Fluorescence In Situ Hybridization Testing Research Articles

CBFB Break-Apart FISH Testing: An Analysis of 1629 AML Cases with a Focus on Atypical Findings and Their Implications in Clinical Diagnosis and Management.

Simple SummaryThe inv(16)/t(16;16) AML is a disease that is considered relatively easy and straightforward to be diagnosed in the clinical laboratories. Up to date, CBFB FISH and/or CBFB-MYH11 RT-PCR are still the major diagnostic assays utilized in the clinical laboratories. However, incidental CBFB FISH findings and their implication in clinical laboratory diagnostics and management, especially in the era of next-generation sequencing (NGS)-based methods, have not been systemically investigated. In this study, we systemically studied over 1600 AML cases tested with CBFB FISH. Over 5% of cases with a confirmed CBFB rearrangement were challenging, including those with discrepant FISH and RT-PCR results and/or atypical FISH findings. Meanwhile, atypical FISH findings usually indicate additional chromosome 16 aberrations (AC16As) overlooked by other methods including RT-PCR and almost all NGS-based methods if following the published parameters. The information revealed in this study will be useful for further workup and interpreting atypical CBFB FISH findings and confirmation of inv(16)/t(16;16) AML diagnosis and related treatment, as well as selection of samples to better validate NGS-based new diagnostic methods.Fluorescence in situ hybridization (FISH) is a confirmatory test to establish a diagnosis of inv(16)/t(16;16) AML. However, incidental findings and their clinical diagnostic implication have not been systemically studied. We studied 1629 CBFB FISH cases performed in our institution, 262 (16.1%), 1234 (75.7%), and 133 (8.2%) were reported as positive, normal, and abnormal, respectively. The last included CBFB copy number changes (n = 120) and atypical findings such as 3′CBFB deletion (n = 11), 5′CBFB deletion (n = 1), and 5′CBFB gain (n = 1). Correlating with CBFB-MYH11 RT-PCR results, totally 271 CBFB rearrangement cases were identified, including five with discrepancies between FISH and RT-PCR due to new partner genes (n = 3), insertion (n = 1), or rare CBFB-MYH11 variant (n = 1) and eight with 3′CBFB deletion. All cases with atypical findings and/or discrepancies presented clinical diagnostic challenges. Correlating FISH signal patterns and karyotypes, additional chromosome 16 aberrations (AC16As) show impacts on the re-definition of a complex karyotype and prognostic prediction. The CBFB rearrangement but not all AC16As will be detected by NGS-based methods. Therefore, FISH testing is currently still needed to provide a quick and straightforward confirmatory inv(16)/t(16;16) AML diagnosis and additional information related to clinical management.

ROS1 rearrangements in lung adenocarcinomas are defined by diffuse strong immunohistochemical expression of ROS1

A small subset of lung adenocarcinomas harbour ROS1 gene arrangements and are amenable to tyrosine kinase inhibitor therapy. Current practice in Australia involves screening for ROS1 rearrangements in adenocarcinomas using ROS1 immunohistochemistry (IHC) followed by confirmatory molecular testing such as fluorescence in situ hybridisation (FISH), if other known genetic driver alterations are absent. The best threshold to determine ROS1 IHC positivity is not well defined, however, and this study aims to determine the optimal threshold for ROS1 IHC screening to identify ROS1-rearranged lung adenocarcinomas. A total of 177 lung adenocarcinomas tested for a ROS1 rearrangement by FISH at our institution between 2017 and 2020 due to presence of ROS1 IHC staining were included in the study. ROS1 IHC staining was assessed by scoring the staining intensity (0, 1, 2, or 3+) and multiplying by the percentage of positive cells to generate an H-score. IHC H-scores were compared with FISH. Of 177 cases, 32 (18%) were ROS1 FISH-positive and 145 (82%) were negative. FISH-positive cases had a median H-score of 300 (range 200-300; mean 290.3) and negative cases had a median H-score of 40 (range 0-300; mean 63). All FISH-positive cases showed strong and diffuse IHC positivity. Using a threshold H-score of 200, the sensitivity of identifying ROS1 rearrangements was 100% and the specificity was 95% amongst cases referred with ROS1 IHC positivity. Adenocarcinomas with a FISH-confirmed ROS1 rearrangement demonstrate diffuse, strong (2-3+) IHC staining. Cases with weak, patchy ROS1 IHC staining are not associated with ROS1 rearrangements and in these cases FISH testing is of little to no utility.

BCR/ABL Fusion Gene by FISH Technique: What is the Appropriate Cut Off for Diagnosis and Monitoring Response to Treatment?

Objective: Fluorescence in situ Hybridisation (FISH) is a widely used and useful cytogenetic technique for diagnosis and monitoring treatment responses in chronic myeloid leukaemia (CML). The positive or negative FISH result is interpreted based on individual standard normal cut off percentage, but universal cut off is unaddressed. We aimed to determine the performance of the FISH technique based on our routine normal cut off. Materials and Methods: A retrospective descriptive and analytical study was conducted on CML patients followed-up at Srinagarind Hospital. Data has been collected from laboratory records over the past ten years. We excluded patients who were not completely tested FISH and chromosome or RQ-PCR. Results: 675 FISH tests from 255 CML patients were analyzed. Specimens were mainly from bone marrow (98.2%) Chromosome analysis (G-banding) showed no metaphase in 31.9%. FISH test was positive in 99 samples with normal cut off at 9.7%. The falsenegative rate of FISH was 2.6% (0.65% by using the standard cut off at 1%) and the false positive rate was 3%. Conclusion: The FISH results should be interpreted carefully. Undue high level of normal cut off causes a high false-negative rate of FISH. The standard normal cut off for double signal FISH (D-FISH) was 1%. However, the cutoff FISH interpretation should be individually set and regularly validated. The FISH result should also be construed with G-banding, and RQ-PCR, which will improve the accuracy and will prevent misdiagnosed of CML. Keywords: Fluorescence in situ Hybridisation (FISH); Chronic Myeloid Leukemia (CML); Cut off; False positive; False negative

The success rate of interphase fluorescence in situ hybridization in plasma cell disorders can be improved using unconventional sources of plasma cells.

