM® registry is to characterize patients with newly diagnosed active myeloma from 200 US sites. Approximately 80% of the patient population will be enrolled from community-based practices and 20% from academic centers. An electronic case report form was developed to collect clinical data, physician choices, patient health-related quality of life (HRQoL) and response, as well as data on survival end points. This is a prospective, observational, longitudinal study with a target accrual of 1,500 patients in 3 years, with a 5 year follow-up from the time of informed consent. There are no mandated treatments or clinical assessments. However, there are data collection requirements for diagnosis and disease monitoring. Results:Since late 2009, 340 patients from 135 sites have been accrued and were included in this interim analysis. Current study demographics include: 60% male, 83% white, and 14% black, with a median age of 67 years. Thus far, 97% have been enrolled from community-based practices. All patients met study enrollment criteria and had active myeloma at entry; prior monoclonal gammopathy of unknown significance (MGUS) was reported in 13% and smoldering MM in 8%. International Staging System (ISS) staging for evaluable patients were 26.3%, 36.4%, 37.3% for stages I, II, and III, respectively. Durie-Salmon Stage (A or B) were 13%, 35%, 52% for stages I, II, and III, respectively. Staging procedures included 82% skeletal survey; 44% computed tomography (CT); 40% magnetic resonance imaging (MRI); 7% positron emission tomography (PET); 2% PET/CT; and 4% had no imaging. International Myeloma Working Group (IMWG) CRAB criteria were assessed in all enrolled patients; 9% had hypercalcemia, 18% renal insufficiency, 36% anemia, and 66% had bone lesions. Median values were: calcium 9.5 mg/dL; serum creatinine 1.1 mg/dL; hemoglobin 10.9 gm/dL. Only 9% of patients had 3 or 4 CRAB features, while 49% had only 1 feature and 26% were asymptomatic (ECOG=0). The incidence of baseline peripheral neuropathy was 6%. Initial pain led to radiation therapy for 10% of patients, with 16% having vertebroplasty or kyphoplasty. Cytogenetic studies were performed at baseline in 64% of patients and fluorescence in situ hybridization (FISH) studies in 54%. Cytogenetics and FISH were normal in 27% of patients, while in 20% both were abnormal in patients who had both performed. FISH was abnormal with normal cytogenetics in 41% and only 2% had normal FISH but abnormal cytogenetics. The most common FISH abnormalities were: 13 q- (31%), 17 p- (28%), t(4; 14) (16%). Freelite® testing was performed in 56% of patients with an abnormal ratio in 94% [rFLC]. Of evaluable patients receiving frontline therapy 98% of patients received a novel agent and only 3 patients (1.4% of treated patients) received melphalan/prednisone. Two drug combinations were used in 53%, 3 drugs in 26%, 4 drugs in 1.3%, and single agents were used in 21% of the patients. The most common regimens were: bortezomib+dexamethasone (28%), lenalidomide+dexamethasone (20%), and bortezomib+lenalidomide+ dexamethasone (15%). Conclusion:These baseline features and treatment choices characterize myeloma patients primarily in community-based practices in the US in 2010. As academic centers enroll more patients, we will be able to further characterize that population. Of particular note, 26% of patients were asymptomatic at baseline but had biochemical evidence of myeloma and met enrollment criteria; conversely 95% had an abnormal rFLC and 73% had abnormal chromosome results. The Connect MM® registry will provide data regarding patient features as they pertain to patterns in testing and treatment in the clinical practice setting, as well as response and survival outcomes. Disclosures:Durie:Celgene & Millennium: Consultancy. Off Label Use: Revlimid (lenalidomide) in combination with dexamethasone is indicated for the treatment of multiple myeloma patients who have received at least one prior therapy. Shah:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Millennium: Research Funding; Novartis: Research Funding. Abonour:Celgene & Millennium: Honoraria. Gasperetto:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Speakers Bureau. Mehta:Celgene: Consultancy, Speakers Bureau; Takeda/Millennium: Speakers Bureau; Onyx: Research Funding. Pashos:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding. Toomey:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees. Swern:Celgene: Employment. Street:Celgene: Employment. Sullivan:Celgene: Employment, Equity Ownership. Rifkin:Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Speakers Bureau; Amgen: Speakers Bureau; Cephalon: Speakers Bureau; Dendreon: Speakers Bureau.

