透明细胞软骨肉瘤临床病理诊断

目的探讨透明细胞软骨肉瘤的临床、影像学、病理学、生物学行为等特征以及病理鉴别诊断。方法报道4例透明细胞软骨肉瘤,对其临床、病理和随访资料作系统整理,并行免疫组化EnVision两步法S-100蛋白、Col Ⅱ和其它与鉴别诊断相关的蛋白标记。结果4例都是中老年男性,均位于长骨骨端,病程较长。X线表现为纯溶骨性骨质破坏,早期边界清楚,后期病灶扩大并向骨干方向延伸,但均未穿透骨皮质,不形成骨外软组织肿块。大体肿瘤灰白色,有弹性,其中2例伴动脉瘤性骨囊肿。镜下瘤细胞边界清楚,有分叶结构,胞质丰富透明或嗜酸性毛玻璃状。透明细胞之间有反应性新骨和破骨细胞样巨细胞增生。其中3例含低级别普通软骨肉瘤成分。免疫组化标记S-100蛋白和Col Ⅱ阳性。临床治疗和随访：例1行右股骨下端病灶刮除后6年原位复发,再作病骨切除和人工膝关节置换。例2行股骨颈肿瘤切除后植骨,缝匠肌骨瓣移植及内固定术,术后放疗。例3行股骨下端肿瘤切除后骨水泥填充,术后放疗。上述3例经2年4个月～7年随访情况均良好。例4行右股骨上端截除手术,术后失访。结论透明细胞软骨肉瘤是一种罕见的低度恶性软骨肉瘤,有不同于普通软骨肉瘤的临床、影像学和病理形态学特点。在病理诊断时既应注意与良性软骨母细胞瘤和骨母细胞瘤鉴别,也应与高度恶性透明细胞型骨肉瘤和转移性透明细胞癌鉴别。     

Cytogenetic analysis and flow cytometric DNA measurement of a human tumor with pronounced hypodiploidy

A case of malignant choroid plexus papilloma of the brain with severe hypodiploidy is presented. The hypodiploidy was estimated by means of flow cytometric measurements of the nuclear DNA content in two investigations with an interval of 21 months. The latter investigation was supplemented with chromosome analyses including quinacrine banding. A modal chromosome number of 34 to 35 was found with a consistent loss of one chromosome Nos. 2, 3, 4, 5, 10, 13, 14, 15, 17, and 18, and no major structural changes. The corresponding calculated DNA content per nucleus correlated very well with the measured content, which was found to be 75% of the male diploid amount. The paper briefly discusses cell survival in extreme hypodiploidy and provides a comparison with cases from the literature in which banding analysis gives comparable information.     

Chondrocytic cell differentiation in clear cell chondrosarcoma

Clear cell chondrosarcoma is a rare mesenchymal neoplasm of unclear differentiation. Besides having a chondrogenic nature, an osteogenic differentiation was also proposed. In this study, expression analysis of extracellular matrix genes, which are specific for different mesenchymal cell differentiation pathways, were used to get a better understanding of origin and differentiation pattern of the clear cell chondrosarcoma tumor cells. Our in situ analysis of two cases shows that (1) chondrocytic cell differentiation as marked by the expression of cartilage collagen type II and proteoglycans is a characteristic feature within the development of the neoplasm, (2) multifocal chondrocyte hypertrophy as shown by the expression of type X collagen does occur, and (3) no significant expression cf collagen type I, the main gene product of osteoblastic cells, is found by the neoplastic cells. Thus, our study indicates that clear cell chondrosarcoma shows a chondrogenic, but not osteogenic, differentiation and represents a true chondrosarcoma. The unusual scarcity of its extracellular and the multifocal expression of type X collagen marks clear cell chondrosarcoma as a chondrosarcoma tumor entity of a particular cell differentiation pattern. The expression of cartilage type collagens represents a distinct marker from bone metastases of clear cell neoplasms of other origins.     

Chromosome 9 alterations and trisomy 22 in central chondrosarcoma: a cytogenetic and DNA flow cytometric analysis of chondrosarcoma subtypes.