Immunomagnetic cell sorting (IMCS) is a preferred technique for the enrichment of plasma cells (PC) before fluorescence in situ hybridization (FISH). Here, we share our real-world experience regarding the success rate of IMCS, its limitations, and the utility of alternate sources to obtain a successful FISH in various PC disorders. A retrospective analysis was performed in patients with a PC neoplasm, who underwent bone marrow (BM) examination, and FISH testing over 30months. In all cases with an unsuccessful IMCS, an attempt was made to identify the cause of failure. Immunomagnetic cell sorting of PCs was successful in 395/450 cases (87.8%; 77/98 cases (78.6%) with <10% PCs and 318/352 (90.3%) with ≥10% PCs in BM aspirate; P=.003). Among cases with unsuccessful IMCS (<10% PCs; n=21 and ≥10% PCs; n=34), an alternate source could be used successfully in 34 (62%) patients and includes air-dried trephine biopsy imprint smears (n=28) with aggregates or sheets of PCs, fine-needle aspiration smears/biopsy from plasmacytoma (n=5), and ascitic fluid (n=1). 284/395 (71.9%) patients with successful IMCS and all 34 cases with an alternate source of PCs showed at least one cytogenetic abnormality on four-probe FISH. Variations in the sample quality together with significant variation in the number of PCs between BM aspirate and the trephine biopsy imprint smears/biopsy reduce the success rate of IMCS in a real-world scenario and necessitate utilization of patient-specific alternate sources of PCs like a trephine biopsy imprint or cytology smears from extramedullary sources for successful FISH testing in PC neoplasms.

DETECTION OF MINIMAL TUMOR CELLS FOR THE DIAGNOSIS AND TREATMENT MONITORING OF CHILDREN WITH NEUROBLASTOMA

Diagnosis, treatment and designing an adequate strategy of neuroblastoma (NB) therapy in children is still a complicated tasks for pediatric oncology and hematology. One of the key aspects of NB control is detection and monitoring of minimal residual disease.The authors make a concise review of the up-to-date methods, such as immunocytochemistry, fluorescent in situ hybridization (FISH), flow cytometry, the methods of qualitative and quantitative polymerase chain reaction (PCR) to estimate mRNA (RT-PCR and QRT-PCR), which are currently used for minimal residual disease detection in patients with NB. Disialoganglioside GD2, a specific NB marker, is traditionally determined by immunocytochemistry with fluorochromes that enhance its specificity, and by flow cytometry, as well. At present, FISH test is a gold standard for evaluation of the MYCN gen status in NB. A widely used multicolor flow cytometry method allows achieving high specificity of the analysis for NB diagnosis. RT-PCR may search for various targets to reveal NB cells, however, at the moment the only accepted immune target is tyrosine hydroxylase mRNA. Moreover, the studies established that quantitative QRT-PCR has more advantages than traditional qualitative RT-PCR, since this method allows a more accurate and quantitative detection of one or several mRNAs in clinical samples. The review discusses advantages and disadvantages of the main methods currently used for minimal residual disease evaluation of NB cells, such as RT-PCR, flow cytometry, FISH, etc. Comparative studies included multicolor flow cytometry with various combinations of CD9/CD81/CD56/CD45/GD2 monoclonal antibodies, conventional RT-PCR and quantitative QRT-PCR to reveal circulating/disseminated NB cells in the clinical samples of cancer patients and healthy volunteers.The authors analyze the results of various studies that compared accuracy and sensitivity of diagnostic methods such as RT-PCR, flow cytometry, FISH and some others. Despite the advantages of each method, the authors emphasize that multicolor flow cytometry is the optimal approach for the rapid and reliable detection of minimal residual disease and micrometastases of NB.

MA14.02 RET Fusion Testing in Advanced Non-Small Cell Lung Carcinoma Patients: the RETING Study

