Biofilms and core pathogens shape the tumor microenvironment and immune phenotype in colorectal cancer.

Extensive research has explored the role of gut microbiota in colorectal cancer (CRC). Nonetheless, metatranscriptomic studies investigating the in situ functional implications of host-microbe interactions in CRC are scarce. Therefore, we characterized the influence of CRC core pathogens and biofilms on the tumor microenvironment (TME) in 40 CRC, paired normal, and healthy tissue biopsies using fluorescence in situ hybridization (FISH) and dual-RNA sequencing. FISH revealed that Fusobacterium spp. was associated with increased bacterial biomass and inflammatory response in CRC samples. Dual-RNA sequencing demonstrated increased expression of pro-inflammatory cytokines, defensins, matrix-metalloproteases, and immunomodulatory factors in CRC samples with high bacterial activity. In addition, bacterial activity correlated with the infiltration of several immune cell subtypes, including M2 macrophages and regulatory T-cells in CRC samples. Specifically, Bacteroides fragilis and Fusobacterium nucleatum correlated with the infiltration of neutrophils and CD4+ T-cells, respectively. The collective bacterial activity/biomass appeared to exert a more significant influence on the TME than core pathogens, underscoring the intricate interplay between gut microbiota and CRC. These results emphasize how biofilms and core pathogens shape the immune phenotype and TME in CRC while highlighting the need to extend the bacterial scope beyond CRC pathogens to advance our understanding and identify treatment targets.

Mesothelioma: morphologic and immunohistochemical findings.

This paper reviews some basic and some new concepts in the diagnosis of mesothelioma. The term "malignant mesothelioma" is no longer recommended; rather, any tumor labeled "mesothelioma" is presumed to be malignant. Clinical and radiologic information is very useful in the diagnosis of mesothelioma; in particular, nodular pleural thickening on CT is usually a marker of malignancy. The literature on markers that separate mesotheliomas from metastatic carcinomas has become very complex and frequently misleading, with many recommended markers actually demonstrating poor specificity. However, newer data show that a combination of HEG1 (clone SKM9-2) and claudin­4 staining provides extremely high accuracy in separating epithelioid mesotheliomas from non-small-cell lung carcinomas with just two immunostains. This combination works at other sites as well, but caution should be used when high-grade serous carcinoma is in the differential, because all "mesothelioma" markers can also stain high-grade serous carcinomas. There are, unfortunately, no sensitive or specific markers for sarcomatoid mesotheliomas. A variety of immunohistochemical and fluorescence in situ hybridization (FISH) markers are useful in separating benign from malignant mesothelial proliferations; immunohistochemal staining for BAP1, MTAP (or CDKN2A FISH), and NF2/Merlin (or NF2 FISH) will enable the diagnosis of most mesotheliomas. Mesothelioma in situ is now recognized as either a single layer of bland cuboidal mesothelial cells that have lost BAP1, and sometimes MTAP, on immunohistochemical staining, or a process that is morphologically identical to a well-differentiated papillary mesothelial tumor that has lost BAP1/MTAP. Mesothelioma in situ probably always progresses to invasive mesothelioma, but this process is often quite slow.

Recommendations for the study of monoclonal gammopathies in the clinical laboratory. A consensus of the Spanish Society of Laboratory Medicine and the Spanish Society of Hematology and Hemotherapy. Part I: Update on laboratory tests for the study of monoclonal gammopathies.

Monoclonal gammopathies (MG) are characterized by the proliferation of plasma cells that produce identical abnormal immunoglobulins (intact or some of their subunits). This abnormal immunoglobulin component is called monoclonal protein (M-protein), and is considered a biomarker of proliferative activity. The identification, characterization and measurement of M-protein is essential for the management of MG. We conducted a systematic review of the different tests and measurement methods used in the clinical laboratory for the study of M-protein in serum and urine, the biochemistry and hematology tests necessary for clinical evaluation, and studies in bone marrow, peripheral blood and other tissues. This review included literature published between 2009 and 2022. The paper discusses the main methodological characteristics and limitations, as well as the purpose and clinical value of the different tests used in the diagnosis, prognosis, monitoring and assessment of treatment response in MG. Included are methods for the study of M-protein, namely electrophoresis, measurement of immunoglobulin levels, serum free light chains, immunoglobulin heavy chain/light chain pairs, and mass spectrometry, and for the bone marrow examination, morphological analysis, cytogenetics, molecular techniques, and multiparameter flow cytometry.

TFEB-translocated and -amplified renal cell carcinoma with VEGFA co-amplification: A case of long-term control by multimodal therapy including a vascular endothelial growth factor-receptor inhibitor.

