Centromeric Probes Research Articles

Pseudoautosomal abnormalities in terminal AZFb+c deletions are associated with isochromosomes Yp and may lead to abnormal growth and neuropsychiatric function.

Are copy number variations (CNVs) in the pseudoautosomal regions (PARs) frequent in subjects with Y-chromosome microdeletions and can they lead to abnormal stature and/or neuropsychiatric disorders? Only subjects diagnosed with azoospermia factor (AZF)b+c deletions spanning to the end of the Y chromosome (i.e. terminal deletions) harbor Y isochromosomes and/or cells 45,X that lead to pseudoautosomal gene CNVs, which were associated with abnormal stature and/or neuropsychiatric disorders. The microdeletions in the long arm of the Y chromosome (Yq) that include the loss of one to threeAZF regions, referred to as Yq microdeletions, constitute the most important known etiological factor for primary spermatogenic failure. Recently, controversy has arisen about whether Yq microdeletions are associated with gain or loss of PAR genes, which are implicated in skeletal development and neuropsychiatric function. We studied a cohort of 42 Chilean patients with complete AZF deletions (4 AZFa, 4 AZFb, 23 AZFc, 11 AZFb+c) from a university medical center, diagnosed over a period of 15 years. The subjects underwent complete medical examinations with special attention to their stature and neuropsychiatric function. All subjects were characterized for Yq breakpoints by PCR, and for CNVs in PARs by multiplex ligation-dependent probe amplification (MLPA), followed by qPCR analysis for genes in PAR1 (SHOX and ZBED1), PAR2 (IL9R) and two single copy genes (SRY and DDX3Y, respectively located in Yp11.3 and AZFa). In addition, karyotypes revision and fluorescence in situ hybridization (FISH) for SRY and centromeric probes for X (DXZ1) and Y (DYZ3) chromosomes were performed in males affected with CNVs. We did not detect CNVs in any of the 35 AZF-deleted men with interstitial deletions (AZFa, AZFb, AZFc or AZFb+c). However, six of the seven patients with terminal AZFb+c deletions showed CNVs: two patients showed a loss and four patients showed a gain of PAR1 genes, with the expected loss of VAMP-7 in PAR2. In these patients, the Yq breakpoints localized to the palindromes P8, P5 or P4. In the four cases with gain of PAR1, qPCR analysis showed duplicated signals for SRY and DDX3Y and one copy of IL9R, indicating isodicentric Yp chromosomes [idic(Y)] with breakpoint in Yq11.22. The two patients who had loss of PAR1, as shown by MLPA, had an additional reduction for SRY and DDX3Y, as shown by qPCR, associated with a high proportion of 45,X cells, as determined by FISH and karyotype. In agreement with the karyotype analysis, we detected DYZ3++ and DYZ3+ cells by FISH in the six patients, confirming idic(Y) and revealing additional monocentric Y chromosome [i(Y)]. Five patients had a history of major depressive disorders or bipolar disorder, and three had language impairment, whereas two patients showed severe short stature (Z score: -2.75 and -2.62), while a man with bipolar disorder was very tall (Z score: +2.56). N/A. The number of males studied with Y-chromosome microdeletions and normozoospermic controls with normal karyotypes may not be enough to rule out an association between AZF deletions and PAR abnormalities. The prevalence of Y isochromosomes and/or 45,X cells detected in peripheral blood does not necessarily reflect the variations of PAR genes in target tissues. This study shows that CNVs in PARs were present exclusively in patients with terminal AZFb+c deletions associated with the presence of Y isochromosomes and 45,X cells, and may lead to neuropsychiatric and growth disorders. In contrast, we show that men with interstitial Yq microdeletions with normal karyotypes do not have an increased risk of PAR abnormalities and of phenotypical consequences. Moreover, our results highlight the importance of performing molecular studies, which are not considered in the usual screening for patients with Yq microdeletions. This work was supported by the National Fund for Scientific and Technological Development of Chile (FONDECYT),grant no. 1120176 (A.C.). The authors declare that no conflicting interests exist.

Circulating Tumor Cells as a Biomarker in Pancreatic Ductal Adenocarcinoma

Background/Aims: Circulating tumor cells (CTCs) are valuable in both basic research and clinical application for cancer management. In the current study, we evaluated the diagnostic value of CTCs in pancreatic ductal adenocarcinoma (PDAC). Methods: In total, 143 blood samples from 95 consecutively diagnosed PDAC patients and 48 healthy donors were collected. Combined data from immunostaining of CD45, DAPI and fluorescence in situ hybridization (FISH) with chromosome 8 centromere (CEP8) probe were used to identify CTCs. Cells with features of CD45-/DAPI+/CEP8>2 were detected as CTCs. Results: CTCs were classified as triploid, tetraploid and multiploid based on chromosome 8 copy number. CTC subtype composition was significantly different among groups. Both subtype number and total CTC number were significantly increased in PDAC patients, compared to healthy controls. Total CTC number had 75.8% sensitivity and 68.7% specificity at a cutoff value of 2 cells/3.2 mL. This study is the first to report that CTC subtype number is also useful in cancer diagnosis. Sensitivity was 53.7% and specificity was 85.4% at a cutoff point of 2 CTC subtypes. The diagnostic value of both total CTC number and CTC subtype number was a little poorer than CA199. Conclusions: Both CTC subtype and total CTC number may serve as potential biomarkers for PDAC.

