Undifferentiated spindle cell carcinoma of the gallbladder: A clinicopathologic, immunohistochemical, and flow cytometric study of 11 cases

Eleven primary spindle cell carcinomas (SpCCs) of the gallbladder are reported. They occurred in eight women and three men ranging in age from 59 to 80 years (mean age, 66.5 years). Histologically, the tumors showed interlacing bundles of atypical spindle cells with eosinophilic cytoplasm, oval to elongated nuclei, and conspicuous nucleoli. Eight SpCCs contained tiny foci of neoplastic glands similar to those seen in adenocarcinoma, and two of these cases also had small foci of neoplastic squamous epithelium. A gradual transition between the squamous cell carcinoma and the spindle cell component was observed in one tumor. Immunohistochemically, all SpCCs were positive for at least one of the epithelial markers (epithelial membrane antigen, nine cases; , nine cases; carcinoembryonic antigen, three cases; and EAB 903, one case), and the tumor cells also were immunoreactive to mesenchymal marker (vimentin, eight cases), muscle markers (alpha-smooth muscle actin, one case; desmin, one case), and histiocytic marker (HAM 56, one case). Abnormalities in tumor suppressor gene p53 expression also were found in two of the 11 SpCC cases using monoclonal antibody PAb 1801. In six cases for which data were available flow cytometry revealed aneuploidy in three SpCCs (50%). The survival curve of the SpCC cases (mean survival, 9 months) was less favorable than that of 224 cases of adenocarcinoma of the gallbladder (mean survival, 81 months) (P = .0011). These results indicate that SpCC of the gallbladder is an epithelial tumor with sarcomatoid components and its prognosis is unfavorable.     

胃癌细胞DNA含量和倍体分析的研究进展

利用流式细胞分析仪和图像细胞分析仪检测胃癌细胞核中DNA含量和倍体已成为病理诊断中的重要手段，DNA含量和倍体分析可用于胃癌前病变及早期胃癌的诊断，与胃癌的临床病理有密切的联系，可用于患者治疗效果的评估和预后的估计。     

The prognostic significance of the DNA content in Ewing's sarcoma: a retrospective cytophotometric and flow cytometric study

The DNA content of the cell nuclei of Ewing's sarcoma was analysed by means of cytophotometry in situ with image analysis in Feulgen-stained sections in 37 patients, and by retrospective flow cytometry according to the method of Hedley in 26 patients. Different histogram patterns were obtained: normal unimodal or bimodal DNA distributions and abnormal DNA distributions with one or two stem lines, or an abnormal DNA distribution with no stem lines. Both methods enabled us to make a distinction between two groups of Ewing's sarcomas with a different prognosis. All patients with aneuploid tumours died within 5 years after the initial diagnosis. Eleven of 19 (58%) patients with a normal DNA distribution in their tumour, as determined by cytophotometry, are still alive and in good health with a mean survival period of 7.5 years, ranging from 2 to 19 years. Of the group of patients in which flow cytometry revealed a normal DNA pattern, eight of 15 (53%) are still alive and in good health, with a mean survival period of 8 years. These results indicate that both techniques are reliable methods for obtaining prognostic information in Ewing's sarcomas. However, cytophotometry in situ yielded a better discrimination for the overall survival ( P < 0.01) than did flow cytometry ( P <0.05). In 19% of the cases there was a discrepancy between the DNA histograms obtained with the two techniques. In five of 26 cases the DNA distributions were classified as normal by one method and aneuploid by the other. Tumour cell representation or selective loss of cells during enzymatic treatment may be responsible for this discrepancy.     

Metastatic mode of gastric carcinoma by flow cytometric and clinicopathologic parameters

We used flow cytometric and conventional clinicopathologic parameters to analyse the metastatic mode of cancer in 113 stomach cancer patients. Liver metastasis was frequent in cases with intestinal-type cancer, cancer located in the distal stomach, positive venous invasion and aneuploid cancer. Lung and pleural metastasis (excluding nodular lung metastasis), however, were frequent in cases with serosal invasion and diploid cancer. Peritoneal metastasis was frequently seen with tumors located in the proximal or whole stomach, diffuse-type cancer and cancer with serosal invasion. All cases developing bone metastasis were positive for lymph node metastasis. DNA ploidy was partially related to the metastatic mode of stomach cancer, but this was not the sole parameter for predicting metastasis. However, prediction may be possible if based on both DNA ploidy and clinicopathologic parameters, suggesting the possibility of the prophylaxis of recurrence by appropriate postoperative adjuvant therapy.     