Introduction: Renal cell carcinoma with TFEB amplification is rare and reportedly aggressive. We herein report a case of renal cell carcinoma with TFEB translocation and amplification in which long-term control was achieved by multimodal therapy including a vascular endothelial growth factor -receptor inhibitor. Case presentation: A 70-year-old man was referred to our institution for the treatment of renal cell carcinoma with multinodal metastases. Open nephrectomy and lymph node dissection were performed. Immunohistochemistry for transcription factor EB was positive, and fluorescent in situ hybridization revealed TFEB rearrangement and amplification. The diagnosis was TFEB-translocated and -amplified renal cell carcinoma. VEGFA amplification was also demonstrated by fluorescent in situ hybridization. The residual and recurrent tumors were treated and controlled for 52 months by vascular endothelial growth factor-receptor target therapy, radiation therapy, and additional surgery. Conclusion: A good long-term response to anti-vascular endothelial growth factor drug therapy may be due to VEGFA amplification and subsequent vascular endothelial growth factor overexpression.

[Clinical value of fluorescence in situ hybridization with MDM2 and DDIT3 probe in diagnosis of liposarcoma].

OBJECTIVE: To investigate the value of using MDM2 amplification probe and DDIT3 dual-color, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique in the diagnosis of liposarcoma. METHODS: In the study, 62 cases of liposarcoma diagnosed in Peking University First Hospital from January 2015 to December 2019 were analysed for clinicopathological information. Of these 62 cases of liposarcoma, all were analysed for MDM2 amplification and 48 cases were analysed for DDIT3 rearrangement using a FISH technique. Our study aimed to evaluate the status of MDM2 and DDIT3 by FISH in liposarcoma and correlate it with diagnosis of different subtypes of liposarcoma. The subtypes of liposarcoma were classified according to the FISH results, combined with the relevant clinicopathological features. RESULTS: The patients aged 31-89 years (mean: 59 years) with a 1.75:1 male to female ratio. Histologically, there were 20 cases of atypical lipomatous tumour/well-differentiated liposarcoma (ALT/WDLPS), 26 cases of dedifferentiated liposarcoma (DDLPS), 13 myxoid liposarcoma (MLPS) and 3 pleomorphic liposarcoma (PLPS). Tumors with DDLPS (23/26) and WDLPS (8/20) were localized retroperitoneally, while both tumours of MLPS and PLPS were localized extra-retroperitoneally, and the difference of sites among the four subtypes of liposarcoma was statistically significant (P < 0.05). Histologically, varied mucoid matrix could be observed in the four subtypes of liposarcoma, and the difference was statistically significant (P < 0.05). MDM2 gene amplification was demonstrated in all cases of ALT/WDLPS and DDLPS (100%, 20/20 and 26/26 respectively); DDIT3 gene rearrangement was noted only in MLPS (100%, 13/13); most cases of DDLPS (96.2%, 25/26) and ALT/WDLPS (83.3%, 5/6, 6 cases selected for detection) demonstrated the picture of amplification of the DDIT3 telomeric tag. According to the instructions of DDIT3 break-apart rearrangement probe, the 5' telomere probe and 3' centromere probe spanned but did not cover the DDIT3 gene itself, on the contrary, the 5' telomere probe covered the CDK4 gene, while the DDIT3 and CDK4 gene were located adjacent to each other on chromosome, therefore, when the amplification signal appeared on the telomeric tag of the DDIT3 rearrangement probe, it indeed indicated the CDK4 gene amplification rather than the DDIT3 gene rearrangement. Then the 10 cases with DDIT3 telomeric tag amplification were selected for CDK4 and DDIT3 gene amplification probe FISH tests, and all the cases showed CDK4 gene amplification (100%, 10/10) and two of the 10 cases demonstrated co-amplification of CDK4 and DDIT3 (20%, 2/10); DDIT3 polysomy detected by DDIT3 gene rearrangement probe was found in 1 case of DDLPS and 2 cases of PLPS (66.7%, 2/3) with morphology of high-grade malignant tumour and poor prognosis. CONCLUSION: Our results indicate that a diagnosis of different subtype liposarcoma could be confirmed based on the application of MDM2 and DDIT3 FISH, combined with clinicopathological findings. It is also noteworthy that atypical signals should be correctly interpreted to guide correct treatment of liposarcomas.

Detection of cryptic balanced chromosomal rearrangements using high-resolution optical genome mapping.

BACKGROUND: Chromosomal rearrangements have profound consequences in diverse human genetic diseases. Currently, the detection of balanced chromosomal rearrangements (BCRs) mainly relies on routine cytogenetic G-banded karyotyping. However, cryptic BCRs are hard to detect by karyotyping, and the risk of miscarriage or delivering abnormal offspring with congenital malformations in carrier couples is significantly increased. In the present study, we aimed to investigate the potential of single-molecule optical genome mapping (OGM) in unravelling cryptic chromosomal rearrangements. METHODS: Eleven couples with normal karyotypes that had abortions/affected offspring with unbalanced rearrangements were enrolled. Ultra-high-molecular-weight DNA was isolated from peripheral blood cells and processed via OGM. The genome assembly was performed followed by variant calling and annotation. Meanwhile, multiple detection strategies, including FISH, long-range-PCR amplicon-based next-generation sequencing and Sanger sequencing were implemented to confirm the results obtained from OGM. RESULTS: High-resolution OGM successfully detected cryptic reciprocal translocation in all recruited couples, which was consistent with the results of FISH and sequencing. All high-confidence cryptic chromosomal translocations detected by OGM were confirmed by sequencing analysis of rearrangement breakpoints. Moreover, OGM revealed additional complex rearrangement events such as inverted aberrations, further refining potential genetic interpretation. CONCLUSION: To the best of our knowledge, this is the first study wherein OGM facilitate the rapid and robust detection of cryptic chromosomal reciprocal translocations in clinical practice. With the excellent performance, our findings suggest that OGM is well qualified as an accurate, comprehensive and first-line method for detecting cryptic BCRs in routine clinical testing.