Unfavorable Prognostic Impact of MYC Increased Copy Number (ICN) in Patients with Diffuse Large B-Cell and High-Grade Lymphoma Treated with Immuno-Chemotherapy (ICT)

Background: MYC rearrangements occur approximately in 5-15% of diffuse large B-cell lymphoma (DLBCL), and are known to unfavorably impact on patients (pts) survival, especially if associated to BCL2 or/and BCL6 rearrangements (double/triple-hit). Previous studies have shown that also a MYC gene ICN may confer a worse prognosis to these pts (Yoon, Histopathology 2008), but only few groups have transferred this observation into the clinical practice. However, this finding may account for those cases that, despite the absence of MYC rearrangements, show a similar aggressive clinical course. For this reason, a careful study of all aberrations involving MYC geneshould be widely applied. We report a single center experience on the clinical outcome of pts with MYC ICN in the setting of aggressive B-cell lymphoma.Methods: In the study period (Aug 2011-Aug 2015) we performed FISH analysis in all consecutive pts with de novo or transformed DLBCL or B-cell lymphoma, unclassifiable (BCLU) that displayed Ki67 >80% or intense bcl2 protein expression. Interphase FISH has been performed on 23 μm thick sections of formalin fixed paraffin embedded tissues, using splitsignal DNA probes (Dako) specific for the following loci: 8q24 (MYC), 18q21 (BCL2) and 3q27 (BCL6). For each sample, 60 evaluable nuclei with complete FISH signals were scored. ICN was considered when 3 or more copies of the gene studied were identified.Almost all pts with a MYC translocation have been treated according to Burkitt lymphoma (BL)-like regimens as first line therapy (FLT); pts with MYC ICN have been treated with BL-like regimens from 2013, whereas standard ICT (R-CHOP or R-CHOPlike) has been used for these pts diagnosed before 2013. Overall survival (OS) was evaluated by Kaplan Meier method.Results: In the 4-year study period, 317 consecutive pts were diagnosed with de novo or transformed DLBCL or BCLU. Twenty-one (7%) showed a translocation of MYC, in single (14%), double (67%) or triple hit (19%), with respect to BCL2 and BCL6 rearrangements. Interestingly, 8 (2.5%) pts with no MYC translocation showed MYC ICN, ranging from 3 up to 10 gene copies per cell. This aberrant gene copy number was observed in > 80% of the analyzed nuclei in 7 cases. The characteristics of the pts with MYC gene rearrangement or ICN are summarized in Table 1.Of the 8 pts with MYC ICN, 5 had de novo DLBCL, 2 had transformed DLBCL and 1 had BCLU. Seven pts also showed BCL2 and/or BCL6 rearrangement/ICN. Three pts were treated with standard ICT and 5 with a BLlike regimen. Two pts (25%) responded to the FLT, showing one complete and one partial remission (CR and PR, respectively); both received BLlike regimen. The patient with PR progressed after 7 months, and received a second line treatment likewise the 6 nonresponders to FLT. None of them had a response and eventually died, with a median OS of 10.5 months. Cause of death was lymphoma in all cases. The patient who obtained a CR was still in remission at 29 months from the end of the FLT. The survival of these pts, compared to pts with MYC rearrangement, is shown in Figure 1 (respectively 12% vs 57% at 2 years, p 0.04).Conclusion: These preliminary data show a dismal prognosis of pts with aggressive B cell lymphoma and MYC ICN, significantly worse than pts harboring a MYC translocation, independently of the treatment received. To better understand the genetic defect underlying this FISH pattern, further analysis using a specific centromere probe for chromosome 8 is ongoing on these and new cases. A previous study distinguished gain from amplification of MYC based on the number of gene copies, identifying 2 groups with different prognosis (Valera, Hematologica 2013). In our hands, there was a wider range of gene copy number in each patient, and the outcome was homogeneously poor. Overall, the biological consequence of this alteration is still to be fully explained, and only a few studies have addressed this point so far (Stasik, Hematologica 2010). Further investigation on larger cohort groups is necessary to confirm the unfavorable prognostic role of MYC ICN in aggressive B cell lymphoma. Such insights would help practitioners to determine more accurate therapeutic approaches, in order to improve the outcome of this specific subset of pts. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Deletion of 18q is a strong and independent prognostic feature in prostate cancer.

