Abnormal FISH Pattern Research Articles

Abstract 3654: Identification of resistance mutations to crizotinib through circulating tumor cell (CTC) analysis in ALK-rearranged non-small-cell lung cancer (NSCLC)

Abstract Background Non-invasive methods including CTCs are crucial to develop for the implementation of precision medicine in the treatment of NSCLC. We recently reported how CTCs with abnormal ALK FISH-patterns monitored on treatment can stratify patients at risk of early-resistance to crizotinib (Pailler et al., Cancer Res, 2017). ALK-rearranged NSCLC patients treated with crizotinib develop resistance which manifest by the upregulation of bypass signaling pathways in two-thirds of cases. Here, we evaluated whether individual CTCs could identify resistance mutations to crizotinib and represent tumor diversity compared to single-site tumor-biopsies. Methods Individual CTCs were isolated from seven ALK-rearranged patients at resistance to crizotinib. Matched tumor-biopsies were available for three. Two isolation strategies were used: (i) ISET-filtration, immunofluorescence (Hoescht/CD45/epithelial-markers/mesenchymal-marker), scanning, laser-microdissection; (ii) RosetteSep, immunofluorescence (Hoescht/CD45/ALK/cytokeratins), cell-sorting. A process including Ampli1 whole-genome amplification, quality controls, multiplex PCR with two panels (Ampli1 CHPCustomBeta cancer panel developed by Silicon Biosystems and a home-made panel targeting all ALK mutations) and Ion Torrent next-generation sequencing (NGS) was established. Ten to 17 pools with 1-5 CTCs and one CD45+ cell pool were analyzed for each patient. A bioinformatic workflow was developed including determination of allele drop-out (ADO), false-positive rate (FPR) and positive predicted value (PPV). Results By comparing germline variants present in constitutional and CD45+ pool DNA, we calculated for each sample the statistics of mean ADO, FPR and PPV which were respectively 19%, 2.6x10-4 and 69%. When variants were present into two pools, the PPV and FPR were 96% and 2.39 10-5 respectively. In the three patients with an available tumor-biopsy, a limited number of shared mutations between CTC and matched tumor-biopsies were identified. In contrast, CTC-private mutations (exclusively present in CTCs and not in matched biopsies) present in at least two CTC pools were identified including in FLT3, SMAD4, KIT, PI3KCA, EGFR, KRAS, PDGFRA genes among which several were COSMIC mutations. A higher degree of mutational diversity was observed in CTCs compared to tumor-biopsies. Conclusion Using a rigorously qualified workflow, we reported for the first time the identification of resistance mutations in individually isolated CTCs of ALK-rearranged patients. In accordance with published series from tumor-biopsies, our CTC results highlighted the role of bypass signaling pathways in resistance to crizotinib. Our study provides important perspectives on using CTCs for precision medicine and informing on tumor heterogeneity in NSCLC patients. Citation Format: Emma Pailler, Vincent Faugeroux, Marianne Oulhen, Claudio Forcato, Mélanie Laporte, Yann Lecluse, Ludovic Lacroix, Maud NgoCamus, Claudio Nicotra, Jordi Remon, Laura Mezquita, David Planchard, Jean-Charles Soria, Nicolò Manaresi, Benjamin Besse, Françoise Farace. Identification of resistance mutations to crizotinib through circulating tumor cell (CTC) analysis in ALK-rearranged non-small-cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3654.

Rearrangements of Tyrosine Kinase (TK) Genes in Chronic Myeloproliferative Disorders (CMPD): A FISH Study