The approval of selective RET inhibitors for patients with advanced RET fusion positive non-small cell lung carcinoma (NSCLC) means that the importance of accurately identifying those patients has never been greater. Due to the co-existance of single-gene testing and next-generation sequencing (NGS), we hypothesized that a comparison of RET fusions testing assays is urgently needed. Along those lines, the performance of RET immunohistochemistry (IHC) and RET fluorescence in situ hybridization (FISH) has recently been challenged. Physicians across 17 hospitals contributed to identify patients with RET fusion positive NSCLC as part of routine clinical care. In addition, RET fusion negative samples from NSCLCs diagnosed at the referral institution were also included. All tumors underwent targeted RNA-based NGS (Oncomine Comprehensive Assay v3), break-apart RET FISH (Vysis) with an automated scanning system (BioView), and RET IHC (clone EPR2871, Abcam). The material available for all tumors had been formalin-fixed and paraffin-embedded. Only cases with enough tissue available (i.e. a minimum of 50 tumor cells, as per FISH test requirements) and sufficient sequencing coverage after NGS were included. FISH and IHC results were interpreted by using previously described criteria. The NGS result was used as the gold-standard. Analyses by the three assays was succesful in all 80 tumors. Thirty-seven RET-positive samples and 43 RET-negative NSCLCs were identified. Signet ring cells and psammomatous calcifications were frequently observed in RET-positive samples (in 30% and 27% of tumors, respectively). The most common partners were KIF5B (29/37, 78%), followed by CCDC6 (6/37, 16%). Other partners included NCOA4 (1/37, 3%) and AKAP13 (1/37, 3%). Thirty-five out of the 37 (95%) NGS-positive samples were FISH-positive (86% with a split pattern and the remaining 14% with a single 3’ signal pattern). Six of those FISH-positive cases (6/35, 17%) displayed very challenging split signals (≤2 signal diameter). The two FISH-negative samples were KIF5B-RET variants that showed a narrow split (≤1 signal diameter) in 36% and 38% of tumor cells, respectively. All 43 NGS-negative tumors were also FISH-negative. Regarding RET IHC, all NGS-positive cases showed cytoplasmic staining with a mean H-score of 210 (median 210, range 5-300). Within the NGS-negative cohort, cytoplasmic staining was observed in 8 cases (19%), with a mean H-score of 9 (median 0, range 0-150). Interestingly, the two RET FISH-negative but NGS-positive samples showed relevant IHC positivity (H-score of 165 and 255, respectively). The most frequent partner in this large multicenter series of RET fusion positive NSCLCs was KIF5B. The concordance between RNA-based NGS and FISH was high, with 0% failure rate. However, the interpretation of RET FISH is very challenging due to the frequent occurrence of narrow split signals (22% in this series). RET IHC should be investigated further as an alternative method to resolve an equivocal FISH or NGS result. Funding This study was mainly funded by Lilly. We also thank Instituto de Salud Carlos III (ISCIII) (Fondos FEDER and Plan Estatal I+D+I 2008–2011 [PI11/02866] and 2013–2016 [PI14-01176 y PI17-01001]) and the iLUNG Program (B2017/BMD-3884) from the Comunidad de Madrid.

FISH diagnostics in plasma cell myeloma: recommendations and own experience

Plasma cell myeloma (PCM) is disease with heterogeneous clinical outcomes. It is increasingly evident that the genetic features of the tumor cells largely dictate the clinical heterogeneity of PCM. Primary chromosomal alterations in myeloma can be divided into hyperdiploid and non-hyperdiploid subtypes. Secondary chromosomal changes occur during progression of disease. Cytogenetic abnormalities are important prognostic markers in PCM and some of them were incorporated into the current prognostic staging system of PCM. The presence of t(4;14), t(14;16), t(14;20), gain of 1q or TP53 deletion is considered to be high-risk myeloma. Detection of these alterations can be performed by interphase fluorescence in situ hybridization (FISH) after separation or identification of the plasma cells. The proper FISH examination in myeloma has to meet further requirements regarding aspirating and timing of samples, probe selection and their cut-off levels, the criteria of accurate analysis and reporting. Based on the literature, we here present technical recommendations regarding FISH in PCM. Furthermore, we share our own experience in FISH diagnostics acquired over 12 years. In this period, we have performed nearly 2,050 FISH tests in 603 myeloma patients and used two different methods of myeloma FISH: FISH on immunolabeled plasma cells, and target FISH with the BioView system.

Reflex ROS1 IHC Screening with FISH Confirmation for Advanced Non-Small Cell Lung Cancer-A Cost-Efficient Strategy in a Public Healthcare System.

ROS1 rearrangements are identified in 1–2% of lung adenocarcinoma cases, and reflex testing is guideline-recommended. We developed a decision model for population-based ROS1 testing from a Canadian public healthcare perspective to determine the strategy that optimized detection of true-positive (TP) cases while minimizing costs and turnaround time (TAT). Eight diagnostic strategies were compared, including reflex single gene testing via immunohistochemistry (IHC) screening, fluorescence in-situ hybridization (FISH), next-generation sequencing (NGS), and biomarker-informed (EGFR/ALK/KRAS wildtype) testing initiated by pathologists and clinician-initiated strategies. Reflex IHC screening with FISH confirmation of positive cases yielded the best results for TAT, TP detection rate, and cost. IHC screening saved CAD 1,000,000 versus reflex FISH testing. NGS was the costliest reflex strategy. Biomarker-informed testing was cost-efficient but delayed TAT. Clinician-initiated testing was the least costly but resulted in long TAT and missed TP cases, highlighting the importance of reflex testing. Thus, reflex IHC screening for ROS1 with FISH confirmation provides a cost-efficient strategy with short TAT and maximizes the number of TP cases detected.

Impact and clinical value of the revised 2019 Chinese HER-2 testing guidelines on the detect result evaluation of invasive breast cancer cases with equivocal HER-2 immunostaining by using fluorescence in situ hybridization

Objective: To investigate the impact and clinical significance of the revised 2019 Chinese HER-2 testing guidelines on the detecting result evaluation of invasive breast cancers with equivocal HER-2 immunostaining by using fluorescence in situ hybridization (FISH). Methods: A total of 569 cases of invasive breast cancers with HER-2 (+ + ) immunostaining evaluated according to the immunohistochemistry (IHC) guidelines of 2014 edition and 2019 edition from May to November 2019 were collected and further detected by FISH. The results of HER-2/CEPl7 double probe were respectively interpreted according to both the 2014 and 2019 Chinese HER-2 testing guidelines and the results were compared. Results: According to the 2014 guidelines, the number of HER-2 positive, equivocal and negative cases were 139 (24.43%), 67 (11.78%), and 363 (63.80%), respectively. Whereas according to the 2019 guidelines, 115 cases (20.21%) were the first group, 9 cases (1.58%) were the second group, 15 cases (2.64%) were the third group, 67 cases (11.78%) were the fourth group, and 363 cases were (63.80%) the fifth group, of which 130 cases (22.85%) were positive and 439 cases (77.15%) were negative by FISH detecting. Compared with the guideline of 2014 edition, the HER-2 positive rate of FISH detection reduced from 24.43% (139/569) to 22.85% (130/559) according to the application of the guideline of 2019 edition, but the difference was not statistically significant (P=0.567), while the negative rate increased from 63.80% (363/569) to 77.15% (439/569), with a statistically significant difference (P<0.05). Forty-three cases with incomplete weak to medium intensity of IHC membrane staining which were HER-2 (+ + ) according to 2014 guideline were changed to IHC (+ ) on the basis of the 2019 guideline. According to the FISH guideline of 2014 edition, 1 case (2.33%) was positive, 6 cases (13.95%) was equivocal and 36 cases (83.72%) was negative, while according to the 2019 FISH guideline, all of the 43 cases were negative. Conclusions: According to the guideline of 2019 edition, a proportion of cases changes from HER-2 (+ + ) to (+ ), and the HER-2 positive rate of FISH test decreases slightly, the negative rate increases, the equivocal result is eliminated, which provides a definite reference for screening patients who will be benefited from the targeted treatment of HER-2.