Deletion of 18q recurrently occurs in prostate cancer. To evaluate its clinical relevance, dual labeling fluorescence in-situ hybridization (FISH) using probes for 18q21 and centromere 18 was performed on a prostate cancer tissue microarray (TMA). An 18q deletion was found in 517 of 6,881 successfully analyzed cancers (7.5%). 18q deletion was linked to unfavorable tumor phenotype. An 18q deletion was seen in 6.4% of 4,360 pT2, 8.0% of 1,559 pT3a and 11.8% of 930 pT3b-pT4 cancers (P < 0.0001). Deletions of 18q were detected in 6.9% of 1,636 Gleason ≤ 3 + 3, 6.8% of 3,804 Gleason 3 + 4, 10.1% of 1,058 Gleason 4+3, and 9.9% of 344 Gleason ≥ 4 + 4 tumors (P = 0.0013). Deletions of 18q were slightly more frequent in ERG-fusion negative (8.2%) than in ERG-fusion positive cancers (6.4%, P = 0.0063). 18q deletions were also linked to biochemical recurrence (BCR, P < 0.0001). This was independent from established pre- and postoperative prognostic factors (P ≤ 0.0004). In summary, the results of our study identify 18q deletion as an independent prognostic parameter in prostate cancer. As it is easy to measure, 18q deletion may be a suitable component for multiparametric molecular prostate cancer prognosis tests.

Clinical Overestimation of HER2 Positivity in Early Estrogen and Progesterone Receptor-Positive Breast Cancer and the Value of Molecular Subtyping Using BluePrint.

PurposeHuman epidermal growth factor receptor 2 (HER2) positivity is an important prognostic and predictive indicator in breast cancer. HER2 status is determined by immunohistochemistry and fluorescent in situ hybridization (FISH), which are potentially inaccurate techniques as a result of several technical factors, polysomy of chromosome 17, and amplification or overexpression of CEP17 (centromeric probe for chromosome 17) and/or HER2. In South Africa, HER2-positive tumors are excluded from a MammaPrint (MP; Agendia BV, Amsterdam, Netherlands) pretest algorithm. Clinical HER2 status has been reported to correlate poorly with molecular subtype. The aim of this study was to investigate the correlation of clinical HER2 status with BluePrint (BP) molecular subtyping.MethodsClinico-pathologic and genomic information was extracted from a prospectively collected central MP database containing records of 256 estrogen receptor–positive and/or progesterone receptor–positive tumors. Twenty-one tumors considered HER2 positive on immunohistochemistry or FISH were identified for this study.ResultsThe median age of patients was 56 years (range, 34 to 77 years), with a median tumor size of 16 mm (3 to 27 mm). Four (19%) tumors were confirmed HER2-enriched subtype, six (29%) were luminal A, and 11 (52%) were luminal B. The positive predictive values of HER2/CEP17 ratio ≥ 2 and HER2 copy number ≥ 6 were only 29% and 40%, respectively. The differences in means for HER2/CEP17 ratio were significant between BP HER2-enriched versus luminal (P = .0249; 95% CI, 0.12 to 1.21) and MP high-risk versus low-risk tumors (P = .0002; 95% CI, 0.40 to 1.06).ConclusionOf the 21 tumors considered clinically HER2 positive, only four were HER2-enriched subtype with BP, indicating an overestimation of HER2 positivity. FISH testing has a poor positive predictive value.

A functional centromere lacking CentO sequences in a newly formed ring chromosome in rice

An awned rice (Oryza sativa) plant carrying a tiny extra chromosome was discovered among the progeny of a telotrisomic line 2n+4L. Fluorescence in situ hybridization (FISH) using chromosome specific BAC clones revealed that this extra chromosome was a ring chromosome derived from part of the long arm of chromosome 4. So the aneuploidy plant was accordingly named as 2n+4L ring. We did not detect any CentO FISH signals on the ring chromosome, and found only the centromeric probe Centromeric Retrotransposon of Rice (CRR) was co-localized with the centromere-specific histone CENH3 as revealed by sequential FISH after immunodetection. The extra ring chromosome exhibited a unique segregation pattern during meiosis, including no pairing between the ring chromosome and normal chromosome 4 during prophase I and pre-separation of sister chromatids at anaphase I.