Application of the crypt isolation technique to the flow cytometric analysis of DNA content in gastric carcinoma

An inevitable limitation of conventional flow cytometric analysis of gastric cancer DNA content is that the preparations of tumor cell nuclei are contaminated with stromal cell nuclei. Using the crypt isolation technique, we separated tumor tissues from stromal tissues and analyzed the DNA content in samples of pure gastric cancer cells (64 intestinal-type and 46 diffuse-type) by flow cytometry. Morphologically, crypts from well-differentiated and moderately differentiated adenocarcinomas usually showed large tube-like or sheet structures, whereas tumor tissues isolated from poorly differentiated adenocarcinomas usually exhibited small tumor cell clumps or clusters of varying sizes. Tumor ploidy was divided into diploid, aneuploid, and multiploid subgroups. Aneuploidy and multiploidy were observed in 12% (13 of 110) and 64% (71 of 110) of gastric cancers, respectively. A high frequency of DNA aneuploidy or multiploidy was associated with intestinal-type tumors, but not with any of the other clinicopathologic variables tested. In contrast, high S-phase fraction values demonstrated a close association with tumors with abnormal ploidy, advanced stage, intestinal type, and late TNM stage. Our results suggest that S-phase fraction may be a more useful indicator of aggressive behavior in gastric cancers than DNA aneuploidy. To our knowledge, the present study is the first to report flow cytometric DNA content in a large number of gastric cancer samples obtained using the crypt isolation technique.     

Immunohistochemical and flow cytometric study of neuroendocrine carcinoma of the skin

Immunohistochemical and flow cytometric analysis using formalin-fixed, paraffin-embedded sections was performed on 10 neuroendocrine carcinomas of the skin (NCS). Grim-elius staining was positive in seven tumors. All tumors showed coexpression of CAM 5.2 and neuron-specific enolase with paranuclear dot-like or diffuse cytoplasmic reactivity. Neuro-filament was positive in five cases, chromogranin in six, calcitonin and carcinoembryonic antigen in two each, and somatostatin and S-100 protein in one each. Eight primary lesions were diploid and the remaining two were aneuploid; however, two diploid NCS presented as aneuploid metastatic tumors. The follow-up periods ranged from 3 to 66 months (mean 13.6). Six patients died of metastatic diseases between 3 and 33 months (mean 9.2) after the diagnosis. There were no significant correlations among histologic features, DNA ploidy, S-phase fraction, and clinical outcome of the patients with NCS. These results indicate that a panel of antibodies may be required for immunohistochemical confirmation of neuroendocrine differentiation and that a flow cytometric analysis is not a good tool to predict the biologic behavior of NCS.     

Clinicopathologic study of 97 cases of small renal cell carcinomas using DNA flow cytometric analyses

The incidence of small renal cell carcinomas (RCC) has Increased recently as diagnostic imaging techniques have improved. The Indications for partial nephrectomy for small RCC confined to the kidney are still controversial. Ninety-seven small RCC 2.5 cm or smaller, Including incidental, occult, and dialysis associated carcinomas obtained at surgery or autopsy were examined. Various clinicopathologic parameters were assessed and survival was analyzed using the Kaplan-Meir method. Seventy-eight cases were incidental carcinomas. Carcinomas caused death in five cases, four of which had Initial signs of metastatic disease. Differences in survival between patients with expansive and invasive (intermediate and infiltrating) growth patterns, solid and other structural patterns, clear and other cell types, grade 1 and higher grades of nuclear atypla, and with and without lymph node metastasis were statistically significant All surgical cases were alive except one who died of another disease. Poor prognostic factors seen in five fatal autopsy cases included invasive growth pattern, solid structural pattern, spindle cells, grade 3 nuclear atypia, and Bellini duct carcinoma. Small RCC may be treated with partial nephrectomy provided they do not show these features, although there is low risk of recurrence or development of de novo cancer.     