Differential and Prognostic Significance of HOXB7 in Gliomas.

Diffuse glioma is the most common primary tumor of the central nervous system. The prognosis of the individual tumor is heavily dependent on its grade and subtype. Homeobox B7 (HOXB7), a member of the homeobox family, is abnormally overexpressed in a variety of tumors. However, its function in glioma is unclear. In this study, HOXB7 mRNA and protein expression levels were analyzed in 401 gliomas from the CGGA RNA-seq database (325 cases) and our hospital (76 cases). HOXB7 expression, at both mRNA and protein levels, were upregulated in glioblastoma (GBM) and isocitrate dehydrogenase 1 (IDH1) wild-type glioma tissues. Kaplan–Meier with log-rank test showed that patients with high HOXB7 expression had a poor prognosis (p < 0.0001). Moreover, HOXB7 protein was deleted in 90.9% (20/22) of oligodendrogliomas and 13.0% (3/23) of astrocytomas. The sensitivity and specificity of HOXB7 protein deletion in oligodendroglioma were 90.9% (20/22) and 87.0% (20/23), respectively. To verify the reliability of using HOXB7 in differentiating oligodendroglioma, we used 1p/19q fluorescence in situ hybridization (FISH) testing as a positive control. The Cohen’s kappa coefficient of HOXB7 immunohistochemistry staining and 1p/19q FISH testing was 0.778 (95% CI: 0.594–0.962, p < 0.001). In conclusion, HOXB7 is an independent predictor of poor prognosis in all grade gliomas. Additionally, HOXB7 is also a highly sensitive and specific indicator to differentiate oligodendroglioma from astrocytoma.

One-Step Polymerase Chain Reaction-Free Nanowire-Based Plasma Cell-Free DNA Assay to Detect EML4-ALK Fusion and to Monitor Resistance in Lung Cancer.

BackgroundNext‐generation sequencing has mostly been used for genotyping cell‐free DNA (cfDNA) in plasma. However, this assay has several clinical limitations. We evaluated the clinical utility of a novel polymerase chain reaction–free nanowire (NW)‐based plasma cfDNA assay for detecting ALK fusion and mutations.Patients, Materials, and MethodsWe consecutively enrolled 99 patients with advanced non‐small cell lung cancer undergoing a fluorescence in situ hybridization (FISH) test for ALK fusion; ALK‐positive (n = 36). The NW‐based assay was performed using 50–100 μL of plasma collected at pretreatment and every 8 weeks during ALK inhibitor treatment.ResultsThere was high concordance between the NW‐based assay and the FISH test for identification of ALK fusion (94.9% with a kappa coefficient value of 0.892, 95% confidence interval [CI], 0.799–0.984). There was no difference in the response rate to the first anaplastic lymphoma kinase inhibitor between the ALK‐positive patients identified by the NW‐based assay and by the FISH test (73.5% vs. 72.2%, p = .931). In the ALK variant analysis, variants 1 and 3 subgroups were detected in 27 (75.0%) and 8 (22.2%) patients, respectively. Among 24 patients treated with crizotinib, variant 3 subgroup was associated with worse median overall survival than variant 1 subgroup (36.5 months; 95% CI, 0.09–87.6 vs. 19.8 months; 95% CI, 9.9–not reached, p = .004]. A serial assessment identified that ALK L1196M resistance mutation emerged before radiologic progression during crizotinib treatment.ConclusionThe newly developed simple NW‐based cfDNA assay may be clinically applicable for rapid diagnosis of ALK fusion with its variant forms and early detection of resistance.Implications for PracticeThe authors developed a novel one‐step polymerase chain reaction–free nanowire (NW)‐based plasma cell‐free DNA (cfDNA) assay. This study evaluated the clinical utility of this novel method for the diagnosis of EML4‐ALK fusion in advanced non‐small cell lung cancer (NSCLC). The NW‐based assay and FISH test showed high concordance rate in 99 patients with advanced NSCLC. Serial cfDNA assessment demonstrated this method provided early detection of resistance before radiologic progression during crizotinib treatment. Taken together, plasma cfDNA genotyping by the NW‐based cfDNA assay may be useful for the rapid diagnosis of ALK fusion, classifying variants, and early detection of resistance.

