Routine Fluorescence In Situ Hybridization Research Articles

ROS1 Translocation Detection in NSCLC: A Comparative Study of PGDx and FISH

Abstract In the United States, lung cancer is the leading cause of cancer-related deaths. In recent decades, the advent of molecularly-targeted therapies in the management of non-small-cell lung cancer (NSCLC) has led to significant improvements in patient outcomes. As such, optimum care depends on timely molecular testing. The mutational spectrum associated with NSCLC, however, is complex and riddled with various point mutations, amplifications, and rearrangements/fusions across multiple genes. Comprehensive testing of NSCLC has historically required multiple testing modalities to detect all clinically-relevant molecular targets. At our institution, a combination of immunohistochemistry, fluorescence in situ hybridization (FISH), and sequencing-based tests are performed on all NSCLCs. Though fragmented, this approach ensures testing for all current targetable genetic alterations is performed. Unfortunately, this style of testing is often incompatible with the limited amount of tumor tissue acquired by biopsy. In 2021, our institution implemented PGDx elio tissue complete (PGDx), introducing redundancy in ROS1 translocation testing, previously assessed by FISH through send-out testing to Mayo Clinic. The objective of this study was to perform a retrospective analysis of our molecular testing experience for NSCLC, with a spotlight on the nuances of ROS1 testing. Since its implementation at our institution, PGDx has been performed on 172 cases of NSCLC. As part of our NSCLC reflex testing agreement, ROS1 translocation FISH testing was also attempted on all cases. Of the 172 cases, one clinically actionable ROS1 translocation was identified by PGDx (0.5%). Interestingly, FISH testing for this case was equivocal. The remaining cases were negative for ROS1 translocations. Notably, FISH failed in six cases (3.5%) due to insufficient tissue. Three additional cases (1.7%) had minimal tumor tissue available for FISH testing (less than 300 cells), providing suboptimal results. Altogether, PGDx was successfully performed on all samples and, in the 166 cases where FISH was successful, there was no discordance between orthogonal tests. This supports the validity and reliability of PGDx in identifying clinically relevant rearrangements/fusions in ROS1. In this study, we demonstrate that PGDx can effectively replace FISH testing for ROS1 translocations in NSCLC. In addition to being technically equivalent to FISH for ROS1 with comparable turnaround times, PGDx adds substantial benefits. Replacing FISH testing with PGDx improves the diagnostic workflow, improves tissue utilization, and eliminates the costs associated with send-out orders. The cost of Mayo ROS1 FISH testing averages $520 per sample and this amount is billed to patient insurance, though reimbursements vary. Further, PGDx can detect the ROS1 gene fusion partners, a feature not possible through routine FISH testing in NSCLC. This added clinical information reduces the ambiguity of equivocal FISH results, exemplified by our study. By implementing this change, our institution will optimize both diagnostic practices and resource utilization in the clinical setting.

MYC translocation is a valuable marker for the development and relapse of extramedullary disease in multiple myeloma.

To study the cytogenetic characteristics of extramedullary disease (EMD) in patients with multiple myeloma (MM) and their impact on prognosis. Patients with newly diagnosed MM (NDMM) at Peking Union Medical College Hospital (Beijing, China) between June 2007 and December 2019 were recruited for this study. Demographic information, clinical data, fluorescence in situ hybridization (FISH) results of marrow and tissue samples, and survival outcome data were collected. A total of 439 patients with NDMM were divided into those without EMD (non-EMD, n = 339), those with EMD with primary paraosseous plasmacytoma (pEMD-B, n = 48), those with primary EMD with soft-tissue involvement (pEMD-S, n = 33), and those with secondary EMD (sEMD, n = 19). The incidence of EMD was 18.5% (81/439) at diagnosis and 22.8% (100/439) throughout the disease course. Comparison of FISH results showed a higher proportion of RB1 deletion (n = 20; 60.0% vs. 20.0%, p = .013) and MYC translocation (n = 12; 44.4% vs. 12.5%, p = .041) in the extramedullary tissues than in the paired bone marrow samples. At diagnosis, the percentage of MYC translocations in the sEMD group was notably higher than that in the non-EMD group (55.6% vs. 15.5%, p = .012). The median overall survival (OS) of patients with pEMD-S (32 months) and sEMD (17 months) was significantly shorter (both p = .001) than that of non-EMD patients (60 months). Soft-tissue EMD can be considered a high-risk condition, even in the era of novel agents. MYC translocation can serve as a valuable marker that correlates with extramedullary spread and relapse in patients with MM and should be considered for inclusion in routine FISH panels in clinical practice.

