Lack of effect of adjuvant chemotherapy on the elimination of single dormant tumor cells in bone marrow of high-risk breast cancer patients.

PURPOSE: There is an urgent need for markers that can predict the efficacy of adjuvant chemotherapy in patients with solid tumors. This study was designed to evaluate whether monitoring of micrometastases in bone marrow can predict the response to systemic chemotherapy in breast cancer. PATIENTS AND METHODS: Bone marrow aspirates of 59 newly diagnosed breast cancer patients with either inflammatory (n = 23) or advanced (> four nodes involved) disease (n = 36) were examined immunocytochemically with the monoclonal anticytokeratin (CK) antibody A45-B/B3 (murine immunoglobulin G(1); Micromet, Munich, Germany) before and after chemotherapy with taxanes and anthracyclines. RESULTS: Of 59 patients, 29 (49.2%) and 26 (44.1%) presented with CK-positive tumor cells in bone marrow before and after chemotherapy, respectively. After chemotherapy, less than half of the previously CK-positive patients (14 of 29 patients; 48.3%) had a CK-negative bone marrow finding, and 11 (36. 7%) of 30 previously CK-negative patients were CK-positive. At a median follow-up of 19 months (range, 6 to 39 months), Kaplan-Meier analysis of 55 assessable patients revealed a significantly reduced overall survival (P =.011; log-rank test) if CK-positive cells were detected after chemotherapy. In multivariate analysis, the presence of CK-positive tumor cells in bone marrow after chemotherapy was an independent predictor for reduced overall survival (relative risk = 5.0; P =.016). CONCLUSION: The cytotoxic agents currently used for chemotherapy in high-risk breast cancer patients do not completely eliminate CK-positive tumor cells in bone marrow. The presence of these tumor cells after chemotherapy is associated with poor prognosis. Thus, bone marrow monitoring might help predict the response to systemic chemotherapy.     

Prognostic impact of micrometastatic tumor cells in the lymph nodes and bone marrow of patients with completely resected stage I non-small-cell lung cancer.

PURPOSE: This study was designed to substantiate the prognostic impact of occult micrometastatic tumor cells in the lymph nodes (LNs) and bone marrow (BM) in stage I non-small-cell lung cancer (NSCLC) patients using cytokeratin (CK) as a micrometastatic marker and the relationship between the micrometastases in the LNs and BM. PATIENTS AND METHODS: A total of 2,432 hilar and mediastinal LNs were removed during surgery from 115 patients with completely resected stage I NSCLC. The LNs were analyzed for micrometastasis using immunohistochemistry with the biclonal anti-CK antibody AE1/AE3. BM aspirates from 115 patients were immunocytochemically stained with the monoclonal anti-CK antibody CK2. RESULTS: CK-positive (CK+) cells were detected in 42 (1.7%) of 2,432 LNs, in 32 (27.8%) of 115 patients, and in 32 (27.8%) of 115 BM aspirates. There was no relationship between the frequencies of CK+ cells in the LNs and in the BM. The patients with CK+ cells in the LNs had a poor prognosis by both univariate (P =.008) and multivariate analyses (P =.01), whereas the presence of CK+ cells in the BM did not allow prediction of survival (P =.32). The prognostic impact of LNs micrometastasis was independent even after adjusting for the status of BM micrometastasis. CONCLUSION: The detection of lymph nodal micrometastatic tumor cells provides an accurate assessment of tumor staging and has powerful prognostic implications for completely resected stage I NSCLC patients.     