CMPDs are heterogeneous hematopoietic stem cell disorders caused by the constitutive activation of specific TK genes induced by mutations or, exceptionally, chromosomal translocations that often escape conventional cytogenetics (CC) identification as they often involve poorly stained regions. The demonstration of these TK gene rearrangements is twofold relevant: diagnostically, as these translocations identify specific entities within the 2016 WHO classification of CMPDs and clinically, as most TK can be targeted with specific TK inhibitors.Thus, this study employed FISH probes specific for TK genes to establish the incidence of these chromosomal translocations, to identify uncommon TK translocation partners, to establish whether eosinophils are part of the clonal cell population and to find any potential correlation with clinical parameters and outcome. From January 2005-December 2015 43 consecutive patients (pts) were analysed; 13 females and 31 males with a median age of 47 years (range 22-72). According to WHO classification, 20 pts were diagnosed as atypical chronic myeloid leukemia (aCML), 22 as chronic eosinophilic leukemia (CEL) and one as AML/T lymphoblastic lymphoma (T-LL). Median follow-up was 39 months (range 8-136). At the time of the study one pt died and one experienced disease progression. FISH probes were obtained from Kreatech (Amsterdam, NL), Abbot Molecular Inc. (Chicago, Il, USA) and from BACPAC Resources Center at C.H.O.R.I. (Oakland, USA) after determining their Mb position using UCSC genome browser on Human Feb. 2009 assembly. The commercial probes, applied according to manufacturer's guidelines were: ON FIP1L1-CHIC2-PDGFRA (4q12) Del, Break; ON PDGFRB (5q33) Break; ON FGFR1 (8p12) Break; ON JAK2 (9p24) Break; LSI BCRABL. The BAC probes RP11-484L21 and RP11-880I16 covering the PCM1 gene were labelled and applied as previously described. i-FISH, cut-off values were obtained from the analysis of 300 nuclei from ten normal samples and were fixed at 10%.An abnormal FISH pattern was revealed in 12 pts (27.9%): 5/20 (25%) with aCML, 6/22 (27.2%) with CEL and one with AML/T-LL. Two aCML pts presented a trisomy 8, one a t(9;13)(q34;q14) which involved the ABL gene and a not yet identified partner gene, one at(9;22)(p24;q11) which involved the ABL and BCR genes and one a t(8;22)(p11;q11) which involved the FGFR1 gene. Interestingly, after one month this last pt progressed to AML and on CC showed a duplication of the derivative chromosome 8. Two CEL pts showed a JAK2 rearrangement: one who carried a t(8;9)(p22;p24) on CC displayed the classical PCM1-JAK2 gene fusion, the other who carried a t(3;8)(?;p24) not revealed by CC harboured a fusion between the JAK2 gene and a not yet identified partner. Three additional CEL pts showed a PDGFRB rearrangement which escaped CC detection too. In these pts who on CC showed a t(1;5)(?;q33) with loss of the reciprocal translocation product, a t(5;8)(q33;?) and a t(5;12)(q33;?), the PDGFRB translocation partner has been not yet identified. The last chromosomally normal CEL pt showed a PDGFRA deletion. Thus, in 4 pts FISH with BAC probes is still on-going in order to search the unknown translocation partners of the JAK2 and PDGFRB genes. Noteworthy, despite the fact that all these pts presented a relevant peripheral eosinophilia (≈65%), FISH performed on peripheral blood smears always revealed a normal pattern. The sole AML/T-LL pt who carried a t(8;13)(p11;q12) which produced the classical FGFR1-ZNF198 gene fusion failed to respond to conventional chemotherapy and died of disease related complications. From a clinical point of view 3 aCML/CEL pts with TK rearrangements responded to TK inhibitors experiencing a haematological improvement including one complete remission (CR); the t(8;22) positive pt entered CR after induction chemotherapy.In conclusion, i) FISH effectively reveals cryptic TK translocations in about 36% of chromosomally normal CMPDs; ii) these rearrangements are more common in CELs than in aCML; iii) peripheral blood eosinophils may show a normal FISH pattern; iv) FISH can effectively be used to monitor the clonal cell population during disease outcome. DisclosuresNo relevant conflicts of interest to declare.

Abstract 856: Number of ALK-amplified circulating tumor cells predicts progression-free survival in ALK-rearranged non-small cell lung cancer patients treated by crizotinib

Abstract The duration of clinical response is unpredictable in ALK-rearranged NSCLC patients treated by crizotinib but all patients invariably develop resistance. Using filter-adapted fluorescence in situ hybridization (FA-FISH) we previously reported ALK-rearrangement detection in circulating tumor cells (CTCs) from ALK-rearranged patients. Here we report the monitoring of ALK-rearranged (≥1×3’/5’, ≥1x(3’ and 5’)) and ALK-amplified (>2×3’/5’) CTCs at baseline and at an early time point under crizotinib therapy in an extended cohort of ALK-rearranged patients. The correlation between CTC subsets harboring distinct FISH patterns and clinical parameters including progression-free survival (PFS) and overall survival (OS) is presented. Forty ALK-rearranged patients were recruited. Blood samples were collected at baseline to crizotinib and at 1-3 months. Abnormal ALK-FISH patterns were examined in CTCs using immunofluorescence staining (DAPI/CD45) combined with FA-FISH after isolation by size of epithelial tumor cells (ISET) filtration. CTCs were defined into five distinct subsets according to the number of FISH ‘break-apart’ signals (3’ and 5’) or isolated red signals (3’) and/or native copies (3’/5’). All ALK-abnormal cells were validated by a cytogenetician. Clinical data was collected retrospectively. Confirming our previous data, ALK rearrangement was detected in CTCs: 32/39 patients (82%) had ≥4 ALK-rearranged CTCs per 1 ml of blood. Age >60 years and smoking status >15 pack years were associated with a higher mean number of ALK-amplified CTCs (p = 0.0423 and p = 0.0407 respectively). No statistically significant correlation was observed between the different CTC subsets with abnormal FISH patterns at baseline and PFS or OS. However we observed a statistically significant correlation (p = 0.0196) between the evolution under crizotinib of the numbers of ALK-amplified CTCs and PFS. An increase in the numbers of ALK-amplified CTCs during treatment is associated with a PFS of 6.5 months, while a decrease in this subset is associated with a PFS of 25 months. There was no corresponding correlation with OS. The evolution under crizotinib therapy of numbers of ALK-amplified CTCs is significantly correlated with PFS. Although not dominant, amplification of the ALK gene has been reported to be one mechanism of acquired resistance to crizotinib therapy in tumor biopsies. CTCs with an amplification of ALK do not express the oncogenic ALK fusion protein and are therefore not targeted by crizotinib. FISH patterns suggested these CTCs may have a high degree of ploidy and chromosomal instability which may contribute to promote the emergence of CTC subclones with a high metastatic potential. Our data suggest that number of ALK-amplified CTCs may be a predictive biomarker allowing prediction of ALK-rearranged NSCLC patients who are at risk of early resistance to crizotinib. Citation Format: Emma Pailler, Marianne Oulhen, Kirsty Ross, Fanny Billiot, Nathalie Auger, Isabelle Borger, Maud NgoCamus, Jordi Remon-Masip, David Planchard, Jean-Charles Soria, Benjamin Besse, Françoise Farace. Number of ALK-amplified circulating tumor cells predicts progression-free survival in ALK-rearranged non-small cell lung cancer patients treated by crizotinib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 856.

