CK-positive Cells Research Articles

Disseminated Tumor Cells in Biologic Subtypes of Stage I–III Breast Cancer Patients

Triple receptor-negative breast cancers (TNBC) are higher grade and more likely to metastasize. Recurrences after 5 years are rare in TNBCs. Conversely, late recurrences are seen in estrogen receptor (ER)-positive (luminal) cancers. Disseminated tumor cells (DTCs) may be responsible for late recurrences. We compared rates of DTCs in basal and luminal subtypes. We evaluated 205 stage I-III patients. DTCs were assessed from bone marrow aspirates using anti-cytokeratin (CK) antibody following cytospin, and the presence of ≥ 1 CK-positive cells was considered positive. Pathologic complete response (pCR) was defined as lack of invasive disease in primary tumor and regional lymph nodes after neoadjuvant chemotherapy (NAC). Statistical analyses used chi-square and Fischer's exact test. Median follow-up (f/u) was 27 months, and 40% of patients had NAC. Forty patients had TNBC, and 148 had luminal cancers. Seventeen percent of TNBC patients, and 27% of those with luminal subtype, had DTCs after NAC (P = NS). Following NAC, pCR occurred in 28% of TNBC and 23% of luminal patients. Luminal A subtypes were less likely to achieve pCR when compared with non-luminal A subtypes (16 versus 41%; P = 0.01). All TNBC patients who achieved pCR had complete eradication of DTCs, whereas 36% of luminal (A and B) subtypes had DTCs. DTCs were found in 29% of stage I-III patients. TNBCs were more likely to have complete eradication of DTCs after pCR. Further study is needed to determine whether DTCs are responsible for late recurrences in patients with luminal cancers.

Quantification of tumor cell burden by analysis of single cell lymph node disaggregates in metastatic prostate cancer

The size of lymph node (LN) metastases in prostate cancer patients represents an important prognosticator, but histological work-up may not reflect the true extent of tumor invasion. We present a novel technique (1) to detect early tumor cell dissemination and (2) to quantify the true tumor burden. Prospectively 232 LN of 20 consecutive patients with prostate cancer after lymph node dissection were longitudinally bisected, one half was subjected to single cell immunocytochemistry for pancytokeratine (CK), the other half underwent routine histopathological work-up and step section analysis. In immunocytochemistry, tumor cell density (TCD) was quantified by calculating the number of CK-positive cells/million leucocytes and compared to routine histopathology and step section analysis. Eight of 20 patients were positive in histopathology and step sectioning, but 14 of 20 patients were positive in single cell analysis. Twenty-five of 232 LN were positive in routine histopathology, whereas 52 of 232 LN were positive in single cell analysis. Median TCD in histopathologically positive LN was 3060.0 x 10(-6) and 9.9 x 10(-6) in histopathologically negative LN (P < 0.0001). Mean TCD of histopathologically negative LN of pN1 patients was significantly higher than the mean TCD of pN0 patients (P < 0.003). Mean TCD per patient correlated with serum-PSA (r(2) = 0.48, P < 0.006). Single cell analysis has an increased detection rate compared to routine histopathology and even to serial step section analysis. The method can detect early tumor dissemination and enables quantification of the tumor burden. The subgroup of histopathologically negative LN with CK-positive cells represents tumor cell dissemination not depicted histologically.

Distribution of hepatic stem cell markers in human liver with massive hepatic necrosis

Background and aims: The origin, niche, and function of human hepatic stem/progenitor cells (HHSPCs) have been longstanding areas of debate. We designed this study to elucidate the distribution of potential HHSPCs during the various stages of regeneration by the expression of known HHSPC markers. Methods: Immunostainings for c-kit, cytokeratin (CK)19, and CD133 were performed in cases of massive hepatic necrosis (MHN) in various regenerating stages. Results: We identified c-kit-positive cells as single cells primarily in the stroma of portal tracts but rarely in the sinusoids. There were no differences in the numbers or distribution of c-kit positive cells according to MHN regeneration stage. The number of CK19-positive cells mostly in a ductular arrangement significantly increased along the interface, and single cells or small clusters of CK19-positive cells were occasionally observed in the sinusoid in cases of MHN. CD133-positive cells were located in ductular structures along the interface in cases of MHN, while no CD133-positive cells were observed in the normal liver. Conclusion: The c-kit- or CD133-positive cells appear first at the limiting plate and are then organized into ductular structures or moved to the sinusoid as single cells or clusters during hepatic regeneration.