Application of fluorescence in situ hybridization (FISH) as a tool to aid cytogenetics in 1,409 fetal samples

To evaluate the technical application of fluorescence in situ hybridization (FISH) as a support to classical cytogenetic in numerical chromosomal aneuploidies studies in samples of amniotic fluid, chorionic villus, and fetal loss. The authois performed cytogenetic analyses in 1,409 patients (678 amniocentesis, 512 chorionic villus samples, and 219 spontaneous abortions) during one year. FISH molecular study aided traditional cytogenetic in 90 cases. These cases were indicated based on the diagnostic hypothesis of each patient or when no cellular growth was obtained. The authors standardized the FISH in discoloured slides. They had 85% positive FISH in amniotic fluid, 70% in chorionic villus, and 90% in abortion material using 13, 18, 21 X and Y centromeric probes. It showed 12% of altered FISH in amniotic fluid (100% trisomies), 10% in chorionic'villus (50% trisomy and 50% X - monosomy), and 22% in abortion material (50% trisomy, 25% X-monosomy, and 25% triploidy). FISH and cytogenetic analysis confirmed the results. This technique revolutionized clinical and research applications of cytogenetics. In this particular paper, FISH was a valuable and reliable technique to promptly identify rapid detection of aneuploidies in interphase cells, metaphase spread and paraffin-embedded samples. It is hoped that, in the future, the economic viability of array CGH and FISH, with the decreasing cost of testing and their genomics advantages can be incorporated as routine and customized in the approach of prenatal diagnosis.

Rapid genomic changes in allopolyploids of Carassius auratus red var. (♀) × Megalobrama amblycephala (♂).

To better understand genomic changes in the early generations after polyploidisation, we examined the chromosomal consequences of genomic merger in allotetraploid hybrids (4 nF1) (AABB, 4n = 148) of Carassius auratus red var. (RCC) (AA, 2n = 100) (♀) × Megalobrama amblycephala (BSB) (BB, 2n = 48) (♂). Complete loss of the paternal 5S rDNA sequence and the expected number of maternal chromosomal loci were found in 4 nF1, suggesting directional genomic changes occurred in the first generations after polyploidisation. Recent studies have reported instability of newly established allotetraploid genomes. To assess this in the newly formed 4 nF1 genome, we performed fluorescence in situ hybridisation on an allotetraploid gynogenetic hybrid (4 nG) (AABB, 4n = 148) and an allopentaploid hybrid (5 nH) (AABBB, 5n = 172) from 4 nF1 (♀) × BSB (♂) with 5S rDNA gene and centromere probes from RCC, the original diploid parent. The expected numbers of maternal chromosomal loci were found in 4 nG, while chromosomal locus deletions and chromosome recombinations were detected in 5 nH. These observations suggest that abnormal meiosis did not lead to obvious genomic changes in the newly established allotetraploid genomes, but hybridisation with the original diploid parent resulted in obvious genomic changes in the newly established allotetraploid genomes, as was found for the maternal genome.

Numerical chromosomal aberrations in acute lymphoblastic leukemia in north Indian population

The cytological diagnosis of cholangiocarcinoma has been significantly aided by applying a 4-probe fluorescence in situ hybridization system on endoscopic retrograde cholangiopancreatography brushing smears, aiming mainly at the detection of hyperdiploidy. However, this approach adds little to our understanding of the genetic background of the disease. With the prospect of obtaining additional data on chromosomal aberrations, we have extended the fluorescence in situ hybridization study, with the application of 4 independent 2-probe systems in 35 patients with documented cholangiocarcinoma. Fluorescence in situ hybridization assays were performed on endoscopic retrograde cholangiopancreatography brushing smears, with probes for the 7q31, 11q13 (CCND1), 17p53 (TP53), and 9p21 (INK4 locus) bands, together with the respective centromeric probe. Hyperdiploidy, involving at least 2 of the 4 chromosomes targeted, was found in 31 patients. 17p13 deletion was detected in 3, and 9p21 deletion, in 5 of the hyperdiploid cases, with the 2 aberrations concurrent in 1. CCND1 amplification was found in 1 case as the sole abnormality and in another together with hyperdiploidy, but in apparently unrelated clones. This work indicates that interphase fluorescence in situ hybridization is a practical and useful tool for the cytogenetic study of cholangiocarcinoma on endoscopic retrograde cholangiopancreatography brushing smears, which is often the only available tissue specimen of the tumor. Apart from hyperdiploidy, it provides additional data on the genetic profile of cholangiocarcinoma, especially regarding structural chromosomal aberrations and clonal diversity. This line of investigation may prove useful in the delineation of oncogenesis and the interpretation of the diverse clinical features of the disease.

MET and PTEN gene copy numbers and Ki-67 protein expression associate with pathologic complete response in ERBB2-positive breast carcinoma patients treated with neoadjuvant trastuzumab-based therapy.