An evaluation of SOX2 and hTERC gene amplifications as screening markers in oral and oropharyngeal squamous cell carcinomas

BackgroundOral and oropharyngeal squamous cell carcinomas (OSCC) are among the most common cancers. The poor survival rate among oral cancer patients can be attributed to several factors, one of them being lack of early detection. A key approach to this problem would be to detect potentially malignant lesion at their early stage. Using the FISH technique, oral brush cytology slides can be an easy and rapid screening approach for malignant cell detection. The present study was designed to detect hTERC and SOX2 amplifications in OSSC exfoliative tumor cells and evaluate whether those two gene amplifications might serve as a supportive biomarker in early detection and diagnosis of oral and oropharyngeal SCC.ResultsBrush biopsies were collected from exophytic and exulcerated oral and oropharyngeal lesions of the oral cavity of 71 patients and 22 healthy controls. FISH techniques using a TERC-specific DNA probe and a SOX2 DNA specific probe both combined with a centromere 3-specific control probe was performed on the cytology slides. A 100 squamous epithelial cell nuclei of the smears per slide were analysed. As abnormal FISH pattern were considered amplified and polyploid patterns.From 71 brush biopsies of oropharynx and other locations in oral cavity analysed by FISH 49 were considered to be abnormal (69%). The over representation of polyploidy and/or TERC/SOX2 amplification in tumour samples was statistically significant when compared to controls (p = 0.01).ConclusionSOX2 and TERC gene amplifications are common in all squamous cell carcinomas and their detection in early stages could be crucial for early detection and more accurate prognosis. Our study strongly suggests that early detection by FISH on cytobrushed samples could be a possible non-invasive screening method even before a tissue biopsy is performed.

Do Unexpected and Cryptic FISH Lesions Of Chromosomally Normal MDS Patients Have Any Prognostic Relevance?

Conventional cytogenetic (CC) still remains a mandatory step in the routine diagnostic work-up of every MDS patient (pt), is one of the major determinant of disease outcome and guides potential treatment decisions. However, CC is not informative in about 50% of chromosomally normal (CN) pts and provides limited information in those with very rare defects even if the revised IPSS cytogenetic categories have tried to overcome this drawback. More sensitive techniques (aCGH, SNP-a and NGS), still used in the research setting only, suggest that CN pts may instead contain novel unexpected chromosomal lesions which prognosis is still undefined.Thus, the principal goal of our study was to establish whether FISH with disease specific probes (i.e. for chromosomal regions most commonly affected in MDS) along with non-disease specific probes (i.e. for regions which alteration in MDS has been demonstrated by aCGH only) may effectively unmask clonal cryptic defects. Other aims were to establish the nature of these defects, to identify the potentially targeted genes and to estimate their possible prognostic relevance.The one-hundred twenty-seven consecutive CN MDS pts of the present study came to our observation in the period January 2003-December 2012. They were forty-nine females and seventy-eight males, median age 66 years (range 24-88). Twenty-one pts were diagnosed as RARS, 29 as RA, one as CRMDS, one as U-MDS, 25 as RCMD, 26 as RAEB-1 and 24 as RAEB-2. On CC 122 pts presented a normal karyotype and five no mitotic figures. Considering the revised IPSS score, 62 pts were considered very low-risk, 32 low-risk, 23 intermediate risk, 8 high-risk and 2 very high-risk. Median follow-up was 22 months (range 1-90). At the time of the study nine pts have died. FISH probes were chosen based on the frequency of their involvement in MDS and their Mb position determined using UCSC genome browser on Human Mar. 2003 assembly. They were obtained from BACPAC Resources Center at C.H.O.R.I. (Oakland, USA), labelled and applied as previously described. These probes were: RP11-912D8 (19q13.2); RP11-196P12 (17q11.2); RP11-269C4 (14q12); RP11-351O1 (10q21.3); RP11-144G6 (10q11.2); RP11-122A11 (7q34); RP11-951K18 (5q13.1); RP11-101K5 (4p14); RP11-544H14 (2q33). i-FISH cut-off values were fixed at 10%.Thirty-one pts (24.4%) presented at least a single defect, always represented by deletions or gains of chromosomal material. Among them 8 pts (25.8%) presented at least two defects. Bands most commonly targeted by deletions/amplifications were 19q13.2 (61.3%), 14q12 (32.2%), 17q11.2 (16.1%), 5q13.1 (12.9%), 7q34 (12.9%), 4p14 (9.6%). Deletions of bands 10q11.2, 10q21.3 and 2p33 were more rare. As the RMD-1 gene, involved in DNA double strand breaks and homologous recombination, maps at band 19q13.2, the most commonly deleted chromosomal area, additional molecular tests are being developed to analyse this gene. An abnormal FISH pattern was observed in 2/21 (9.5%) RARS, in 7/29 (24.1%) RA, in 5/25 (20.0%) RCMD, in 8/26 (30.6%) RAEB-1 and in 9/24 (37.5%) RAEB-2. Considering IPSS, an abnormal FISH pattern was revealed in 7/62 (11.3%) very low-risk, in 8/32 (25%) low-risk, in 10/23 (43.4%) intermediate risk, in 5/8 (62.5%) high-risk and in 1/2 very high-risk patients. Disease evolution occurred in a total of 34 pts (3 RARS, 7 RA, 5 CRMD, 11 RAEB-1 and 8 RAEB-2), 16 (one RARS, 3 RA, 2 CRMD, 6 RAEB-1 and 4 RAEB-2) with an abnormal FISH pattern. All the 8 patients with at least two chromosomal deletions experienced disease progression.In conclusion, i) FISH reveals novel unexpected karyotype defects, most commonly deletions pinpointing genes involved in DNA repair, in about 24.4% of CN MDS; ii) band 19q13.2 deletion is the most common defect, frequently associated with disease evolution; ii) an abnormal FISH pattern is correlated with an advanced disease stage and an intermediate/high revised IPSS score; iii) >two lesions are associated with an increased risk of disease progression. Disclosures:No relevant conflicts of interest to declare.