Bone Marrow-Derived Cells Promote Liver Regeneration in Mice With Erythropoietic Protoporphyria

Bone marrow transplantation can reverse hepatic protoporphyrin accumulation and prevent the hepatobiliary complications characteristic of erythropoietic protoporphyria. The aim of this study was to assess the recruitment capacity of bone marrow cells in the damaged liver and their possible contribution to the improved or recovered hepatic function in a murine model of erythropoietic protoporphyria (EPP). Lethally irradiated female EPP mice were transplanted with bone marrow cells from healthy male mice and were monitored during 12 or 36 weeks. Two groups of animals killed 12 weeks after transplant were also treated with granulocyte colony-stimulating factor. Cell transplantation decreased porphyrin contents in erythrocytes and liver. Improved hepatic structure and function and reduced hepatic fibrosis were observed, especially 36 weeks after transplant. Bone marrow-derived cells (22%-35%) were identified in the liver of recipient mice by means of fluorescence in situ hybridization (chrY-FISH) or green fluorescent protein staining and were characterized by immunofluorescence staining. The livers of recipients contained 20% to 30% myofibroblasts (alpha-smooth muscle actin-positive cells), 40% CK19-positive cells, and 10% to 28% hepatocytes (albumin-positive cells) derived from the donor bone marrow. Bone marrow-derived cells play a significant role in restoring and regenerating hepatic tissue in EPP mice. Hepatic repair was associated with fibrogenesis, enhanced by granulocyte colony-stimulating factor treatment, and almost normal liver structure and function was observed in the long term (36 weeks posttransplant).

Immunostaining as a Diagnostic Aid in Cytopathologic Study of Upper Urinary Tract Urothelial Carcinoma

To evaluate preoperative diagnosis of low-grade urothelial carcinoma (LGUC) and urothelial neoplasms of unknown malignant potential (UMP UN) of the upper urinary tract (UUT) and its role in disease management, especially in the context of nephron-sparing treatment possibilities.Wash and brush ureteral specimens of LGUC/UMP UN of the UUT with histopathologic correlation were retrieved at our institution for 7 years and studied along with 7 ureteral specimens from nonneoplastic ureteral lesions.Of 30 specimens from 25 LGUC/UMP UN, 5 were negative for tumor cells and 3 showed cytologic atypia. The remaining 22 contained tumor cells with characteristic features of urothelial carcinoma, including hard and soft criteria. The 4 hard criteria included branching stromal cores, dyshesive cell networks, 3-dimensional papillary clusters with stromal core and atypia associated with CK20-positive cells. The 2 soft criteria were hypercellularity and atypia in CK20-negative cells. All LGUC/UMP UN of the UUT were associated with at least 1 hard criterion or both soft criteria.Branching stromal cores, 3-dimensional papillary clusters, dyshesive cell networks and CK20-positive atypia immunostaining appear specific for LGUC/UMP UN of the UUT but are seen in few cases. Combined soft and hard criteria will increase sensitivity to 83%.

Circulating cytokeratin 18 fragments and activation of dormant tumor cells in bone marrow of cancer patients (Review)