Normal Fish Cytogenetics and 13q Deletions Unveil Marked Biological and Clinical Heterogeneity In Chronic Lymphocytic Leukemia

AbstractAbstract 2692Cytogenetic aberrations are considered major prognostic indicators for predicting the survival of chronic lymphocytic leukemia (CLL) patients (pts) [Dohner et al, 2000]. Given the difficulties in obtaining abnormal metaphases in CLL, fluorescent in situ hybridization (FISH) with specific probes is generally used to detect the most frequent abnormalities. Interphase FISH (I-FISH) is a rapid and sensitive technique for analysis of chromosome aberrations in CLL, but the limited number of DNA probes available to screen B-CLL results in a high number of cases presenting normal cytogenetics. Moreover, deletion 13q14 on FISH analysis which is the most common cytogenetic abnormality in CLL is a favourable prognostic biomarker when detected as a sole abnormality, but a higher percentage of 13q- nuclei was found to be associated with significantly shorter time to treatment (P<0.001), [van Dike et al, 2010]. On this line, the primary endpoints of our research were: 1) to determine progression free survival (PFS) and overall survival (OS) within the normal FISH cytogenetics subset on the basis both of ZAP-70, CD38, CD69 expression by flow cytometry and IgVH status; 2) to correlate percentages of 13q- nuclei (<50% or ≥50%) with PFS and OS and finally 3) to confirm I-FISH as an independent prognostic factor. We investigated 400 pts, median age 65 years (range 33–89), 235 males and 165 females. With regard to modified Rai stages, 115 pts had a low stage, 266 an intermediate stage and 19 a high stage. One hundred forty-two pts (35.5%) exhibit a normal karyotype, 133 pts (33.2%) showed an isolated 13q-, 69 pts (17.3%) presented trisomy 12, 30 pts (7.5%) 11q deletion, 20 (5%) 17p deletion and 6 (1.5%) other chromosome abnormalities. Clearly, pts with intermediate/poor cytogenetic abnormalities (trisomy 12, del11q-, del17p-) showed significant shorter PFS and OS (8% vs 33% at 16 years, P<0.0001 and 41% vs 65% at 16 years, P=0.0001) in comparison with normal or del13q- pts. Therefore, in order to better define the apparently homogenous subset of the patients with normal cytogenetics, we analysed ZAP-70, CD38, CD69 and IgVH status within this subgroup. Interestingly, we observed significant longer PFS and OS for ZAP-70 negative pts (60% vs 29% at 10 years, P<0.0001 and 92% vs 23% at 16 years, P=0.0006), for CD38 negative pts (54% vs 22% at 10 years, P=0.0008 and 83% vs 0% at 14 years, P=0.03), for CD69 negative pts (60% vs 23% at 10 years, P=0.0003 and 83% vs 63% at 14 years, P=0.02) and finally also for IgVH mutated cases (64% vs 35% at 10 years, P=0.0002 and 95% vs 45% at 14 years, P=0.04). Moreover, pts with isolated 13q- in <50% of nuclei (60 pts) showed a longer PFS and OS (68% vs 6% at 16 years, P=0.0003 and 92% vs 0% at 18 years, P=0.002; Figure) compared to those with ≥50% of nuclei (72 pts). No significant correlation was found between 13q- percentages and CD38 or ZAP-70 or IgVH status, while it was found with CD69 expression (P=0.01). In multivariate analysis of PFS, FISH cytogenetics was confirmed to be an independent prognostic factor (P=0.002) together with Rai stages (P=0.005), ZAP-70 (P=0.0001), IgVH status (P=0.0002) and CD69 (P=0.007). Therefore, in the clinical practice, ZAP-70, CD38, IgVH status and CD69 are irreplaceable in order to define prognosis and treatment within the large series of CLL pts with normal cytogenetics, probably presenting abnormalities undetectable by FISH. Besides, the percentage of nuclei exhibiting 13q- has to be considered an important predictor of the outcome and the clinical implications of 13q- in CLL seem to appear more complex than originally considerated. In conclusion, a comprehensive scoring prognostic system based on a combination of clinical, genetic, phenotypic parameters and IgVH status improves the separation of prognostic subgroups in CLL already early in the course of the disease. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