ALK Rearrangements Are Mutually Exclusive with Mutations in EGFR or KRAS: An Analysis of 1,683 Patients with Non–Small Cell Lung Cancer

Anaplastic lymphoma kinase (ALK) gene rearrangements define a distinct molecular subset of non-small cell lung cancer (NSCLC). Recently, several case reports and small series have reported that ALK rearrangements can overlap with other oncogenic drivers in NSCLC in crizotinib-naïve and crizotinib-resistant cancers. We reviewed clinical genotyping data from 1,683 patients with NSCLC and investigated the prevalence of concomitant EGFR or KRAS mutations among patients with ALK-positive NSCLC. We also examined biopsy specimens from 34 patients with ALK-positive NSCLC after the development of resistance to crizotinib. Screening identified 301 (17.8%) EGFR mutations, 465 (27.6%) KRAS mutations, and 75 (4.4%) ALK rearrangements. EGFR mutations and ALK rearrangements were mutually exclusive. Four patients with KRAS mutations were found to have abnormal ALK FISH patterns, most commonly involving isolated 5' green probes. Sufficient tissue was available for confirmatory ALK immunohistochemistry in 3 cases, all of which were negative for ALK expression. Among patients with ALK-positive NSCLC who acquired resistance to crizotinib, repeat biopsy specimens were ALK FISH positive in 29 of 29 (100%) cases. Secondary mutations in the ALK kinase domain and ALK gene amplification were observed in 7 of 34 (20.6%) and 3 of 29 (10.3%) cases, respectively. No EGFR or KRAS mutations were identified among any of the 25 crizotinib-resistant, ALK-positive patients with sufficient tissue for testing. Functional ALK rearrangements were mutually exclusive with EGFR and KRAS mutations in a large Western patient population. This lack of overlap was also observed in ALK-positive cancers with acquired resistance to crizotinib.

A retrospective analysis of the prevalence of EGFR or KRAS mutations in patients (pts) with crizotinib-naïve and crizotinib-resistant, ALK-positive non-small cell lung cancer (NSCLC).

