Fluorescence In Situ Hybridization Detection Research Articles

Clinical, imaging and pathological and molecular characteristics of simple bone cyst

Objective: To investigate the radiologic, pathologic, and molecular features of simple bone cysts (SBC), and their differential diagnoses. Methods: Fourteen cases of SBC were collected at the Department of Pathology, the First Affiliated Hospital of Nanjing Medical University from 2017 to 2022, and fluorescence in situ hybridization (FISH) was performed for retrospective analysis. Results: There were 14 patients, including 7 females and 7 males, with age range of 7 to 45 (median 29) years. The most common complaint was pain, including 4 cases with pathological fracture and 5 with history of previous trauma. The tumor size ranged from 3.4 to 13.5 (median 5.6) cm. The lesion involved the femur (n=4), humerus (n=5) and iliac bone (n=5). Radiologic diagnoses included SBC, aneurysmal bone cyst, and giant cell tumor of the bone or its combination with aneurysmal bone cyst-like region and fibrous dysplasia. Histologically, the cyst walls of the lesions were composed of fibrous tissue, fibrin-like collagen deposits, bone-like matrix and occasional woven bone. The lesional cells were spindled to ovoid, with scattered osteoclast-like giant cells, foamy histiocytes, hemosiderin deposits and cholesterol clefts. In 6 cases there were nodular fasciitis-like areas. Immunohistochemically, the spindled to ovoid cells were positive for SMA, EMA and SATB2 in varying degrees. FISH detection was performed in all 14 cases and EWSR1/FUS rearrangement were found in 9 cases. One case of FUS::NFATC2 fusion was detected by next-generation sequencing. Nine cases of SBC with the rearrangement were more cellular, and there were more mitotic figures in the recurrent FUS::NFATC2 fusion tumor. Clinical follow-up was obtained in all 14 cases with the time ranging from 5 to 105 (mean 46) months. Amongst them, the tumor with FUS::NFATC2 rearrangement had local recurrence twice after the first local excision, but had no more recurrence or metastasis 34 months after the subsequent segmental resection. The other 13 cases had no recurrence. Conclusions: EWSR1 or FUS rearrangement is most commonly identified in SBC, suggesting that SBC might be a neoplastic disease. In cases where the radiologic appearance and histomorphology are difficult to differentiate from aneurysmal bone cyst, FISH detection can aid in the definitive diagnosis.

Detection of MDM2 gene amplification by fluorescence in situ hybridization and its diagnostic value in low-grade osteosarcoma

Objective: To investigate the diagnostic value of detecting MDM2 gene amplification by fluorescence in situ hybridization (FISH) in low-grade osteosarcoma (LGOS). Methods: Thirty cases of parosteal osteosarcoma (POS) and 14 cases of low-grade central osteosarcoma (LGCOS) from April 2009 to August 2022 at Beijing Jishuitan Hospital, Capital Medical University were analyzed for the presence of MDM2 gene amplification by FISH. Fifty-eight additional cases were used as negative controls (including 28 cases of fibrous dysplasia, 5 cases of giant cell tumor, 4 cases of conventional osteosarcoma, 2 cases each of periosteal osteosarcoma, reparative changes after fracture, pleomorphic undifferentiated sarcoma, low grade myofibroblastic sarcoma, fibrous dysplasia with malignant transformation, one case each of leiomyosarcoma, sclerosing epithelioid fibrosarcoma, malignant peripheral nerve sheath tumor, desmoplastic fibroma of bone, solitary fibrous tumor, aneurysmal bone cyst, clear cell chondrosarcoma, osteofibrous dysplasia, and 3 cases of unclassified spindle cell tumor). Results: Among the 30 patients with POS, 15 were male and 15 were female, ranging in age from 10 to 59 years (mean 35 years, median 30.5 years). Among the 14 patients with LGCOS, four were male and 10 were female, ranging in age from 15 to 56 years (mean 37 years, median 36 years). All except one case were successfully detected by FISH. MDM2 gene amplification was detected in 27 cases of POS (27/29,91.3%) and 8 cases of LGCOS (8/14). All the negative controls were negative for MDM2 gene amplification. The positive rate of MDM2 gene amplification was significantly different between the case group and the control group (P<0.05). The sensitivity and specificity of MDM2 gene amplification in diagnosing POS and LGCOS were 91.3% and 100.0%; and 57.1% and 100.0%, respectively. The sensitivity and specificity of MDM2 gene amplification in diagnosing LGOS (including POS and LGCOS) were 81.3% and 100.0%, respectively. In cases where MDM2 gene was amplified, the MDM2 amplified signal was clustered. Nine cases showed increased CEP12 signal different from polyploidy which was displayed as small and weak signal points or cloud flocculent and cluster signals. Conclusions: Detection of MDM2 gene amplification by FISH is a highly sensitive and specific marker for LGOS. The interpretation criteria for FISH detection of MDM2 amplification are currently not unified. The signal characteristics need more attention when interpreting.