Immunocytochemical monitoring of micrometastatic disease: Reduction of prostate cancer cells in bone marrow by androgen deprivation

Occult dissemination of tumor cells mainly determines the prognosis of patients with primary prostate cancer. The effect of androgen deprivation on micrometastatic tumor cells in these patients is currently unknown. We therefore used an immunocytochemical assay with monoclonal antibodies (MAbs) directed against epithelial cytoskeleton proteins (i.e., cytokeratins) to monitor the concentration of isolated tumor cells in the bone marrow of 36 prostate cancer patients (stage C), who underwent hormonal androgen deprivation with Flutamide and Leuproreline acetate. Tumor cells in cytologic bone marrow preparations were detected using an assay that employed the MAb CK2 directed against cytokeratin (CK) 18 and the alkaline anti-alkaline phosphatase staining method. Prior to therapy, we detected between 1 and 38 CK-positive cells per sample of 2 × 10⁴ nucleated cells in 21 patients, while the remaining 15 patients displayed tumor-free marrow samples. There was no significant correlation between the concentration of CK-positive cells and the volume of hypo-echogenic lesions as an indicator of the primary tumor volume or the serum level of prostate-specific antigen (PSA). After androgen deprivation, 20 of the 21 initially positive patients either became negative (n = 16) or showed at least a reduction in the concentration of CK-positive cells (n = 4). Moreover, only 2 of the 15 patients with negative pre-treatment findings became positive. All of the 7 patients with remaining tumor cells in the bone marrow after therapy showed no detectable amounts of PSA in their serum. Our findings suggest that serum PSA concentration is no indicator of micrometastatic disease in bone marrow. Neoadjuvant androgen deprivation appears to eliminate disseminated CK-positive tumor cells present in bone marrow, a preferred site of overt metastasis in prostate cancer patients. Int. J. Cancer 71:521-525, 1997. © 1997 Wiley-Liss, Inc.     

Prognostic impact of hematogenous tumor cell dissemination in patients with stage II colorectal cancer

Adjuvant chemotherapy is not routinely recommended in patients with colorectal cancer stage UICC II. Some of these patients, however, develop recurrent disease. Therefore, valid prognostic criteria are needed to identify high-risk patients who might benefit from adjuvant therapy. Disseminated tumor cells, detected in blood and bone marrow, may prove to be a valid marker, however, the prognostic relevance of these cells remains debated. In our study, we examined the prognostic significance of disseminated tumor cells in blood and bone marrow of patients with stage II colorectal cancer. Ninety patients with potentially curative (R0) resection of colorectal cancer stage II were prospectively enrolled into the study. Bone marrow and blood samples were examined for disseminated tumor cells by CK 20 RT-PCR. Patient, tumor and treatment factors were analyzed as prognostic factors. Multivariate analysis confirmed tumor cell detection in blood (hazard ratio 2.1, p = 0.03) and T-category (hazard ratio 2.2, p = 0.02) to be independent prognostic factors for relapse-free survival. Tumor cell detection in postoperative blood samples (hazard ratio 7.7, p < 0.001) and number of removed lymph nodes (hazard ratio 6.4, p < 0.001) were independent prognostic factors for disease-specific survival. Detection of circulating tumor cells in blood samples of patients with stage II colorectal cancer identifies patients with poor outcome. This finding should be confirmed by further studies and could then be used as a basis for conducting a randomized trial evaluating the effect of adjuvant chemotherapy in stage II patients.     

Detection and enrichment of disseminated renal carcinoma cells from peripheral blood by immunomagnetic cell separation

We have established an immunomagnetic separation procedure for the detection of circulating tumor cells in the peripheral blood based on the magnetic cell sorting (MACS) technique. In previous in vitro experiments, renal-cell carcinoma (RCC) cells were mixed with peripheral blood. In dilutions of 1:200 to 1:107 tumor cells per mononuclear blood cells, an average recovery rate of 84% of tumor cells was determined. In our study, 104 peripheral blood samples from 59 renal carcinoma patients were analyzed. MACS resulted in significant depletion of leukocytes, permitting a search for tumor cells on just 1 slide. Analyzing 8 ml of peripheral blood per patient, 19/59 RCC patients carried disseminated tumor cells (32%) in the range of 1 to 38 cells (median 8). Interestingly, for the cytokeratin-positive (CK+) patient group, we found a correlation between tumor cell number and grading (G2 vs. G3) and an increased number of CK+ patients with advanced tumor stage. MACS appears to be an efficient technique to detect disseminated tumor cells in peripheral blood.     