BackgroundPathologic complete response (pCR) after neoadjuvant chemotherapy for breast cancer is associated with improved prognosis in aggressive tumor subtypes, including ERBB2- positive tumors. Recent adoption of pCR as a surrogate endpoint for clinical trials in early stage breast cancer in the neoadjuvant setting highlights the need for biomarkers that, alone or in combination, help predict the likelihood of response to treatment.MethodsBiopsy specimens from 29 patients with invasive ductal carcinoma treated with trastuzumab-based therapy prior to definitive resection and pathologic staging were evaluated by dual color bright field in situ hybridization (dual ISH) using probes for MET, TOP2A, PTEN, and PIK3CA genes, each paired with centromeric probes to their respective chromosomes (chromosomes 7, 17, 10, and 3). Ki-67 expression was assessed by immunohistochemistry (IHC). Various parameters describing copy number alterations were evaluated for each gene and centromere probe to identify the optimal parameters for clinical relevance. Combinations of ISH parameters and IHC expression for Ki-67 were also evaluated.ResultsOf the four genes and their respective chromosomes evaluated by ISH, two gene copy number parameters provided statistically significant associations with pCR: MET gain or loss relative to chromosome 7 (AUC = 0.791, sensitivity = 92 % and specificity = 67 % at optimal cutoff, p = 0.0032) and gain of PTEN (AUC = 0.674, sensitivity = 38 % and specificity = 100 % at optimal cutoff, p = 0.039). Ki-67 expression was also found to associate significantly with pCR (AUC = 0.726, sensitivity = 100 % and specificity = 42 % at optimal cutoff, p = 0.0098). Combining gain or loss of MET relative to chromosome 7 with Ki-67 expression further improved the association with pCR (AUC = 0.847, sensitivity = 92 % and specificity = 83 % at optimal cutoffs, p = 0.0006).ConclusionsAn immunogenotypic signature of low complexity comprising MET relative copy number and Ki-67 expression generated by dual ISH and IHC may help predict pCR in ERBB2-positive breast cancer treated with neoadjuvant chemotherapy and trastuzumab. These findings require validation in additional patient cohorts.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2743-x) contains supplementary material, which is available to authorized users.

Sorting of chromosomes on FACSAria(TM) SORP for the preparation of painting probes.

High purity chromosome sorting can be performed on instruments such as MoFlo MLS and BD influx, which are stream-in-air sorters equipped with water-cooled high power lasers. The FACSAria is a true fixed alignment, low laser powered instrument with a quartz flow cell gel-coupled to the collection optics. However, whether high purity mouse and human chromosomes can be obtained by sorting on the BD FACSAria(TM) Special Order Research Product (FACSAria SORP) remains to be determined. Here, we report that the high resolution flow karyotype of mouse lymphocytes and normal male human peripheral blood mononuclear cells (hPBMCs) can be obtained on the FACSAria SORP using laser power settings of 50mW for 355nm and 20mW for 444nm excitation. Furthermore, the use of Fluorescence in situ hybridization (FISH) confirmed that chromosome paints prepared from the sorted chromosomes demonstrated high purity and signal specificity. Notably, human chromosome 12 was separated from the chromosome 9-12 cluster in the flow karyotype, and its identity was confirmed using FISH in trisomy 12 human ES cell lines B2-C7 and B2-B8. In addition, multicolor FISH (mFISH) with human chromosome painting probes to 13,18, 21, and sex chromosomes X and Y showed high signal specificity in hPBMCs. Taken together, our findings demonstrated that high resolution flow karyotype can be obtained using FACSAria SORP. Moreover, a FISH analysis confirmed high purity of the sorted chromosomes. Additionally, in contrast to centromeric satellite probes, chromosome painting probes with high specificity are more suitable for detection of chromosome aberrations, such as deletions and translocations, in prenatal diagnosis. © 2016 International Society for Advancement of Cytometry.

Identification of small marker chromosomes using microarray comparative genomic hybridization and multicolor fluorescent in situ hybridization.

BackgroundMarker chromosomes are small supernumerary chromosomes that cannot be unambiguously identified by chromosome banding techniques alone. However, the precise characterization of marker chromosomes is important for prenatal diagnosis and proper genetic counseling. In this study, we evaluated the chromosomal origin of marker chromosomes using a combination of banding cytogenetics and molecular cytogenetic techniques including diverse fluorescence in situ hybridization (FISH) assays and array comparative genomic hybridization (array CGH).ResultsIn a series of 2871 patients for whom cytogenetic analysis was requested, 14 cases with small supernumerary marker chromosomes (sSMCs) were identified. Nine sSMCs were mosaic, and five nonmosaic. Of the nine cases with known parental origins, four were identified as de novo, and four and one were maternally and paternally inherited, respectively. Six sSMCs were identified by FISH using centromeric probes; three sSMCs were derived from chromosome 15, including two heterochromatic sSMC(15)s and a large sSMC(15) spanning 15q11.1q13.1, and three sSMCs originated from chromosome 14 or 22. Array CGH revealed two cases with derivatives of chromosome 2 and whole chromosome painting multicolor-FISH (M-FISH) identified three cases with derivatives of chromosome 6, 16, and 19, respectively. One maker chromosome in Turner syndrome was characterized as sSMC(X) by preferential application of a centromeric probe for X-chromosome. In addition, one sSMC composed of genomic materials from chromosomes 12 and 18 was identified in parallel with parental karyotype analysis that revealed the reciprocal balanced translocation.ConclusionsThis report is the largest study on sSMCs in Korea and expands the spectrum of sSMCs that are molecularly characterized.Electronic supplementary materialThe online version of this article (doi:10.1186/s13039-016-0273-5) contains supplementary material, which is available to authorized users.