Chondrosarcomas are malignant cartilaginous tumors. Most are located in the medullar cavity (central chondrosarcoma), and a minority develop in a preexisting osteochondroma (peripheral chondrosarcoma). The authors present karyotypes for 37 central, peripheral, juxtacortical, and dedifferentiated chondrosarcomas. Using loss of heterozygosity (LOH) analysis and DNA flow cytometry, the authors previously showed that central and peripheral chondrosarcomas probably evolve by different genetic mechanisms. Peripheral chondrosarcoma is characterized by genetic instability, as was previously shown by a high percentage of LOH and a broad range in DNA ploidy. The authors now show that all peripheral chondrosarcomas tested are aneuploid, combined with many nonspecific chromosomal aberrations. Two juxtacortical chondrosarcomas showed normal chromosome numbers combined with limited structural alterations, substantiating that juxtacortical and peripheral chondrosarcomas are two clinicopathologically different entities with a different genetic background. Central chondrosarcomas were previously found to be peridiploid with limited LOH, most frequent at 9p21. In the current study, chromosome 9 was involved in five of seven central chondrosarcomas compared with only one of four peripheral chondrosarcomas. Three central tumors showed involvement of the 9pl2-22 region, suggesting an important role for chromosome 9 in the oncogenesis of central chondrosarcoma. Moreover, trisomy 22 was found in four central chondrosarcomas only.     

Anaplastic large cell lymphoma: a flow cytometric analysis of 29 cases

We report our experience with flow cytometric (FC) analysis of 29 cases of anaplastic large cell lymphoma (ALCL). Morphologic analysis of processed cytocentrifuged preparations demonstrated neoplastic cells in 28 cases. In 25 of these, an aberrant lymphoid population was detected by FC analysis. The majority showed high orthogonal light scatter, similar to monocytes or granulocytes. Of the antigens CD2, CD3, CD4, CD5, and CD7, 5 cases expressed 1, 8 expressed 2, 6 expressed 3, 3 expressed 4, and 3 expressed all 5. CD4 was expressed most commonly (20/25 [80%]), followed by CD2 (18/25 [72%]), CD3 (10/25 [40%]), and CD5 and CD7 (8/25 [32%] each). CD45 was expressed in 23 of 25 cases and CD13 in 7 of 9. Of 21 cases, 13 were anaplastic lymphoma kinase (ALK)+, all of which were CD4+, vs 5 of 8 ALK - cases (P = .042). Most ALCLs can be detected and characterized by multiparameter FC analysis. However, light scatter gating on typical lymphoid regions may yield false-negative results in a substantial number of cases.     

Cytogenetic findings in a case of dedifferentiated chondrosarcoma

Cytogenetic analysis of a case of dedifferentiated chondrosarcoma with a malignant fibrous histiocytoma (MFH) component revealed a near-triploid karyotype with multiple copies of chromosome 7, deletion of chromosome arm 9p, and a derivative chromosome 19 [add(19p)] in each metaphase. Whereas numerical anomalies of chromosome 7 have been frequently reported in chondrosarcoma, deletion 9p and add (19p) have been observed in MFH. A putative MFH tumor suppressor gene could be present in a critical region on chromosome bands 9p21~p22.     

Genetic characterization of mesenchymal, clear cell, and dedifferentiated chondrosarcoma