Molecular characteristics and clinical outcomes of complex ALK rearrangements identified by next-generation sequencing in non-small cell lung cancers

BackgroundComplex kinase rearrangement, a mutational process involving one or two chromosomes with clustered rearrangement breakpoints, interferes with the accurate detection of kinase fusions by DNA-based next-generation sequencing (NGS). We investigated the characteristics of complex ALK rearrangements in non-small cell lung cancers using multiple molecular tests.MethodsSamples of non-small cell lung cancer patients were analyzed by targeted-capture DNA-based NGS with probes tilling the selected intronic regions of fusion partner genes, RNA-based NGS, RT-PCR, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH).ResultsIn a large cohort of 6576 non-small cell lung cancer patients, 343 (5.2%) cases harboring ALK rearrangements were identified. Fourteen cases with complex ALK rearrangements were identified by DNA-based NGS and classified into three types by integrating various genomic features, including intergenic (n = 3), intragenic (n = 5) and “bridge joint” rearrangements (n = 6). All thirteen cases with sufficient samples actually expressed canonical EML4-ALK fusion transcripts confirmed by RNA-based NGS. Besides, positive ALK IHC was detected in 13 of 13 cases, and 9 of 11 cases were positive in FISH testing. Patients with complex ALK rearrangements who received ALK inhibitors treatment (n = 6), showed no difference in progression-free survival (PFS) compared with patients with canonical ALK fusions n = 36, P = 0.9291).ConclusionsThis study firstly reveals the molecular characteristics and clinical outcomes of complex ALK rearrangements in NSCLC, sensitive to ALK inhibitors treatment, and highlights the importance of utilizing probes tilling the selected intronic regions of fusion partner genes in DNA-based NGS for accurate fusion detection. RNA and protein level assay may be critical in validating the function of complex ALK rearrangements in clinical practice for optimal treatment decision.

Two Cases of Ocular Manifestations in Patients with Microdeletion of the Chromosome 7 Long Arm

Purpose: We report ocular manifestations in two patients with 7q microdeletion. Case summary: (Case 1) A 62-day-old male infant was admitted to the ophthalmology outpatient department for ocular examination after being diagnosed with microdeletion of chromosome seven (7q36.2q36.3 deletion) in DNA microarray comparative genomic hybridization (DNA microarray CGH) and fluorescence in situ hybridization (FISH) tests. Fundus examination showed optic disc hypoplasia in both eyes and retinopathy of prematurity, accompanied by retinal hemorrhage in his right eye. Around the age of 24 months, the patient was diagnosed with intermittent exotropia with anisometropia and was prescribed spectacles. (Case 2) A 3-year-old male infant was referred to the ophthalmology clinic to evaluate poor fixation, which was found during rehabilitation therapy for cerebral palsy and developmental delay. Fundus examination showed an increased cup/disc ratio bilaterally. A flash visual evoked potential test indicated a decrease in amplitude in his right eye. Intermittent exotropia of forty prism diopters was observed. DNA microarray CGH and FISH tests performed at another hospital revealed microdeletion of chromosome seven (7q35 microdeletion) and CNTNAP2 gene loss. Conclusions: When genetic anomalies associated with ocular development are identified, it is necessary to detect the ophthalmic abnormalities early and provide the appropriate treatment to allow for the development of normal visual function.

Diagnostic workup of multiple myeloma in resource-constrained setting

Background Monoclonal protein detection and characterization is integral component of myeloma workup and is at times used as the first step toward diagnosis. Costs of these tests are major concern in developing countries. Recent studies have emphasized on the prognostic importance of risk stratification with cytogenetic in patients with myeloma. Aims We intend to analyze additional information obtained by two costly tests, namely, serum free light chain assay (sFLC) and fluorescent in situ hybridization (FISH), for high-risk cytogenetic abnormalities during baseline workup of patients with myeloma and their necessity. Patients and methods We retrospectively analyzed laboratory data of 130 patients with myeloma enrolled at our institute between years 2016 and 2019. Subset analyses of 71 patients who underwent all three tests, namely, serum protein electrophoresis (SPEP), immunofixation electrophoresis (IFE), and sFLC for monoclonal protein detection, were done. Patterns of cytogenetic abnormalities were noted in 70 patients undergoing FISH testing for high-risk cytogenetic abnormalities and serum lactate dehydrogenase (LDH) level. International staging system (ISS) and revised international staging system (RISS) categories were compared among this group of patients. Statistical analysis used A proportion test was used. Results SPEP detected and quantified M protein in 55 (77.4%) patients. IFE detected M protein in 68 (95.7%) patients and typing was done. sFLC ratio was abnormal in 66 (92.6%) patients. IFE and sFLC were more sensitive in detecting monoclonal protein as compared with SPEP (P=0.001 and 0.009, respectively) at diagnosis. In 70 patients with FISH and LDH results available, seven (10%) patients had high-risk karyotype on FISH, and LDH was raised in five (7.1%). Based on these results, three (4.2%) patients who were ISS stage I were upgraded to RISS stage II and 21 (30%) ISS III patients were downgraded to RISS II category, thereby helping in 34.2% patients for accurate prognostication. Conclusion In resource-constrained setting, using sFLC sequentially after SPEP is noninformative, but UPEP/IFE shows M band, which can be a viable approach at diagnosis. For better prognostication, FISH for high-risk karyotypic abnormalities is required.