Comparative analysis of individual chromosome involvement in micronuclei induced by mitomycin C and bleomycin in human leukocytes.

BackgroundMicronucleus (MN) assay is a well standardized approach for evaluation of clastogenic/aneugenic effects of mutagens. Fluorescence in situ hybridization (FISH) is successfully used to characterize the chromosomal content of MN. However, the relationships between nuclear positioning, length, and gene density of individual chromosomes and their involvement in MN induced by different mutagens have not been clearly defined.ResultsChromosomal content of MN was characterized in human leukocytes treated with mitomycin C (MMC) and bleomycin (BLM) by FISH using centromeric (cep) and whole-chromosome painting (wcp) probes. Involvement of chromosomes 8, 15 and 20 in MMC-induced and chromosomes 1, 9 and 16 in BLM-induced MN was studied, and correlated with chromosome size, gene density and interphase position. The results obtained were analyzed together with previous own data on the frequencies of inclusion of chromosomes 3, 4, 6, 7, 9, 16, 17, 18, and X in MMC-induced MN. It could be shown that MMC- and BLM-induced MN could contain material derived from all chromosomes investigated. Involvement of whole chromosomes 8, 15 and 20 in MMC-induced MN negatively correlated with gene density; however, analysis together with earlier studied chromosomes did not confirm this correlation. Inclusion of chromosomes 8, 15 and 20 in MMC-induced MN does not depend on their size and interphase position; the same result was found for the twelve overall analyzed chromosomes. In BLM-treated cells significant correlation between frequencies of involvement of chromosomes 1, 9 and 16 in MN and their size was found.ConclusionsOur results clearly revealed that BLM differs from MMC with respect to the distribution of induced chromosome damage and MN formation. Thus, DNA-damaging agents with diverse mechanism of action induce qualitatively different MN with regard to their chromosomal composition. Also this study demonstrates the utility of combined sequential application of cep and wcp probes for efficient detection of MN chromosomal content in terms of centric and acentric fragments.

1 - Direct FISH, Cultured Chromosome Analysis, and Direct SNP Microarray Analysis in Identifying Ring Chromosomes on Prenatal Analysis: A Case of 45,X/46,X,r(X)

We report the results of an amniotic fluid analysis on a 41.5 year old female presenting with a circulating cell free DNA (ccfDNA) abnormal for monosomy X. Complete ultrasound at 16 weeks 3 days revealed appropriately sized fetus without evidence of fetal abnormalities or aneuploidy markers within limitations of early gestational age. Ultrasound for nuchal translucency was not performed. Amniocentesis was performed same day at 16 weeks 3 days gestation. Fluorescent in situ hybridization analysis performed on direct amniotic fluid showed 22% of nuclei positive for one copy of the X chromosome using the X centromere probe. The remaining 78% of cells showed two centromere X hybridization signals by FISH and was reported as positive for monosomy X mosaicism. However, initial G-banded chromosome analysis of in situ cultures showed only a 45,X karyotype. No structurally abnormal X or two copies of an X was observed. Additional FISH analysis, using a centromere specific probe performed on a de-stained in situ cultured slide (previously used for G-banded chromosome analysis) showed two metaphase cells out of ten metaphase cells with two X centromere signals. A review of the DAPI FISH images of the two metaphases showed that one X chromosome appeared to be a ring chromosome. In addition 14 of 100 interphase nuclei showed two chromosome X centromere signals while the remaining cells showed a single X chromosome signal. An additional two slides from a sub-culture were also analyzed and showed one G-banded metaphase cell with a ring X chromosome with the remaining 27 cells showing a 45,X count. The mosaic ring X detection discrepancy between the cultured cells in vitro and the aneuploidy FISH on direct amniotic fluid in vivo cells (previously reported with two X hybridization signals in 78% of interphase cells) is likely due to the ring X chromosome poorly dividing and preferentially selected against in the in vitro cultures.

C-Met expression and MET amplification in malignant pleural mesothelioma.