Clear cell, mesenchymal, and dedifferentiated chondrosarcoma are rare, cartilaginous tumors with limited treatment options other than surgery. Conventional chondrosarcomas have been extensively studied at the genetic level, but for rare chondrosarcoma subtypes, this is merely restricted to case reports. Information on the genetics of rare chondrosarcomas may provide insight into the etiology of these specific disease subtypes and possible alternative treatment strategies. Therefore, the aim of this study was to genetically characterize this subset of rare tumors. Using array CGH, we gathered genomic information of 30 rare cartilaginous tumors. In addition, we constructed tissue microarrays with 2 mm cores of 23 clear cell, 23 mesenchymal, and 45 dedifferentiated chondrosarcomas, in triplicate. Using immunohistochemistry, we investigated expression of R132H IDH1, and p53 and retinoblastoma pathways. Results were verified and further investigated with a methylation assay and MLPA for CDKN2A/p16, and IDH1/2, and TP53 mutation analysis. Array‐CGH showed numerous genomic alterations in all subtypes. However, only a limited number of recurrent alterations were detected, none of which seemed to be associated with the subtypes. The IDH1/2, p53, and retinoblastoma pathways were affected in 0, 9, and 95% of clear cell chondrosarcomas, in 0, 39, and 70% in mesenchymal chondrosarcomas, and in 50, 59, and 85% of dedifferentiated chondrosarcomas, respectively. Our results suggest an important role for the retinoblastoma pathway in all three rare chondrosarcoma subtypes investigated. © 2012 Wiley Periodicals, Inc.     

Four-color flow cytometric analysis of peripheral blood donor cell chimerism

Passenger leukocytes have been demonstrated to play significant roles in initiating and also regulating immune reactions after organ transplantation. Reliable techniques to detect donor leukocytes in recipients after organ transplantation are essential to analyze the role, function, and behavior of these leukocytes. In this report we describe a simple, reliable method to detect donor cells with low frequencies using peripheral blood samples. Detection of small numbers of major histocompatibility complex (MHC) mismatched cells was first studied using four-color flow cytometry in artificially created cell mixtures. By selecting the CD45(+) population and simultaneous staining with several leukocyte lineage markers (CD3, CD4, CD8, CD56, and CD19), MHC-mismatched leukocytes were consistently detected in cell suspensions prepared from directly stained whole blood samples with a threshold sensitivity as low as 0.1%-0.2%. When the fresh peripheral blood mononuclear cells were separated by conventional Ficoll gradient purification, similar, but slightly lower levels of donor cells were detected. Blood samples obtained 1-5 months after liver, kidney, and intestine transplants revealed that the kind of organ allograft influenced levels and lineage pattern of the circulating donor cells. This procedure provided a simple and reliable method in determining early chimerism in transplant recipients. However, the detection of MHC-mismatched leukocytes of all lineages was much lower when frozen peripheral blood mononuclear cells were used.     

DNA flow cytometric and immunohistochemical analysis of proliferative activity in spindle cell haemangioendothelioma

Although spindle cell haemangioendothelioma was initially described as a low-grade angiosarcoma, recent reports have suggested that it is a reactive or benign vascular proliferation. In order to assess the proliferative activity in spindle cell haemangioendothelioma, 12 cases, one of which was associated with Maffucci's syndrome, were immunohistochemically analysed with antibodies against proliferating cell nuclear antigen (PCNA), Ki-67 and p53. DNA flow cytometry was performed on six of the 12 cases. Seven of the 12 patients had multiple nodules or papules. Although two cases recurred once and twice, respectively, after surgery, there was no evidence of metastasis. Immunohistochemically, the percentages of PCNA, Ki-67 and p53 positive tumour cells ranged from 0.1% to 6.4% (mean 3.3%), 0.1% to 14.9% (3.5%) and 0.1% to 2.8% (1.1%), respectively, indicating a low proliferative activity and a low p53 expression in this tumour. All seven lesions from the six cases examined flow cytometrically were DNA diploid with proliferative indices (S + G₂/M-phase fractions) ranging from 4.9% to 19.5% (mean, 10.9%). These findings are compatible with a bland-looking histological picture and an indolent clinical course of spindle cell haemangioendothelioma.     

Clinical flow cytometric reticulocyte analysis.

The flow cytometric (FCM) analysis of reticulocytes in a clinical laboratory can be accomplished using acridine orange (AO), thiazole orange (TO), auramine O, thioflavin T, pyronin Y, dimethyl-oxacarbocyanine or transferrin receptor assays. AO and TO are vital stains, show good correlation with microscopic reticulocyte determinations and, when compared to each other, give an excellent correlation. The coefficient of variation is below 5% and batch analysis on blood samples stored for 96 h further reduces manpower needs. Finally we have used the mean fluorescent intensity of TO as a reticulocyte maturity index (RMI). In bone marrow transplant patients, the RMI has been the earliest indicator of marrow engraphment. Using the RMI we have been able to define three patterns of engraphment: early, delayed and failed. Although a variety of standards will be required, the clinical FCM reticulocyte analysis promises to be the preferred and accepted method in the clinical laboratory.     