Correlation of MYB/NFIB gene fusion with the grade and prognosis of head and neck adenoid cystic carcinoma and the concordance of two detection methods

Objective: To explore the correlation between MYB/NFIB gene fusion and clinicopathological features such as tumor grade and prognosis of head and neck adenoid cystic carcinoma (ACC), and to assess the concordant rate of fluorescent in situ hybridization (FISH) with MYB and NFIB immunohistochemistry. Methods: FISH detection of MYB/NFIB gene fusion was performed on 48 head and neck ACC cases and 15 non-ACC salivary gland tumors at National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China during April 2014 and January 2020. ACC cases were divided into grade Ⅰ-Ⅱ, grade Ⅲ and high-grade transformation, according to pathological grading criteria. Prognosis, FISH results and other clinicopathological characteristics were analyzed. MYB and NFIB immunohistochemistry was performed on the 48 ACC and 15 non-ACC cases. The diagnostic accuracy of FISH and immunohistochemistry was compared. Results: FISH detected MYB/NFIB gene fusion in 41.7% (20/48) of the ACC. Its positive rate was inversely correlated with higher pathological grades (P=0.036). The higher histological grade was linked to worse progression-free survival (P=0.024), whereas there was no correlation between the status of gene fusion detected by FISH and progression-free survival (P=0.536). FISH didnot detect MYB/NFIB gene fusion in 15 non-ACC salivary gland tumors The specificity of diagnosing ACC is 100% for both FISH detection of gene fusion and immunohistochemical detection of MYB expression. However, the sensitivity for both methods was only about 41.7%, respectively. By combining FISH and MYB immunohistochemistry, the sensitivity for diagnosing ACC was increased to 66.7%. Conclusions: MYB/NFIB gene fusion has a lower detection rate in grade Ⅲ ACC and high-grade transformation ACC. Meanwhile gene fusion status is not correlated with prognosis. The sensitivity for diagnosing ACC can be improved by combining FISH and MYB immunohistochemistry.

UroVysion™ fluorescence in situ hybridization (FISH) possibly has a high positive rate in carcinoma of non-urothelial lineages.

Background: Positive UroVysion™ fluorescence in situ hybridization (FISH) is generally considered as urothelial carcinoma (UC). We clarify if UroVysion™ FISH can be positive in carcinoma of non-urothelial lineages (CNUL), and verify the consistency of urine FISH and histological FISH in CNUL. Methods: All CNUL subjects detected by urine FISH assay due to haematuria from Tongji Hospital were screened. Meanwhile, 2 glandular cystitis and 2 urothelial carcinoma were served as negative or positive control. Paraffin-embedded tissue sections of all subjects were sent to the pathology department for histological FISH detection. Results: A total of 27 patients were included in this study, including 9 with adenocarcinomas, 11 with squamous cell carcinomas, and 7 with other tumour types. The overall positive rate in urine FISH was 64.00% (16/25) in patients with CNUL, 77.78% (7/9) in those with adenocarcinoma and 54.55% (6/11) in those with squamous carcinoma. There was a significant difference in the GLP p16 gene deletion rate between UC and CNUL (100% vs. 8.00%, p = 0.017). Histological FISH results showed that the histological results of 19 patients were consistent with their urine FISH results, and only one patient with stage Ⅲa urachal carcinoma had inconsistent histological FISH results (positive) and urine FISH (negative) results. Conclusion: We demonstrated for the first time the application value of FISH in CNUL on urine samples. Positive urine FISH tests indicate not only UC, but also CNUL. UroVysion™ FISH possibly has a high positive rate in CNUL. CNUL and UC have different genetic changes shown by FISH.

