Carcinoma Cell Clusters Research Articles

Machine learning reveals mesenchymal breast carcinoma cell adaptation in response to matrix stiffness.

Epithelial-mesenchymal transition (EMT) and its reverse process, mesenchymal-epithelial transition (MET), are believed to play key roles in facilitating the metastatic cascade. Metastatic lesions often exhibit a similar epithelial-like state to that of the primary tumour, in particular, by forming carcinoma cell clusters via E-cadherin-mediated junctional complexes. However, the factors enabling mesenchymal-like micrometastatic cells to resume growth and reacquire an epithelial phenotype in the target organ microenvironment remain elusive. In this study, we developed a workflow using image-based cell profiling and machine learning to examine morphological, contextual and molecular states of individual breast carcinoma cells (MDA-MB-231). MDA-MB-231 heterogeneous response to the host organ microenvironment was modelled by substrates with controllable stiffness varying from 0.2kPa (soft tissues) to 64kPa (bone tissues). We identified 3 distinct morphological cell types (morphs) varying from compact round-shaped to flattened irregular-shaped cells with lamellipodia, predominantly populating 2-kPa and >16kPa substrates, respectively. These observations were accompanied by significant changes in E-cadherin and vimentin expression. Furthermore, we demonstrate that the bone-mimicking substrate (64kPa) induced multicellular cluster formation accompanied by E-cadherin cell surface localisation. MDA-MB-231 cells responded to different substrate stiffness by morphological adaptation, changes in proliferation rate and cytoskeleton markers, and cluster formation on bone-mimicking substrate. Our results suggest that the stiffest microenvironment can induce MET.

Clinicopathological implications to micropapillary bladder urothelial carcinoma of the presence of sialyl Lewis X-decorated mucin 1 in stroma-facing membranes

ObjectivesBladder urothelial carcinoma (UC) comprises more than 90% of all bladder cancers. Among several UC variants, micropapillary UC (MPUC) is a rare one with high potential for lymphovascular invasion and subsequent lymph node metastasis. Histologically, MPUC is characterized by the presence of small papillary carcinoma cell clusters surrounded by lacunar spaces. Immunohistochemically, the outer circumference of these clusters, that is, the stroma-facing membrane of carcinoma cells, is reportedly almost invariably positive for mucin 1 (MUC1) protein and to a lesser extent for sialyl Lewis X (sLeX) carbohydrates; however, the clinicopathological implications of these expression patterns have not been fully investigated. Materials and methodsWe performed immunohistochemical analysis of MPUC (n = 11) and conventional UC (n = 57) for MUC1 and sLeX to determine whether these factors immunolocalized. Dual immunofluorescence staining was also carried out to assess MUC1 and sLeX colocalization. We also performed Western blot analysis of Chinese hamster ovary cells misexpressing both recombinant epitope-tagged MUC1 and glycosyltransferases enabling sLeX biosynthesis. ResultsMPUC samples preferentially exhibited both MUC1 protein and sLeX carbohydrate expression on the stroma-facing membrane of carcinoma cells. Based on univariate analysis, MUC1 expression in that pattern was positively correlated with tumor extension, lymphovascular invasion, lymph node metastasis, disease stage, and relatively poor patient prognosis. A comparable sLeX expression pattern also correlated positively with tumor extension and nodal metastasis. Based on multivariate analysis, localization of MUC1 and sLeX on the stroma-facing side of the membrane was positively correlated with lymph node metastasis. ConclusionsOverall, our immunofluorescence findings as well as immunoprecipitation analyses of Chinese hamster ovary cell transfectants strongly suggest that MUC1 is a potential scaffold protein for sLeX carbohydrates in MPUC. Both MUC1 and sLeX may cooperatively contribute to MPUC histogenesis and clinicopathological characteristics.