In cancer patients detection of systemic disease is of great importance to obtain prognostic information and to guide therapy. Bone marrow (BM) seems to be a common homing tissue for the early spread of tumor cells from various epithelial tumors; however, verification of the prognostic significance of BM-disseminated tumor cells (BM-DTCs), is restricted to breast cancer so far. These cells may be dormant for a long time, and signals triggering their activation leading to recurrence remain to be characterized. A recent study involving metastatic breast cancer patients reported that the shortest disease-free survival is correlated with cytokeratin (CK)-negative BM aspirates and that CK-positive BM-DTCs correspond to dormant tumor cells. Soluble CK fragments in serum including CK18 and 19 (measured as TPS and CYFRA 21-1, respectively) and caspase-cleaved CK18 are widely used to monitor tumor progression and response to therapy, actually indicating proliferation and/or necrotic/apoptotic cell death. In order to assess the source of the CK fragments, we used determinations of CK18 and caspase-cleaved CK18 fragments in serum samples before and after radical tumor surgery in colon cancer patients. Elevated serum concentrations of CK18 were found to persist in patients with a high incidence of BM-DTCs, and high perioperative levels of caspase-cleaved CK18 fragments were detected in patients with early relapses, respectively. These results indicate that in some patients at increased risk of recurrence disseminated cell populations exist that are responsible for the release of the bulk of CK fragments after removal of the apparently nonmetastatic tumor. In good agreement with the results in metastatic breast cancer patients, release of CK18 or 19 fragments by BM-DTCs seem to indicate disseminated tumor cells mainly in a dormant state, whereas caspase-cleaved CK18 may indicate skipping of this latent phase and early progression. Therefore, caspase cleavage of CKs in intact tumor cells seems to accompany or is involved in the differentiation leading from dormant to progressively active disseminated tumor cells. Release of respective CK fragments would result in an apparent clearing of CK-positive cells in BM, leaving malignant cells that have possibly undergone an epithelial-mesenchymal transition. Micrometastatic cancer cell lines derived from breast cancer patients were found to display loss of epithelial CK8, 18 and 19 as well as ectopic expression of vimentin as in mesenchymal cells. In conclusion, degradation of CKs may represent a marker indicating reactivation of dormant tumor cells in BM.

Can the condition of the cell microenvironment of mediastinal lymph nodes help predict the risk of metastases in non-small cell lung cancer?

Background: The aim of this study was to analyze the properties of the immune cell microenvironment of regional lymph nodes (LNs) positive for lung cancer. Methods: Twenty-four patients operated on for stages T1 and T2 of the NSCLC, were enrolled in the study. Peripheral blood and LN tissue were obtained from different lymph node sites and levels. As a control, LN tissue was taken from patients diagnosed with emphysema or pneumothorax. The cells from randomly chosen LN were tested by multi-color flow cytometry. Separate portions of LN were snap-frozen and examined for the presence of cytokeratin positive cells (CK). Propensity for apoptosis, level of TCR zeta chain expression of T cells and the number and maturation status of dendritic cells were confronted with the presence of CK-positive cells. Results: The presence of metastases correlated with the downregulation of TCR zeta, especially CD8(+) T cells. The most striking feature was the reduction in the number of myeloid CD11c(+) dendritic cells in the LN of patients with LN metastases. This could be a reflection of the immunodeficient state observed in lung cancer patients. Even in the absence of metastases in the regional LN, the same type of changes in the LN microenvironment were observed in those LN located nearer the primary tumor. Conclusions: The preliminary results of this study suggest that this approach may be helpful as an independent tumor staging factor. It is also worth noting that part of the staging process could also be based on features describing the immune cells in the peripheral blood.

297 Epidemiology and prognosis of breast cancer in very young population

Metastasis–the spread of tumour cells from a primary lesion to distant organs–is the main cause of cancer-related death, and bone marrow (BM) is a frequent site for the settlement of disseminated tumour cells. Many BM samples harbour isolated tumour cells, whereas tumour cell clusters, as the potential precursors of solid distant metastases, are rarely detected after current enrichment procedures. We have analysed BM samples from 43 patients with carcinomas of the breast, colon and ovaries; 41 of these patients had no clinical signs of overt metastases (stage M0). Tumour cells in BM were enriched with immunomagnetic beads coupled to monoclonal antibodies against both EpCAM and HER2/neu. After enrichment, tumour cells were identified by immunostaining with the anti-cytokeratin antibody A45-B/B3. In total, 886 CK-positive cells were detected in 16 (35%) samples after immunomagnetic enrichment as compared to 34 cells in 9 (21%) samples using Ficoll density centrifugation previously used as the standard enrichment technique. Most remarkably, clusters of 2 to 10 CK-positive cells were found in 75% of CK-positive samples enriched by immunobeads, whereas no CK-positive cell clusters were detected after Ficoll enrichment. The method described offers an excellent tool for the enrichment of micrometastatic tumour cell clusters; these clusters may represent the initial stage of development from a single disseminated tumour cell towards an overt metastasis.