Clinical value of fluorescence in situ hybridization with MDM2 and DDIT3 probe in diagnosis of liposarcoma / 北京大学学报(医学版)

OBJECTIVE@#To investigate the value of using MDM2 amplification probe and DDIT3 dual-color, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique in the diagnosis of liposarcoma.@*METHODS@#In the study, 62 cases of liposarcoma diagnosed in Peking University First Hospital from January 2015 to December 2019 were analysed for clinicopathological information. Of these 62 cases of liposarcoma, all were analysed for MDM2 amplification and 48 cases were analysed for DDIT3 rearrangement using a FISH technique. Our study aimed to evaluate the status of MDM2 and DDIT3 by FISH in liposarcoma and correlate it with diagnosis of different subtypes of liposarcoma. The subtypes of liposarcoma were classified according to the FISH results, combined with the relevant clinicopathological features.@*RESULTS@#The patients aged 31-89 years (mean: 59 years) with a 1.75:1 male to female ratio. Histologically, there were 20 cases of atypical lipomatous tumour/well-differentiated liposarcoma (ALT/WDLPS), 26 cases of dedifferentiated liposarcoma (DDLPS), 13 myxoid liposarcoma (MLPS) and 3 pleomorphic liposarcoma (PLPS). Tumors with DDLPS (23/26) and WDLPS (8/20) were localized retroperitoneally, while both tumours of MLPS and PLPS were localized extra-retroperitoneally, and the difference of sites among the four subtypes of liposarcoma was statistically significant (P < 0.05). Histologically, varied mucoid matrix could be observed in the four subtypes of liposarcoma, and the difference was statistically significant (P < 0.05). MDM2 gene amplification was demonstrated in all cases of ALT/WDLPS and DDLPS (100%, 20/20 and 26/26 respectively); DDIT3 gene rearrangement was noted only in MLPS (100%, 13/13); most cases of DDLPS (96.2%, 25/26) and ALT/WDLPS (83.3%, 5/6, 6 cases selected for detection) demonstrated the picture of amplification of the DDIT3 telomeric tag. According to the instructions of DDIT3 break-apart rearrangement probe, the 5' telomere probe and 3' centromere probe spanned but did not cover the DDIT3 gene itself, on the contrary, the 5' telomere probe covered the CDK4 gene, while the DDIT3 and CDK4 gene were located adjacent to each other on chromosome, therefore, when the amplification signal appeared on the telomeric tag of the DDIT3 rearrangement probe, it indeed indicated the CDK4 gene amplification rather than the DDIT3 gene rearrangement. Then the 10 cases with DDIT3 telomeric tag amplification were selected for CDK4 and DDIT3 gene amplification probe FISH tests, and all the cases showed CDK4 gene amplification (100%, 10/10) and two of the 10 cases demonstrated co-amplification of CDK4 and DDIT3 (20%, 2/10); DDIT3 polysomy detected by DDIT3 gene rearrangement probe was found in 1 case of DDLPS and 2 cases of PLPS (66.7%, 2/3) with morphology of high-grade malignant tumour and poor prognosis.@*CONCLUSION@#Our results indicate that a diagnosis of different subtype liposarcoma could be confirmed based on the application of MDM2 and DDIT3 FISH, combined with clinicopathological findings. It is also noteworthy that atypical signals should be correctly interpreted to guide correct treatment of liposarcomas.

[Recurrent intracerebral haemorrhage in a 24-year-old female patient]. / Rezidivierende intrazerebrale Blutungen bei einer 24-jährigen Patientin.

A 24-year-old female patient from Sierra Leone was referred to the authors' hospital after several unclear intracerebral bleeding events and an echogenic structure on the aortic valve. The patient was receiving oral anticoagulation therapy due to paroxysmal atrial fibrillation and left ventricular noncompaction. Fluorescence in situ hybridization in combination with polymerase chain reaction and sequencing revealed infective endocarditis of the mitral and aortic valve caused by Bartonella quintana. In retrospect, the intracerebral bleeding events could be identified as septic emboli with secondary haemorrhagic transformation under anticoagulation therapy. The patient showed significant clinical improvement and no further bleeding events occurred after receiving biological mitral and aortic valve replacement and several weeks of doxycycline and gentamicin antibiotic therapy.