Association of c-Met phosphorylation with micropapillary pattern and small cluster invasion in pT1-size lung adenocarcinoma

Lung adenocarcinomas with micropapillary pattern (MPP) are associated with frequent nodal metastasis. However, little is known about the mechanisms that underlie MPP-associated nodal metastasis. We have previously reported that pT1 lung adenocarcinomas with MPP are significantly associated with small cluster invasion (SCI) and lymphatic involvement. SCI is defined as markedly resolved acinar–papillary tumor structures with single or small clusters of carcinoma cells invading stroma within fibrotic foci. In this study, we hypothesized that c-Met activation may be involved in the MPP-SCI sequence, given that the c-Met tyrosine-kinase receptor and its ligand hepatocyte growth factor (HGF), play important roles in tumor cell motility and invasion. We analyzed 125 pT1-size lung adenocarcinomas for immunohistochemical expression of phosphorylated c-Met and its correlation with MPP, SCI, lymphatic involvement and prognosis. SCI was significantly more frequent in the MPP-positive group (P<0.0001) and associated with lymphatic involvement (P<0.0001) and nodal metastasis (P=0.021). c-Met protein was detected in all tumors by immunohistochemistry as membranous and cytoplasmic staining. Phospho-c-Met (pc-Met) was positive in 119/125 tumors (95%) and expressed at high levels in 27 cases (22%). A high level of pc-Met expression was significantly associated with MPP (P=0.01) and SCI (P=0.0059). Moreover, in tumors with MPP or SCI, those expressing high levels of pc-Met were significantly more associated with lymphatic involvement. In p-Stage IA lung adenocarcinomas (n=99), patients in the high pc-Met expression group showed significantly worse survival than patient in the low expression group (P=0.0313). These results suggest that activation of c-Met through phosphorylation may be involved in MPP and SCI.

One-step method of bone marrow aspiration and biopsy applied in diagnosis of the bone marrow metastatic cancer

This study was aimed to explore the value of one-step method of bone marrow aspiration and biopsy applied in diagnosis of the bone marrow metastatic cancer. The total of 46 cases of bone marrow metastatic cancer were analyzed retrospectively, and the results of the bone marrow smear and the bone marrow biopsies were compared. The results indicated that the success rate of one-step method of bone marrow aspiration and biopsy was 95.7%. Metastatic carcinoma cells in clumps or clusters with morphological changes were observed in the bone marrow biopsies of the 45 patients (97.8%), but the metastatic carcinoma cell clusters were observed only in bone marrow smears of the 25 patients (54.3%). There were mild to moderate hyperplasia of fibrous tissue in the patients with metastatic cancer. In addition to the 83.3% diagnostic rate of esophageal cancer, the rest diagnostic rate of bone marrow biopsy for metastatic carcinoma was 100%. The diagnostic rate of bone marrow smear for ovarian cancer, lung cancer, gastric cancer, thoracic tumor, sigmoid colon cancer, esophageal cancer and metastatic cancer of unknown primary cancers were 33.3%, 50%, 72.2%, 60%, 50%, 33.3% and 25%, respectively. The diagnostic rate of bone marrow biopsy was higher than that of bone marrow smear. It is concluded that the success rate of drawing specimen by one-step method of bone marrow aspiration and biopsy is high. The bone marrow biopsy is better than that of aspiration in diagnosis of metastatic cancer. Combining biopsy with aspiration can improve the accuracy of diagnosis.