Improved functions of human hepatocytes on NH3 plasma-grafted PEEK-WC–PU membranes

PEEK-WC–PU membranes were modified with an NH3 glow discharge process to graft N-containing functional groups at their surface in order to improve the maintenance of human hepatocytes. Native and modified membrane surfaces were characterized with XPS, ToF-SIMS and WCA measurements. We have investigated morphological behaviour and specific functions of primary human hepatocytes on native and modified PEEK-WC–PU membranes in a small-scale gas-permeable bioreactor. N-containing groups grafted at the surface of the membranes improved the initial steps of adhesion and the maintenance of phenotype and differentiated functions of cells. Confocal microscopy of cell morphology evidenced human hepatocytes exhibiting a polygonal shape and organizing a 3D structure. The presence of CK19 positive cells, a marker of biliary duct epithelium, was also found on native and modified membranes. Liver specific functions, investigated in terms of urea production, albumin synthesis and diazepam biotransformation, were maintained at high levels up to 19 days, particularly on surface-modified membranes.

Cytokeratins 8 and 18 in adult human corneal endothelium

The aim of this study was to determine if cytokeratins (CKs) 8 and 18 – typical epithelial cell markers – are constitutively expressed in adult human corneal endothelium. Cryosections, paraffin-embedded sections and corneal endothelial imprints obtained from eleven adult human corneal discs not suitable for transplantation were used. Different fixative solutions were applied before indirect immunofluorescent or enzymatic staining was performed with antibodies against CK8 (Chemicon), CK18 (Dako and Sigma) and CK8/18 (Novocastra). Semi-quantitative RT-PCR and Western blotting (mRNA or proteins were isolated from Millicell membranes) were used to determine cytokeratin mRNA and protein levels. Approximately 50% of the corneal endothelial cells were positive for CK8 (Chemicon), CK18 (Sigma) and the CK pair 8/18 (Novocastra) in the endothelium when acetone was used for fixation. Four and 52% CK18-positive cells were observed using immunofluorescent and enzymatic immunohistochemistry, respectively, when the CK18 antibody from Dako was used. No signal was detected when 4% formalin or 10% paraformaldehyde was used as a fixative, irrespective of the antibody used. CK8 and CK18 proteins and mRNAs were detected in the endothelium of all tested corneas by Western blotting or semi-quantitative RT-PCR, respectively. We detected both CK8 and CK18 in the endothelium of all specimens at both the protein and mRNA levels. These results clearly demonstrate that cells of the corneal endothelium express CKs 8 and 18 and share some features with simple epithelia.

Prognostic significance of occult axillary lymph node metastases after chemotherapy‐induced pathologic complete response of cytologically proven axillary lymph node metastases from breast cancer