8083 Background: Anaplastic lymphoma kinase (ALK) gene rearrangements define a distinct molecular subset of NSCLC. Recently, several studies have reported that ALK+ pts occasionally harbor concomitant mutations in other oncogenic drivers. Methods: We retrospectively analyzed tumor genotyping data from 1,683 pts with NSCLC seen at 3 U.S. centers from 2009 – 2012 to determine rates of overlapping alterations in EGFR, KRAS and ALK. Mutations in EGFR and KRAS were mainly identified using the SNaPshot multiplexed assay (>95% of cases). ALK FISH was performed in all cases. To determine if this prevalence is impacted by crizotinib, we also updated our earlier analysis (Katayama et al., Sci Transl Med, 2012) of a series of repeat biopsy specimens from 34 crizotinib-resistant, ALK+ pts. Resistant specimens were examined using ALK FISH, SNaPshot, and direct sequencing of the ALK tyrosine kinase domain (TKD). Results: Screening identified 301 (17.8%) EGFR mutations, 465 (27.6%) KRAS mutations, and 75 (4.4%) ALK rearrangements. EGFR mutations and ALK rearrangements were mutually exclusive. 4 pts with KRAS mutations also had abnormal ALK FISH patterns, involving isolated 5’ green probes (3/4 cases) and an isolated 3’ red probe that was unusually small (1/4 cases). Sufficient tissue was available for confirmatory ALK immunohistochemistry (clone 5A4, Novacastra, UK) in 3 of these cases, all of which were negative for ALK expression. Among pts with ALK+ NSCLC and acquired crizotinib resistance, repeat biopsy specimens remained ALK fusion positive in 28/28 (100%) cases. Secondary mutations in the ALK TKD (1151Tins, L1196M, G1202R, S1206Y, and G1269A) were identified in 7/34 (20.6%) cases. L1196M was the most common secondary mutation (3/34, 8.8% cases). ALK gene amplification was present in 3/28 (10.71%) pts. No EGFR or KRAS mutations were identified in 23 crizotinib-resistant, ALK+ pts with sufficient tissue for testing. Conclusions: Functional ALK rearrangements were mutually exclusive with EGFR and KRAS mutations in a large Western patient population. This lack of overlap was also observed in ALK+ pts with acquired resistance to crizotinib.

Do FISH Lesions Have Any Role in the Pathogenesis and Outcome of Chromosomally Normal MDS?

Abstract 1472 Background:The chromosomal pattern is one of the most relevant parameters to make an accurate diagnosis and predict disease outcome of MDS patients. However, in about 40–50% of patients, mostly low-risk MDS, conventional cytogenetics (CC) does not reveal any defect and thus it is not informative. In this MDS subset FISH with specific probes may identify clonal defects and improve CC results. In addition, recent aCGH data suggest that these patients may present novel lesions affecting unsuspected chromosomal regions harbouring genes with a crucial role in MDS pathogenesis. Objectives:Based on these findings the principal goal of the present study was to establish whether FISH with probes specific for chromosomal regions most commonly involved in MDS along with additional probes exploring chromosomal areas which alteration has been revealed by recent aCGH studies was truly able to unmask clonal cryptic defects in chromosomally normal MDS. Additional aims were to establish whether these defects consisted in either gains/losses or balanced rearrangements, to identify the potential affected genes and to evaluate any possible influence on OS and progression free interval (PFI). Methods:The ninety-eight consecutive chromosomally normal MDS patients analysed in the present study came to our observation in the period January 2005-December 2010. They were thirty-seven females and sixty-one males, median age 64 years (range 22–77). Eighteen patients were classified as RARS, 27 as RA, one as CRMDS, 15 as RCMD, 16 as RAEB-1 and 21 as RAEB-2. Considering IPSS score, 38 patients were considered low-risk, 36 intermediate-1 risk and 18 intermediate-2 risk and 6 as high-risk. Median follow-up was 33 months (range 1–84). At the time of the study four patient have died. FISH probes were chosen based on the frequency of their involvement in MDS and their Mb position determined using UCSC genome browser on Human Mar. 2003 assembly. They were obtained from BACPAC Resources Center at C.H.O.R.I. (Oakland, USA), labelled and applied as previously described. The probes applied were the followings: RP11-912D8 (19q13.2); RP11-196P12 (17q11.2); RP11-269C4 (14q12); RP11-351O1 (10q21.3); RP11-144G6 (10q11.2); RP11-122A11 (7q34); RP11-951K18 (5q13.1); RP11-101K5 (4p14); RP11-544H14 (2q33). i-FISH cut-off values were fixed at 10%. Results:An abnormal FISH pattern was revealed in 30 patients (33.7%). A single defect was revealed in 31 patients (31.6%) and more than two defects in 8 (26.7). Twenty-two patients (22.4%) presented a single defect, whereas 9 (9.1%) more than two defects. Band 19q13.2, 14q12, 4p14, 5q13.1, 7q34, 17q11.2, 7q22, 10q11.2, 10q21.3 and 2p33 deletions had an incidence of 54.8%, 25.8%, 16.1%, 12.9%, 12.9%, 12.9%, 9.6%, 6.4% and 3.2%. The RP11-196P12 covers the RDM-1 gene which encodes for a motif found in the RAD52 protein involved in DNA double strand breaks and homologous recombination and the RP11-144G6 covers the ANXA8L1 gene over-expressed in AML. Thus, these two genes are now analysed with additional molecular tests in order to check whether they may be affected by mutations. An abnormal FISH pattern was observed in 4/18 (22.2%) RARS, in 5/27 (18.5%) RA, in 4/15 (26.6%) RCMD, in 6/16 (37.5%) RAEB-1 and in 12/21 (57.1%) RAEB-2. Considering IPSS, an abnormal FISH pattern was revealed in 7/38 (18.4%) low-risk, in 12/36 (33.3%) intermediate-1 risk, in 9/18 (50%) intermediate-2 risk and in 3/6 (50%) high-risk patients. Disease evolution occurred in a total of 21 patients (2 RARS, 5RA, 6 RAEB-1 and 7 RAEB-2), 11 (one RARS, 2 RA, 5 RAEB-1 and 2 RAEB-2) presented an abnormal FISH pattern. At least two chromosomal deletions were observed in 7/11 patients. Conclusions:i) FISH reveals novel not expected karyotype defects, mostly deletions, in about 32% of chromosomally normal MDS; ii) some deletions pinpoint genes involved in DNA repair; iii) an abnormal FISH pattern correlates with advanced and int-2, high-risk MDS; iii) the presence of more than two lesions seems to associate with an increased risk of disease progression. Disclosures:No relevant conflicts of interest to declare.