Low-Grade Clear Cell Renal Cell Carcinoma Mimicking Hemangioma of the Kidney: A Series of 4 Cases

Clear cell renal cell carcinoma (CCRCC) has a rich, sinusoid-like vascularity frequently used as a diagnostic criterion. CCRCC with predominantly vascular architecture has not been described. To describe 4 unusual CCRCC cases, primarily presenting with hemangioma-like morphologic pattern. Clinicopathologic and selected immunohistochemical analysis of 4 cases of CCRCC mimicking hemangioma. Cases were seen in 1 woman and 3 men (average age, 48.8 years; range, 40-66 years). Grossly, tumors were red-brown (3 of 4) with scant bright-yellow foci in 1. The average tumor size was 4 cm (range, 2.5-5.5 cm). Microscopically, all were composed of varying proportions of a rich, arborizing, sinusoid-like vasculature with focal hobnail appearance of endothelial cells. Entrapment of renal tubules between blood vessels was seen at the periphery of the tumors. This morphology was reminiscent of anastomosing hemangioma. Isolated tumor cells resembling lymphocytes with clear halos were sparsely interspersed between vessels. Cytokeratin immunostain confirmed the diagnosis of CCRCC. Extensive sampling and immunohistochemical workup of what is deemed to be a benign vascular neoplasm of the kidney is needed to rule out the presence of individual carcinoma cells or small viable carcinoma cell clusters.

Five-Year Survival after Surgery for Invasive Micropapillary Carcinoma of the Stomach

Invasive micropapillary carcinoma (IMPC) of the breast, urinary bladder, ovary, and colon has been reported. However, few reports have described IMPC of the stomach. In addition, IMPC has been described as a histological indicator for lymphatic invasion and nodal metastasis, resulting in poor prognosis. We report a case of 5-year survival after surgery for IPMC of the stomach. A 69-year-old woman was admitted to our hospital with symptoms of upper abdominal pain. Upper gastrointestinal endoscopy revealed a tumor at the antrum of the stomach. Histological examination of the biopsy specimen indicated poorly differentiated adenocarcinoma. The patient underwent distal gastrectomy with lymph node dissection. Microscopic examination of the specimen revealed that the tumor consisted of an invasive micropapillary component. Carcinoma cell clusters were floating in the clear spaces. The patient recovered uneventfully and remains alive without recurrence 5 years after surgery.

A case report of pigmented mammary Paget’s disease mimicking nevus of the nipple

A 43-year-old Japanese woman consulted our hospital for a pigmented lesion on her right nipple. Two years later, the lesion became enlarged, measuring 5×5mm. It was dark brown, had an irregular shape and relatively clear borders. Incisional biopsy yielded a pathological diagnosis of junctional nevus of the skin. An additional 2years later, a small mass developed under the right nipple area and core needle biopsy yielded a pathologic diagnosis of invasive ductal carcinoma. Partial resection of the right EC areas included the skin of the nipple and sentinel lymph node biopsy was performed. Histologically, the skin of the nipple demonstrated small clusters of pigmented carcinoma cells that were low molecular weight cytokeratin (CAM5.2) positive. Most of the carcinoma cells were small and did not have abundant cytoplasm, but nuclear enlargement and prominent nucleoli indicated malignancy, and the cytoplasm was pale compared with that of the surrounding squamous epithelial cells. Scattered dendritic melanocytes were identified by S-100 protein and HMB-45 immunohistochemically. In the upper dermis, carcinoma cells also involved the lactiferous ducts. A small focus of carcinoma cells that invaded the fat tissues did not contain melanin pigment. The final diagnosis was pigmented mammary Paget's disease. Pigmented lesions on the nipple should be carefully examined, because pigmented mammary Paget's disease sometimes mimics malignant melanoma or junctional nevus.

Small cluster invasion: a possible link between micropapillary pattern and lymph node metastasis in pT1 lung adenocarcinomas