c-Met is a receptor tyrosine kinase shown to be overexpressed in malignant pleural mesothelioma (MPM). Whereas MET mutations have been identified in 3%-16% of MPMs, MET amplification has recently been reported in a single epithelioid MPM. We studied c-Met expression and MET amplification in a large MPM cohort and correlated results with morphologic and clinical features. We report the first case of MET amplification in sarcomatoid MPM. MPMs from surgical pathology files (1989-2014) were reviewed. c-Met immunohistochemistry was performed. Staining intensity and distribution were multiplied (H-score). Staining localization (cytoplasmic and/or membranous) was noted. Fluorescence in situ hybridization was performed using probes for MET and centromere 7. One hundred forty-nine patients (median age, 68.0years; interquartile range, 61-75) had epithelioid (n=97), biphasic (n=18), or sarcomatoid (n=34) MPM. Median follow-up was 10.1months (range, 0.1-222.5). One hundred thirty patients died of disease; 2 were alive with disease. c-Met was expressed in 147 MPMs. c-Met staining intensity, distribution, and H-score differed among the histologic subtypes (P=.015; P=.0001, and P=.0005, respectively), but none were predictive of survival. Epithelioid subtype had greater c-Met expression. MET amplification was identified in 1 sarcomatoid MPM and MET duplication in 1 epithelioid MPM; both had poor outcomes. Chromosome 7 aneusomy was observed in 54 of 144 (37.5%) MPMs and associated with decreased overall survival in sarcomatoid MPMs (hazard ratio=2.81; 95% confidence interval, 1.21-6.51; P=.01). In conclusion, c-Met is expressed in MPM, with significant differences in expression among histologic subtypes. MET amplification is a rare event in MPM, making it an unlikely common pathogenesis for c-Met expression.

Micro fluorescence in situ hybridization (μFISH) for spatially multiplexed analysis of a cell monolayer.

We here present a micrometer-scale implementation of fluorescence in situ hybridization that we term μFISH. This μFISH implementation makes use of a non-contact scanning probe technology, namely, a microfluidic probe (MFP) that hydrodynamically shapes nanoliter volumes of liquid on a surface with micrometer resolution. By confining FISH probes at the tip of this microfabricated scanning probe, we locally exposed approximately 1000 selected MCF-7 cells of a monolayer to perform incubation of probes — the rate-limiting step in conventional FISH. This method is compatible with the standard workflow of conventional FISH, allows re-budgeting of the sample for various tests, and results in a ~ 15-fold reduction in probe consumption. The continuous flow of probes and shaping liquid on these selected cells resulted in a 120-fold reduction of the hybridization time compared with the standard protocol (3 min vs. 6 h) and efficient rinsing, thereby shortening the total FISH assay time for centromeric probes. We further demonstrated spatially multiplexed μFISH, enabling the use of spectrally equivalent probes for detailed and real-time analysis of a cell monolayer, which paves the way towards rapid and automated multiplexed FISH on standard cytological supports.Electronic supplementary materialThe online version of this article (doi:10.1007/s10544-016-0064-0) contains supplementary material, which is available to authorized users.

Implications of the Updated 2013 American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations on Human Epidermal Growth Factor Receptor 2 Gene Testing Using Immunohistochemistry and Fluorescence In Situ Hybridization for Breast Cancer.

Human epidermal growth factor receptor 2 (HER2/neu) amplification is used as a predictive marker for trastuzumab treatment in breast cancer. Both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) testing algorithms have been based on the 2007 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. In late 2013, the guidelines were updated with new scoring criteria. To assess the impact of the revised ASCO/CAP recommendations on both IHC and FISH results by using the dual-color HER2/neu and centromeric FISH probes. Retrospective analysis of 590 invasive carcinomas with concurrent IHC and dual-color HER2/neu and centromeric 17 (CEP17) FISH results, based on 2007 ASCO/CAP guidelines, was conducted from July 2011 to June 2013. With the revised guidelines, patients were recategorized and concordance rates between the 2 assays were recalculated. Overall concordance rates for FISH and IHC decreased from 94.9% to 93.8% with reclassification. Negative FISH cases decreased from 79.1% to 69.3%. However, equivocal FISH cases were significantly increased from 0.7% to 9.5%, leading to more retesting. Both positive IHC and FISH cases were also noted to be increased, leading to more patients being eligible for trastuzumab treatment, especially those patients with concurrent HER2/neu and CEP17 polysomy. Approximately 1% of patients with initial FISH negative results were reclassified as having positive results when both the ratios and average copy number of HER2/neu were considered under the revised guidelines. The revised 2013 ASCO/CAP guidelines can potentially lead to more patients being eligible for trastuzumab therapy but additional retesting is to be expected owing to an increased number of equivocal FISH cases.