Diagnostic and prognostic values of flow cytometry in diffuse large B-cell lymphoma with bone marrow involvement

Objective: To analyze the diagnostic and prognostic values of flow cytometry (FC) in diffuse large B cell lymphoma (DLBCL) with bone marrow involvement (BMI). Methods: The clinical data of 412 patients with newly diagnosed DLBCL, including 243 males and 169 females, aged 64 (28-92) years old, in the Department of Hematology at Peking University Third Hospital from December 2012 to June 2022 were retrospectively analyzed. All patients underwent bone marrow biopsy (BMB) and bone marrow FC. The patients with BMI by FC were further detected by fluorescence in situ hybridization (FISH) for gene analysis. The positive rates and consistency of BMI detected by BMB and FC were evaluated. According to the results of BMB and FC examinations, all patients were divided into four groups: the BMB+FC+group (115 cases), the BMB-FC+group (50 cases), the BMB+FC-group (8 cases, the results did not include in statistical analysis because of small sample size), and the BMB-FC-group (239 cases). The clinical features, treatment response rates, 5-year survival rates, and immunophenotype characteristics by FC in different groups were analyzed. Results: Among the 412 patients with DLBCL, the positivity rates of BMB and FC for BMI detection were 29.9% (123/412) and 40.0% (165/412), respectively. Good consistency between BMB and FC was found (Kappa=0.841, P=0.001). The numbers of extranodal involvement≥2, splenomegaly, huge mass, higher Ki-67 score, higher international prognostic index (IPI) score, thrombocytopenia, and elevated lactate dehydrogenase level were more prevalent in the BMB+FC+group than those in the BMB-FC+group and the BMB-FC-group (all P<0.05). The treatment response rate in BMB+FC+group was 63.5% (73/115), which was lower than those in BMB-FC+group (88.0%, 44/50, P=0.048) and BMB-FC-group (90.0%, 215/239, P=0.032), respectively. The 5-year overall survival rates in three groups were (53.6±9.7) %, (72.5±8.6) %, and (75.2±7.6) %, respectively, with a statistically significant difference (P=0.037). According to the FISH results of bone marrow, 102 cases were diagnosed as not otherwise specified (NOS), 48 cases were diagnosed as double hit lymphoma (DHL), and 15 cases were diagnosed as triple hit lymphoma (THL). Compared with NOS subtypes, the tumor cells in DHL or THL subtypes had higher proportion of increased side scatter (SSC), higher positive rates of CD10 expression, CD38 strong expression and CD56 expression, and lower proportion of surface immunoglobulin light chain restriction (all P<0.05). Conclusions: FC is well consistent with BMB in diagnosing DLBCL with BMI. Combined with FISH detection, FC can contribute to the auxiliary diagnosis and risk stratification for DHL and THL, and provide reference for the prognostic evaluation in DLBCL with BMI.

Significance of YAP1–MAML2 rearrangement and GTF2I mutation in the diagnosis and differential diagnosis of metaplastic thymoma

Background Metaplastic thymoma (MT) is a very uncommon thymoma type, with biphasic differentiation as one of its histological characteristics. This histological pattern, however, can also be mistaken for type A thymoma and the A component in type AB thymoma. Methods Postoperative specimens were collected from five MT and four type A thymomas with a retrospective analysis involving immunohistochemistry, fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS). Results The histological morphology of the MT overlapped with that of the type A thymoma. With immunostains, the former’s spindle cell components expressed vimentin and EMA, but not CD20. In MT, 3/5 cases had the nuclear expression of YAP1. The spindle cell component of the type A thymoma was found to express CD20. In all five cases of MT, FISH detection revealed YAP1–MAML2 fusion, which was not found in any type A thymoma cases. NGS sequencing confirmed YAP1–MAML2 rearrangement in all five cases of MT, and mutations in POLE and HRAS were also found in two cases, respectively. GTF2I c.74146970 T > A mutations were found in all cases of type A thymoma, and HRAS and NRAS mutations were found in two cases, but no YAP1–MAML2 rearrangement was evident. Conclusions For the diagnosis and differential diagnosis of challenging cases, the YAP1–MAML2 rearrangement and GTF2I mutation were both significant molecular events specific to MT and type A thymoma, respectively.