Immuno-gold labeling for flow cytometric analysis

Colloidal gold particles coated with goat anti-mouse immunoglobulin antibodies were used to analyse surface antigens on various cell types by flow cytometry. The gold-labeled cells showed an increasing signal amplification in the 90 degree light scatter with increasing wavelength of the incident laser light, reaching a more than 10-fold amplification at 632.8 nm. This wavelength was provided by a 0.5 mW helium-neon laser. The magnitude of the signal amplification due to the gold label as well as the specificity of the label was sufficient for quantitative discrimination between positive and negative cells. Cell viability was not affected by the gold label. Mouse spleen cells were labeled with various combinations of FITC- and gold-conjugated antibodies. It was found that the gold and fluorescent labels did not interfere with each other. Colloidal gold may thus be used as an additional label for multiparameter cell analysis and sorting. Biparametric cell analysis/sorting of surface antigen-labeled cells (label versus low-angle scatter) becomes possible even with a low energy helium-neon laser.     

Morphometric and flow cytometric analysis of small cell undifferentiated carcinoma of the bladder.

Eighteen cases of primary small cell carcinoma of the bladder were studied. Three patients survived for two years and one survived for five years, which was significantly worse when compared with poorly differentiated transitional cell carcinoma (WHO grade 3). Aggressive tumour behaviour was independent of the presence of neuroendocrine characteristics. Morphometric analysis showed that the nuclear size, which was comparable with that reported in pulmonary small cell carcinoma, was significantly smaller than in poorly differentiated transitional cell carcinoma. Nuclear morphometry may therefore help identify small cell bladder carcinoma, especially in the absence of neuroendocrine differentiation characteristics. DNA flow cytometry of paraffin wax embedded specimens showed aneuploidy in 14 tumours--five were peritetraploid and two multiple aneuploid--and only three were diploid. Aggressiveness of small cell bladder carcinoma usually coincides with aneuploidy in most cases, but diploid tumours may also follow a rapid, lethal clinical course.     

Multiparameter flow cytometric analysis of colon polyps

Sixty-eight colonic polyps of various histologic types were studied using retrospective flow cytometric (FCM) DNA analysis to determine the prevalence of aneuploid cell populations and whether they were associated with any particular histologic features. Overall, 13 of 68 polyps (19%) contained DNA-aneuploid cells, including 3 of 9 villous and 4 of 10 villoglandular polyps with histologic features of carcinoma in situ (CIS), 4 of 11 villous and 1 of 18 villoglandular polyps without CIS, and 1 of 12 adenomatous polyps. Eight hyperplastic polyps were diploid. These results show retrospective FCM analysis can detect DNA aneuploidy in polyps; DNA aneuploidy may occur before histologic evidence of invasive carcinoma; and this change is more frequent in types of polyps thought to have increased malignant potential (i.e., those with villous morphology and/or CIS).     