Primary systemic chemotherapy has been a standard of care for the management of locally advanced breast cancer (LABC) patients and has increasingly been used for patients with large operable breast cancer. Pathologic complete response (pCR) of axillary lymph node metastases predicted an excellent probability of long-term disease-free and overall survival. Although the clinical significance of occult lymph node metastases in patients with breast cancer was extensively studied, their prognostic value in patients with LABC after primary chemotherapy was not known. This study evaluated the detection rate and clinical significance of occult lymph node metastases in lymph nodes that contained metastatic carcinoma at the time of initial diagnosis and converted to negative based on routine pathologic examination after primary systemic chemotherapy. Fifty-one patients with LABC and cytologically involved axillary lymph nodes that converted to negative after preoperative chemotherapy were identified from 2 prospective clinical trials. All lymph node sections were reviewed, 1 deeper level hematoxylin and eosin-stained section of each lymph node was obtained and immunohistochemical staining for cytokeratin (CK) was performed. A total of 762 lymph nodes were evaluated for occult metastases. Kaplan-Meier survival curves were used for calculating disease-free and overall survival times. Occult axillary lymph node metastases were identified in 8 of 51 (16%) patients. In 6 patients, occult metastases were found in only 1 lymph node. In 7 patients, only isolated CK-positive cells were identified. In all cases, occult carcinoma cells were embedded within areas of fibrosis, foreign body giant cell reaction, and extensive histiocytosis. Patients with occult lymph node metastases tended to have a higher frequency of residual primary breast tumors than those without occult metastases (4 of 8 vs 7 of 43, respectively). There was no statistically significant difference in disease-free or overall survival times between patients with and without occult metastases after a median follow-up 63 months. Persistent occult axillary lymph node metastases were not uncommon in patients with axillary lymph node-positive LABC who experienced a pCR in involved lymph nodes after preoperative chemotherapy. However, such occult metastases did not adversely affect the good prognosis associated with axillary lymph node pCR. Therefore, routine lymph node CK evaluation was not recommended after primary chemotherapy.

Estrogen Receptor Immunohistochemistry for Confirmation of Sentinel Lymph Node Metastasis in Cases With Equivocal Cytokeratin Positivity

Differentiation of true metastases from cytokeratin (CK)-positive nonepithelial cells by immunohistochemistry occasionally may be difficult in the evaluation of sentinel lymph nodes (SLNs) for occult breast carcinoma metastases. In this study, we evaluated estrogen receptor (ER) immunostaining superimposed on CK as a method for the confirmation of metastasis when CK immunostaining alone was equivocal. We performed sequential ER staining on previously CK-stained slides on 15 axillary SLNs from breast cancer patients: 5 SLNs with known metastatic carcinoma (positive controls), 6 known negative SLNs (negative controls), and 4 test cases (3 SLNs in which CK-positive cells were equivocal for malignancy and 1 SLN in which metastasis was obvious, but contained focal weakly CK-positive signet ring cells). The primary tumor in all cases expressed ER in >50% of cells. Only 3 of 5 positive controls showed metastatic cells with dual CK/ER staining. CK-positive reticulum cells in all negative controls were ER negative. Three test cases showed dual CK/ER staining in the equivocal cells. The case with signet ring cells showed strong ER staining in the nonsignet ring cells and weaker staining in the signet ring cells. We conclude that dual CK/ER staining can be useful in SLNs when CK staining alone is equivocal, particularly when the primary tumor is known to have high expression of ER. Although dual ER/CK positivity helps to confirm metastasis, negative ER staining does not exclude metastatic disease.

Epithelial cells within the human pancreas do not coexpress mesenchymal antigens: epithelial–mesenchymal transition is an artifact of cell culture

Pancreatic mesenchymal stem cells (MSCs) may be derived from human β-cells undergoing reversible epithelial–mesenchymal transition (EMT), suggesting that they could be a potential source of new β-cells. In this study we sought to determine the origin of pancreatic MSCs in the nonendocrine pancreas. Double immunofluorescent (IF) staining and flow cytometry were used to assess the cell phenotype of nonendocrine pancreas tissue following islet procurement, during in vitro expansion of MSCs, and after differentiation. IF staining of paraffin-embedded pancreatic biopsy sections was used to assess cell phenotype in vivo. In this study we demonstrated that: (1) pancreatic epithelial cells do not express MSC antigens in vivo; (2) following islet isolation EpCAM- and CK19-positive epithelial cells coexpressed the MSC antigens CD44 (32±8% and 38±10%) and CD29 (85±4% and 64±4%); (3) during in vitro expansion the number of single-positive epithelial and double-positive epithelial/MSCs decreased whereas the number of single-positive MSCs increased and (4) differentiated MSCs do not revert to a true epithelial cell phenotype in our culture conditions, as epithelial cell surface markers (EpCAM, CK19 and E-Cadherin) are not reexpressed, although the MSC phenotype is altered. This study demonstrates that MSCs may be derived in vitro via a pancreatic epithelial cell undergoing EMT, however it is more likely that a small percentage of MSCs that reside in the adult pancreas are proliferating whereas the epithelial cells are negatively selected by the experimental culture conditions.