Paired Capture and FISH Detection of Individual Virions Enable Cell-Free Determination of Infectious Titers.

Early detection of viruses can prevent the uncontrolled spread of viral infections. Determination of viral infectivity is also critical for determining the dosage of gene therapies, including vector-based vaccines, CAR T-cell therapies, and CRISPR therapeutics. In both cases, for viral pathogens and viral vector delivery vehicles, fast and accurate measurement of infectious titers is desirable. The most common methods for virus detection are antigen-based (rapid but not sensitive) and polymerase chain reaction (PCR)-based (sensitive but not rapid). Current viral titration methods heavily rely on cultured cells, which introduces variability within labs and between labs. Thus, it is highly desirable to directly determine the infectious titer without using cells. Here, we report the development of a direct, fast, and sensitive assay for virus detection (dubbed rapid capture fluorescence in situ hybridization (FISH) or rapture FISH) and cell-free determination of infectious titers. Importantly, we demonstrate that the virions captured are "infectious," thus serving as a more consistent proxy of infectious titers. This assay is unique because it first captures viruses bearing an intact coat protein using an aptamer and then detects genomes directly in individual virions using fluorescence in situ hybridization (FISH); thus, it is selective for infectious particles (i.e., positive for coat proteins and positive for genomes).

Application of fluorescence in situ hybridization technique to verify the clonalities of non-clonal cytogenetic abnormalities identified in Myelodysplastic syndrome

To assess the value of fluorescence in situ hybridization (FISH) technique for the verification of the clonalities of non-clonal cytogenetic abnormalities (n-CCA) identified by conventional chromosome banding analysis (CBA) in patients with Myelodysplastic syndrome (MDS). Clinical data and results of karyotyping and FISH assays for 91 patients of MDS with n-CCA identified by CBA were retrospectively analyzed. In total 94 non-clonal +8, 5q-, -7/7q- or 20q- were detected by CBA, among which 43 (45.7%) were verified to be clonal abnormalities by FISH. The detection rates for +8, 5q-, -7/7q- and 20q- by FISH were 47.6% (30/63), 25% (2/8), 41.7% (5/12), 40% (2/5) and 66.7% (4/6), respectively, with the positive cells accounting for 4% to 90% of all counted cells, with a median value of 7%. The 91 patients were divided into three groups including ≥ 20, 10 ~< 20 and < 10 based on the numbers of metaphase cells in CBA, and the detection rates by FISH for the three groups were 43.7% (31/71), 33.3% (3/9) and 63.6% (7/11), respectively, which showed no statistically difference (P > 0.05). Continuous CBA and FISH surveys were conducted for 26 patients who received supportive treatment, and the results revealed that 91.7% (11/12) of FISH-verified positive abnormalities had persisted, whereas 92.9% (13/14) of the n-CCA verified as negative by FISH was transient. Nearly half of the CBA identified n-CCA have been verified as clonal aberrations by FISH, and the FISH detection rate showed no correlation with the number of metaphase cells. FISH test is strongly recommended for verifying the clonalities of n-CCA detected by CBA, and continuous cytogenetic survey of the patients with MDS is necessary.

Is t(11;14)(q13;q32) good or bad for newly diagnosed multiple myeloma?

Is t(11;14)(q13;q32) good or bad for newly diagnosed multiple myeloma?

Clinicopathological features and genomic profiles of a group of secretory breast carcinomas in which progressive cases have more complex genomic features