Consideration of tumor size improves the accuracy of TNM predictions in patients with gastric cancer after curative gastrectomy

ObjectiveTo investigate whether addition of tumor size improves the prognostic accuracy of the UICC 7th TNM staging system in gastric cancer patients who underwent radical surgery (R0 resection).MethodsThe clinical and pathological data and postoperative 5-year survival rate of 507 patients with gastric cancer who underwent radical surgery (R0 resection) in our department from January 2004 to June 2006 were evaluated retrospectively. The prognostic accuracy of conventional UICC 7th TNM staging was compared with that of UICC 7th TNM staging plus tumor size. The ability of tumor size to improve the 95% confidence interval (CI) of postoperative 5-year survival rate in gastric cancer patients was assessed.ResultsOf the 507 patients, 470 (92.7%) were followed up. The five-year survival rate of these patients was 50.4%. The survival rates of patients with pT1, pT2, pT3, and pT4 stage tumors were 89.3%, 72.4%, 36.9%, and 23.7%, respectively (P < 0.05), and the survival rates of patients with pN0, pN1, pN2, and pN3 stage tumors were 75.2%, 68.8%, 46.7%, and 21.3% (P < 0.05). Depth of invasion, lymph node metastasis stage, metastatic lymph node ratio (MLR), lymphatic invasion and tumor size were independent predictors of patient prognosis. The accuracy of UICC 7th TNM staging in predicting 5-year survival was 75.4% and the accuracy of tumor size plus the UICC 7th TNM staging was 77.9% (P < 0.05). This combination improved the 95% CI of postoperative 5-year survival rate in gastric cancer patients.ConclusionTumor size can improve the accuracy of UICC 7th TNM staging in predicting survival in gastric cancer patients following radical surgery (R0 resection). Tumor size is likely to be another important indicator in future UICC-TNM staging systems for gastric cancer patients.     

Occult tumor cells in bone marrow of patients with locoregionally restricted ovarian cancer predict early distant metastatic relapse.

PURPOSE: Based on conventional tumor staging, primary ovarian cancer is viewed as an intraperitoneal disease that rarely spreads to extraperitoneal organs. However, autopsy studies reveal a much higher rate of occult metastasis, indicating that extraperitoneal spread occurs with much greater frequency than previously appreciated. Consequently, we investigated the incidence of early hematogenous dissemination and its association with distant disease-free and overall survival. PATIENTS AND METHODS: Bone marrow aspirates from 108 patients newly diagnosed with International Federation of Gynecology and Obstetrics stage I to III ovarian cancer were prospectively analyzed with the novel anti-cytokeratin (CK) antibody A45-B/B3. We investigated the frequency of CK-positive tumor cells in bone marrow and their effect on prognosis in relation to established risk factors for tumor progression. RESULTS: Tumor cells in bone marrow were detected in 32 (30%) of 108 patients. A CK-positive finding was unrelated to established risk parameters, except for poor nuclear grading of the primary tumor. At a median follow-up of 45 months (range, 12 to 77 months), the presence of occult metastatic cells in bone marrow was associated with the occurrence of clinically overt, extraperitoneal (predominantly extraskeletal) distant metastasis (relative risk [RR], 16.5; 95% confidence interval [CI], 6.2 to 56.9; P < .0001) and death from cancer-related causes (RR, 2.3; 95% CI, 1.2 to 4.3; P = .01). Multivariate analysis identified a positive bone marrow finding as an independent prognostic factor of reduced distant disease-free survival for all patients (RR, 13.8; 95% CI, 5.4 to 52.9; P < .0001) and for the 64 stage R0-1 patients (RR, 7.3; 95% CI, 1.5 to 56.8; P = .0021). CONCLUSION: Our data signal that hematogenous dissemination of tumor cells occurs early and throughout all stages of ovarian cancer. The clinical significance of our findings is supported by the unfavorable prognosis in association with the presence of occult metastatic cells, especially in those patients who received an effective locoregional therapy.     