Clinical Significance of Cytokeratin 20-Positive Circulating Tumor Cells Detected by a Refined Immunomagnetic Enrichment Assay in Colorectal Cancer Patients

Current immunomagnetic enrichment method can only detect general epithelial antigens of circulating tumor cells (CTC). Further characterization of the CTCs to provide specific information on the tumor type is not possible. We attempted to overcome this drawback by developing the methodology for using a gastrointestinal-specific anti-cytokeratin (CK) 20 antibody to detect CTCs in colorectal cancer patients' blood. The protocol was validated using a colorectal cancer SW480 cell line. The clinical significance of findings in colorectal cancer was investigated by detecting CK20-positive CTCs (pCTC) in patients with colorectal cancer, other common cancers, colorectal adenoma, benign colorectal diseases, and normal subjects. Moreover, the malignant nature of CK20 pCTCs was examined by comparing chromosome 17 aberration patterns with those from the corresponding primary tumors. The assay successfully showed CK20-positive SW480 cells. When applied in patient samples, the detection rates were 62% (132 colorectal cancer patients; median number = 11 CTCs), 0% (120 patients with other common cancers), 6% (50 colorectal adenoma patients), 0% (120 patients with benign colorectal diseases), and 0% (40 normal subjects). Furthermore, statistical analysis showed that CK20 pCTC numbers were associated with tumor-node-metastasis stage and lymph node status. Using the median CK20 pCTC numbers as the cutoff points, stratified groups of colorectal cancer patients had significant differences in their recurrence, metastasis, and survival. Finally, chromosome 17 aneusomy in 90% of colorectal cancer patients with CK20 pCTCs matched with those from the primary tumors. Detection of CK20 pCTCs using the new protocol could generate clinically important information for colorectal cancer patients.

Multicenter study on the detection of disseminated tumor cells in primary breast cancer.

Abstract Abstract #5021 Background: Detection of disseminated tumor cells (DTC) in bone marrow (BM) has prognostic value in primary breast cancer (BC) and is currently under investigation for therapy stratification and monitoring. Anti-cytokeratin (CK) antibodies (Abs) have been established as markers for detection of such DTC in patients with BC. Here we present a direct comparison of the two Abs most frequently used for DTC detection over the past 10 years.&amp;#x2028; Material and Methods: We screened BM samples from 391 BC patients of which 64 were node-positive (N+). All patients were free of any signs of overt metastases (stage M0, pT1-3, pN0-3). Tumor cells in BM were enriched by Ficoll-Hypaque gradient and the mononuclear cell (MNC) fraction was spun on slides. For each patient 2*106 MNC were analyzed per Ab, using A45-B/B3 (A45) and AE1/AE3 (AE) Abs in parallel. A45 staining was, however, performed a couple of years later than AE staining, on cytospins stored at -80°C. All samples were screened automatically for the presence of CK-positive cells. By morphological evaluation only TC-compatible cells were scored positive. Median follow-up times ranged from 5 to 118 months.&amp;#x2028; Results: For both Abs, the number of DTC revealed was 5 or less. Of the 391 patients, 24 patients (6.1%) were positive for A45, and 41 patients (10.5%) for AE, respectively. Concordance obtained by cross-analysis for the two Abs was 84.4%, whereas the overlap in positive cases was only 3.2%. Regarding immunohistopathological characteristics AE- and A45-positive BM were found significantly more frequent in tumors of higher nodal status (p=0.003 and p=0.026), and for AE also in hormone receptor negative (HR-) compared to HR+ patients (p=0.006). Positivity for AE and/or A45 staining in BM correlated with high nodal status and large tumor size (p=0.001 and p= 0.016, respectively). Survival analyses revealed shorter relapse-free survival for A45-positive patients (p=0.026). In node-positive patients, a significantly worse prognosis regarding overall and BC specific survival was observed for AE-positive (p=0.0245 and p=0.0125) and for AE- and/or A45-positive patients (p=0.035 and p=0.044, respectively). In node-negative patients, A45-positivity was correlated to a significantly shorter relapse-free survival (p=0.032). Immunocytochemical double staining of BM samples from 26 patients (M+ and M0) revealed subpopulations of DTC labeled with both Abs (in 8/26 cases) and other DTC exclusively stained for either A45 or AE (4/26).&amp;#x2028; Discussion: Our results indicate that the two anti-cytokeratin Abs most frequently used for DTC detection in BM do not give identical results. They appear to identify at least partially different subpopulations of DTC in BC patients. This implies a possible advantage of the combined use of these Abs in future trials using DTC diagnostics. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5021.