BackgroundSecretory breast carcinoma (SBC) is a rare malignant breast neoplasm with distinct histological features, including solid, microcystic, tubular, and rarely papillary structures, traditionally characterized by a t (12;15) (p13:q25) translocation, which usually leads to ETV6-NTRK3 fusion, suggesting an early event in tumorigenesis. Due to the rarity of this disease, very few genome sequencing studies have been performed on a series of cases, especially progressive cases.MethodsSeven lesions from 5 patients diagnosed at the Third Affiliated Hospital of Soochow University from 2007 to 2021 were included. Clinicopathological features and prognosis/survival data were collected. Next-generation DNA sequencing was performed on six of the seven lesions.ResultsIn total, 3/7 (42.9%) lesions demonstrated estrogen receptor (ER) expression, including weak, moderate to strong staining, and no lesion demonstrated progesterone receptor (PR) expression. There were no cases of human epidermal growth factor (HER2) overexpression, and the Ki-67 index was low. S-100 and pan-TRK protein were diffusely positively expressed in all cases. All lesions were characterized by a t(12;15) (p13:q25) translocation, leading to ETV6-NTRK3 fusion confirmed by fluorescence in situ hybridization (FISH). The sequencing results showed that ETV6-NTRK3 fusion was the main driver of early tumorigenesis, while SBC with invasive biological behavior had more complex genomic variation in which TERT promoter mutation was detected.ConclusionsImmunohistochemical staining of a biomarker panel, including ER, PR, HER2, Ki-67, S-100 and pan-TRK, can be used as an auxiliary diagnostic tool, and FISH detection can be used as a diagnostic tool. ETV6-NTRK3 gene fusion involving multiple sites may drive tumorigenesis, while mutations in the TERT promoter region may be a factor driving tumor progression.

A novel method for making cell blocks with higher cellular yield.

Cytopathology is an important part of pathology that is used to diagnose disease on the cellular level. The application of the cell block (CB) technique plays a vital role in cytological diagnosis, as blocks and slides can be further used for special stains, immunohistochemistry (IHC), and molecular pathological analysis. Several methods for making CBs have been reported, but their procedures and cellular yield are still deemed unsatisfactory. In this article, we used gellan gum (GG) as an adjuvant for CBs, which resulted in higher cellular yield with simpler procedures. CBs were prepared by using GG, copper sulfate, plasma/thrombin, or pregelatinized starch methods. The procedures of each of these four methods were then compared. CB sections were stained with hematoxylin and eosin (H&E), and the background and morphological features seen by H&E staining were compared. A preliminary IHC and fluorescence in situ hybridization (FISH) study was performed using cytology specimens from elevenand five cases, respectively. The expression of immunocomplex by IHC and the molecular signals detected by FISH were compared in CB sections made by the four methods and a section derived from the biopsy specimen block from the same patient. Feulgen staining, Alcian blue staining, and Masson trichrome staining were performed on the CB sections from 3 cases of pleural fluid. The cellular yield of CB sections from 83 cases according to the four methods was compared using NDP analysis software. The results demonstrated that sections derived from CBs made with GG had a clear background and good morphological features by H&E staining. The expression of immunocomplex by IHC and the molecular signals of FISH detection in the sections from CBs made by GG were accurately located just as those in biopsy sections from the same patient. The DNA, acidic mucus, and fibrin could be clearly identified through special stains in the CB sections. The procedures involved in the GG method were easily controllable and the coagulated gel increased the ease by which the CB was embedded and sectioned. Specifically, sections from CBs made by the GG method contained higher cellular yield because cells could be concentrated on the bottom of the gel after centrifugation. This novel method for making CBs is a practical, simple method that can result in higher cellular yield. This method is therefore worth promoting in clinical applications.

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma.

Fluorescence in situ hybridization (FISH) detection is an indispensable method in genetic risk stratification in multiple myeloma (MM), which is one of the most common hematological malignancies. The identifying characteristic of MM is accumulated malignant plasma cells in bone marrow. FISH reports for MM mainly focus on purified or identified clonal plasma cells, rather than all nucleated cells, by sorting with anti-CD138 magnetic beads or marking with cytoplasmic immunoglobulin light chain κ or λ. Bone marrow interphase nuclei are usually obtained from fresh bone marrow cells. However, satisfactory enrichment of plasma cell specimens requires large amounts of fresh heparin anti-coagulated bone marrow, which cannot be obtained in the case of difficult bone marrow extraction or a bone marrow dry tap. Herein, we establish a novel method to improve the success of FISH detection on stained or unstained bone marrow smears. Bone marrow smears are easier to obtain than anticoagulated bone marrow specimens.

Preparation of long single-strand DNA concatemers for high-level fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) is a powerful tool to visualize transcripts in fixed cells and tissues. Despite the recent advances in FISH detection methods, it remains challenging to achieve high-level FISH imaging with a simple workflow. Here, we introduce a method to prepare long single-strand DNA concatemers (lssDNAc) through a controllable rolling-circle amplification (CRCA). Prepared lssDNAcs are used to develop AmpFISH workflows. In addition, we present its applications in different scenarios. AmpFISH shows the following advantages: 1) enhanced FISH signal-to-noise ratio (SNR) up to 160-fold compared with single-molecule FISH; 2) simultaneous detection of FISH signals and fluorescent proteins or immunofluorescence (IF) in tissues; 3) simple workflows; and 4) cost-efficiency. In brief, AmpFISH provides convenient and versatile tools for sensitive RNA/DNA detection and to gain useful information on cellular molecules using simple workflows.