NOTCH1 PEST domain mutation is an adverse prognostic factor in B‐CLL

<i>NOTCH1</i> PEST domain mutation is an adverse prognostic factor in B‐CLL

Fluorescence in situ hybridization to visualize genetic abnormalities in interphase cells of acinar cell carcinoma, ductal adenocarcinoma, and islet cell carcinoma of the pancreas.

To use fluorescence in situ hybridization (FISH) to visualize genetic abnormalities in interphase cell nuclei (interphase FISH) of acinar cell carcinoma, ductal adenocarcinoma, and islet cell carcinoma of the pancreas. Between April 4, 2007, and December 4, 2008, interphase FISH was used to study paraffin-embedded preparations of tissue obtained from 18 patients listed in the Mayo Clinic Biospecimen Resource for Pancreas Research with a confirmed diagnosis of acinar cell carcinoma, ductal adenocarcinoma, islet cell carcinoma, or pancreas without evidence of neoplasia. FISH probes were used for chromosome loci of APC (see glossary at end of article for expansion of all gene symbols), BRCA2, CTNNB1, EGFR, ERBB2, CDKN2A, TP53, TYMP, and TYMS. These FISH probes were used with control probes to distinguish among various kinds of chromosome abnormalities of number and structure. FISH abnormalities were observed in 12 (80%) of 15 patients with pancreatic cancer: 5 of 5 patients with acinar cell carcinoma, 5 of 5 patients with ductal adenocarcinoma, and 2 (40%) of 5 patients with islet cell carcinoma. All 3 specimens of pancreatic tissue without neoplasia had normal FISH results. Gains of CTNNB1 due to trisomy 3 occurred in each tumor with acinar cell carcinoma but in none of the other tumors in this study. FISH abnormalities of all other cancer genes studied were observed in all forms of pancreatic tumors in this investigation. FISH abnormalities of CTNNB1 due to trisomy 3 were observed only in acinar cell carcinoma. FISH abnormalities of genes implicated in familial cancer, tumor progression, and the 5-fluorouracil pathway were common but were not associated with specific types of pancreatic cancer.

Usefulness of FICTION Method for the Evaluation of Response after Treatment in Multiple Myeloma