T-cell response to p53 tumor-associated antigen in patients with colorectal carcinoma

Despite the radical surgical resection performed in patients with colorectal carcinoma, there is a high rate of tumor recurrence. Over an observation period of 3 years, 18% of the patients in our collective suffered a tumor relapse with local or distinct metastases after initial R0-resection. Some evidence suggests that this may be due to suppression of anti-tumor responses, a phenomenon that might be attributed to regulatory T cells. The aim of our study was to investigate the tumor-specific immune response depending on the UICC stage of patients with colorectal cancer. The cellular immune responses against defined antigens that are overexpressed in most of the patients with colorectal cancer were characterized. For this purpose, the tumor suppressor gene, p53, was chosen as the tumor-associated antigen that exhibits mutations and overexpression in up to 60% of colorectal carcinoma. We observed that p53 induced both IFN-gamma and IL-10 secretion. The predominance of IL-10 production indicated that regulatory T cells directly participate in modulating the anti-tumor immune response. IL-10 levels in the blood as well as the expression of regulatory T-cell specific genes at the tumor site correlate with the UICC stage of the disease. These results may provide an explanation for the poor prognosis and increased recurrence rate in patients with advanced carcinoma.     

The expression of low density lipoprotein receptor-related protein in colorectal carcinoma

Low density lipoprotein receptor-related protein (LRP) is a multifunctional cell surface receptor binding many different ligands including proteinases and their inhibitors, some of which are known to be involved in tumor biology. We studied the expression of LRP and its putative role in colorectal carcinoma. Tissue samples were obtained from 50 patients with colorectal carcinoma and fixed in formalin and embedded in paraffin. Immunohistochemical staining was performed using antibodies directed against LRP, cathepsin B and urokinase-type plasminogen activator (u-PA). The expression of LRP was further studied by polymerase chain reaction. The TNM stage was determined according to UICC guide lines and was based upon histological analysis. LRP was primarily expressed in stroma cells [36 patients (72%)] and less frequently in tumor cells [6 patients (12%)]. In 22% of all cases LRP was prominent at the invasion front. Cathepsin B was found both in the tumor stroma [50 (100%)] and in the tumor cells [46 (92%)]. u-PA was present in the tumor stroma [44 (88%)] and in the tumor cells [44 (88%)]. In stromal cells the expression of LRP correlated significantly with the expression of u-PA (p=0.043). Furthermore, the expression of LRP and of u-PA in tumor cells correlated with the tumor stage according to UICC (p=0.038 and 0.018, respectively). We provide evidence that LRP is expressed in colorectal cancer. As LRP forms complexes with u-PA and its inhibitor, we suspect that LRP can influence the known effects of u-PA on tumor biology.     

Up-regulation of the peripheral benzodiazepine receptor during human colorectal carcinogenesis and tumor spread.

The peripheral benzodiazepine receptor (PBR) is overexpressed in a variety of cancers. In Unio Internationale Contra Cancrum (UICC) III colorectal cancers, a high level of PBR overexpression correlates with poor prognosis. However, little is known about the role of PBR in the development and progression of colorectal cancer. This study addresses the up-regulation of PBR during colorectal carcinogenesis and tumor spread. One hundred sixteen consecutive patients undergoing surgery for colorectal cancer with either regional (59 patients) or distant metastases (57 patients) were followed-up for 5 years or until death. Twenty-four of the 59 patients with initial UICC stage III cancers later developed distant metastases. PBR overexpression in tumor specimens was determined by immunohistochemistry. UICC stage III patients with colorectal primaries highly overexpressing PBR developed metastases significantly more often than patients with low PBR overexpression in their primary carcinoma. In 54 of the 116 patients adenomas and/or metastases and/or recurrences were available to be studied for PBR up-regulation during colorectal carcinogenesis and tumor spread. PBR was found to be overexpressed in 86% of early and late adenomas. Furthermore, 85% of primaries and of 86% of metastases displayed PBR overexpression. PBR overexpression was also detected at the mRNA level as revealed by real-time PCR. The extent of PBR protein overexpression was equivalent in colorectal adenomas and carcinomas but slightly increased in metastases. These data suggest a functional role of PBR during colorectal carcinogenesis and tumor spread. Thus, PBR qualifies as a target for innovative diagnostic and therapeutic approaches.     