Lung adenocarcinomas with micropapillary pattern (MPP) are associated with frequent nodal metastasis. However, little is known about the mechanisms that underlie MPP-associated nodal metastasis. In this study, we investigated how small micropapillary clusters of carcinoma cells present in tumoral alveolar spaces lead to increased lymph node metastasis. We analyzed 146 cases of pT1 lung adenocarcinomas with reference to the presence of MPP, small cluster invasion (SCI), and lymphatic involvement. SCI was defined as markedly resolved acinar-papillary tumor structures with single or small clusters of carcinoma cells invading stroma within fibrotic foci. The MPP-positive group (88/146 cases) was associated with significantly more frequent nodal metastasis and significantly worse survival. Moreover, SCI was significantly more frequent in the MPP-positive group (71/88 cases) than MPP-negative group (10/58 cases) and was significantly associated with lymphatic involvement (p < 0.0001) and nodal metastasis (p = 0.0073). The SCI-positive group showed significantly worse survival (5-year survival, 70%) than the SCI-negative group (91%, p = 0.0017). Carcinoma cells undergoing SCI demonstrated the same characteristic MUC-1 expression on the outer surface of cell clusters as those undergoing MPP. Thus, SCI could link MPP to nodal metastasis; carcinoma cells with MPP tend to undergo SCI in scars and invade lymphatics in pT1 lung adenocarcinomas.

Characteristic cytological features of histological grade one (G1) breast carcinomas in fine needle aspirates

To analyse the spectrum of nuclear features as well as dissociation pattern found in fine needle aspirates (FNAC) from histological grade 1 breast carcinomas and evaluate the critical cytological features of these lesions. The material consisted of FNAC smears from 494 histologically confirmed grade 1 breast carcinomas. All smears were revaluated for cell dissociation pattern, nuclear size, cell uniformity, nucleoli, nuclear margin and chromatin pattern. All features were compared with the histological subtype and cytological grading. 73.9% of the cases were cytological grade 1, 24.3% were grade 2 and 1.8% were grade 3. The majority of the cases had a cell dissociation pattern showing both a population of single carcinoma cells and cell clusters (65.9%). Practically all tumours had a granular chromatin pattern (94.7%) and a slightly irregular nuclear margin with folds and grooves (94%) irrespective of histological subtype and cytological grading. Nucleoli were mostly indistinct or small (74%), whereas 24.3% were noticeable and 1.7% abnormal. Practically all cases revealed some degree of pleomorphism with 74.3% showing mild and 22.4% a distinct pleomorphism. A small subgroup of IDC was classified as monomorphic (3.3%). Almost all tumours had nuclear sizes in the range of 2-4 x RBC (96.9%). Not all histological grade 1 carcinomas are cytological grade 1. About 25% were grade 2, and a small subpopulation reached grade 3. The typical/average findings in FNAC from grade 1 breast carcinomas were a population of both groups and single cells showing mild pleomorphism, granular chromatin, slightly irregular nuclear margin, indistinct nucleolus and nuclear size 2-4 x RBC.

Primary invasive micropapillary carcinoma of the stomach

Reported herein is the case of a 74-year-old man with an unusual gastric carcinoma that developed at the lesser curvature of the stomach. The tumor consisted of small clusters of carcinoma cells surrounded by clear spaces, with histopathology similar to invasive micropapillary carcinoma (IMPC) of the breast. The carcinoma cells, which had downregulation of E-cadherin expression, invaded the subserous tissue and metastasized to the perigastric lymph nodes. IMPC, an unusual subtype of invasive breast carcinoma, is known to have frequent lymph node metastases, resulting in a poor clinical outcome. Although IMPC has been reported in breast, urinary bladder, ureter, lung, salivary gland and colon, to the best of the authors' knowledge this is the first report of IMPC arising in the stomach. Presented here are the clinicopathological features of primary IMPC of the stomach.