Are There More HER2 FISH in the Sea? An Institution’s Experience in Identifying HER2 Positivity Using Fluorescent In Situ Hybridization in Patients with HER2 Negative Immunohistochemistry

BackgroundApproximately 20% of breast cancers express HER2-positive receptors in the USA. HER2 receptor immunohistochemistry (IHC) staining with equivocal (2+) results commonly undergoes fluorescence in-situ hybridization (FISH) for further classification. Current guidelines do not recommend routine FISH testing in IHC-negative (0 or 1+) cases. This study investigates an institution that performs both IHC and FISH testing on all cases to identify the true HER2-positive rate.Patients and MethodsA retrospective chart review from 2015 to 2021 was conducted at an institution where both HER2 IHC and FISH testing were performed at the time of diagnosis for all invasive breast cancers. The rate of true HER2-positive patients was determined, and patient and tumor characteristics were further explored.ResultsA total of 1835 invasive breast cancer cases were primarily treated at this institution. A total of 289 cases were HER2 positive on IHC and FISH testing (15.7%). An additional 38 cases were identified as HER2 negative on IHC, but reclassified as HER2 positive on reflex FISH testing. Total HER2 positive cases increased from 289 (15.7%) to 327 cases (17.8%) with reflex FISH testing.ConclusionsThe additional HER2-positive cases after completing FISH testing on IHC-negative tumors suggests there may be a role for routine FISH testing in addition to standard IHC staining to determine HER2 status for breast cancer. The ethical, prognostic and even benefits of a correct diagnosis outweigh the added expense of FISH testing.

Fluorescence In Situ Hybridization in Primary Diagnosis of Biliary Strictures: A Single-Center Prospective Interventional Study

Background and aims: Diagnosis of the biliary stricture remains a challenge. In view of the low sensitivity of brush cytology (BC), fluorescence in situ hybridization (FISH) has been reported as a useful adjunctive test in patients with biliary strictures. We aimed to determine performance characteristics of BC and FISH individually and in combination (BC + FISH) in the primary diagnosis of biliary strictures. Methods: This single-center prospective study was conducted between April 2019 and January 2021. Consecutive patients with unsampled biliary strictures undergoing first endoscopic retrograde cholangiopancreatography in our institution were included. Tissue specimens from two standardized transpapillary brushings from the strictures were examined by routine cytology and FISH. Histopathological confirmation after surgery or 12-month follow-up was regarded as the reference standard for final diagnosis. Results: Of 109 enrolled patients, six were excluded and one lost from the final analysis. In the remaining 102 patients (60.8% males, mean age 67.4, range 25-92 years), the proportions of benign and malignant strictures were 28 (27.5%) and 74 (72.5%), respectively. The proportions of proximal and distal strictures were 26 (25.5%) and 76 (74.5%), respectively. In comparison to BC alone, FISH increased the sensitivity from 36.1% to 50.7% (p = 0.076) while maintaining similar specificity (p = 0.311). Conclusions: Dual-modality tissue evaluation using BC + FISH showed an improving trend in sensitivity for the primary diagnosis of biliary strictures when compared with BC alone.

Cryptic COL1A1-PDGFB fusion in dermatofibrosarcoma protuberans: a clinicopathological and genetic analysis

Objective: To investigate the clinicopathological and cytogenetic features of cryptic COL1A1-PDGFB fusion dermatofibrosarcoma protuberans (CC-DFSP). Methods: Three cases of CC-DFSP diagnosed in West China Hospital, Sichuan University, Chengdu, China from January 2021 to September 2021 were studied. Immunohistochemistry for CD34 and other markers, fluorescence in situ hybridization (FISH) for PDGFB, COL1A1-PDGFB and COL1A1, next-generation sequencing (NGS), reverse-transcriptase polymerase chain reaction (RT-PCR) and Sanger sequencing were performed. Results: There were three cases of CC-DFSP, including two females and one male. The patients were 29, 44 and 32 years old, respectively. The sites were abdominal wall, caruncle and scapula. Microscopically, they were poorly circumscribed. The spindle cells of the tumors infiltrated into the whole dermis or subcutaneous tissues, typically arranging in a storiform pattern. Immunohistochemically, the neoplastic cells exhibited diffuse CD34 expression, but were negative for S-100, SMA, and Myogenin. Loss of H3K27me3 was not observed in the tumor cells. The Ki-67 index was 10%-15%. The 3 cases were all negative for PDGFB rearrangement and COL1A1-PDGFB fusion, whereas showing unbalanced rearrangement for COL1A1. Case 1 showed a COL1A1 (exon 31)-PDGFB (exon 2) fusion using NGS, which was further validated through RT-PCR and Sanger sequencing. All patients underwent extended surgical resection. Except for case 3 with recurrence 2 years after surgical resection, the other 2 cases showed no recurrence or metastasis during the follow-up. Conclusions: FISH has shown its validity for detecting PDGFB rearrangement and COL1A1-PDGFB fusion and widely applied in clinical detection. However, for cases with negative routine FISH screening that were highly suspicious for DFSPs, supplementary NGS or at least COL1A1 break-apart FISH screening could be helpful to identify cryptic COL1A1-PDGFB fusions or other variant fusions.