Laparoscopic vaginal radical trachelectomy: a treatment to preserve the fertility of cervical carcinoma patients

BACKGROUND: Cervical carcinoma occurs frequently in young women who would like to preserve their childbearing potential. For those with early stage invasive lesions, the authors designed and performed radical trachelectomy, a surgical procedure that preserves the functions of the uterus. METHODS: Radical trachelectomy combines laparoscopic (for pelvic lymphadenectomy) and transvaginal approaches. Between April 1987 and December 1996, 56 patients were scheduled for this procedure, and 47 underwent it. The charts of these patients were retrospectively reviewed for medical and obstetric history, characteristics and complications of surgical procedures, pathologic findings, postoperative obstetric results, and cancer recurrences. RESULTS: The mean durations of the laparoscopic and vaginal steps of the procedure were 62 and 67 minutes, respectively. One intraoperative complication (cystotomy) and seven postoperative complications (drainage of pelvic collection) were observed. The pathologic tumor classification was International Union Against Cancer (UICC) pT1a1 (International Federation of Gynecology and Obstetrics [FIGO] Stage pIA1) in 5 cases, UICC pT1a2 (FIGO Stage pIA2) in 13 cases, UICC pT1b (FIGO Stage pIB) in 25 cases, UICC pT2a (FIGO Stage pIA2) in 1 case, and UICC pT2b (FIGO pIIB) in 3 cases. The mean follow-up was 52 months. Two recurrences (4%) were observed (one lateropelvic and one distant), and one patient died of disease progression. Despite a 25% rate of late miscarriages, 13 normal children were born after radical trachelectomy. CONCLUSIONS: In young patients affected by early invasive cervical carcinoma, radical trachelectomy does not appear to increase the rate of recurrence. It carries a relative risk of infertility and late miscarriage but makes it possible for some patients to become pregnant and give birth to normal newborns. Thus, it seems reasonable to offer this procedure in selected cases, provided that each patient is fully informed and the surgeon properly trained.     

The role of CA 15-3 and MCA monoclonal antibody assays in the detection of primary and recurrent breast cancer

CA 15-3 and MCA assays were tested in 103 operable patients (preoperative determination) and 100 patients with advanced breast cancer. Normal CA 15-3 and MCA values were determined in a series of 68 healthy women. The negative/positive cut-off was set at 28.8 U/ml and 15.5 U/ml respectively for CA 15-3 and MCA (mean value + 2SD). Results were analyzed in the two groups and with respect to T and N pathological categories in the preoperative series. In pT1 (59 pts), pT2 (30 pts), pT3 + pT4 (14 pts), pNO (58 pts), pN1 (45 pts) and overall preoperative series CA 15-3 and MCA sensitivities were respectively 25%, 40%, 57%, 22%, 42%, 30% and 27%, 30%, 35%, 21%, 33%, 26%. In the patients affected by widespread disease, sensitivity was 92% and 80% for CA 15-3 and MCA. Results were significantly different among normal, preoperative and advanced patients (P less than 0.05). Our results suggest that CA 15-3 and MCA levels are correlated with the tumor mass. Nevertheless, the low sensitivity in pT1 and pNO cases indicates that these two assays have no role in the diagnosis of early breast cancer. In the advanced patients, too, the results can be questioned: in the present study, in fact, recurrent cases were characterized by gross disease with multiple site involvement and cannot be considered as an example of early diagnosis of breast cancer recurrence.     

Comparative analysis of micrometastasis to the bone marrow and lymph nodes of node-negative breast cancer patients receiving no adjuvant therapy.