FISH Scoring for CLL: Comparison of Methods That Assess Round Versus Non-Round Nuclei,

Abstract 3538 Background:Cytogenetics detection of common recurring genetic defects by FISH is an important clinical prognostication tool in CLL and is frequently used by clinicians for counseling and risk stratification. Thus, any improvement in FISH technology is a welcome advance in our care for CLL patients. Mayo Clinic CLL FISH analyses are prepared directly from buffy coat samples rather than mononuclear cells or lymphocytes. FISH probes (deletion 6q, 11q, 13q, 17p, +12 & IGH/CCND1) are scored among consecutive nuclei without selection for nuclear morphology (so called clinical scoring). CLL B-cells are observed, microscopically, primarily as round lymphocytes with round nuclei, and we found (ASH 2010, #3606) that the % abnormal nuclei in the CLL round cell population is significantly higher than that of the non-round nuclei population. In 79 of 87 (91%) patients, the % abnormal nuclei was greater in round vs unselected nuclei (mean difference 24%; range 1.5 – 57%). Round nuclei (p = 0.004) correlated better with % B-cells by flow cytometry than either consecutive unselected nuclei (p = 0.046) or the lobed (non-round) nuclei (p = 0.40). Utilizing this “Poor Man’s” sorting technique, we proposed that scoring consecutive round nuclei would improve sensitivity of detection of CLL FISH defects in patients, especially those with low B-cell counts. In a retrospective study of Mayo Clinic patients with normal baseline FISH who had a FISH defect in follow-up, we showed that by re-scoring 28 baseline cases for round nuclei exclusively, 12 originally normal samples remained normal but 16 (57%) were abnormal using the “Poor Man’s” scoring technique. In the present study, we prospectively compared FISH scoring on unsorted samples using the “Poor Man’s” technique vs the clinical technique. Methods:After IRB approval, we collected 63 consecutive unsorted Mayo Clinic patient blood samples referred for CLL FISH testing with an absolute lymphocyte count (ALC)< 30K × 109/L, and scored each sample using both the “Poor Man’s” round nuclei method and the standard clinical method. The kappa statistic was used to adjust for chance agreement. Bland Altman plots of difference (“Poor Man’s” result minus clinical result) vs the average of the two methods were generated. Results:Median age of the 40 males (64%) and 23 females (36%) was 66 years (range 36–87), and median ALC was 6.803 (range 0.980–29.750). The most common FISH defects were 11q-, trisomy 12 & 13q- by clinical technique (14%, 22% & 46% respectively) as well as by “Poor Man’s” method (16%, 24% & 49% respectively). A high degree of agreement was observed between the two methods when positive/negative comparisons were made (p<0.0001). Additional FISH defects were observed in 12 of 63 (19%) patients (one 11q-, one +12; six 13q-, and four 17p-) when we utilized the “Poor Man’s” method that were not observed by the clinical method. Discussion:The “Poor Man’s” sorting method compares well with the clinical method for the presence and absence of FISH defects. The abnormal CLL FISH patterns are truly confined to round nuclei. The “Poor Man’s” method is more sensitive than the clinical method, resulting in higher reported abnormal cells and detection of more defects. Importantly, additional FISH defects were identified in 12 patients via “Poor Man’s” method that were not observed by clinical technique and 6 of these involved a change in the patients’ hierarchical (Döhner) classification due to detection of the more unfavorable prognostic markers of trisomy 12 or deletion 11q or 17p. Thus, selectively scoring FISH in the round nuclei increases the sensitivity for detection of a genetic defect in CLL and provides a practical framework for prognostication in CLL patients with low lymphocyte counts and, we would speculate, including those in clinical remission or with minimal residual disease. Disclosures:Kay:Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity's Board of Directors or advisory committees.