Risk of Central Nervous System Involvement in High-Grade B-Cell Lymphoma with MYC and BCL2 Rearrangements: Analysis of a Population-Based Cohort with Routine Fluorescence in Situ Hybridization Testing in British Columbia

Introduction: Secondary central nervous system (CNS) involvement in aggressive B-cell lymphoma (SCNSL) is associated with dismal outcomes, highlighting the need to identify at-risk patients (pts). High-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2) is a cytogenetically-defined entity associated with poor outcomes, partly attributed to a high rate of SCNSL. However, historic estimates of SCNSL vary widely across studies, confounded by selective fluorescence in situ hybridization (FISH) testing. The CNS International Prognostic Index (CNS-IPI) is a robust model used to estimate the risk of CNS relapse in de novo diffuse large B-cell lymphoma (DLBCL). However, it has not yet been applied to HGBCL-DH-BCL2. Here we performed a population-based analysis to determine the rates of SCNSL in HGBCL-DH-BCL2 identified by non-selective FISH testing in British Columbia (BC) stratified by the CNS-IPI. Methods: All HGBCL-DH-BCL2 pts diagnosed in BC since the introduction of routine FISH testing in all de novo aggressive B-cell lymphoma biopsies and provincial guidelines implementing treatment with dose-adjusted etoposide, doxorubicin, vincristine, cyclophosphamide, prednisone and rituximab (DA-EPOCH-R) with intrathecal methotrexate (IT-MTX) CNS prophylaxis for appropriately fit HGBCL-DH-BCL2 pts aged ≤75 years (y) (DA-EPOCH-R era: 2015-2020) were included. The cohort was supplemented with HGBCL-DH-BCL2 pts from a historic province-wide cohort of aggressive B-cell lymphoma with FISH performed on all evaluable diagnostic biopsies in a research setting (R-CHOP era: 2005-2010). Time to CNS relapse was measured from the date of diagnosis accounting for the competing risk of death from other causes. Biopsies underwent institutional pathology review for consensus assignment to DLBCL or high-grade (HG) morphology. Results: 191 pts with HGBCL-DH-BCL2 were identified (DA-EPOCH-R era n =125, R-CHOP era n = 66). Median (interquartile range) follow up of alive pts was 3.3y (2.3-4.7y) in the DA-EPOCH-R era and 11.9y (11.4-13.2y) in the R-CHOP era. Baseline characteristics are shown in Table 1. Five (3%) pts had CNS involvement at diagnosis (4 DLBCL and 1 HG morphology). Excluding pts with CNS involvement at diagnosis, the 2y cumulative risk of CNS relapse in all HGBCL-DH-BCL2 pts treated with curative intent was 6% (Figure 1), and estimates were 5.5% and 11% in DLBCL and HG morphologies, respectively (p= 0.2). Leptomeningeal involvement was found in 8/10 (80%) of CNS relapses. High CNS-IPI (> 4) was observed in 44% of pts and was associated with a significantly higher rate of CNS relapse (12% high vs < 5% low/intermediate (int) CNS-IPI, p = 0.04). CNS-IPI was also predictive in those with DLBCL morphology (p=0.02). Limited by a small number of events, of the 3/27 pts with HG morphology that developed CNS relapse, 2 had high CNS-IPI and 1 had int CNS-IPI. CNS prophylaxis did not impact CNS relapse risk in either the entire group or when restricting the analysis to pts with high CNS-IPI. CNS relapse was similar in those treated with R-CHOP or DA-EPOCH-R (p = 0.6). To minimize potential bias introduced by treatment selection, we performed an era-on-era comparison in pts aged ≤75y at diagnosis to determine whether the introduction of DA-EPOCH-R with integrated IT-MTX, was associated with a decrease in CNS relapse risk. 79% of pts received R-CHOP in the R-CHOP era whereas 66% of pts received DA-EPOCH-R in the DA-EPOCH-R era. There were no significant differences in CNS-IPI distribution or morphology proportions between the two eras. Competing risk regression analysis found no significant difference in CNS relapse risk between the two eras after adjusting for morphology (hazard ratio and 95% confidence interval of DA-EPOCH-R vs R-CHOP era: 0.47 (0.12-1.84), p = 0.3). Conclusions: In the largest series to date examining SCNSL in HGBCL-DH-BCL2 identified through routine FISH testing, SCNSL rates are considerably lower than previously reported. For those with CNS relapse, leptomeningeal involvement was frequent. The CNS-IPI remains predictive of SCNSL in this entity, suggesting that the overall risk is driven by additional clinical risk factors. Further, as the CNS relapse risk is <5% in pts with low/int CNS-IPI, CNS-directed procedures and prophylaxis may not be required in this subset. Larger series are required to examine the impact of treatment regimen on CNS relapse risk. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Unilateral Primary Breast Dermatofibrosarcoma Protuberans with Negative COL1A1-PDGFB Fusion on Fluorescence In Situ Hybridization