Caracterización clínica y citogenética de una cohorte de pacientes con leucemia promielocítica aguda atendidos en un Hospital Universitario en Medellín, Colombia / Clinical and cytogenetic characterization of a cohort of patients with acute promyelocytic leukemia treated at a University Hospital in Medellín, Colombia

Introducción. La leucemia promielocítica aguda (LPA) es un subtipo poco frecuente de leucemia mieloide aguda (LMA), que se caracteriza por un comportamiento clínico particularmente agresivo, y en ausencia de tratamiento, su curso generalmente es fatal. El objetivo de este trabajo fue determinar las características clínicas y citogenéticas de una cohorte de pacientes con LPA, con la finalidad de evaluar su relación con las complicaciones, el pronóstico y el desenlace de estos pacientes. Metodología. Se realizó un estudio observacional, descriptivo, retrospectivo de los pacientes mayores de 15 años con diagnóstico de LPA, atendidos en el Hospital Universitario San Vicente Fundación, entre los años 2012 a 2020. Resultados. Un total de 32 pacientes fueron incluidos. La edad media del diagnóstico fue 37 años. El 84,4% de los pacientes tenía la traslocación (15;17) en el cariotipo, y el 93,75% tenían FISH positivo. El 12,5% de los casos tenían cariotipo complejo. La mortalidad en los primeros 30 días fue del 15,6%, siendo el sangrado la causa de muerte más frecuente. Todos los pacientes que sobrevivieron alcanzaron la remisión completa (84,3%). En un promedio de seguimiento de 24 meses, el 14,8% de los casos recayeron. En el análisis bivariado se encontró relación entre sexo masculino y tener cariotipo complejo (p=0,015). No se encontró relación entre cariotipo complejo y mortalidad temprana (p=0,358), tampoco entre cariotipo complejo y recaída (p=0,052). Conclusiones. Se presentan las características clínicas y citogenéticas de una cohorte de pacientes con LPA en Colombia. El sangrado en el sistema nervioso central fue la principal causa de mortalidad temprana, todos los pacientes que sobrevivieron alcanzaron la remisión completa con la terapia de inducción. Las tasas de mortalidad, remisión completa y recaída fueron similares a las reportadas por otras series latinoamericanas, pero inferiores a estudios provenientes de países europeos. Contrario a lo reportado en otros estudios, no se encontró relación entre el cariotipo complejo y la mortalidad temprana o recaída.

Introduction. Acute promyelocytic leukemia (APL) is a rare subtype of acute myeloid leukemia (AML), characterized by a particularly aggressive clinical behavior, that in the absence of treatment is usually fatal. The objective of this work was to determine the clinical and cytogenetic characteristics of a cohort of patients with APL, in order to evaluate their relationship with the outcome and prognosis of these patients. Methodology. An observational, descriptive, retrospective study of patients older than 15 years with a diagnosis of APL treated at the Hospital Universitario San Vicente Fundación, between 2012 and 2020, was carried out. Results. A total of 32 patients were included. The mean age at diagnosis was 37 years, 84.4% of the patients had the t(15;17) in the karyotype, and 93.75% had positive FISH. 12.5% of cases had a complex karyotype. Mortality in the first 30 days was 15.6%, with bleeding being the most common cause of death. All patients who survived achieved complete remission (84.3%). In an average follow-up of 24 months, 14.8% of cases relapsed. In the bivariate analysis, a relationship was found between the male sex and having a complex karyotype (p<0.015). No relationship was found between complex karyotype and early mortality (p=0.358), nor between complex karyotype and relapse (p=0.052). Conclusions. We present the clinical and cytogenetic characteristics of a cohort of patients with APL in Colombia. Central nervous system bleeding was the main cause of early mortality, with all surviving patients achieving complete remission on induction therapy. Mortality, complete remission and relapse rates were similar to those reported by other Latin American series, but lower than studies from European countries. Contrary to what has been reported in other studies, no relationship was found between complex karyotype and early mortality or relapse

[Epigenetics of Prader-Willi Syndrome: A good example of translational medicine]. / Epigenética del síndrome de Prader-Willi: un buen ejemplo de medicina traslacional.

In this editorial the author presents a study, concerning Prader-Willi syndrome, which is paradigmatic for translational medicine, given that it creates a synergy between genetics and molecular biology, in order to improve the care for patients suffering from this syndrome.

En el presente editorial el autor expone un estudio, relacionado con el síndrome de Prader-Willi, que es paradigmático para la medicina traslacional, dado que crea una sinergia entre la genética y la biología molecular, a fin de mejorar la atención a los pacientes que padecen este síndrome.

[Prader-Willi and Angelman syndromes: case series diagnosed by MS-MLPA assay]. / Síndromes Prader-Willi y Angelman: serie de casos diagnosticados mediante la técnica de MS-MLPA.