Flow cytometric DNA analysis of 199 histologically favourable or unfavourable non-Hodgkin lymphomas

We have studied the nuclear DNA content of histologically favourable (n = 82) or unfavourable (n = 117) non-Hodgkin lymphomas (NHLs) diagnosed between 1957 and 1978 in the Tampere University Central Hospital. The DNA analysis was done by applying a trypsin digestion method to archival tumour samples. DNA aneuploidy was seen in 40 per cent of the unfavourable cases and in 10 per cent of the favourable cases, but varied considerably between different histological subtypes. The unfavourable cases showed high proliferative activity (S-phase fraction, SPF), while considerable variation in the SPF among the favourable NHLs was noted. Among the unfavourable NHLs, cases with DNA-aneuploid tumours had significantly (P less than 0.01) worse prognosis than stage and treatment matched cases with DNA-diploid tumours. In general, survival of the patients who had high SPF tumours was significantly lower compared with patients with low SPF tumours (P less than 0.01). However, SPF was not related to the prognosis in the unfavourable NHLs. We conclude that the flow cytometric DNA analysis revealed characteristic features in the favourable and unfavourable NHLs and may be useful in predicting the clinical outcome of patients.     

Epithelioid sarcoma: An electron-microscopic,immunohistochemical and DNA flow cytometric analysis

Summary Eight epithelioid sarcomas (ES) were studied by electron microscopy, immunohistochemistry, and DNA flow cytometry. Ultrastructurally, the tumour cells showed desmosome-like intercellular junctions and numerous microvilli, in addition to whorled arrangements of intermediate filaments. Tumour cells were positive for epithelial membrane antigen, cytokeratin, and vimentin, and negative for carcinoembryonic antigen and desmin. All seven cases examined by flow cytometry showed diploid or hyperploid (near diploid) DNA content. This seems to correspond to the relatively long clinical course and low-grade malignant nature of ES. Although the histogenesis of ES is still uncertain, the results of this study suggest that it is a tumour of primitive mesenchymal cells with the capacity to show epithelial differentiation.     

Fibrolamellar hepatocellular carcinoma: a report of a resected case with an electron microscopic and flow cytometric analysis [corrected

A resected case of fibrolamellar (FLC) and hepatocellular (HCC) combined carcinoma arising in a non-cirrhotic liver of a 29 year old female is reported, including results of the preoperative percutaneous aspiration biopsy, which suggested FLC, and postoperative electron microscopic and flow cytometric analysis. Sections of the resected massive tumor of the left lobe of the liver showed hepatocellular carcinoma accompanying the fibrolamellar carcinoma element which was composed of tumor cells with eosinophilic granular cytoplasm and unique cytoplasmic vacuoles (pale bodies). Lamellar fibrosis was present in the stroma, while no macroscopic central scar was demonstrated. Electron microscopy showed typical features of FLC and flow cytometric DNA analysis indicated diploid DNA pattern with a low proliferation rate. A common HCC element with trabecular structure also existed at the periphery of the tumor. No apparent etiologic agent for the development of hepatic neoplasm was indicated in the history of this patient. She had been without recurrence for about 3 years after extended left lobectomy, when local recurrence was revealed. The recurrence has been treated with chemoembolization and percutaneous ethanol infusion several times up till the present. This case reconfirms the importance of the pathological diagnosis of FLC to promote surgical intervention.     

Spindle cell hemangioendothelioma: An immunohistochemical and flow cytometric study of six cases

Six cases of spindle cell hemangioendothelioma (SCH) are presented with immunohistochemical and flow cytometric analyses. One case was associated with Maffucci's syndrome. All lesions were solitary or multifocal in the extremities, and prepresentation duration ranged from years to decades. One case recurred but none had metastases. Histologically, in four of the six cases the main lesions appeared to arise within vessels, predominantly muscular vessels. All lesions consisted of cavernous hemangiomalike areas and solid cellular areas resembling Kaposi's sarcoma. Cellular atypia was minimal. At the periphery of the lesions, a cluster of large thick or thin walled, and probably malformed, vessels were observed. Immunohistochemically, factor-VIII related antigen, CD34, vimentin, and lectin binding Ulex europaeus agglutinin 1 stained endothelial cells lining vascular channels, and vacuolated, or epithelioid cells. Spindle cells in the solid areas were negative for these endothelial markers except for vimentin, but showed divergent positive immunoreactions of HHF35, alpha-smooth muscle actin, desmin, and collagen type IV. Five cases were diploid and one was aneuploid. There was no significant correlation among DNA ploidy, S-phase fraction, and local recurrence in SCH although the number of cases examined was small. These results suggest SCH may be a benign lesion, probably a reactive process, rather than a low-grade angiosarcoma.     