Abstract Introduction/Objective Dermatofibrosarcoma protuberans (DFSP) is a superficial and locally aggressive cutaneous soft tissue sarcoma. In most patients, DFSP arises from the trunk and proximal extremities with breast being an uncommon location. Although they rarely metastasize (&amp;lt;5%), DFSP variants tend to recur locally. Most molecularly characterized cases have been defined by the translocation t(17;22)(q21.3;q13.1), which yields a fusion of the COL1A1 and PDGFB genes. In routine practice, dual fusion or break-apart fluorescence in situ hybridization (FISH) for the COL1A1-PDGFB fusion gene is the most widely used screening method for DFSP. However, in ~4% of typical DFSP cases, routine FISH screening is negative. In some of the DFSP cases negative for COL1A1-PDGFB fusion gene by FISH, RNA sequencing has identified two novel gene fusions, COL6A3-PDGFD and EMILIN2-PDGFD, with the former having a predilection for the female breast. Methods/Case Report We present a 32-year-old woman who underwent a biopsy for a 3.4 cm echogenic firm mass in her right breast. Histology showed a cytologically-uniform spindle cell neoplasm with fibroblastic morphology. The neoplastic cells displayed a storiform growth pattern and honeycomb-like infiltration of the adipose tissue. Immunohistochemically, the neoplastic cells expressed CD34 and vimentin and were negative for AE1/AE3, p63, S100, CD31, SMA, β-catenin, and STAT6. PDGFB break-apart FISH was negative. Based on the histologic and immunohistochemical findings, a diagnosis of DFSP was rendered. FISH for PDFGD rearrangements or RNA sequencing targeting PDGFD was not performed at this time. Results (if a Case Study enter NA) NA. Conclusion Awareness is warranted for the rare variant of DSFP with a predilection for female breast, which is characterized by a novel gene fusion not detectable by routine FISH used for DFSP screening.

Molecular determinants of clinical outcomes in a real-world diffuse large B-cell lymphoma population

AbstractMolecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) underlies the variable outcomes achieved with immunochemotherapy. However, outcomes of gene expression profiling (GEP)–defined molecular subgroups in a real-world DLBCL population remain unknown. Here we examined the prevalence and outcomes of molecular subgroups in an unselected population of 1149 patients with de novo DLBCL in British Columbia, Canada. Evaluable biopsies were profiled by fluorescence in situ hybridization (FISH), immunohistochemistry, and digital GEP to assign cell-of-origin and the so-called “double-hit signature” (DHITsig)—a signature originally described as being characteristic for high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2). DHITsig was expressed in 21% of 431 germinal center B-cell-like (GCB)–DLBCL and all 55 Burkitt lymphomas examined. Reflecting this latter finding, DHITsig has been renamed the “dark zone signature” (DZsig). DZsigpos-DLBCL, non-DZsigpos GCB-DLBCL and activated B-cell-like (ABC)–DLBCL were associated with a 2 year overall survival of 57%, 89%, and 71%, respectively. 62% of DZsigpos tumors were negative for HGBCL-DH-BCL2 by FISH, but were associated with outcomes similar to HGBCL-DH-BCL2. A small group of HGBCL-DH-BCL2 that lacked DZsig expression had different molecular features compared with DZsig-expressing HGBCL-DH-BCL2 and were associated with favorable outcomes comparable to DLBCL, not otherwise specified. DZsigpos and ABC-DLBCL had a shorter diagnosis-to-treatment interval (DTI) than GCB-DLBCL, with this metric being associated with outcome. In conclusion, DZsig expression extends beyond HGBCL-DH-BCL2 and captures a poor-prognosis DLBCL subgroup with short DTI, including patients unidentifiable by routine FISH testing, that should be considered for treatment intensification or novel therapies in prospective trials.

IGH Translocations in Chinese Patients With Chronic Lymphocytic Leukemia: Clinicopathologic Characteristics and Genetic Profile.

Immunoglobulin heavy chain translocations (IGH-t) have occasionally been reported in Chinese patients with chronic lymphocytic leukemia (CLL). The objective of the present study was to identify the clinicopathologic features of patients with IGH-t CLL and compare them with those of patients with non-IGH-t CLL. We performed fluorescence in situ hybridization (FISH) based on a routine CLL prognostic FISH panel using IGH, IGH-BCL2, BCL3, IGH-CMYC, and BCL6 FISH probes. Furthermore, we retrospectively evaluated the clinical features of 138 newly diagnosed CLL patients via chromosome banding analysis (CBA), FISH, and targeted next-generation sequencing. IGH-t was identified in 25 patients (18.1%). Patients with IGH-t CLL had lower flow scores than those with non-IGH-t CLL. The most frequent translocation was t(14;18) (10 patients), followed by t(14;19) (3 patients), and t(2;14)(p13;q32), t(7;14)(q21.2;q12), t(9;14)(p13;q32) (3 patients). The remaining nine patients included three with abnormal karyotypes without translocation involving 14q32, four with a normal karyotype, and two who failed CBA. The most frequently concomitant FISH-detected aberrations were 13q deletion, followed by +12 and TP53 deletion, while one case involved ATM deletion. Complex karyotypes were detected in five patients with IGH-t CLL, in whom all partner genes were non-BCL2. Available mutational information indicated that KMT2D mutation was the most frequent mutation among tested 70 patients, while TP53 mutation was the most frequent mutation in the IGH-t group. Moreover, the IGH-t group had higher FBXW7 (P=0.014) and ATM (P=0.004) mutations than the non-IGH-t group, and this difference was statistically significant. Our study demonstrates that IGH-t is not uncommon among Chinese CLL patients, and that its partner genes are multiple. The gene mutational profile of the IGH-t group was distinct from that of the non-IGH-t group, and the concomitant chromosomal abnormalities within the IGH-t CLL group differed. Thus, identification of IGH-t and its partner genes in CLL patients may help further refine risk stratification and strengthen the accurate management in CLL patients.