Evaluation of Tumor Cell Dissociation as a Predictive Marker of Lymph Node Metastasis in Submucosal Invasive Colorectal Carcinoma

Tumor cell dissociation-the histologic finding of small solid carcinoma cell clusters and groups of dissociated dedifferentiated carcinoma cells at the invasive front-is related to tumor metastasis and patient prognosis. However, few previous reports have examined tumor cell dissociation in submucosal invasive colorectal carcinoma. We investigated the relation between tumor cell dissociation and lymph node metastasis in submucosal invasive colorectal carcinoma. We also examined immunohistochemical expression of E-cadherin and beta-catenin in submucosal invasive colorectal carcinoma. Submucosal invasive colorectal carcinoma tissue samples from 20 patients with lymph node metastasis and 100 patients without lymph node metastasis were evaluated. Sections stained with hematoxylin and eosin were evaluated for tumor cell dissociation. Immunohistochemistry was used to determine the expression and cellular distribution of E-cadherin and beta-catenin. Tumor cell dissociation was more frequently identified in submucosal invasive colorectal carcinoma cases with lymph node metastasis than in those without lymph node metastasis (P = 0.0001). Decreased membranous expression of E-cadherin occurred more frequently in submucosal invasive colorectal carcinoma cases with lymph node metastasis than in those without lymph node metastasis (P = 0.025). Nuclear expression of beta-catenin tended to be present in submucosal invasive colorectal carcinoma cases with lymph node metastasis (P = 0.063). Decreased membranous expression of E-cadherin occurred more frequently in submucosal invasive colorectal carcinoma cases with tumor cell dissociation than in those without tumor cell dissociation (P = 0.0023). Our results suggest that there is a relation between tumor cell dissociation and lymph node metastasis in submucosal invasive colorectal carcinoma. Tumor cell dissociation formation might be related to abnormal expression patterns of E-cadherin and beta-catenin in submucosal invasive colorectal carcinoma. Tumor cell dissociation and decreased membranous expression of E-cadherin would be important predictive markers for lymph node metastasis in submucosal invasive colorectal carcinoma.

Markedly elevated cell turnover is characteristic of small, deeply invasive carcinomas of the colorectum

Small, deeply invasive carcinomas invading the muscularis propria or deeper and measuring < or = 2 cm in greatest dimension (S-ADV) are rare in comparison with their larger counterparts (NS-ADV), and their clinicopathologic features are obscure. S-ADV and NS-ADV cases as well as cases of submucosal carcinoma (SM-CA) were comparatively assessed for: 1) clinicopathologic findings; 2) Ki-67, mitotic, and apoptotic indices; 3) cathepsin G, p53, and bcl-2 immunoreactivities; and 4) c-Ki-ras mutations. S-ADV and SM-CA, which both are significantly smaller than NS-ADV, did not differ in size, but the frequency of moderately and poorly differentiated carcinoma elements at the leading edges was observed to be higher than in the central cores only in S-ADV, as was tumor "budding" of small clusters of undifferentiated carcinoma cells. The frequency of severe lymphatic involvement in S-ADV was as high as in NS-ADV, and significantly greater than in SM-CA. The Ki-67, mitotic, and apoptotic indices for S-ADV were significantly increased compared with those for NS-ADV and/or SM-CA. Expression of cathepsin G in S-ADV tumor and stromal cells was significantly decreased compared with NS-ADV and/or SM-CA cases. No significant differences in the expression of either p53 or bcl-2 or the incidence of c-Ki-ras mutations were observed among the three groups. S-ADV can be considered a distinct type of deeply invasive carcinoma, presenting with poor tumor differentiation at the leading edge, and with increased tumor cell proliferation despite its small size.

Markedly elevated cell turnover is characteristic of small, deeply invasive carcinomas of the colorectum