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.

Mesenchymal chondrosarcoma in central nervous system: a clinicopathological analysis

Objective: To investigate the clinicopathological features of central nervous system (CNS) mesenchymal chondrosarcoma (MCS). Methods: Nine cases of CNS MCS were collected at the First Affiliated Hospital of Fujian Medical University from September 2010 to September 2020. The clinical,imaging,histopathological and immunohistochemical features were reviewed. NCOA2 gene rearrangement was evaluated by fluorescence in situ hybridization (FISH). Results: There were three male and six female patients, with age range of 1 to 59 years (median 31 years). Six cases were intracranial and three cases were intraspinal, and the tumors showed dural attachment. They were often diagnosed as meningioma basing on preoperative imaging. Microscopically, the tumors showed a characteristic biphasic histologic pattern composed of undifferentiated mesenchymal small cells and well-differentiated hyaline cartilage islands. The small cells area were positive for SOX9 (9/9), CD99 (8/9), and without BRG1 and INI1 deletion. The cartilaginous component expressed SOX9 (9/9) and S-100 protein (8/9). NCOA2 gene break apart signal was identified in five cases (5/5). Eight patients were followed up for 4-124 months. Three patients (3/8) had recurrences within one year and two patients died of the tumor. Conclusions: CNS MCS is an extremely rare malignant neoplasm with a propensity to dural involvement. Preoperative imaging has low diagnostic accuracy. CNS MCS should be differentiated from other CNS small round cell tumors and chondrosarcoma. FISH detection of NCOA2 gene rearrangement will assist the diagnosis of MCS.

The Cytogenetic Aberrations and Their Prognostic Value in 181 Untreated Patients with Lymphoplasmacytic Lymphoma/ Waldenström Macroglobulinemia in China

Background: lymphoplasmacytic lymphoma/ Waldenström macroglobulinemia (LPL/WM) is a rare B cell proliferative malignancy characterized by immunoglobulin M monoclonal gammopathy and /or bone marrow infiltration by lymphoplasmacytic cells. Cytogenetic aberrations are now the main prognostic predicators in some B-NHLs such as CLL. However, their prognostic valve in LPL/WM remains controversial and recurrently chromosomal abnormalities have rarely been reported. Furthermore, the genetic basis of LPL/WM in China is also unclear.Methods: A panel of DNA probes was used to detect cytogenetic aberrations by fluorescence in situ hybridization (FISH), including RB1/D13S25 at 13q14, ATM at 11q22, TP 53 at 17p13, CEP12 and IGH translocation at 14q32, MYB at 6q23. Chromosomes were identified by G-banding and karyotypes were described according to the International System for Human Cytogenetics Nomenclature (ISCN, 1995). Totally, 181 untreated LPL/WM patients were enrolled in this study.Results: The median age of 181 patients was 61 years (range 32-87). The male to female ratio was 2.75 (133/48). 90% and 15.7% patients showed an elevated serumβ2 microglobulin and LDH. The percentage of hepatomegaly, splenomegaly, lymphadenopathy and B symptom were 15.3%, 35.2%, 50% and 27.3%, respectively. Almost half patients were in the high risk ISSWM group. The chromosomal karyotypes were successful in 153/181 (84.5%) cases, of which 15 patients showed clonal abnormalities. 8 of the 15 patients with abnormal chromosome were complex. The main aberrations of chromosomal karyotypes were 6q deletion (3/15, 20%), and loss of sex chromosomes (3/15, 20%). The other abnormalities include trisomy 12, trisomy 4, trisomy 3, 20q deletion, 17p deletion and so on. All the 6q deletions patients were in the complex karyotype group. Moreover, the trisomy 12 detected by FISH were always accompanied with chromosomal karyotypes abnormalities. Of 181 patients, 139 patients had FISH detection, of which 25 patients (18%) had FISH abnormalities. The most common aberrations were 6q deletion (6/28, 21.4%), trisomy 12 (5/59, 8.5%), 17p deletion (8/131, 6.1%). 13q deletion, gain 1q21and 11qdeletion were 4.6% (6/130) and 2.8% (3/36) and 2% (2/99), respectively. About 5.4% (7/130) patients had IGH gene translocation. Although it was higher in our study, this aberration always accompanied with other clones. Besides, 17p deletion was also more frequent in complex clone group, while trisomy 12 was more common in the single clone group. In LPL/WM, the chromosomal abnormities was an adverse predictor for both progress-free survival (PFS) and overall survival (OS). The median PFS and OS for patients with this abnormity was 62 months (62m vs 69m, p=0.022) and 75 months (75m vs 109m, p=0.033), which was significantly higher than normal patients. In different FISH groups, only TP 53 deletion had a poor effect on both PFS and OS in LPL/WM patients. The median PFS and OS were significantly shorter than those without the abnormality (median PFS 34m vs 77m, p=0.008; median OS 34m vs 135m, p=0.006). However, the 6q deletion and trisomy 12 did not significantly influence both PFS and OS. In addition, we found that the number of different clones had an effect on the survival of patients. The complex clone group showed shorter PFS and OS than the single clone group (p<0.05).Conclusion: Although the detection of cytogenetic aberration is rare in Chinese patients, it plays an essential role in diagnosis and prognostication. DisclosuresNo relevant conflicts of interest to declare.