Population-Wide Introduction of Dose-Adjusted EPOCH-R Is Associated with Improved Outcome of High Grade B-Cell Lymphoma with MYC and BCL2 Rearrangements with Diffuse Large B-Cell Lymphoma Morphology

Population-Wide Introduction of Dose-Adjusted EPOCH-R Is Associated with Improved Outcome of High Grade B-Cell Lymphoma with MYC and BCL2 Rearrangements with Diffuse Large B-Cell Lymphoma Morphology

Applying fluorescence in situ hybridization to aquatic systems with cyanobacteria blooms: Autofluorescence suppression and high‐throughput image analysis

AbstractCyanobacterial Harmful Algal Blooms (CyanoHABs) are expanding geographically in both fresh and marine water bodies due to coastal eutrophication and global climate change and are restructuring the microbial ecology of these systems. Cyanobacterial autofluorescence can pose a significant impediment to accurately identifying prokaryotic taxonomic groups in environmental samples using fluorescence in situ hybridization (FISH). This can hinder our ability to accurately quantify, and therefore fully understand ecological changes. As abundances of FISH target cells and autofluorescent cells can often be of the same the order of magnitude, simply subtracting average autofluorescent cell concentrations—determined from enumerating unhybridized samples—yields apparent concentrations of target cells with unacceptably large analytical uncertainty. Here we present a CuSO4/EtOH chemical pretreatment protocol that significantly reduces undesirable autofluorescence in hybridized environmental samples. We apply a novel data filtration routine to FISH images that efficiently removes residual autofluorescent cells from final cell counts. We then subject images to an automated image analysis routine that accurately enumerates probe‐positive cells. This method is inexpensive and easy to implement as part of a routine FISH workflow. By applying this method to cyanobacteria rich samples, we can better understand how microbial community changes are contributing to globally changing biogeochemical cycles.

The Impact of Fluorescence in situ Hybridization on the Staging of Upper Tract Urothelial Carcinoma

Objectives: The aim of the study was to evaluate the impact of fluorescence in situ hybridization (FISH) diagnostics on the T stage in final histology specimen of patients undergoing radical nephroureterectomy (RNU) due to upper tract urothelial carcinoma (UTUC) at a large tertiary care center. Methods: We retrospectively analyzed patients who underwent RNU at our center between 2008 and 2020. Inclusion criteria were RNU due to UTUC. Urine cytologies were used for FISH analysis to detect chromosomal abnormalities. Pre-FISH group was defined as patients without access to routine preoperative urinary FISH testing (2008–2014), and FISH group was defined as patients with access to routine FISH testing. Primary outcome was T stage in final histology. Statistical analysis was performed by χ² test and Mann-Whitney U test. Results: Out of 212 patients who underwent RNU at our center between 2008 and 2020, 155 patients were included into the final analysis. The median age was 71 (range 33–90) years, and 108 (69.7%) patients were male and 47 (30.3%) female. Age and gender were not differently distributed in both groups (age: p = 0.925; gender: p = 0.682). Organ-confined disease was found in 37/72 patients in the pre-FISH cohort and in 48/83 patients in the FISH cohort (p = 0.422). Within organ-confined disease, 18/37 patients revealed a T stage smaller than T1 in the pre-FISH cohort and 35/48 patients in the FISH cohort (p = 0.022). Conclusions: Preoperative FISH diagnostics add important information to preoperative diagnostic workup of patients with UTUC. Within organ-confined disease, a significant shift toward T stages lower than T1 is observed. Further research is required to determine the impact of this shift on survival in UTUC.

Comparative assessment of conventional chromosomal analysis and fluorescence in situ hybridization in the evaluation of suspected myelodysplastic syndromes: a single institution experience.

Background:Myelodysplastic syndromes (MDSs) are a heterogeneous group of clonal hematopoietic neoplasms, roughly half of which harbor cytogenetic abnormalities with diagnostic, prognostic, and therapeutic significance. Fluorescence in situ hybridization (FISH) for the most commonly seen abnormalities (5/5q, –7/7q, +8, and –20/20q–) is routinely performed alongside conventional cytogenetics (CC) in the evaluation of suspected MDS despite conflicting reports of its relative contribution compared to CC alone.Objectives:To assess the additional diagnostic and prognostic value of performing concurrent FISH versus CC alone in cases of suspected MDS.Materials and Methods:A total of 127 bone marrow samples submitted to our cytogenetic laboratory with a presumptive diagnosis of MDS were evaluated by concurrent CC and an MDS FISH panel.Results:CC was used as the gold standard method with 100% sensitivity in detecting suspected MDS-associated cytogenetic abnormalities. FISH alone had a sensitivity of 76%, whereas CC alone achieved a sensitivity of 97%. The addition of FISH did not change the diagnosis nor change the Revised International Prognostic Scoring System score in any patient. Moreover, in 12 cases identified as positive by both CC and FISH, CC identified multiple chromosomal aberrations of clinical significance not interrogated by the FISH probe panel.Conclusion:CC alone is sufficiently sensitive in detecting suspected MDS-associated cytogenetic abnormalities that influence clinical decision-making. Routine FISH testing does not provide a significant increase in test sensitivity when an adequate karyotype is obtained. Therefore, FISH testing is best reserved for suspected MDS cases lacking sufficient metaphases.

FISH analysis of selected soft tissue tumors: Diagnostic experience in a tertiary center.