BACKGROUND: Prader Willi syndrome (PWS) and Angelman syndrome (AS) are neurodevelopmental disorders caused by deletions or methylation defects, making a loss of expression of imprinted genes located in the 15q11-q13 region, and these can be assessed by different cytogenomic and molecular techniques. We report a case series of patients with PWS and AS evaluated through the MS-MLPA assay. CLINICAL CASES: We studied four patients with a clinical diagnosis of PWS and another with AS, evaluated as far as possible with karyotype and FISH, and with MS-MLPA assay for the 15q11-q13 region in all cases. In patients with PWS, neonatal hypotonia was the main reason for consultation and in three of them we identified a deletion of 15q11-q13 by MS-MLPA, also confirmed by FISH; and in the other one, an abnormal methylation pattern consistent with a maternal uniparental disomy. The patient with AS presented with a typical picture which led to the identification of a deletion in 15q11-q13 by MS-MLPA, also confirmed by FISH. CONCLUSIONS: The use of the MS-MLPA assay for the 15q11-q13 region was very useful for the diagnosis and identification of the genomic and epigenetic defects involved in either PWS and AS.

INTRODUCCIÓN: el síndrome Prader-Willi (SPW) y el síndrome de Angelman (SA) son trastornos del neurodesarrollo producidos por deleciones o defectos de metilación que producen pérdida de expresión en los genes improntados de la región 15q11 q13, mismos que pueden ser evaluados por diferentes técnicas citogenómicas y moleculares. Presentamos una serie de pacientes con SPW y SA en los que se identificó el tipo de defecto de la región 15q11-q13 mediante la técnica de MS-MLPA. CASOS CLÍNICOS: estudiamos cuatro pacientes con diagnóstico clínico de SPW y uno con SA, evaluados en lo posible con cariotipo, FISH y todos con ensayo MS-MLPA para la región 15q11-q13. En los pacientes con SPW, la hipotonía neonatal fue el motivo principal de consulta. En tres de ellos se identificó deleción de 15q11-q13 por MS-MLPA, confirmada por FISH, y en uno el patrón de metilación anormal fue compatible con una disomía uniparental materna. El paciente con SA presentó un cuadró típico y también se identificó una deleción en 15q11-q13 por MS-MLPA, confirmada por FISH. CONCLUSIONES: confirmamos que el uso de la técnica de MS-MLPA para la región 15q11 q13 mostró ser de gran utilidad para identificar los mecanismos genómicos y epigenéticos implicados en el SPW y el SA.

Analysis of Siewert classification, microsatellite instability and HER2 expression in 242 cases of adenocarcinoma of esophagogastric junction / 国际外科学杂志

Objective:To explore the characteristics of Siewert classification and microsatellite instability(MSI) and HER2 expression in adenocarcinoma of esophagogastric junction (AEG).Methods:The clinicopathological data of gastric adenocarcinoma from May 2019 to November 2020 were retrospectively analyzed. The patients were divided into two groups: AEG group and non AEG group. The composition ratio of Siewert type of AEG was counted, and the relationship between tumor size and Siewert type was analyzed. The MSI status and HER2 expression status of AEG and non AEG were statistically compared. The measurement data of normal distribution were expressed as mean ± standard deviation( Mean± SD), the comparison between groups were by t test, the comparison of count data between groups were by Chi-square test. Results:A total of 328 consecutive cases of gastric adenocarcinoma were collected, including 242 cases of AEG and 86 cases of non AEG. The Siewert classification of AEG was mainly type Ⅱ (151 cases, 62.40%), followed by type Ⅲ (86 cases, 35.54%) and type Ⅰ (5 cases, 2.07%). The analysis of the relationship between the size of the tumor length and the number of Siewert type showed that the number of Siewert type Ⅱ cases decreased and the number of Siewert type Ⅲ cases increased with the increase of the tumor size. MSI status was detected non selectively in 192 cases of gastric adenocarcinoma (140 cases of AEG and 52 cases of non AEG). There were 12 cases of MSI (6.25%), 2 cases of MSI-H (1.04%) and 10 cases of MSI-L (5.21%). There was no significant difference in MSI ratio between AEG group and non AEG group ( P>0.05). All MSI cases were negative or weakly positive for PMS2. The expression of HER2 was detected by immunohistochemistry in 313 cases of gastric adenocarcinoma, except 15 cases of PTIS/T1a. There were 30 cases (9.58%) with HER2 expression 3+ . Thirty-two cases (10.22%) expressed HER2 (2+ ), of which 7 cases were detected by fluorescence in situ hybridization (FISH), and 3 cases were positive. The proportion of HER2 (3+ ) in AEG was significantly higher than that in non AEG group ( P<0.05). Conclusions:The main type of AEG was Siewert type Ⅱ. AEG may mostly occur between 1 cm above the esophagogastric junction and 2 cm below the esophagogastric junction; For endoscopic screening of early AEG, more attention should be paid to this area of stomach. Siewert type Ⅲ may be derived from the development of Siewert type Ⅱ. The incidence of microsatellite instability in gastric cancer is low. Compared with other gastric adenocarcinoma, AEG has no specificity in MSI. The MSI of AEG was mainly the expression defect of PMS2. Compared with other gastric adenocarcinoma, there are more HER2 overexpression in AEG.