Introduction: According to the new uniform response criteria of International Myeloma Working Group (IMWG), stringent complete response (sCR) is defined as a condition of normal free light chain (FLC) ratio and absence of clonal cells in the bone marrow (BM) by immunohistochemistry or immunofluorescence, in addition to the CR condition. The kappa/lambda ratio is assessed to identify clonal cells in the BM and a minimum of 100 plasma cells is required for analysis of clonal cells. However, the evaluation of kappa/lambda ratio by immunohistochemistry or immunofluorescence may be inaccurate, because it is difficult practically to countthe number of anti-kappa/lambda antibodies-stained cells in the BM section and in case of low percentage of residual plasma cells, flow cytometric evaluation is also difficult. To investigate whether FICTION (Fluorescence Immunophenotyping and interphase Cytogenetics as a Tool for the Investigation Of Neoplasms) technique can be used as a tools for evaluation of clonal cells after treatment in multiple myeloma, we performed FICTION on BM cells in follow-up patients with myeloma and compared the results of FICTION with other parameters.Method: 18 myeloma patients, whose BM examination and serum free light chain were checked at the same time after treatment, were enrolled in Seoul National University Hospital. We performed FICTION for the fluorescence in situ hybridization (FISH) items that were abnormal at initial BM examination of each patient. The selected probes were LSI 1q25/1p36/1p subtelomere probe (Vysis, Downers Grobe, IL, USA) for trisomy 1q25, LSI 13 (RB1) 13q14 probe (Vysis) for RB1 deletion, LSI IGH probe (Vysis) for IGH rearrangement and LSI p16 (9p21)/CEP 9 probe (Vysis) for p16 deletion. The FICTION results were reported by the percentage of plasma cells with abnormal FISH signals among plasma cells stained with anti-kappa and lambda antibodies labeled with fluorescein isothiocyanate (FITC). We compared FICTION results with response parameters, such as % plasma cells in BM aspirates, serum FLC ratio, M-component in serum and/or urine protein electrophoresis (PEP) and FISH results.Results: Among 18 patients in follow-up, 5 (28%) showed <5% plasma cells by light microscope based differential count in BM aspirates and normal FISH results below the cut-off level. However, these patients turned out to have clonal cells in BM by FICTION techniques. Among these 5 patients, 3 patients showed abnormal serum FLC ratio and the other 2 patients showed M-component in serum PEP with normal serum FLC ratio. One patient with plasma cells fewer than 5% in BM aspirates, no cytogenetic abnormality in FISH and no M-component in serum, showed abnormal serum FLC ratio and abnormal FICTION results; 2 (25%) of 8 plasma cells showed RB1 deletion in FICTION. After 2 years, this patient progressed to plasma cell leukemia.Conclusion: Results of FICTTON correlated with FLC ratio and/or M-component in serum, while the percentage of plasma cells in BM aspirates or FISH results did not. The assessment of percentage of plasma cells in bone marrow aspirates might be inaccurate due to poor aspiration technique including dilution and focal infiltration of plasma cells. Also, FISH results based on the percentage of abnormal cells among all bone marrow nucleated cells do not reflect clonal plasma cells. In clonclusion, we suggest that FICTION technique is more sensitive method for identification of residual malignant plasma cells with clonalityalong with the evaluation of response after treatment in multiple myeloma.

Primary Cutaneous T-Cell Lymphomas Do not Show Specific NAV3 Gene Deletion or Translocation

The mapping of a balanced t(12;18)(q21;q21.2) translocation in a Sézary syndrome (SS) case led Karenko et al. to identify NAV3 gene (12q21-22) deletion by interphase fluorescence in situ hybridization (FISH) in 15/21 patients with mycosis fungoides (MF) or SS. To determine whether the NAV3 deletion is the result of a specific gene breakpoint, we used FISH with dual-color split or break-apart bacterial artificial chromosome (BAC) probes covering the NAV3 locus. A total of 31 samples (18 skin, 11 blood, 1 lymph node, and 1 spleen) from 24 patients with advanced MF/SS (18 with large-cell transformation) were studied. Chromosome 12 imbalances were analyzed by comparative genomic hybridization (CGH) array with a 3K BAC probes in 24 samples from 22 patients. Both normal FISH and CGH array patterns were observed in 22 samples from 18 patients. In 6 patients, abnormal patterns were observed with an abnormal number of chromosome 12 set in 5 of them. Chromosome 12 structural abnormalities were seen in four of these six patients. An imbalanced FISH pattern between NAV3 and pericentromeric control probes was seen in three patients in accordance with CGH array data (one with a pericentromeric deletion and two with a large 12q deletion including NAV3). No NAV3 specific breakpoint or partial deletion was detected.