Fluorescence in situ hybridization (FISH) is an important ancillary tool for the classification of bone/soft tissue (BST) tumors. The aim of this study was to evaluate the contribution of FISH to the final classification of common BST entities in the molecular pathology department of the Royal Prince Alfred Hospital (RPAH), which is one of the most important referral centers for the management of sarcomas in Australia. All routine diagnostic FISH tests performed on BST formalin-fixed paraffin embedded (FFPE) tissue specimens at the RPAH in a 5-year period (February, 2010-November, 2015) were reviewed. FISH analyses presented in this study include commercial break-apart probes (SS18, FUS, DDIT3, FUS, USP6, PDGFB, TFE3 and ALK) and a single enumeration (MDM2) probe. There were 434 interpretable FISH assays on BST samples including MDM2 (n=180), SS18 (n=97), FUS (n=64), DDIT3 (n=37), USP6 (n=30), PDGFB (n=13), TFE3 (n=8) and ALK (n=5). Discrepancies between the histopathological diagnosis and the FISH results were seen in 12% of the cases. In this subset of discordant cases, FISH contributed to the re-classification of 7% of cases originally diagnosed as synovial sarcoma (SS18) and 6% of adipocytic neoplasms (MDM2) based on the presence or absence of the expected gene alteration. Our study confirms that paraffin FISH is a sensitive and specific ancillary tool in the diagnosis of BST neoplasms when used in the appropriate clinicopathological context. These findings highlight the need for further ancillary molecular tools in the diagnosis and characterization of challenging cases.

UroVysion Multi-Target Fluorescence in situ Hybridization Assay for the Detection of Malignant Bile Duct Brushing Specimens: A Comparison with Routine Cytology

Objective: Routine bile duct brushing cytology is an important diagnostic tool in the evaluation of bile duct stricture. The purpose of this study was to evaluate the performance of the UroVysion fluorescence in situ hybridization (FISH) assay for the detection of malignant bile duct brushing specimens. Study Design: Thirty-five bile duct brushing specimens were included in the study. The FISH assay utilized the commercially available UroVysion probes. The indeterminate cytology results were considered as negative for statistical analysis. Results: Twenty-two of 35 patients were diagnosed as having malignancy based on tissue diagnosis or clinical progression of disease by image assessment. The sensitivity of routine cytology and FISH for the detection of malignancy was 14% (3/22) and 55% (12/22), respectively (p = 0.003). The specificity of routine cytology and FISH was 100% (13/13) and 62% (8/13), respectively (p = 0.025). The false-positive rate for routine cytology and FISH was 0% (0/13) and 38% (5/13), respectively. Conclusions: Our study shows that FISH is significantly more sensitive than routine cytology for the detection of malignancy in bile duct brushing specimens. However, in our study, the specificity of FISH was poor compared to the excellent specificity of routine cytology. The compromised specificity of FISH may limit its utility in the detection of malignant bile duct brushing specimens.

Baseline characteristics, chromosomal alterations, and treatment affecting prognosis of deletion 17p in newly diagnosed myeloma.

Deletion 17p13, del(17p), is associated with poor outcome in myeloma but some patients show long-term survival. With the current study we intended to identify factors impacting outcome of such high risk patients. We analyzed 110 newly diagnosed, symptomatic patients with del(17p) detected by fluorescence in situ hybridization (FISH) in CD138-purified myeloma cells to identify prognostic factors for survival. Age >65 years, ISS III, and elevated LDH negatively impacted survival. Patients with subclonal (10-60% of plasma cells) del(17p) had longer progression-free survival (PFS) than patients with del(17p) in >60% of plasma cells (26 vs. 19 months, P = 0.03). Additional gain of 1q21 was associated with shorter PFS (17 vs. 25 months, P = 0.01). Hyperdiploidy did not ameliorate impact of del(17p), but gain 19q13 predicted longer PFS (30 vs. 18 months, P = 0.01) and overall survival (50 vs. 29 months, P = 0.01). Multivariate analysis in transplant eligible patients (≤65 years) revealed better survival for patients treated with upfront autologous transplantation (hazard ratio, [95% confidence interval]: 0.15 [0.04, 0.58], P = 0.006). Application of maintenance therapy was associated with better survival in transplant-eligible patients (0.30 [0.09, 0.99], P = 0.05). We demonstrate heterogeneous outcome of patients with del(17p) according to baseline characteristics and treatment. 19q13 should be included in routine FISH panel, since gains were associated with better survival. Am. J. Hematol. 91:E473-E477, 2016. © 2016 Wiley Periodicals, Inc.