Real-World Testing and Treatment Patterns in Chronic Lymphocytic Leukemia (CLL) in the United States during the Modern Era: A SEER Patterns of Care Analysis

Introduction: The use of fluorescence in situ hybridization (FISH) and IgVH mutation analyses were initially used to help prognosticate CLL patients in clinical trials. In the modern era of Bruton tyrosine kinase inhibitors and improved response rates in patients with deletion 17p, and demonstrated long-term remissions among mutated IgVH patients treated with fludarabine, cyclophosphamide and rituximab (FCR), the use of testing prior to treatment is now considered standard of care to determine appropriate therapy. Several therapies were FDA approved for CLL since 2008 through 2014, including bendamustine and novel therapeutics (obinutuzumab, ibrutinib, and idelalisib). We aimed to look at patterns of real-world testing frequencies and treatment patterns by comparing two United States population based samples of untreated and treated CLL patients in 2008 and 2014.Methods: The National Cancer Institute sponsored Patterns of Care (POC) study was conducted in a population of CLL patients, randomly sampled from 13 Surveillance, Epidemiology, and End Results (SEER) program registries, diagnosed in 2008 and again in 2014. Trained abstractors collected data on demographics, diagnostics, testing, and treatments from patient medical records within 24 months post-diagnosis (along with a physician verification survey). Patient characteristics were described by age, sex, race, Charlson comorbidity index, area-based socioeconomic status, insurance status, receipt of systemic therapy, and type of treatments by diagnosis year. Patient characteristics and frequency of testing (FISH and IgVH mutation analyses) in 2008 and 2014 were compared using Rao-Scott chi-square tests. Logistic regression taking into account sampling weights was used to estimate odd ratios and 95% confidence intervals for receipt of FISH testing, both unadjusted and adjusted for age, sex, race, insurance status, and receipt of systemic treatment. Adjusted models were run overall and stratified by age at diagnosis (<65 and ≥65).Results: This study included 1,008 CLL patients diagnosed in 2008 and 1,367 CLL patients diagnosed in 2014. The mean age was 69.6 years in 2008, and 68.0 years in 2014. Among those with a known status for FISH testing, the proportion of patients who underwent testing was 51.3% in 2008, which increased to 59.6% in 2014 (p<0.01). Among those with a known status for IgVH mutation analyses, 8.5% underwent testing in 2008, which increased to 15.1% in 2014 (p<0.01). Older age was a strong predictor for not receiving FISH testing in both 2008 and 2014 (adjusted p-trend <0.01 for both years). In 2014, Hispanic patients were less likely to undergo testing (aOR 0.68, 95% CI: 0.48-0.96) compared to non-Hispanic whites, but no significant difference was found among non-Hispanic blacks. Among patients 65 or older, those with Medicare without private insurance were less likely to receive testing compared to Medicare patients with private insurance (aOR 0.49, CI: 0.32-0.74).Most CLL patients were untreated within the first two years from diagnosis (74.3% in 2008, and 70.6% in 2014). FISH testing occurred in a majority but not all patients who received therapy (64.5% of treated patients in 2008, increased to 74.7% in 2014). In 2008, the four most frequently used regimens for first treatment were 1) FCR, 2) rituximab monotherapy, 3) FR, and 4) chlorambucil based regimens. This is in contrast to 2014, where the regimens were 1) bendamustine and rituximab (BR), 2) rituximab monotherapy, 3) FCR, and 4) ibrutinib. When evaluating the use of novel therapies (obinutuzumab, ibrutinib, and idelalisib) for those diagnosed in 2014, there were no significant differences in distribution by age, race/ethnicity, comorbid conditions, SES, or insurance type.Conclusions: There has been an increase in frequency of FISH testing and IgVH mutation analyses from 2008 to 2014. FISH testing is more common compared to IgVH mutation analyses which was modest in both years. Patients of older age, Hispanic ethnicity, and Medicare with no private insurance were also less likely to receive FISH testing in 2014. There are significant differences in the treatment patterns of patients in 2008 compared to 2014, with the use of bendamustine being more common at the expense of FCR. These results highlight rapidly changing practices within CLL in the modern era. DisclosuresNo relevant conflicts of interest to declare.

Real-World Characteristics of CLL Patients Receiving Front-Line Therapy, Using the Flatiron Health Database

Introduction: Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the United States (US) with an estimated 18,960 new cases for 2016 [ American Cancer Society. Cancer Facts & Figures 2016 ]. CLL is a disease of the elderly; the median age at diagnosis in the US is 71 years, and most patients do not reach criteria for treatment until years after their diagnosis [ SEER Stat Fact Sheets: Chronic Lymphocytic Leukemia ]. Several front-line combination therapies and monotherapies are approved for the treatment of CLL; however, few studies have examined the prevalence of their use in the real world or differences in patient characteristics across regimens. Thus, the current analysis characterizes the real-world prevalence of CLL front-line regimens, as well as patient characteristics across front-line regimens.Methods: CLL patients (ICD-9: 204.1x or ICD-10: C91.1x, C83.0x) receiving front-line therapy between 11/01/2008 and 5/31/2017 were identified in the Flatiron Health electronic health records database [ Flatiron Health Data, New York, NY (5, 31,2017)] . Differences among cohorts were examined using general linear modeling for normally distributed variables and non-parametric tests (ie, chi-square and Kruskal Wallis) for non-normally distributed variables.Results: A total of 38 different front-line therapies in 5124 patients were identified. The top four regimens received by 3089 of the patients included: bendamustine plus rituximab (BR; n = 1157); rituximab monotherapy (n = 939); ibrutinib monotherapy (n = 620); and fludarabine, cyclophosphamide, and rituximab (FCR; n = 373). The mean age at the time of treatment of the sample was 69.32 years (SD = 10.06; median = 70) and males comprised 64% of the sample. The BR (68.52 years; SD = 9.13) and FCR (61.23 years; SD = 9.75) cohorts were significantly younger than the rituximab (72.18 years; SD = 9.59) or ibrutinib (71.37 years; SD = 9.75) cohorts. A significantly lower proportion of females relative to males received BR, FCR, or ibrutinib, compared with the cohort who received rituximab. No differences in overall mortality were observed among the BR, ibrutinib, or FCR cohorts; however, both the BR and ibrutinib cohorts had significantly (P < .05) lower mortality rates than the rituximab cohort. Patients in the FCR began therapy significantly sooner after diagnosis relative to the BR, rituximab, and ibrutinib cohorts. No differences in severity based on Rai stage were found. IGHV analysis was conducted in less than 25% of the sample, and fluorescence in situ hybridization (FISH) testing was conducted in approximately 60% of the sample. Patients in the rituximab cohort received less mutational testing, FISH testing, and immunophenotyping prior to starting therapy.Conclusions: Results from long term real-world data suggest that while several front-line CLL therapies exist, the majority of patients during the time period of this study (2008-2017) were treated with one of four regimens: BR, rituximab monotherapy, ibrutinib, or FCR. Patients receiving FCR and BR are significantly younger compared with those receiving rituximab monotherapy, and FCR patients were significantly younger than BR patients. CLL has a known male predominance [ American Cancer Society. Cancer Facts & Figures 2016 ], but notably, there was a significant male/female difference identified in this study in selection of front-line therapy, with a lower proportion of females starting therapy with FCR or ibrutinib and a greater proportion starting on rituximab. Older age, higher mortality rate, and less IGHV and FISH testing were observed in the rituximab monotherapy cohort, suggesting that this cohort may have included less fit patients. Low rates of IGHV mutational status and FISH testing in these front-line patients is in line with other published reports [ eg, Mato A, et al. Br J Haematol 2016;175:892-903 ], and may fail to identify favorable-risk patients who can have extended remissions with chemoimmunotherapy, as well as high-risk patients who benefit from novel therapeutic approaches. [Display omitted] DisclosuresTang:Teva Pharmaceuticals: Employment. Szabo:Teva Pharmaceuticals: Employment. Brander:Teva Pharmaceuticals, Genentech, AbbVie, Pharmacyclics: Consultancy; Genentech: Consultancy; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Genetic abnormality and protein expression of C-MYC and PD-L1 in ALK-negative anaplastic large cell lymphoma