两种不同染色法在流式细胞术中检测细胞周期的探讨

目的比较碘化丙啶（PI）和4,6-联脒-2苯基吲哚（DAPI）两种不同染色方法在流式细胞术中对细胞周期检测的影响。方法 A549细胞分别采用PI和DAPI染色法进行染色,于染色后0和24h进行流式细胞仪检测细胞G0/G1期、S期和G2/M期的DNA含量,比较两种方法的测定结果,同时进行细胞计数,观察细胞浓度变化。结果PI和DAPI法染色后0h上机测定,结果分别为：CV值（7.72±0.19）、（5.92±0.09）,G0/G1期含量（69.63±1.16）%、（69.87±1.28）%,S期含量（24.53±0.47）%、（24.43±0.86）%,G2/M期含量（5.85±1.04）%、（5.72±0.65）%,两种方法检测细胞周期的结果基本一致（P〉0.05）;染色后24h重新测定,结果分别为：CV值（8.82±0.05）、（6.09±0.30）,G0/G1期含量（58.50±0.90）%、（70.47±0.81）%,S期含量（31.73±0.75）%、（23.67±0.45）%,G2/M期含量（9.51±0.47）%、（5.86±0.46）%,两种方法检测细胞周期的结果差异有统计学意义（P〈0.05）。DAPI法染色后0和24h测定结果基本一致（P〉0.05）,而PI法检测结果差异有统计学意义（P〈0.05）。细胞计数结果显示,放置24hPI法细胞损耗为63.14%,DAPI法细胞损耗为12.50%,两种方法结果差异有统计学意义（P〈0.05）。染色24h后PI法细胞开始崩解,镜下见较多的细胞碎片,而DAPI法细胞形态良好。结论应用流式细胞术检测细胞周期,DAPI染色法优于PI染色法。     

Cytogenetic aberrations and their prognostic impact in chondrosarcoma.

Chondrosarcoma is the second most common primary malignancy of bone. Cytogenetic data are available from close to 100 cases, including all subtypes of chondrosarcoma. Specific chromosomal rearrangements have been identified only in extraskeletal myxoid chondrosarcoma (EMC). Strong prognostic factors are largely missing, although size and, in particular, histologic tumor grade have been implicated. In the present study, we investigated the genomic aberrations in 59 chondrosarcomas (six grade 1, 24 grade 2, and 29 grade 3, including dedifferentiated tumors), excluding EMC, by chromosome banding analysis and DNA flow cytometry and correlated the findings with clinical outcome. Hyperhaploid to near-diploid karyotypes were found in half of the cases, and there was a good correlation between cytogenetics and flow cytometry data; discrepancies were seen primarily in cases with normal karyotypes and in those with -Y as the sole anomaly. Abnormal karyotypes, excluding those with -Y as the only change, were found in 36 cases. No recurrent structural aberration was found, but a nonrandom pattern of aberrations was seen. Total or partial gains and losses were the dominant karyotypic features. Genomic imbalances found in at least 10 cases included -1p36, -1p13-p22, -4, -5q13-q31, -6q22-qter, +7p13-pter, -9p22-pter, -10p, -10q24-qter, -11p13-pter, -11q25, +12q15-qter, -13q21-qter, -14q24-qter, -18p, -18q22-qter, +19, +20pter-q11, +21q, and -22q13. At the latest follow-up, 19 patients had experienced distant metastases, and the 5-year metastasis-free survival rate was 0.69. By univariate analysis, malignancy grade and loss of material from 6q, 10p, 11p or 11q, 13q, and 22q were associated with impaired metastasis-free survival. Only -13q was an independent prognostic factor for metastasis, regardless of tumor grade or size.     

透明细胞脑膜瘤的临床病理分析

透明细胞脑膜瘤(clear cell meningioma,CCM)是一种少见的脑膜瘤亚型,组织学较温和,但临床过程具有侵袭性,复发率高.2007年WHO中枢神经系统肿瘤分类已将其定位为Ⅱ级,迄今国内外相关文献报道较少,我们报道5例经病理证实的颅内CCM,分析其临床表现及病理特点,旨在加深对该病的认识,为这一疾病的诊治提供参考.     

Cytogenetic analysis of a juvenile granulosa cell tumor.

We report chromosomal changes in a juvenile granulosa cell tumor with complex chromosomal rearrangements. These tumors have not been subjected previously to cytogenetic analysis.     

Platelet-associated IgG assay using flow cytometric analysis

In this study we describe an immunofluorescent assay system to measure platelet-associated immunoglobulin G levels using flow cytometric analysis. This semi-quantitative system allows ready distinction of immune from non-immune related thrombocytopenias. It is simple to perform, highly reproducible and has the advantage of requiring a minimum concentration of 5000 platelets/microliter per assay sample.