Flow cytometric evaluation of nuclear DNA content in hepatoblastoma: Further evidence for the inhomogeneity of the different subtypes

The DNA ploidy pattern of nine hepatoblastomas and three normal liver specimens was investigated by flow cytometry. Areas of unlike differentiation in the same tumor were investigated separately. Normal liver tissue and the fetal subtype were always diploid. Aneuploidy was found in the embryonal subtype. The one case of small cell tumor was also diploid. When the fetal and embryonal areas of the same tumor were analyzed together, there was always an aneuploid peak in the histogram. There are obvious differences in DNA ploidy among the various hepatoblastoma subtypes. Differences in methods used, including the failure in some studies to evaluate areas of unlike differentiation in the same tumor separately, probably account to some extent for the conflicting results of previously reported studies. When flow cytometric analysis of the DNA content of a hepatoblastoma is performed, it is important to ensure that the various types of differentiation in the tumor are represented adequately in the material investigated, especially when conclusions about the prognosis are to be based on these findings.     

Nuclear grading and flow cytometric DNA pattern in fine-needle aspirates of primary breast cancer

Fine-needle aspiration (FNA) biopsy is increasingly used in the diagnosis and biological characterization of breast carcinomas in patients who receive preoperative chemotherapy. In this context, nuclear cytologic grade supplemented by DNA content could play an important role in the morphologic assessment of breast cancer. In this study, DNA ploidy pattern, analyzed by flow cytometry on FNAs from 92 primary breast carcinomas, was related to cytologic nuclear grade. Twenty-seven samples were cytologic grade 1, 33 were grade 2, and 32 were grade 3. Ploidy correlated with cytologic nuclear grade (P = 0.0001). Thirty percent of grade 1, 55% of grade 2, and 84% of grade 3 tumors were DNA aneuploid. For 30 of the 92 FNAs, it was possible to compare nuclear cytologic grade with the corresponding histologic grade using the Scarff, Bloom, and Richardson system. A high concordance (80%) between nuclear grade on FNAs and histologic grade was found. DNA flow cytometry in combination with nuclear cytologic grade might represent additional information for the characterization of breast cancer diagnosed by FNA. Diagn Cytopathol 1996;15:116–120. © 1996 Wiley-Liss, Inc.     

Correlation between DNA flow cytometric and nucleolar organizer region data in non-Hodgkin's lymphomas

The argyrophilic staining (AgNOR) technique, novel in histopathology, was applied to a series of 20 non-Hodgkin's lymphomas (NHL) of established Kiel subtype. The method demonstrates nucleolar organizer regions (NORs) by virtue of sulphydryl groups on their associated proteins and the enumeration of AgNOR foci has been previously shown to discriminate between NHL of low- and high-grade histological types. This finding was confirmed and the results were compared with those obtained by means of DNA flow cytometry performed on paraffin wax-embedded tissue from the same lymphomas. There was a very good linear correlation between the mean numbers of AgNOR sites per nucleus and the percentage of S-phase cells for each case, both values being high in high-grade NHL and low in low-grade lesions. Conversely there was no significant correlation between the DNA index, representing DNA aneuploidy, and AgNOR counts. It is suggested that the numbers of AgNORs in a lymphoma may be related to the dividing fraction of cells rather than, as might be expected, to ploidy alone. It is also proposed that the AgNOR technique, which is rapid, simple, and inexpensive, may provide, at least, an adjunct to DNA flow cytometry in the assessment of neoplasm in histopathology.     

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.     

鼻咽泡状核细胞癌DNA含量与PCNA计数的研究

应用流式细胞仪和免疫组化技术对１２例鼻咽泡状核细胞癌和３３例低分化鳞ＤＮＡ含量及增殖细胞核抗原（ＰＣＮＡ）进行分析。结果显示：泡状核细胞癌的ＤＮＡ指数，非整倍体率和ＰＣＮＡ阳性率均高于低分化鳞癌，表明泡状核细胞癌分化程度低于后者，并初步探讨ＤＮＡ与ＰＣＮＡ所反映的泡状核细胞癌生长生物学特征与预后的联系。     

A simultaneous flow cytometric assay for c-myc oncoprotein and DNA in nuclei from paraffin embedded material

A simultaneous flow cytometric assay for the c-myc oncoprotein and DNA in nuclei extracted from archival paraffin wax embedded clinical biopsies is presented. The nuclei were extracted by pepsin digestion after dewaxing 20 micron sections. The c-myc oncoprotein was probed with a mouse monoclonal antibody. This was raised against a synthetic peptide corresponding to a hydrophilic region of the protein predicted from the amino acid sequence. The technique is illustrated with biopsies from patients with testicular cancer and with benign and malignant neoplasms of the colon.     