Objective: To investigate the genetic abnormality and protein expression of C-MYC and PD-L1 in the patients with ALK-negative anaplastic large cell lymphoma (ALK－ALCL), and to explore their roles in the pathogenesis of ALK－ALCL and their relationship with clinicopathological characteristics. Methods: Thirty-seven cases of ALK－ALCL diagnosed at Fujian Provincial Hospital from January 2003 to January 2017 were selected. Fluorescence in situ hybridization (FISH) was used to detect the genetic abnormality of C-MYC and PD-L1. The expression of C-MYC and PD-L1 proteins was detected by immunohistochemistry. The relationship between C-MYC and PD-L1 genes' abnormalities and protein expression was analyzed, as well as their associations with various clinicopathological parameters. Results: Among the 37 ALK－ALCL patients, 17 (45.9%) were positive for C-MYC protein, and 14 (37.8%) were positive for PD-L1 protein. There was a significant correlation between C-MYC protein and PD-L1 protein (r=0.990,P=0.014). The protein expression of C-MYC and PD-L1 (versus negative) was associated with the clinical stage of ALK－ALCL, respectively. The international prognosis index (IPI) in high-risk group was higher than that in the low-risk group (P<0.05). FISH test showed that 9 (24.3%) of the 37 cases had amplification of C-MYC gene, and no translocation of C-MYC gene was found in any of the cases. Amplification of PD-L1 gene was found in only 2 cases (5.4%). The 3-year overall survival rate of the C-MYC or PD-L1 immunohistochemistry-positive cases was significantly lower than those of the C-MYC or PD-L1 negative cases (P<0.01 and P<0.05), respectively. Conclusion: The expression of C-MYC and PD-L1 proteins are related to the clinical stage, IPI and overall survival rate of ALK－ALCL. Thus, it can be used to assess the disease's aggressiveness and to predict the prognosis of ALK－ALCL. The expression of PD-L1 in ALK－ALCL may be regulated by C-MYC, thus suggesting a possible design of combined C-MYC targeted therapy and immune checkpoint blocking for some ALK－ALCL patients.

A hypodiploid karyotype in childhood B-cell precursor acute lymphoblastic leukemia

The detection of genetic markers associated with poor prognosis is crucial to the selection of an appropriate treatment plan for B-cell precursor acute lymphoblastic leukemia (BCP-ALL). A hypodiploid karyotype in patients with BCP-ALL has an unfavorable impact and serves as a criterion for the stratification of patients into a high-risk group. However, the survival rates of patients with a hypodiploid karyotype remain poor. Russian treatment protocols for childhood acute lymphoblastic leukemia do not include a hypodiploid karyotype in risk stratification criteria. In order to determine the prognostic value of a hypodiploid karyotype and the clinical characteristics of BCP-ALL in patients with a hypodiploid karyotype, we analyzed the survival rates of 2,700 patients included in a multicenter study. Our study was approved by the Independent Ethics Committee and the Scientific Council of the D. Rogachev NMRCPHOI of the Ministry of Healthcare of the Russian Federation. All patients underwent karyotyping and fluorescence in situhybridization (FISH) testing. A hypodiploid karyotype was detected in 27 patients. Eighteen out of 27 patients had a hypoploid clone (according to karyotyping results), 2 patients had a doubled near-haploid clone (according to karyotyping and FISH results); in 7 patients with a normal karyotype or in the absence of mitosis, hypodiploidy was determined only by FISH test. BCP-ALL with hypodiploidy is usually associated with increased WBC count at disease onset. The median WBC count in the study group was 24.2 (3.4–206.0) × 109/l vs 10.3 (0.2–1290.0) × 109/l in the control group. The number of patients with initial leukocytosis &lt; 30 × 109/l in the study group was significantly lower than in the control group (p&lt; 0.062). Remission was achieved in 26/27 patients. The event-free survival rates in patients with hypodiploidy were significantly lower than in those without hypodiploidy: 50 ± 11% vs 72 ± 8% (p&lt; 0.0001). The overall survival was 64 ± 10% and 90 ± 1%, respectively (p&lt; 0.0001). The cumulative incidence of relapse in patients with a hypodiploid karyotype was higher (42.6 ± 10.9%) than in the controls (22.3 ± 8.1%) (p&lt; 0.0001). The patients who received more intense treatment for intermediate- and high-risk groups showed better survival rates than those in the standard-risk group: 62 ± 13% vs 40 ± 15% (р= 0.59); the cumulative incidence of relapse according to the risk group was 26.4 ± 12.1% and 60 ± 16.9%, respectively (р= 0.19).The highest risk of relapse was observed in a group that included patients with near-haploidy and low hypodiploidy (26–39 chromosomes; 52.9 ± 14.4%). The event-free survival in this group was 36 ± 13%. The results of treatment of patients with BCP-ALL and hypodiploidy according to the national guidelines turned out to be comparable to the international ones. Patients with BCP-ALL and hypodiploidy should be initially stratified to the most intense treatment arm. In order to identify patients with hypoploidy, standard karyotyping is required; where needed, it can be supplemented by FISH analysis