Application values of multiple detection methods of bone marrow in newly diagnosed multiple myeloma / 白血病·淋巴瘤

Objective:To investigate the application values of bone marrow morphology, bone marrow immunohistochemistry, flow cytometry, fluorescence in situ hybridization (FISH) and cytogenetic testing in newly diagnosed multiple myeloma.Methods:A total of 280 patients with multiple myeloma who were newly diagnosed in Tianjin KingMed Diagnosis Center from September 2018 to August 2019 were collected. The bone marrow biopsy was carried out according to the routine method, and bone marrow morphology, bone marrow immunohistochemistry, flow cytometry immunophenotyping, FISH and cytogenetic testing were performed. The detection results of each method were compared.Results:In 280 patients, the bone marrow immunohistochemistry results showed that the median ratio of plasma cells was higher than those of bone marrow morphology (20 cases, 0.675 vs. 0.300) and flow cytometry (47 cases, 0.650 vs. 0.147), and the differences were statistically significant ( Z = -3.883, P < 0.01; Z = -5.947, P < 0.01). Flow cytometry results showed that the positive rates of CD38, CD138, κ, λ, CD56 and CD19 were 100.0% (280/280), 100.0% (280/280), 57.5% (161/280), 42.5% (119/280), 62.1% (174/280) and 19.3% (54/280); bone marrow immunohistochemistry results showed that the positive rates of CD38, CD138, κ, λ and CD56 were 98.9% (277/280), 98.2% (275/280), 57.5% (161/280), 42.5% (119/280) and 62.1% (174/280); there was no statistical difference between the two detection methods in the detection coincidence rate of the same detection index (all P > 0.05). Among patients who underwent FISH detection, the detection rate of gene abnormalities was 69.9% (93/133); the detection rate of abnormalities by direct fluorescence in situ hybridization (D-FISH) was 42.9% (57/133); the detection rate of abnormalities by CD138 immunomagnetic sorting myeloma cells (MACS)-FISH was 82.7% (110/133). Among patients who underwent G-band karyotyping, the detection rate of abnormal karyotype was 38.5% (85/221). FSIH, especially MACS-FISH, had a higher detection rate of cytogenetic abnormalities than G-band karyotyping, and the difference was statistically significant ( χ2 = 65.697, P < 0.05). Conclusion:The comprehensive application of bone marrow morphology, bone marrow immunohistochemistry, flow cytometry, FISH (especially MACS-FISH), cytogenetic testing and other detection methods is more helpful for the diagnosis of multiple myeloma, and may be useful for prognostic judgment.

[Reevaluation of equivocal HER2 status detected by immunohistochemistry according to the 2019 guidelines for HER2 detection].

Objective To understand the effects and clinical significance of the 2019 guidelines for the human epidermal growth factor receptor 2 (HER2) detection. Methods: According to the 2014 guidelines, 548 cases of invasive breast cancer with equivocal HER2 (2+) detected by immunohistochemistry (IHC) in Taizhou Enze Medical Center, Zhejiang Province, China from 2013 to 2019 were selected. The results of IHC and HER2/CEPl7 double-probe were reevaluated and divided into groups according to the 2019 guidelines for the comparative analysis. Results: Among the 548 IHC HER2 (2+) invasive breast cancers, the number of positive, equivocal and negative cases for HER2 were 96 (17.52%), 81 (14.78%) and 371 (67.70%), respectively, according to the 2014 guidelines. However, according to the 2019 guidelines, 10 cases (1.82%) were reclassified as IHC 1+, 2 cases in the group 2 were reclassified as negative, and all the originally equivocal cases in group 4 were reclassified as negative. Finally, the total number of positive and negative cases for HER2 were 94 (17.15%) and 454 (82.85%), respectively. Conclusions: After applying the 2019 guidelines, the number of IHC 2+ cases decreases, and the positive rate for HER2 also decreases slightly due to the reevaluation change in groups 2 and 4, leading to reclassification of the cases that were deemed equivocal according to the 2014 guidelines. In general, the new 2019 guidelines are more reasonable and easier to use.

[Ancillary values of fluorescence in situ hybridization with different gene combination in diagnosis of malignant melanoma].