ERalpha-status of disseminated tumour cells in bone marrow of primary breast cancer patients.

IntroductionIsolated disseminated tumour cells (DTC) are regarded as surrogate markers for minimal residual disease in breast cancer. Characterisation of these cells could help understand the known limitations of adjuvant therapy. Of particular interest is their oestrogen-receptor (ER) status because endocrine adjuvant therapy remains a cornerstone of breast cancer treatment.MethodsBone marrow (BM) aspirates from 254 patients with primary breast cancer were included in this study. A double immunofluorescence staining procedure was established for the identification of cytokeratin (CK) positive/Erα-positive cells. ERα status of the primary tumour was assessed immunohistochemically using the same antibody against ERα.ResultsIn 107 of 254 (42%) breast cancer patients, CK-positive cells could be detected in the BM. More than one DTC in the BM was observed in 38 of the 107 patients. The number of detected cells ranged between 1 and 55 cells per 2 × 106 mononuclear cells. DTCs demonstrated ERα positivity in 12% of the patients. The ERα expression was heterogeneous in 10 of the 38 (26%) patients with more than one DTC. The concordance rate of ERα status between primary tumour and DTC was 28%. Only 12 of 88 patients with ERα-positive tumours also had ERα-positive DTCs.ConclusionsPrimary tumours and DTCs displayed a concordant ERα status in only 28% of cases. Most of the DTCs were ERα negative despite the presence of an ERα-positive primary tumour. These findings further underline the distinct nature of DTCs and may explain the failure rates seen in conventional endocrine adjuvant therapy.

Histopathological work-up and interpretation of sentinel lymph nodes for vulvar squamous cell carcinoma

Identification / removal of sentinel lymph nodes (SLN) aims at reduction of long-term morbidity from complete lymphadenectomies e.g. inguinal seromas in vulvar squamous cell carcinoma (SCC). Correct histopathological work-up of SLN is imperative, as patients with inguinal recurrence / disease have a high risk to die from systemic disease. Work-up and interpretation of SLN from vulvar SCC differs from that in other organs, in particular from breast cancer. Methods: The experience with SLN dissection and work-up of 48 patients with vulvar SCC at the Institute of Pathology, Medical University Graz, Austria, is presented. Results: All lymph nodes identified after injection of radioactive tracers / blue dye (1–4) were removed in a “same-day procedure“. After frozen section, all SLN were formalin-fixed, sectioned entirely at 250µm intervals for 3 HE-stains and 1 unstained slide for immunohistochemistry / millimeter. In the absence of metastases on HE sections, ALL unstained slides are submitted for immunohistochemistry with antibody to cytokeratin (CK). SCORING IS POSITIVE EVEN WHEN ONLY INDIVIDUAL CK-POSITIVE CELLS &amp; DEBRIS ARE IDENTIFIED. 13 SLN from 48 SCC (34 pT2, 9 pT1b, 5 pT1a) had obvious metastases on HE stains. After CK-staining, 28/35 SLN remained negative and 4 SLN revealed micro-metastases. 3 SLN showed only individual single CK-positive cells &amp; debris. In 1 of these 3 patients, the SLN was called “negative“ and the patient developed conglomerate metastases within 9 months. All metastasis to SLN were from pT2 SCC. Conclusion: SLN dissection for vulvar SCC is a safe procedure for the patient, but requires a careful and complete histopathological work-up of all removed SLN and a correct interpretation of the staining results.