Genetic and cytogenetic screening of autistic spectrum disorder: Genotype-phenotype profiles

Autism, a pervasive developmental disorder typically characterized by repetitive behaviour, social skills deficit (or a deficit in social communication), speech and language impairments. Our prime focus is to analyze the clinical features and phenotypical behavioural changes using the diagnostic and statistical manual of mental disorders, fourth edition, text revision (DSM IV-TR), and locating the biomarkers associated with specific autistic characters using karyotyping and fluorescence in situ hybridization (FISH) techniques. The prevalence rate of the neurexin 1 (NRXN1) gene polymorphism was also assessed in the current study. The study group involved 196 samples with 98 autistics, and equal age-matched (±2) controls based on their birth order and carrier. The participants include 35.2% males (n = 69) and 14.8% females (n = 29). The autistic and control participants were categorized based on their ages as group I (<12 yrs) with n = 62; males n = 41 (20.9%); females n = 21 (10.7%) and group II (≥12 yrs)-n = 36; males n = 28 (14.2%); females n = 08 (4.08%). Karyotyping was done for autism participants (n = 98) and the results showed that 90% of autistic participants were either the only child or the first child with a low perception and frequency in both the groups. Subsequently, we carried out the FISH assay on participants (n = 37) with higher DSM-IV TR score (≥30). Only 30 FISH tests were negative for subtelomeric deletions with NRXN1 polymorphism genotypic frequency as 62.50%, 25% and 25% for A/A, A/G and G/G genotype respectively. Our study suggests the link between a haplotype with clinical signs of autism for the single nucleotide sequence (SNP rs9636391) and links autistic characters and gene among autistic children according to their birth order, age and gender in India.

Phase II two-arm study of tepotinib plus osimertinib in patients with EGFR-mutant NSCLC and acquired resistance to first-line osimertinib due to MET amplification: INSIGHT 2.

TPS9136 Background: METamp is a mechanism of acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). METamp occurs in ̃30% of patients who progress on EGFR TKI therapy as measured using fluorescence in situ hybridization (FISH). There is an unmet need for targeted treatment options in these patients. Combination treatment with a MET TKI may overcome MET-related osimertinib resistance. Tepotinib is an oral, once daily (QD), highly selective, potent MET TKI. In the INSIGHT study (NCT01982955), the combination of tepotinib and the EGFR TKI gefitinib improved outcomes in patients with EGFR-mutant METamp NSCLC and EGFR TKI resistance compared to chemotherapy (INSIGHT). Median progression-free survival (PFS) was 16.6 vs 4.2 months (hazard ratio [HR] = 0.13; 90% confidence interval [CI]: 0.04, 0.43) and median overall survival (OS) was 37.3 vs 13.1 months (HR = 0.08; 90% CI: 0.01, 0.51). Methods: INSIGHT 2 is a global, open-label, Phase II trial of tepotinib + osimertinib in patients with advanced EGFR-mutant NSCLC. Following a protocol amendment in Apr 2020, the study is enrolling patients with acquired resistance to 1L osimertinib (radiological documentation of disease progression following previous objective clinical benefit) due to METamp by FISH (GCN ≥5 or MET/CEP7 ratio ≥2). Patients must be ≥18 years old, have an Eastern Cooperative Oncology Group performance status of 0/1, and normal organ function. Both tissue and liquid biopsy, obtained at the time of progression to osimertinib, will be sent for central confirmation of METamp. Liquid biopsy samples will also be used for exploratory biomarker evaluation. Enrollment is allowed based on local FISH testing while awaiting central confirmation of METamp. Patients will receive 500 mg QD (450 mg active moiety) tepotinib + 80 mg QD osimertinib until disease progression, unacceptable toxicity, or consent withdrawal. The study is anticipated to enroll 120 patients. The primary endpoint is objective response rate (ORR) by independent review (RECIST v1.1) in patients with METamp, centrally confirmed by FISH. Secondary endpoints include ORR by investigator assessment, duration of response, disease control, PFS, OS, pharmacokinetics, health-related quality of life, tolerability, and safety. An exploratory tepotinib monotherapy arm will enroll 12 patients to assess the contribution of tepotinib to the activity of the combination. At progression (determined by independent review committee), monotherapy patients can switch to combination treatment. These patients will be analyzed separately. Recruitment is ongoing, with &gt; 300 patients prescreened. Approximately 100 sites in 17 countries in Europe, Asia, and North America are expected to participate. Approximately 15 sites will recruit patients in the US. Clinical trial information: NCT03940703.