BACKGROUND Small, deeply invasive carcinomas invading the muscularis propria or deeper and measuring ≤ 2 cm in greatest dimension (S-ADV) are rare in comparison with their larger counterparts (NS-ADV), and their clinicopathologic features are obscure. METHODS S-ADV and NS-ADV cases as well as cases of submucosal carcinoma (SM-CA) were comparatively assessed for: 1) clinicopathologic findings; 2) Ki-67, mitotic, and apoptotic indices; 3) cathepsin G, p53, and bcl-2 immunoreactivities; and 4) c-Ki-ras mutations. RESULTS S-ADV and SM-CA, which both are significantly smaller than NS-ADV, did not differ in size, but the frequency of moderately and poorly differentiated carcinoma elements at the leading edges was observed to be higher than in the central cores only in S-ADV, as was tumor “budding” of small clusters of undifferentiated carcinoma cells. The frequency of severe lymphatic involvement in S-ADV was as high as in NS-ADV, and significantly greater than in SM-CA. The Ki-67, mitotic, and apoptotic indices for S-ADV were significantly increased compared with those for NS-ADV and/or SM-CA. Expression of cathepsin G in S-ADV tumor and stromal cells was significantly decreased compared with NS-ADV and/or SM-CA cases. No significant differences in the expression of either p53 or bcl-2 or the incidence of c-Ki-ras mutations were observed among the three groups. CONCLUSIONS S-ADV can be considered a distinct type of deeply invasive carcinoma, presenting with poor tumor differentiation at the leading edge, and with increased tumor cell proliferation despite its small size. Cancer 1999;85:796–802. © 1999 American Cancer Society.

Expression of the 67 kD laminin receptor in human ovarian carcinomas as defined by a monoclonal antibody, MLuC5.

Previous immunohistochemical data from our laboratory have demonstrated that expression of the 67 kD laminin receptor (67LR), a cancer-associated, high-affinity laminin-binding protein, is upregulated in ovarian carcinoma cells compared with normal serosal cells, and that this increased expression in cancer cells could be related to patient outcome. The aim of this study was to validate MLuC5, a monoclonal antibody that recognises the 67LR, as a tool to perform future immunohistochemical studies on larger populations of ovarian carcinoma patients. Expression of the 67LR was determined in 51 primary human ovarian carcinoma samples using immunohistochemistry and MLuC5. The 67LR was detected in ovarian carcinoma cell clusters of variable extent. Analysis of the data determined that 67LR expression was significantly increased in the samples from patients with disease progression, compared with those with no evidence of disease after completion of primary therapy, and in pooled grade 2 and 3 tumours compared to borderline and grade 1 tumours (P < 0.05, chi-squared test). No other significant correlation between 67LR expression and other clinicopathological parameters could be established. These data suggest that the 67LR is correlated to ovarian tumour progression. Detection of the 67LR using this monoclonal antibody could constitute an interesting parameter in prognosis determination of ovarian cancer.

Histopathological study of lymphatic invasion in squamous cell carcinoma (O-1N) with high potential of lymph node metastasis

The process of lymph node metastasis was studied in an animal model (termed O-1N) that was successfully established using a metastatic tumor to the submandibular lymph node from a chemically induced squamous cell carcinoma of the hamster tongue. The model has been maintained by serial transplantation of metastatic tumors into the buccal pouch. Lymphovascular invasion of transplanted O-1N in the tongue was examined in serial histologic sections. Lymphatic vessels were distinguished from blood vessels by Masson's trichrome stain for vascular smooth muscle, BSA-I lectin binding for vascular endothelium, and laminin and type IV collagen immunostaining for the vascular basement membrane. Transplanted tumors enlarged progressively with invasion of surrounding tissues of the tongue and resulted in lymph node metastasis in all animals with successful takes. Local growth of the tumors in the tongue was accompanied by stromal proliferation with abundant dilated lymphatic vessels which contained clusters of tumor cells. On serial sections, the carcinoma cell clusters in lymphatics in the close proximity of tumor nests were in continuity with adjacent tumor nests, whereas such continuity was not recognized in those occurring apart from tumor nests. The formation of isolated carcinoma cell clusters resulting from disintegration of elongated processes of tumor nests with invasion of lymphatics and subsequent transport in lymphatics and deposition in lymph nodes in clusters were well demonstrated in other serial sections. The key step of lymph node metastasis therefore appears to be direct invasion of lymphatic vessels by tumor cells, similar to their invasion of adjacent tissues but different from the way that blood cells escape through vessel walls. Proliferation of lymphatics around tumor nests and transport of tumor cells in clusters would also contribute to the production of metastatic deposits in lymph nodes.