Comparison of clinical features and liver histology in acute and chronic autoimmune hepatitis

We investigated the clinical and morphological features between acute and chronic autoimmune hepatitis (AIH) with or without acute exacerbation. Serum total bilirubin on average was elevated to 12 mg/dL in acute AIH, alanine aminotransferase and aspartate aminotransferase peaked to more than 1000 U/L, and serum gamma-glutamyl transpeptidase was higher in the acute type compared with the chronic type without exacerbation. Serum immunoglobulin G was lowest in all other types of AIH. A liver biopsy showed interface or lobular hepatitis with lympho-plasmacytic infiltration, and rosette formations were frequently seen in acute AIH. There were morphological changes of central necrosis with plasmacytic infiltration and giant cell hepatitis. CK19-positive cholangiolar cells had proliferated in the periportal area with massive necrosis, and bile duct injuries were seen in acute AIH more frequently than in the chronic type. Laboratory data and liver histology in acute AIH differed from those of chronic AIH and were clarified for the diagnosis of acute AIH.

Isolated Tumor Cells and Circulating CK20 mRNA in pN0 Colorectal Cancer Patients

Micrometastases in lymph nodes and blood may provide important prognostic information. In this study, cytokeratin 20 (CK20) positive cells in lymph nodes and circulating CK20 mRNA were studied using 57 paraffin-embedded lymph node specimens and blood from 24 patients with pN0 colorectal cancer (CRC), respectively. Results showed that 29 out of 56 (52%) lymph node specimens had CK20-positive cells (range: 1-35). Follow-up of the patients for 12 months indicated that 4 patients (7%) had CRC metastases to liver, lung, and bone. In addition, 8 out of 24 (33%) samples had at least 2-fold circulating CK20 mRNA expression higher than the pooled normal sample. This study provides evidence that CK20-positive cells were found in the lymph nodes and differentially expressed circulating CK20 mRNA was also detected in the blood from patients with pN0 CRC. Long-term follow-up is necessary to study their prognostic use in patients with non-metastatic CRC.

Obesity-Induced Lymphocyte Hyperresponsiveness to Chemokines: A New Mechanism of Fatty Liver Inflammation in Obese Mice

Hepatic lipid retention (steatosis) predisposes hepatitis. We investigated the mechanisms of lymphocyte homing to fatty liver and the role of lipopolysaccharide (LPS) in the onset of inflammation in ob/ob mice. We decreased intestinal bacterial compounds by oral antibiotic treatment to test the role of endogenous LPS in liver inflammation. Adoptive transfer of lymphocytes was used to study the respective contributions of steatosis and lymphocytes to liver inflammation. We tested lymphocyte response to chemokines by in vitro chemotaxis assays in ob/ob, their lean controls, and "non-obese ob/ob" mice, generated by controlling caloric intake to distinguish between the effects of obesity and leptin deficiency. Antibiotic treatment decreased liver infiltration with CD4(+) T, CD8(+) T, natural killer (NK)T, B, and NK cells. Adoptive transfer of lymphocytes from ob/ob or control mice showed that (1) steatosis increased lymphocyte recruitment to the liver; (2) CD4(+) T, CD8(+) T, and B cells from ob/ob mice had a greater propensity to migrate specifically to the liver. This migration was enhanced by LPS. These results were also observed in a model of high-fat diet-induced obesity. CD4(+) T and B cells were hyperresponsive to CXCL12 and CXCL13, respectively. Weight normalization in "non-obese ob/ob" mice decreased liver inflammation, lymphocyte response to chemokines, and homing to the liver. Our study provides the first evidence that liver inflammation in mice with genetic or diet-induced obesity results from both steatosis and lymphocyte hyperresponsiveness to chemokines expressed in the liver. These abnormalities are reversible with weight normalization.