Nodular fasciitis of the breast: a clinicopathological and genetic analysis of seven cases

Objective: To investigate the clinicopathological and genetic features of nodular fasciitis of the breast (NFB). Methods: The clinical and histologic features of seven NFBs were retrospectively reviewed. Immunohistochemistry, fluorescence in situ hybridization (FISH) and reverse transcription-polymerase chain reaction (RT-PCR) were performed. Results: All the seven patients were female, with a mean age of 36 years (range from 15 to 51 years). The duration of the lesion ranged from 10 days to 2 years. There was no history of trauma for all patients. The lesions occurred in the upper quadrant (4 cases), the lower quadrant (2 cases) and the axillary tail region (1 case). The maximum diameter was 1.0-3.5 cm. All cases showed similar morphology as nodular fasciitis occurring elsewhere in the body. They were composed of plump spindle cells arranged in short bundles or fascicles within a loose collagenous/myxoid stroma. Erythrocyte extravasation, mixed chronic inflammatory cells infiltration and microcystic changes were typically seen. Mitoses were present, with no atypical mitoses observed. The spindle cells were positive for smooth muscleactin(SMA, 6/6), CD10(2/3), and negative for desmin, β-catenin, CD34, CKpan, EMA, S-100, p63 and ALK-1.The Ki-67 index were 5%-15%. USP6 gene rearrangement was found in six cases and MYH9-USP6 gene fusion in two cases. Local resection was performed in six cases. Spontaneous regression was observed in one case. Follow-up of all seven cases revealed no recurrence or metastasis. Conclusions: Although rare, NFB can mimic breast cancer clinically, radiologically and histologically. It should be always considered in the differential diagnosis for the spindle cell proliferations of the breast. A diagnosis of NFB can be achieved basing on the typical clinicopathological presentation. FISH detection of USP6 gene rearrangement in challenging cases is of great value.

High Expression of MDM2 and the p53 Protein is Predictive Biomarkers for Poor Prognosis of Oesophageal Squamous Cell Carcinoma.