Abstract 1374: Isolation and analysis of pure intact tumor cell populations from FFPE: Implications for more precise HER2 FISH testing in breast cancer

Abstract Body: Guidelines worldwide focus on the importance of precise, reproducible, and quality assurance of Fluorescent In Situ Hybridization (FISH) methods for testing companion diagnostic markers, including Human Epidermal Growth Factor Receptor 2 (HER2) gene amplification in breast cancer. Despite these guidelines, variations in test results due to pre-analytical sampling and tissue processing are observed. In this study, we demonstrate a unique approach to isolating pure and intact tumor cells from breast cancer Formalin-Fixed, Paraffin-Embedded (FFPE) samples for precise subsequent FISH analysis. Methods: Fifty-micron thick FFPE curls from both HER2 non-amplified breast cancer tumors (n = 4; each with a reported HER2/CEP17 ratio 1.8) and positive control SKBr3 breast cancer cells were tested. Isolation of ∼250 pure and intact cytokeratin-positive/vimentin-negative/DAPI positive tumor cells from each sample was achieved using the DEPArray™ platform, an automated system enabling image-based cell sorting with single-cell resolution for pure cell population isolation and collection. Recovered cells were then cyto-spun onto poly-L coated glass slides prior to standard dual-color HER2/CEP17 FISH (Path Vysion Abbott/Vysis) analysis. Results: Positive HER2 amplification levels for the FFPE derived control SKBr3 cells were observed (HER2/CEP17 ratio &amp;gt;4.4) and consistent with levels reported in the literature. Among the patient samples, ≥75% of the DEPArray™ isolated tumor cells were recovered onto slides prior to FISH. Through routine FISH scoring, an expected non-amplified result was observed for each patient sample, with observed HER2/CEP17 ratios only ranging from 1.1 to 1.4. Conclusion: We demonstrate feasibility in performing FISH for HER2/CEP17 on pure and intact tumor cells isolated from breast cancer derived FFPE using the DEPArray™ platform. Using a 50-micron section permitted recovery of whole, intact, tumor cells based on immunostaining for cytokeratin, vimentin, and DAPI. Efficient recovery of the DEPArray™ sorted cells onto slides further permitted routine FISH analysis of only tumor cells. These preliminary results imply the possibility of more precise FISH analysis when standard FISH results are inconclusive or when insufficient tumor content prohibits downstream analysis. Evaluation of larger numbers of patient samples is underway. Citation Format: Amanda Gerber, Trisky Clarin, Ambica Bhandari, Valeria Sero, Chiara Bolognesi, Gianni Medoro, Nicolò Manaresi, Mathew Moore, Philip D. Cotter, Farideh Bischoff. Isolation and analysis of pure intact tumor cell populations from FFPE: Implications for more precise HER2 FISH testing in breast cancer. [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 1374.

Image-based collection to allow isolation and analysis of pure intact tumor cell populations from FFPE: Implications for more precise HER2 FISH analysis.

e12094 Background: Guidelines worldwide focus on the importance of precise and accurate Fluorescent In Situ Hybridization (FISH) methods for testing companion diagnostic markers, including Human Epidermal Growth Factor Receptor 2 (HER2) gene amplification in breast cancer. Despite these guidelines, variations in test results are observed due to pre-analytical sampling. In this study, we demonstrate a unique approach to isolating pure and intact tumor cells from breast cancer Formalin-Fixed, Paraffin-Embedded (FFPE) samples for precise FISH analysis. Methods: Fifty-micron thick FFPE curls from both HER2 non-amplified breast cancer tumors (n = 4; each with a reported HER2/CEP17 ratio 1.8) and positive control SKBr3 breast cancer cells were tested. Isolation of ∼250 pure and intact cytokeratin-positive/vimentin-negative/DAPI positive cells from each sample were captured using the DEPArray platform, an automated system enabling image-based cell sorting with single-cell resolution for pure cell population isolation and collection. Standard dual-color HER2/CEP17 FISH (Vysis PathVysion) analysis was performed on recovered cells. Results: Positive HER2 amplification levels for the FFPE derived control SKBr3 cells were observed HER2/CEP17 ratio > 4.4. Among the patient samples, ≥ 75% of the DEPArray isolated tumor cells were recovered onto slides. Through routine FISH scoring, an expected non-amplified result was observed for each patient sample, with HER2/CEP17 ratios ranging from 1.1 to 1.4. Conclusions: We demonstrate feasibility in performing FISH on pure and intact tumor cells isolated from breast cancer derived FFPE using the DEPArray platform. Using a 50-micron section permitted recovery of whole, intact, tumor cells based on immunostaining for cytokeratin, vimentin, and DAPI. Efficient recovery of the DEPArray sorted cells onto slides further permitted routine FISH analysis of only tumor cells. These preliminary results imply the possibility of more precise FISH analysis when standard FISH results are inconclusive or when insufficient tumor content prohibits downstream analysis. Evaluation of larger numbers of patient samples is underway

An Optimized Set of Fluorescence In Situ Hybridization Probes for Detection of Pancreatobiliary Tract Cancer in Cytology Brush Samples