Objective: To investigate the clinical value of the first multicolor fluorescence in situ hybridization (FISH) assay on multiple genes, and combined with 9p21 and 8q24 evaluation in the differential diagnosis of melanoma. Methods: Fifty-six melanomas and 36 benign melanocytic nevi diagnosed in Fudan University Shanghai Cancer Center from 2017 to 2019 were included. Each specimen was examined by first multicolor FISH assay targeting 6p25 (RREB1), 6q23 (MYB), 11q13 (CCND1) and CEP6, as well as 9p21 (CDKN2A) and 8q24 (MYC). The results of FISH assay in all cases were recorded according to Gerami's criteria. Basing on the sensitivity and specificity of the first FISH assay, the refinement of diagnosis by adding combined 9p21 and 8q24 probes was further evaluated, as well as their association with different clinicopathological features. Results: In 86 cases, the FISH signals were adequate for analysis. Of the 56 melanoma cases, 52 cases were adequate for analysis; 36 cases (69.2%) were positive in the first FISH assay. The most frequent chromosomal anomaly was gain of RREB1 (30/52, 57.7%), followed by gain of CCND1 (20/52, 38.5%), loss of MYB relative to CEP6 (18/52, 34.6%) and gain of RREB1 relative to CEP6 (17/52, 32.7%). The frequency of homozygous deletions in 9p21 was 15.4% (8/52) and gain of 8q24 was 36.5% (19/52). Among the 36 melanocytic nevi cases, FISH results could be accurately evaluated in 34 cases, and none showed a positive result in the first FISH assay or 9p21 and 8q24 FISH analysis. Compared with the first FISH assay, the sensitivity of combination with 9p21 and 8q24 FISH analysis increased from 69.2% to 76.9% (40/52) and the specificity remained 100.0%. Statistical data showed that the rates of FISH positivity in patients with acral-lentiginious melanoma and nodual melanoma subtypes were higher than that in patients with superficial spreading melanoma and lentigo maligna melanoma subtypes, and patients with Breslow thickness>2.0 mm had higher positive FISH frequency than patients with Breslow thickness ≤2.0 mm. Conclusion: Multisite FISH analysis is a highly effective ancillary tool for the differentiation of unequivocal malignant from benign melanocytic lesions. By combining the first FISH assay with CDKN2A and MYC assay, the clinical utility of FISH analysis is further optimized in differential diagnosis of melanoma. Patients with Breslow thickness>2.0 mm, or acral-lentiginious melanoma and nodual melanoma subtypes tend to have higher FISH positivity. There remains a need to further explore the ancillary value of FISH analysis in diagnosis of ambiguous lesions.

The Utility of MDM2 and CDK4 Immunohistochemistry and MDM2 FISH in Craniofacial Osteosarcoma.

Craniofacial osteosarcoma is rare (2-10% of all osteosarcomas). Most low grade fibroblastic osteosarcomas of the long bones are characterized by amplification of chromosome12q including MDM2 and CDK4 genes. This study aims to investigate the utility of MDM2 and CDK4 immunostains as well as MDM2 FISH in craniofacial osteosarcomas as a means of distinguishing them from benign fibro-osseous lesions. Cases of primary osteosarcoma and benign fibro-osseous lesions of the craniofacial bones were identified in the diagnostic pathology archives. MDM2 (SMP14 and/or IF2) and CDK4 (D9G3E and/or DCS-31) immunostains were performed on a representative block from each osteosarcoma and benign case. Fluorescence in situ hybridization (FISH) for MDM2 was performed on non-decalcified osteosarcomas. In osteosarcomas, the rate of expression of either MDM2 IF2, MDM2 SMP14, CDK4 DCS-31, or CDK4 D9G3E was 72.7% (8/11 cases), usually focal and weak. Using the MDM2 IF2 clone and the CDK4 DCS-31 clone, MDM2 and CDK4 were negative in lesional cells in all 14 benign fibro-osseous lesions. Using the IF2 and SMP14 clones, MDM2 nuclear expression was present in associated osteoclast-like giant cells in both benign and malignant cases. Of 4 successful cases, 1 high grade osteosarcoma was positive for MDM2 amplification. MDM2 or CDK4 expression or MDM2 amplification may aid in a diagnosis of head and neck osteosarcoma. However, when absent, sarcoma is not excluded. Due to focal weak expression of MDM2 in tumor cells in conjunction with nuclear expression in associated giant cells, caution should be exercised when interpreting positive stains.

[Comparison of radiological and clinicopathological features of craniofacial fibro-osseous lesions].