Pleomorphic carcinoma of the gallbladder: case report and ultrastructural study

Carcinomas of the gallbladder with pleomorphic spindle cell histology are unusual. We report such an occurrence in a 47-year-old male with a neoplasm showing both a mixed squamous and a pleomorphic spindle cell pattern. Ultrastructural studies supported the likelihood that this lesion represented a pleomorphic carcinoma and revealed unique paracrystal-line structures intimately associated with tonofila-ments. For this reason we believe that this and some other previously diagnosed sarcomas and carcinosarcomas of the gallbladder are in reality poorly differentiated carcinomas of either adeno, squamous, or mixed type. This illustrates the need for critical ultrastructural assay to determine the nature of poorly defined and differentiated neoplastic growths.     

Peripheral primitive neuroectodermal tumour and extra-osseous Ewing's sarcoma; a histological,immunohistochemical and DNA flow cytometric study

Although peripheral primitive neuroectodermal tumour (pPNET) and extra-osseous Ewing's sarcoma (EES) are thought to be closely related neoplasms, their clinical behaviour differs considerably. To determine the clinical relevance of the Schmidt classification scheme for differentiating pPNET and EES, 20 tumour specimens of poorly differentiated round cell tumours were evaluated. In addition, the diagnostic value of several neural markers and the prognostic value of quantitative morphological variables (DNA ploidy, S-phase fraction, and the mitotic activity) were assessed. Homer-Wright rosettes were present in 9 tumours. Neuron specific enolase (NSE) was expressed in 11 tumours, 8 of which expressed a second neural marker (CD57, S100, or neurofilament). According to the Schmidt classification, 11 pPNET and 5 EES were distinguished. HBA-71 was exclusively expressed in pPNET and EES. The remaining tumours were classified as sarcoma not otherwise specified (n=2), rhabdomyosarcoma (n=1), and desmoplastic tumour with divergent differentiation (n=1). EES611 patients fared significantly better than the pPNET patients (100% versus 42% 5-year survival). Neither DNA ploidy nor S-phase fraction assessed in 12 evaluative histograms (9 pPNET and 3 EES), nor mitotic activity yielded information of additional prognostic value. On the basis of this study and the Schmidt classification scheme, it can be concluded that if the diagnosis of EES and pPNET is based on light microscopy (Homer-Wright rosettes) and/or immunohistochemistry (at least two neural markers, i.e. NSE, S-100, CD57, and neurofilament), the classification provides important clinical information. Furthermore, positivity for HBA-71 is helpful in differentiating pPNET and EES from all other small round cell tumours.     

Evaluation of flow cytometric immunophenotyping and DNA analysis for detection of malignant cells in serosal cavity fluids

The serosal cavities are frequent sites of tumor metastasis. The distinction between carcinoma cells, inflammatory cells, and reactive or malignant mesothelial cells can be difficult in cytology. Multicolor flow cytometry (FCM) provides the opportunity to evaluate multiple antigens simultaneously, making it possible to characterize various cell populations. In this study, we aimed to assess the diagnostic accuracy of FCM immunophenotyping and DNA in comparison with serum tumor markers and classic cytology for detection of malignant cells in pleural and ascitic fluids. One hundred and nineteen samples of body cavity fluids were analyzed. Immunophenotyping was performed by four‐color immunofluorescent staining using monoclonal antibodies against Ber‐EP4, cytokeratin, CD3, and CD45. The DNA analysis by FCM was also performed. In addition, serum CA19‐9, CEA, AFP, and CA125 were analyzed. Ber‐EP4 marker had the highest sensitivity (73%) and specificity (95.5%) in the detection of carcinoma cells in serous fluid and correlated with cytology in most of cases (73%). The mean of DI differed statistically in patients with malignant effusions than in benign one. DI showed no difference in fluids due to infiltration of malignant epithelial cells or hematopoitic malignancy or due to hepatocellular carcinoma developing in cirrhotic liver. Thus, flow cytometry appears to aid not only in the detection of malignant cells but also in the characterization of cell type. On the other hand, although DNA ploidy examination had better sensitivity; it had no advantage over conventional cytopathological examination in identification of malignant cells. Diagn. Cytopathol. 2009. © 2009 Wiley‐Liss, Inc.