4.16 Identification of Minor Proportion p53 Mutations Selected Later by Therapy

fixed cell pellet) were re-scored for % abnormal nuclei among 200 consecutive round nuclei. Scores were compared to the original clinical FISH analyses (200 consecutive nuclei not selected for nuclear morphology). Results: After re-scoring by the round cell approach, 12 originally normal samples remained normal while 16 (57%) were abnormal. The average number of days between the diagnostic and follow-up sample was 826 for those that remained normal and 601 for those abnormal by scoring round cells only. In one case, 12 had originally been scored in 0.5% but round cell approach showed 10% 12. Another 2 previously normal cases with the unusual combination of 11qand 17pbecame abnormal by the round cell approach. Among previously normal samples, 3 were abnormal for multiple FISH probes by the round cell approach and 13 were abnormal for 1 FISH probe. Discussion: The present experiment shows, as in our past study, that abnormal CLL FISH patterns are confined to round nuclei. Thus, utilization of the round cell approach for scoring FISH in CLL increases the sensitivity to detect a genetic defect. We believe restricting CLL FISH panel scoring to the leukemic B-cell population will be a powerful adjunct to cytogenetic analysis for CLL patients with low lymphocyte counts, including those in clinical remission or with minimal residual disease.

FISH Redoux — A Novel Way to Emulate CLL Leukemic Cells From Buffy Coat Samples

AbstractAbstract 3606 Background:Cytogenetic abnormalities are important prognostic indicators in CLL. At Mayo Clinic, CLL FISH analyses are derived from buffy coat samples not purified for lymphocytes. Scoring is therefore performed on consecutive qualifying nuclei regardless of cell size, shape or morphology. This scoring may not reflect the percent of abnormal nuclei of the affected cell type, but rather the entire population of cells present in the sample. We sought to determine whether the percent abnormal nuclei in only the affected cell type (e.g. lymphocytes) of patients with CLL differed from that of the general cell population. Scoring only B-lymphocytes could increase the sensitivity of the test in patients with low B-cell counts, either early in their disease or after treatment. Cell sorting techniques could be used to reach this goal but can be expensive, labor intensive and add to completion time. We propose a “Poor Man's Cell Sorting” technique based on cell morphology when stained with 4',6-diamidino-2-phenylindole (DAPI). In CLL samples, both round and so called “lobed” cells are seen by DAPI staining but we hypothesize that the CLL B-cells typically present as perfectly round in shape (Fig. 1). Thus our hypothesis: if only the round cells are scored for a genetic defect, would this more accurately represent the malignant leukemic B-cell population and allow for enhanced disease status by FISH? [Display omitted] Methods:After IRB approval, we identified 87 CLL patients (75% male; 25% female, mean age 62.4 y, range 43–89 y) in the Mayo Clinic CLL Database, who were seen at Mayo Clinic between March, 2002 - July, 2010. We selected patients known to have low level FISH abnormalities and who therefore should have a significant population of both lymphocytes and neutrophils. Selection criteria included <20×106 B-cells per microliter (by flow cytometry) of peripheral blood and ≤40% of interphase nuclei expressing a specific FISH abnormality. Although most of the 87 patients exhibited more than one FISH abnormality, we focused on only one FISH defect per patient for this study. The original specimen (slide or fixed cell pellet) was rescored for % abnormal nuclei among 100 consecutive round and 100 consecutive lobed (or multilobular) nuclei, using DAPI to identify nuclear architecture. These scores were compared to each other and to the original clinical FISH analysis (scored for 200 consecutive nuclei and not selected for nuclear morphology). Results:Among 87 cases, FISH signals were scored for 6q-(1), 11q-(9), +12(15), 13q-(58), 17p-(2) and 2 IGH rearrangements. In 86/87 cases, the abnormal lobed nuclei did not have FISH defects. One patient exhibited 35% +12 by clinical assay, 52% +12 in the round nuclei, and 11% by lobed nuclei. For all cases, the mean percentage of abnormal nuclei was greater in the round cells (46%) vs original scoring (23.6%). In 79/87 patients (91%), the % of abnormal nuclei was greater in the round cells vs original FISH (mean increase 24.3%; range 1.5–57%). One patient's score was the same in round cells vs original, and 7 patients exhibited fewer abnormal nuclei in the round cells vs original. For these 7 cases, the mean % abnormal nuclei was 5.6% for round nuclei vs 8.4% for original score (score differences ranged from 1 – 5.5%). Overall, by univariate regression analysis, round cells (p=0.0043) have a better correlation with % B-cells, ascertained by flow cytometry, than either the current clinical approach (p=0.0462) or the lobed-cell approach (p=0.4058). Discussion: In virtually all cases (99%), the abnormal FISH patterns were confined to the round nuclei, and the lobed nuclei were virtually always normal by FISH. For patients with <20×106 lymphocytes, selecting for round nuclei uniformly resulted in identification of a higher percentage of abnormal cells. Further studies, including comparison of our round nucleus approach to CLL FISH analysis to actual sorted B-lymphocyte cell selection and the association of this leukemic restricted estimation of abnormal FISH levels to clinical outcome are necessary. Given our findings, we believe that estimating FISH defects restricted to the leukemic B-cell population will become an important adjunct to cytogenetic analysis for patients with low lymphocyte counts, including those in clinical remission and those with minimal residual disease. Disclosures:No relevant conflicts of interest to declare.

FISH Reveals Abnormalities of Unsuspected Regions in Chromosomally Normal De Novo Myelodysplastic Syndromes (MDS).