Objective: To investigate the clinicopathological and radiological features of benign fibro-osseous lesion (BFOL). Methods: Sixty-five cases of craniofacial BFOL, eight cases of peripheral ossifying fibroma (POF) and one case of low-grade central osteosarcoma diagnosed at Sichuan Provincial People's Hospital between January 2010 and March 2019 were collected. The clinicopathologic features, hematoxylin-eosin and immunohistochemical (IHC) staining and radiographic features were analyzed. MDM2 gene amplification was detected by FISH in difficult borderline cases. Results: This cohort of BFOLs included 50 cases of fibrous dysplasia (FD), 12 cases of ossifying fibroma (OF), and three cases of juvenile psammomatoid ossifying fibroma (JPOF). The average ages of patients with FD,OF and JPOF were 31.7, 39.2 and 26.0 years respectively. The male to female ratio was 1.0∶1.8.The average age of POF was 47.0 years, with male to female ratio of 1∶7. Patient of low-grade central osteosarcoma was a 48-year-old man. Twenty-seven cases of FD were located in the jaw, and 23 cases were in other craniofacial bones. Nine cases of OF were located in the jaw, and three cases were in the nasal cavity. Two cases of JPOF were in the nasal sinus, and one was in the jaw. All POF were located in the gingiva, and low-grade central osteosarcoma was located in the mandible. The imaging features of FD were luffa-like or ground-glass like signal shadows with poorly defined borders with expansion. OF had clear borders or sclerosing margins. Both JOF and low-grade central osteosarcoma were expansile intraosseously and with focally invasive nodular masses with ground-glass like signal shadows; and POF showed soft tissue mass with bone formation. Histological features of BFOLs showed mixed fibrous and irregular osteoid lesions. FD had no clear relationship with the host bone and no osteoblasts surrounded the bone trabeculae. Osteoblasts rimming was found in OF, and the boundaries of the host bone were clear. JPOF and low-grade central osteosarcoma infiltrated the host bone focally, and the latter showed mild cellular atypia. MDM2 amplification was detected in low-grade central osteosarcoma. Conclusions: BFOLs are a group of fibro-osseous lesions with similar morphology in the head and neck and face, but their clinical features and prognosis are different; and their imaging and histological characteristics are also slightly different. Attentions should be given to the combination of clinical, imaging and pathologic features of BFOLs, especially the differential diagnosis between BFOLs and low-grade central osteosarcoma. Molecular detection could be used to assist the diagnosis in difficult cases.

[Alveolar soft part sarcoma in children: a clinicopathological study of 13 cases].

Objective: To investigate the clinicopathological manifestations, molecular genetic, diagnostic histology and differential diagnosis of alveolar soft part sarcoma (ASPS) in children. Methods: A total of 13 cases of ASPS diagnosed at Beijing Children's Hospital from August 2009 to November 2018 were collected. HE staining, histochemical staining for PAS and D-PAS, immunohistochemical (IHC) staining for TFE3, INI1 and CD68 and florescence in situ hybridization (FISH) for TFE3 gene translocation were performed. Results: There were four males and nine females, age ranged from 1 year and 2 months to 13 years and 8 months (mean 7.8 years); and four patients were under 5 years old. Histologically, the tumors showed a distinctive and characteristic nested or organoid growth pattern (11 cases) or solid, diffuse growth (2 cases). The tumor cells possessed abundant eosinophilic, or glycogen-rich and clear to vacuolated cytoplasm. The chromatin was relatively dispersed, with prominent and pleomorphic nucleoli; mitotic figures were rare. Vascular invasion was frequently seen. IHC staining showed specific nuclear TFE3 staining. The tumor cells were also positive for INI1,CD68 and vimentin; but were negative for MyoD1, Myogenin, CK and S-100 protein. Seven cases showed PAS and D-PAS staining, with fuchsia acicular or rod-shaped crystals in tumor cytoplasm. Nine cases showed TFE3 break-apart signals by FISH. Conclusions: ASPS is a rare soft tissue sarcoma in children. Compared with ASPA in adults, it has both similarities and unique clinicopathologic characteristics. The diagnosis needs to be confirmed by combining clinical, pathologic, IHC and genetic testing.

[Clinicopathological features of dedifferentiated liposarcomas with meningothelial-like whorls: report of six cases].

Objective: To investigate the histopathologic, immunohistochemical, molecular genetic characteristics of dedifferentiated liposarcomas with meningothelial-like whorls(DDLMW). Methods: Six cases of DDLMW diagnosed at Jiangsu Province Hospital(the First Affiliated Hospital of Nanjing Medical University) from March 2012 to August 2018 were enrolled. The cases were analyzed by routine HE staining, immunohistochemistry(MDM2, CDK4 and p16) and fluorescent in-situ hybridization(FISH) on MDM2 gene. Related literatures were also reviewed. Results: Three of the 6 patients were male.The patient ages ranged from 40 to 77 years (mean, 58 years). Four tumors occurred in the retroperitoneum and two in the mediastinum. Histologically, the tumors showed, in addition to foci of well-differentied liposarcoma, characteristic, scattered meningothelial-like concentrical whorls. The whorls were composed of tightly, concentrically arranged, spindle to ovoid cells with mild to mederate cytological atypia. Metaplastic bone was present within or in their immediate vicinity in four case. The tumors cell also showed strong and diffuse immunoreactivity to MDM2, CDK4 and p16, but no immunoreactivity to S-100 protein, SMA, SOX10, EMA, CD21, CD23 or CD35. The Ki-67 labeling indexes were low, while FISH showed high levels of MDM2 amplification in all cases. Conclusions: DDLMW is a rare morphologic variant of dedifferentiated liposarcoma. The whorls in DDLMW do not represent perineurial or follicular dendritic differentiation. Recognition and familiarity with its existence, as well as combined application of immunohistochemical staining and MDM FISH, are important to avoid confusion with other lesions.