Background and ObjectiveIn the present study, we detected the expression of MDM2 and p53 in oesophageal squamous cell carcinoma (OSCC) specimens, studied their relationship with the survival of OSCC patients, and explored the potential of MDM2 and p53 to serve as predictive OSCC tumour markers.Patients and MethodsThrough immunohistochemistry and fluorescence in situ hybridization (FISH), we detected the expression of MDM2 and the p53 protein in 157 OSCC specimens that met the inclusion and exclusion criteria. After scoring the results, Pearson’s chi-square test and Cox regression were used for analysis.ResultsThe results showed that the rates of high MDM2 and p53 expression in OSCC tissues were 60.5% and 51.0%, respectively. The expression levels of MDM2 and p53 in OSCC were significantly positively correlated (p<0.001, r=0.414). In addition, the pathological metastasis (M) status and MDM2 protein expression in OSCC were significantly correlated (p=0.027), and high expression of the p53 protein was positively correlated with OSCC transfer (p=0.005), pathological node status (p=0.008), and clinical stage (p=0.003). Kaplan-Meier survival analysis showed that the high expression of MDM2 and p53 was significantly related to the poor prognosis of OSCC. Moreover, subgroup analysis of the TNM staging of OSCC patients showed that the high expression of MDM2 and p53 was significantly correlated with poor OS and DFS of OSCC patients in either stage I–II or III–IV patients. Both univariate and Cox multivariate analyses showed that p53 and MDM2 can be used as independent factors for the prognosis of OSCC patients. Finally, our FISH detection results for MDM2 showed that the high expression of MDM2 was significantly correlated with the amplification of MDM2 (p=0.015).ConclusionThis study shows that MDM2 and p53 can be used as independent predictors of the prognosis of patients with oesophageal squamous cell carcinoma.

Fluorescence in Situ Hybridization for Detecting Molecular Cytogenetic Abnormalities of Chronic Lymphocytic Leukemia

To investigate the value of fluorescence in situ hybridization (FISH) in the diagnosis and prognosis evaluation of patients with chronic lymphocytic leukemia (CLL). Ninty-three patients with newly diagnosed CLL were tested by five probes including RB1 (13q14.1), D13S25 (13q14.3), p53(17p13.1), ATM( 11q22.3) and CSP12, while conventional cytogenetics (CC) was used for karyotype analysis. Then the correlation of the molecular cytogenetic abnormalities with the clinical Binet stages, Rai stages and the other related laboratory examinations was analyzed. The detection rate of chromosome abnormality in 93 patients was 79.6%, out of which detection rate of 13q (13q- was the highest and accounted for 45.2%), followed by trisomy 12 (+12) 26.9%, p53 deletion (17p-) 19.4% and ATM deletion (11p-) 17.2%. There were 27 cases (29.0%) with 2 or more abnormalities, including 13 cases with 13q-/17q-, 5 with 13q-/11q-, and 4 with 13q-/+12. Compared with CC test results, the positive rate of FISH detection was significantly higher (χ2=32.127, P＜0.01). There was no significant correlation between FISH results and Rai stages (P＞0.05), meanwhile 17p- highly correlated with later stage of the Binet stages (P=0.012). The molecular cytogenetic abnormalities significantly correlated with age, absolute value of peripheral lymphocyte count and CD38 expression level (P＞0.05). The incidence of 13q- in female (65.4%) was statistically significantly higher than that in male (37.3%) (P=0.015). The unmutated IGHV rate of CLL patients with a 17p- was significantly higher than that in patients without this genetic abnormality (P=0.013). The expression of CD38 was detected among 29.0% of the patients, which significantly correlated with Binet stages (P=0.027) and unmutated IGHV (P=0.006). FISH can greatly increase the detection rate of molecular cytogenetic abnormalities in CLL patients, which, as a powerful supplement to the conventional cytogenetics, can be applied for the clinical staging and prognosis evaluation of CLL patients.

3D Immuno-DNA Fluorescence In Situ Hybridization (FISH) for Detection of HIV-1 and Cellular Genes in Primary CD4+ T Cells.

Fluorescence in situ hybridization (FISH) is a powerful, broadly used microscopy-based technique that leverages fluorescently labeled nucleic acid probes to detect parts of the genome inside metaphase or interphase cell nuclei. In recent years, different methodologies developed to visualize genome topology and spatial relationships between genes have gained much attention as instruments to decode the relationship between chromatin structure and function. In addition to chromosome conformation capture-based techniques, highly multiplexed forms of FISH combined with high-throughput and super-resolution microscopy are used to map and spatially define contact frequencies between different genomic regions. All these approaches have strongly contributed to our knowledge of how the human genome is packed in the cell nucleus.In this chapter, we describe detailed step-by-step protocols for 3D immuno-DNA FISH detection of genes and Human immunodeficiency virus 1 (HIV-1) provirus in primary CD4+ T cells from healthy donors, or cells infected in vitro with the virus. Our multicolor 3D-FISH technique allows, by using up to three fluorophores, visualization of spatial positioning of loci inside a 3D cell nucleus.