Molecular characterization of chromosome 7 in AML and MDS patients

Myelodysplastic syndromes (MDS) share many features with acute myeloid leukemias (AML) and in fact 20 - 40% of the patients eventually develop a picture of full blown AML. Chromosome 7 has been a focus of attention as a site harboring tumor suppressor genes whose loss of function contributes to leukemia transformation or tumor progression. Abnormalities of chromosome 7 are frequently encountered in AML and MDS. The aim of the present study was to detect the molecular abnormalities of chromosome 7 in Egyptian AML and MDS patients using the FISH technique and whether the abnormality has an implication on the prognosis of the disease after a period of one year follow up. Fluorescence in-situ hybridization (FISH) was performed for chromosome 7 using a locus specific probe for 7q31 and a centromeric probe from 7p11.1-q11.1 in a series of 30 patients diagnosed as: AML (20 patients) and MDS (10 patients) according to the FAB criteria. Aberrations of Chromosome 7 were found in 36.6% of AML patients: 3 cases showing monosomy with a mean positivity of 17.3%, 2 cases showing 7q deletion with a mean positivity of 11%. While both monosomy and deletion were detected in 3 cases. However, in MDS patients; monosomy for chromosome 7 was the only abnormality detected and was found in 30% of cases. Genetic abnormality of chromosome 7 showed a significant association with poor prognostic criteria. Patients who had normal FISH results showed a higher percentage (31.6%) of complete remission (CR) versus 0% in patients with monosomy or deletion who showed a higher percentage (100%) of death or poor response to therapy (NR). Although AML patients had a worse prognosis when compared to MDS patients, patients with genetic abnormalities showed the worst outcome. African Journal of Health Sciences Vol. 13 (3-4) 2006: pp. 33-42

Analysis of the evolution of chromosome abnormalities in human embryos from Day 3 to 5 using CGH and FISH

The use of interphase fluorescent in situ hybridization (FISH) has shown that a large number of human embryos exhibit chromosomal abnormalities in vitro. The most common abnormality is mosaicism which is seen in up to 50% of preimplantation embryos at all stages of development. In this study, comparative genomic hybridization (CGH) was used to analyse 1-2 cells biopsied on Day 3 of development while the rest of the embryo was cultured until Day 5. Embryos were spread on Day 5 and analysed by FISH using probe combinations that varied depending on the CGH result, to investigate the progress of any abnormalities detected on Day 3. A total of 37 frozen-thawed embryos were analysed in this study. One gave no CGH or FISH results and was excluded from analysis. Six embryos failed to give any FISH result as they were degenerating on Day 5. Thirty embryos provided results from both techniques. According to the CGH results, the embryos were divided into two groups; Group 1 had a normal CGH result (13 embryos) and Group 2 an abnormal CGH result (17 embryos). For Group 1, three embryos showed normal CGH and FISH results, while 10 embryos were mosaic after FISH analysis, with various levels of abnormalities. For Group 2, FISH showed that all embryos were mosaic or completely chaotic. The combination of CGH and FISH enabled the thorough investigation of the evolution of mosaicism and of the mechanisms by which it is generated. The main two mechanisms identified were whole or partial chromosome loss and gain. These were observed in embryos examined on both Day 3 and 5.

Influence of Chromosomal Aberrations on Response to First Line Therapy in B-Cell Chronic Lymphocytic Leukemia: Interim Analysis of PALG-CLL3 Randomized Study Comparing Cladribine Plus Cyclophosphamide vs Fludarabine Plus Cyclophosphamide.