Pancreatobiliary cancer is detected by fluorescence in situ hybridization (FISH) of pancreatobiliary brush samples with UroVysion probes, originally designed to detect bladder cancer. We designed a set of new probes to detect pancreatobiliary cancer and compared its performance with that of UroVysion and routine cytology analysis. We tested a set of FISH probes on tumor tissues (cholangiocarcinoma or pancreatic carcinoma) and non-tumor tissues from 29 patients. We identified 4 probes that had high specificity for tumor vs non-tumor tissues; we called this set of probes pancreatobiliary FISH. We performed a retrospective analysis of brush samples from 272 patients who underwent endoscopic retrograde cholangiopancreatography for evaluation of malignancy at the Mayo Clinic; results were available from routine cytology and FISH with UroVysion probes. Archived residual specimens were retrieved and used to evaluate the pancreatobiliary FISH probes. Cutoff values for FISH with the pancreatobiliary probes were determined using 89 samples and validated in the remaining 183 samples. Clinical and pathologic evidence of malignancy in the pancreatobiliary tract within 2 years of brush sample collection was used as the standard; samples from patients without malignancies were used as negative controls. The validation cohort included 85 patients with malignancies (46.4%) and 114 patients with primary sclerosing cholangitis (62.3%). Samples containing cells above the cutoff for polysomy (copy number gain of ≥2 probes) were classified as positive in FISH with the UroVysion and pancreatobiliary probes. Multivariable logistic regression was used to estimate associations between clinical and pathology findings and results from FISH. The combination of FISH probes 1q21, 7p12, 8q24, and 9p21 identified cancer cells with 93% sensitivity and 100% specificity in pancreatobiliary tissue samples and were therefore included in the pancreatobiliary probe set. In the validation cohort of brush samples, pancreatobiliary FISH identified samples from patients with malignancy with a significantly higher level of sensitivity (64.7%) than the UroVysion probes (45.9%) (P < .001) or routine cytology analysis (18.8%) (P<.001), but similar specificity (92.9%, 90.8%, and 100.0%respectively). Factors significantly associated with detection of carcinoma, in adjusted analyses, included detection of polysomy by pancreatobiliary FISH (P < .001), a mass by cross-sectional imaging (P < .001), cancer cells by routine cytology (overall P= .003), as well as absence of primary sclerosing cholangitis (P= .011). We identified a set of FISH probes that detects cancer cells in pancreatobiliary brush samples from patients with and without primary sclerosing cholangitis with higher levels of sensitivity than UroVysion probes. Cytologic brushing test results and clinical features were independently associated with detection of cancer and might be used to identify patients with pancreatobiliary cancers.

Abstract P6-08-53: Her2 FISH amplification in ER/PR/Her2 IHC negative breast cancer

Abstract Background: Triple negative (ER negative, PR negative, Her2 negative) breast cancer is an aggressive cancer that is not likely to respond to endocrine or anti-Her2 therapy. While Her2 positivity is a poor prognostic factor, patients with Her2 positive tumors are eligible for anti-Her2 therapy. Her2 positivity is based on either immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) result. Her2 IHC scores of 0+ and 1+ are negative, 2+ is equivocal, and 3+ is positive. The Her2 gene is amplified when the Her2/CEP17 ratio is ≥2.0. While most tumors will show IHC/FISH concordance, some tumors may show discordant findings between IHC staining and FISH. Previous studies have shown benefit from anti-Her2 therapy in tumors with discordant IHC/FISH results. The purpose of this study was to determine the frequency of Her2 amplification in triple IHC negative breast cancer to determine whether routine Her2 FISH testing is necessary in this subset of patients. Methods: After Institutional Review Board approval, the pathology databases were searched for cases of triple IHC negative breast cancer from 2003-August 2013. All cases of triple IHC negative with corresponding Her2 FISH testing were included in this study. The results of Her2 FISH were correlated with Her2 IHC. The clinical history and IHC slides for all discordant Her2 cases were reviewed when available. Results: A total of 659 triple IHC negative breast cancer cases from 516 patients were found. The patients’ age at diagnosis ranged from 26-92. The cases were classified using the ratio of Her2/CEP17 per the 2013ASCO/CAP guidelines: non-amplified &amp;lt; 2.0, amplified ≥ 2.0. As the copy number for Her2 was not always available, the 2013 ASCO/CAP criterion for amplification based on the copy number was not used in reclassifying the cases. Based on the ratio, 20 tumors were amplified, 631 tumors were not amplified, and 8 tumors had insufficient tissue for Her2 FISH testing. The Her2/CEP17 ratio for amplified cases ranged from 2.0 to 11.1 and for non-amplified cases from 0.5 to 1.9. Of these 20 patients, 10 received some trastuzumab treatment, 6 did not receive any trastuzumab, and in 4 patients, there was no additional information available regarding treatment with trastuzumab. Of the 10 who received trastuzumab treatment, 2 died of disease (DOD), 26 months and 45 months after their diagnosis. The overall survival of the other patients in this group was 23-106 months. Of the 6 who did not receive trastuzumab, one DOD after 76 months. The other 5 patients have overall survival ranging from 15-93 months. Of the patients with unknown Her2 targeted therapy status, one is deceased, the status of 2 is unknown, and one patient is alive at 110 months. Discussion: Our study showed 3% of triple IHC negative breast cancers to be Her2 FISH amplified. Of the 20 tumors, 19 showed Her2/CEP17 between 2.0 to 4.2 with one case showing Her2/CEP17 of 11.1. Firm conclusions on the efficacy of trastuzumab treatment in this small subset of patients with Her2 amplification but no protein overexpression cannot be drawn. However, the overall survival of those treated and not treated is similar. Based on the small number of cases with Her2 IHC/FISH discordance, a compelling case for routine testing of all Her2 IHC negative tumors cannot be made. Citation Format: Sunati Sahoo, Helena Hwang. Her2 FISH amplification in ER/PR/Her2 IHC negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-08-53.