Abstract A59: Prognostic relevance of PTEN and TP53 deletion in breast cancer

Abstract Inactivation of PTEN and TP53 have been suggested to promote unfavorable phenotypes in various cancers, including breast cancer. Chromosomal deletion is one well established mechanism for inactivation of these genes. In breast cancer, deletions of TP53 and PTEN are known to occur, but systematic analyses of its clinical relevance are lacking. We thus analyzed a tissue microarray (TMA) with 2,197 breast cancers by fluorescence in-situ hybridization (FISH) using specific probes against TP53 and PTEN in combination with respective centromere probes. We found, that TP53 deletion (29%/1026 cancers) occur more frequent in breast cancers than deletions of PTEN (19%/1,239 cancers). The frequency of PTEN deletion varied between histological subtypes with highest deletion rate in medullary cancers (18 of 39, 46%) and lowest deletion rate in mucinous cancers (1 of 28; 3%; p&amp;lt;0.0001) for mucinous vs. medullary). PTEN deletions were significantly linked to various unfavorable tumor features, including advanced tumor stage (p=0.0054), high-grade (p&amp;lt;0.0001), high tumor cell proliferation (Ki67 Labeling Index; p&amp;lt;0.0001), and shortened overall survival (p=0.0090). Deletions of TP53 were significantly linked to high-grade (p&amp;lt;0.0001) and high tumor cell proliferation (p=0,0193) but unrelated to histological subtypes, tumor stage, nodal status, and patient survival. Deletion of PTEN and TP53 were unrelated to each other (p=0.6469). Combined analysis of PTEN and TP53 deletions showed that TP53 deletion had no additional effect on patient prognosis in cancers with or without PTEN deletion. In conclusion, our data support a role of PTEN and TP53 deletions in breast cancer. However, only PTEN deletion is inked to aggressive tumor behavior, while TP53 deletion has no prognostic relevance in breast cancer. Citation Format: Martina Kluth, Anette Lebeau, Isabell Witzel, Sven Mahner, Linn Woelber, Stefan Geist, Peter Paluchowski, Christian Wilke, Uwe Heilenkoetter, Volkmar Mueller, Ronald Simon, Guido Sauter, Luigi Terracciano, Rainer Krech, Albert von der Assen, Eike Burandt, Patrick Lebok. Prognostic relevance of PTEN and TP53 deletion in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A59.

Molecular cytogenetic analysis of dicentric chromosomes in acute myeloid leukemia

Dicentric chromosomes (DCs) have been described in many hematological diseases, including acute myeloid leukemia (AML). They are markers of cancer and induce chromosomal instability, leading to the formation of other chromosomal aberrations and the clonal evolution of pathological cells. Our knowledge of the roles and behavior of human DCs is often derived from studies of induced DCs and cell lines. It is difficult to identify all the DCs in the karyotypes of patients because of the limitations of metaphase cytogenetic methods. The aim of this study was to revise the karyotypes of 20 AML patients in whom DCs were found with conventional G-banding or multicolor fluorescence in situ hybridization (mFISH) with (multi)centromeric probes and to characterize the DCs at the molecular cytogenetic level. FISH analyses confirmed 23 of the 29 expected DCs in 18 of 20 patients and identified 13 others that had not been detected cytogenetically. Fourteen DCs were altered by other chromosomal changes. In conclusion, karyotypes with DCs are usually very complex, and we have shown that they often contain more than one DC, which can be missed with conventional or mFISH methods. Our study indicates an association between number of DCs in karyotype and very short survival of patients.

High HER2/Centromeric Probe for Chromosome 17 Fluorescence In Situ Hybridization Ratio Predicts Pathologic Complete Response and Survival Outcome in Patients Receiving Neoadjuvant Systemic Therapy With Trastuzumab for HER2-Overexpressing Locally Advanced Breast Cancer.

The present study was performed to determine whether the human epidermal growth factor receptor-related 2 (HER2)/centromeric probe for chromosome 17 fluorescence in situ hybridization (FISH) ratio is a predictor of a pathologic complete response (pCR), recurrence-free survival (RFS), and/or overall survival (OS) in patients receiving neoadjuvant systemic treatment (NST) with trastuzumab (NST-T) for HER2+ locally advanced breast cancer (LABC). The present retrospective study included 555 patients with HER2+ LABC who had undergone NST and definitive surgery (1999-2012); 373 had concurrently received trastuzumab. HER2-positivity was considered present with an immunohistochemical score of 3+ and/or HER2 FISH ratio of ≥2.0. We used logistic regression analysis and Cox proportional hazard modeling to determine whether a high HER2 FISH ratio, either as a continuous variable or with a cutoff of ≥7.0, would predict for pCR (no invasive disease in the breast and no tumor in the ipsilateral axillary lymph nodes), RFS, and/or OS. The pCR group's median HER2 FISH ratio was significantly higher than that of the non-pCR group (6.4 vs. 5.2; p = .003). The logistic regression model demonstrated that the independent predictors of pCR included a high HER2 FISH ratio as a continuous variable (p = .04). The multicovariate Cox proportional hazard model showed that a high HER2 FISH ratio (with a cutoff of ≥7.0 or as a continuous variable) was a significant prognostic indicator of longer RFS time (p = .047 and p = .04, respectively). Similarly, a high HER2 FISH ratio of ≥7.0 was associated with longer OS (p = .06). A high HER2 FISH ratio is a predictor of pCR in patients with HER2+ LABC who receive NST-T. This study demonstrated the optimal predictive and prognostic value of a HER2/centromeric probe for chromosome 17 FISH ratio for primary HER2+ breast cancer treated with trastuzumab combined with neoadjuvant systemic treatment (NST-T). This suggests that a high HER2 FISH ratio is a potential indicator for a high pathologic complete response rate and a better prognosis when patients are treated with NST-T.