Impact of cytogenetic abnormalities on treatment with different purine nucleoside analogs in patients (pts) with B-cell chronic lymphocytic leukemia (B-CLL) is largely unknown. One of objectives of PALG-CLL3 trial, comparing cladribine plus cyclophosphamide (CC) with fludarabine plus cyclophosphamide (FC) in previously untreated progressive B-CLL, was to verify the response to treatment in subsets of pts characterized by common cytogenetic abberrations. Chromosomal abnormalities were assessed using fluorescence in situ hybridization (FISH) on interphase nuclei of lymphocytes on whole blood smears prior to the start of the study treatment. Pts were screened for trisomy 12, deletions (del) 11q, del 13q and del 17p using DNA probes: CEP12, LSI: ATM, D13S319 and p53 (Vysis), respectively. For the purpose of the present interim analysis complete cytogenetic results were available in 133 pts out of 423 pts included to the study. In this group the chromosomal aberrations were detected in 102 pts (77%) including single abnormalities observed in 69 pts (52%) and two or more aberrations in 33 pts (25%). Thirty-one pts (23%) exhibited a normal interphase FISH pattern. The most frequent single abnormality was del 13q found in 38 pts (29%), while del 17p, trisomy 12 and del 11q were identified in 14 pts (11%), 11 pts (8%), and 6 pts, (5%), respectively. The most frequently observed associations of chromosomal aberrations were: del 13q with del 11q (11 pts, 8%) and del 13q with del 17p (10 pts, 8%). Four pts (3%) revealed three chromosomal abnormalities including association of trisomy 12/del 11q/del 13q in two pts, trisomy 12/del 11q/del 17p in one pt and del 11q/del 13q/del 17p in one pt. Overall, treatment was completed and response assessed in 113 out of 133 pts with known FISH pattern. In this group of pts del 17p was the only chromosomal abnormality that correlated significantly with treatment outcome. Pts with del 17p (21, 19%) had lower probability to achieve a complete response (CR) (0.044). Interestingly, in independent analyses of both treatment arms, the negative impact of 17p was seen in pts treated with FC (p=0.002), but not in pts treated with CC (p=0.6). Moreover, comparing response rates between treatment arms we found that CC was superior to FC in terms of complete response in pts with del 17p (57% CR in CC v 14% CR in FC arm, p=0.04). In conclusion, chromosomal abnormalities can be detected in majority B-CLL pts requiring treatment. Our preliminary results suggest that CC combination may have some advantage in terms of CR achievement in B-CLL pts harboring del 17p.

Monosomy 7 Detected by FISH at Disease Presentation Is a Marker for Non-Response to Immunosuppression.

About fifteen percent of patients with severe aplastic anemia (sAA) who undergo immunosuppression therapy (IST) develop clonal disease in the decade following treatment. Of 122 patients treated with horse ATG/CsA at NIH, 13 developed cytogenetic abnormalities (monosomy 7 in 9 patients, 7p deletion in one, and trisomy 8 in 2) (Rosenfeld et al: JAMA 289: 1130; 2003). Monosomy 7 usually occurs in patients with sAA who are unresponsive to IST. Factors responsible for clonal progression in bone marrow failure are still unclear. We and others have reported that monosomy 7 may be detected by fluorescent in situ hybridization (FISH) months before conventional cytogenetics and that high levels of GCSF foster preferential expansion of the monosomy 7 clone in vitro (Sloand E et al; Proc. Natl. Acad. Sci 2006; 103:14483). We hypothesized that a clone of monsomy 7 cells might indicate an underlying stem cell disorder unlikely to respond to immunosuppression or reflect more severe disease marked by higher endogenous GCSF levels. FISH was undertaken on 40 bone marrow samples obtained from aplastic anemia patients at presentation and before immunosuppressive treatment. Bone marrow mononuclear cells were hybridized with centromeric probes specific for chromosome 7 and chromosome 8 (to control for hybridization efficiency); samples were assessed on duplicate slides by three investigators who were unaware of the diagnoses and outcomes. Twenty-five healthy controls were tested concurrently. The upper limit of normal was set at 6% based on control data. Of twenty-one patients with > 6% monosomy 7 cells, response to IST was observed in 12 (57%), while 6 of 19 (84%) with normal FISH responded to IST (p=0.08). Thirteen patients had monosomy 7 frequencies of >10%. Of these 5 (38%) responded to IST, compared with 23/27 (85%) with ≤10% monosomy 7. The proportion of monosomy 7 cells in the bone marrow correlated with age (R2 =0.6, p<0.001). Two of the 23 non-responding patients later developed clonal progression with monosomy 7 or 7q-; both had >10% monosomy 7 by FISH at presentation, but normal cytogenetics. The two responding patients tested with >6% monosomy 7 prior to IST demonstrated <2% following successful IST, suggesting that the high endogenous levels of G-CSF present before IST response may have facilitated expansion of the clone. Significant monosmy 7 clones may reflect a stem cell disorder in patients not responding to IST.