Distribution of type-VII collagen in xenografted human carcinomas.

The distribution of type-VII collagen, the main molecular component of the anchoring fibrils (AF) attaching the basal lamina (BL, lamina densa of the basement membrane) to the surrounding connective tissue, was investigated in four xenografted human carcinomas of the hypopharynx (H-Stg 1), the lung (L 261), the sigmoid colon (CA 1), and the rectum (R 85). The studies were performed with a recently prepared, affinity-purified and highly specific antibody to type-VII collagen by using the indirect immunofluorescence and the APAAP (alkaline phosphatase anti-alkaline phosphatase) techniques. For comparison, the localization of the intrinsic BL components laminin and type-IV collagen were additionally analyzed in all four carcinomas. It was shown that type-VII collagen usually colocalized to laminin and type-IV collagen and was deposited at the borderline between carcinoma cell clusters and the surrounding strands of connective tissue in a similar, but more diffuse and less continuous distribution than both intrinsic BL components. In the squamous cell carcinoma H-Stg 1 and the adenocarcinoma L261, type-VII collagen was additionally accumulated in enlarged extracellular spaces between carcinoma cells, away from the contact zone to the connective tissue and again colocalized to laminin and type-IV collagen. Numerous carcinoma cells of both xenografts showed remarkable intracytoplasmic immunoreactivity for the antibody to type-VII collagen. Even in the case of the gastrointestinal carcinomas CA 1 and R 85, faint immunoreactivity for type-VII collagen was found at the contact zone between the mucosal epithelium and the surrounding connective tissue. These results confirm that epithelial carcinoma cells are obviously involved with the synthesis of the main molecular component of AF usually attaching the BL to the adjacent connective tissue and hint at a possible correlation between the localization of type-VII collagen and the observed pattern of the BL. However, it cannot be decided whether there is a direct causal relation between both phenomena or whether they are both the consequence of an independent but common cause, such as abnormal cellular differentiation of carcinoma cells. In no case, can the discontinuities in the distribution of type-VII collagen be explained by active tumor cell invasion since xenografted human carcinomas neither invade nor metastasize.

Synthesis and release of glycoconjugates bearing N-linked oligosaccharides by ovarian carcinoma cells isolated from effusions.

Ovarian carcinoma cell clusters were isolated from patient effusions. The cell isolates were incubated in vitro with radioactive glycoconjugate precursors. Radiolabelled glycoconjugates released to culture media were analyzed for molecular mass heterogeneity and lectin binding activity. From 10 to 50% of the released glycoconjugates were present as a heterogeneous array of glycoconjugates of molecular mass greater than 250 kilodaltons. The remaining glycoconjugates were dispersed in a molecular mass range extending down to approximately 15 kilodaltons. Concanavalin A-Sepharose affinity chromatography revealed the presence of N-linked oligosaccharides. Interaction of glycoconjugates with lentil and pea lectins indicated the presence of L-fucose residues linked to asparagine-bound N-acetylglucosamine. Precipitation of glycoconjugates with ricinus communis agglutinin I showed the presence of nonreducing terminal N-acetyllactosamine residues. Collectively, the data indicate that ovarian carcinoma cells release to culture medium fucosylated glycoconjugates bearing complex-type oligosaccharides. The synthesis and release of these glycoconjugates showed no significant differences among different histologic types of ovarian carcinoma; however, modulation as a function of tumor progression may occur.

Isolation and morphologic characterization of human ovarian carcinoma cell clusters present in effusions.