Abstract 2599Poster Board II-575In de novo MDS the chromosomal pattern is a mandatory step for an accurate diagnosis, predicts overall survival (OS) and the risk of MDS/AML evolution, guides therapeutic decisions. However, conventional cytogenetics (CC) studies show a normal un-informative chromosomal pattern in about half of MDS patients, especially in low-risk disease. FISH with probes pinpointing the chromosomal regions most frequently affected in MDS can increase the incidence of abnormal karyotypes up to 60%, but the percentage of normal karyotypes remains high and makes the search of novel cytogenetic/molecular markers a urgent need. A fundamental contribution to overcome CC and FISH shortcomings, has been recently provided by array CGH (aCGH) studies which have revealed that, independently of the cytogenetic pattern, MDS patients may harbour novel abnormalities involving unsuspected chromosomal regions.Based on this assumption, we decided to investigate whether FISH with probes already employed in aCGH studies can truly unmask cryptic lesions in chromosomally normal MDS patients, whether these defects are either chromosomal gains/losses or balanced rearrangements and whether these chromosomal abnormalities influence OS and disease evolution. FISH analyses were carried out in thirty-five patients examined between January 2005 and June 2008. There were thirteen females and twenty-two males, whose median age was 66 years (range 24–78). According to WHO classification, 6 patients were classified as RA, 13 as RAEB-1 and 16 as RAEB-2. According to IPSS score, 7 patients were considered low-risk, 14 intermediate-1 risk and 14 intermediate-2 risk. Median follow-up was nine months (range 1–46). At the time of the analyses no patients has died; 6 have progressed to RAEB-2 and 3 to AML. Probes for FISH analysis were chosen following two criteria: the frequency of their involvement in chromosomal abnormalities identified by aCGH studies and their Mb position on Human Mar. 2003 assembly according to the UCSC genome browser. All probes, obtained from BACPAC Resources Center at C.H.O.R.I. (Oakland, USA), were labelled and applied as previously reported. The following probes were applied: RP11-912d8 (19q13.2); RP11-196p12 (17q11.2); RP11-269c4 (14q12); RP11-351o1 (10q21.3); RP11-144g6 (10q11.2); RP11-122a11 (7q34); RP11-951k18 (5q13.1); RP11-100m20 (4p14); RP11-544h14 (2q33). The cut-off values for interphase FISH (i-FISH) were obtained from the analysis of 300 nuclei from ten normal samples and were fixed at 10%. An abnormal FISH pattern was revealed in eighteen patients (51.4%). It was observed in 3/6 RA patients, in 7/13 RAEB-1 and in 8/16 RAEB-2 and in 2/7 IPSS low-risk, in 7/14 intermediate-1 risk and in 9/14 intermediate-2 risk MDS patients. Seven presented a 19q13.2 deletion, three a 14q12 deletion, four an amplification of band 4p14, two a defect of band 10q21.3, two a potential amplification and one a deletion of band 10q11.2, two a deletion of band 5q13.1 and one a deletion of band 17q11.2. Cryptic defects were also revealed in six of the nine patients who experienced disease evolution on FISH analyses. This event occurred in 2/3 RA, in 2/7 RAEB-1 and in 2/8 RAEB-2 patients with an abnormal FISH pattern. Despite these data, the prognostic significance of an abnormal FISH pattern needs to be assessed on additional patients.In conclusion, our data show that i) FISH can truly reveal novel lesions involving unsuspected chromosomal regions in 51% of MDS patients with a normal karyotype; ii) most of these lesions consist of chromosomal gains/losses; iii) an abnormal FISH pattern seems to correlate with disease progression, but this correlation needs to be tested on additional patients. Disclosures:No relevant conflicts of interest to declare.

Exploring polycythaemia vera with fluorescence in situ hybridization: additional cryptic 9p is the most frequent abnormality detected.

Between 1986 and 2001, 220 patients with polycythaemia vera (PV) were studied using conventional cytogenetics. Of 204 evaluable patients, 52 (25.4%) had clonal abnormalities. The recurrent chromosomal rearrangements were those of chromosome 9 (21.1%), del(20q) (19.2%), trisomy 8 (19.2%), rearrangements of 13q (13.4%), abnormalities of 1q (11.5%), and of chromosomes 5 and 7 (9.6%). Subsequent analysis of 32 patients, performed at follow-up of up to 14.8 years, revealed new clonal abnormalities in five patients and the disappearance of an abnormal clone in four. Eleven patients remained normal up to 11.5 years and seven patients maintained an abnormality for over 10 years. Fifty-three patients were studied retrospectively using interphase fluorescence in situ hybridization (I-FISH), utilizing probes for centromere enumeration of chromosomes 8 and 9, and for 13q14 and 20q12 loci. Conventional cytogenetics demonstrated clonal chromosome abnormalities in 23% of these 53 patients. The addition of I-FISH increased the detection of abnormalities to 29% and permitted clarification of chromosome 9 rearrangements in an additional 5.6% of patients. FISH uncovered rearrangements of chromosome 9 in 53% of patients with an abnormal FISH pattern, which represented the most frequent genomic alteration in this series.