Serous, mucinous, endometrioid and clear cell human ovarian carcinoma cells were isolated as multicellular aggregates from patient effusions by filtration on nylon mesh of defined porosity and examined by light microscopy. The cell clusters ranged from compact to loosely adherent groups of cells to spheroids with a central lumen surrounded by a cell monolayer. There was considerable variation in cluster morphology between effusions from different patients as well as within effusion from the same patient. Apparent budding of clusters was observed as well as different stages of cluster growth and development. This was observed for all histologic types studied. Electron microscopy of serous, mucinous and clear cell types showed that cells forming clusters were attached to each other by desmosomes, demonstrating that cluster formation did not result from a nonspecific stickiness of cells. Irregular microvilli were present on the external periphery of the various carcinoma cells and a prominent glycocalyx was present on the surface of mucinous carcinoma cells. Extensive interdigitation of cytoplasmic extensions and extended villi was present in mucinous and serous clusters which appeared to strengthen cluster cohesiveness. Nuclei were irregular with prominent nucleoli frequently present. The cell clusters usually remained intact and viable in culture but generally did not attach to glass or plastic substrata, whereas mesothelial cells and nonactivated histiocytes rapidly attached. When carcinoma cell clusters did attach, they were resistant to detachment by trypsin-EDTA treatment, in contrast to the nonmalignant cells.

Monoclonal Antibody Against Human Ovarian Tumor-Associated Antigens

Mouse monoclonal antibodies (OV-TL 3) were raised against human ovarian tumor-associated antigens for diagnostic purposes. A cloned hybridoma cell line was obtained by fusion of murine myeloma cells with spleen lymphocytes from BALB/c mice immunized with a tumor cell suspension prepared from an ovarian endometrioid carcinoma. The antibodies were initially screened for their ability to bind on frozen sections of human ovarian carcinoma tissue and a negative reaction on gastric carcinoma tissue by indirect immunofluorescence. The reactivity of the selected OV-TL 3 clone (IgG1 subclass) was studied on normal and neoplastic tissues as well as on a cell line derived from the original tumor cell suspension used for immunization. OV-TL 3 antibodies stained frozen sections of human ovarian carcinomas of the following histological types: serous, mucinous, endometrioid, and clear cell. No reaction was found with breast cancers or other nongynecological tumors. No differences in staining pattern were observed between primary and metastatic ovarian carcinomas. OV-TL 3 antibodies brightly stained ovarian carcinoma cell clusters in ascitic fluids and left unstained mesothelial cells and peripheral blood cells. The OV-TL 3-defined antigen also remained strongly expressed on a cell line derived from the endometrioid ovarian carcinoma originally used for generation of OV-TL 3 clone. Reactivity was weak and irregular in a few ovarian cysts, while traces of fluorescence were sometimes detected in epithelial cells lining the female genital tract. In only 3 specimens of 15 endometrium carcinomas was weak focal reactivity with OV-TL 3 antibodies observed. The results of the immunofluorescence study were confirmed by the more sensitive avidin-biotin method and by 125I-labeled OV-TL 3 antibodies. Thus OV-TL 3 recognizes a common antigen for most ovarian carcinomas and may be a useful tool for rapid diagnosis of ovarian carcinomas.

A histopathologic study of benign and malignant lymphoepithelial lesions of the parotid gland

Twenty-three cases of benign and malignant lymphoepithelial lesions without Sjögren's syndrome were examined: 21 were classical benign lymphoepithelial lesions, and were mostly found in males older than 50 years of age. The other two cases were identified as undifferentiated carcinoma arising from benign lymphoepithelial lesion (8.7%; 2/23). Histologically, this carcinoma is characterized by the formation of cell nests of variable size and by marked lymphoplasmacytic infiltrations into the stromal tissue. In immunofluorescence, an increase of plasma cells containing IgA and IgG was noted in the marginal areas of carcinoma cell clusters. Electron microscopy disclosed that the carcinoma was composed of poorly differentiated cells with occasional squamous differentiation. No dysplastic characteristics were found in the infiltrating lymphocytic components. These two cases of carcinoma were found in middle-aged females.