PURPOSE: In node-negative patients, of whom up to 30% will recur within 5 years after diagnosis, markers are still needed that identify patients at high enough risk to warrant further adjuvant treatment. In the present study we analyzed whether a correlation exists between microscopic tumor cell spread to bone marrow and to lymph nodes and attempted to determine which route is clinically more important. PATIENTS AND METHODS: According to a prospective design, bone marrow aspirates and axillary lymph nodes of level I (n = 1,590) from 150 node-negative patients with stage I or II breast cancer were analyzed immunocytochemically with monoclonal anticytokeratin (CK) antibodies. We investigated associations with prognostic factors and the effect of micrometastasis on patients' prognosis. RESULTS: CK-positive cells in bone marrow aspirates were present in 44 (29%) of 150 breast cancer patients, whereas only 13 patients (9%) had such positive findings in lymph nodes; simultaneous microdissemination to bone marrow and lymph nodes was seen in merely two patients. No correlation of bone marrow micrometastases with other risk factors was assessed. Reduced 4-year distant disease-free and overall survival were each associated with a positive bone marrow finding (P =.032 and P =.014, respectively) but not with lymph node micrometastasis. Multivariate analysis revealed an independent prognostic effect of bone marrow micrometastasis on survival, with a hazards ratio of 6.1 (95% confidence interval, 1.2 to 31.3) for cancer-related death (P =.031) in our series. CONCLUSION: Immunocytochemical detection of micrometastatic cells in bone marrow but not in lymph nodes is an independent prognostic risk factor in node-negative breast cancer that may have implications for surgery and stratification into adjuvant therapy trials.     

Therapeutic efficacy of transcatheter arterial chemoembolization as compared with hepatic resection in hepatocellular carcinoma patients with compensated liver function in a hepatitis B virus-endemic area: a prospective cohort study.

PURPOSE: Identifying a special subgroup of hepatocellular carcinoma (HCC) patients who may benefit from transcatheter arterial chemoembolization (TACE) when compared with the standard treatment of hepatic resection (HR) warrants research in Asian countries. PATIENTS AND METHODS: From January 1993 to December 1994, 182 patients with operable HCC (Child-Pugh class A and International Union Against Cancer [UICC] stage T1-3N0M0) were enrolled. After initial TACE and lipiodol computed tomography, 91 received HR and 91, who refused the operation, received repeated sessions of TACE. After stratification according to the tumor stage (UICC and Cancer of the Liver Italian Program [CLIP]) and lipiodol retention pattern, the survival rates of the two treatment groups were compared. The median follow-up period was 83 months. RESULTS: As of December 31, 2000, 48 patients who underwent HR and 68 patients who underwent TACE had died. In a subgroup analysis according to tumor stage, the HR group survival rate was significantly higher than the TACE group in both UICC T1-2N0M0 (P =.0058) and CLIP 0 (P =.0027) subgroups. However, there was no significant difference in either UICC T3N0M0 (P =.7512) or CLIP 1-2 (P =.5366) subgroups. Even in patients with UICC T1-2N0M0 HCC, when lipiodol was compactly retained, the survival rate of the HR group was comparable to that of the TACE group (P =.0596). CONCLUSION: TACE proved to be as effective as HR in the subpopulations with UICC T3N0M0 or CLIP 1-2 HCC and adequate liver function, and even with UICC T1-2N0M0 HCC when lipiodol was compactly retained in the tumor. In such cases, the choice of treatment modality between TACE and HR may be left to the patient's preference.     

Minimal residual disease in gastric cancer: evidence of an independent prognostic relevance of urokinase receptor expression by disseminated tumor cells in the bone marrow.

PURPOSE: To study the invasion-related molecule urokinase-type plasminogen activator receptor (u-PAR) expressed by disseminated tumor cells as a biologic predictor of poor survival in a large prospective series of patients with gastric cancer. PATIENTS AND METHODS: In 156 gastric cancer patients (prospective series), disseminated tumor cells in the bone marrow and the u-PAR expressed by these tumor cells were determined by cytokeratin (CK) 18 immunocytochemistry and u-PAR/CK18 double immunocytochemistry. RESULTS: In contrast to the mere detection of disseminated tumor cells at primary surgery, the additional evidence of u-PAR on these cells correlated significantly with pathologic T stage (P =.0474) and the expression of u-PAR (P =.0093) and plasminogen-activator inhibitor 1 (P =.0145) in the primary tumor (immunohistochemistry, chi(2)). Kaplan-Meier analysis revealed no association with prognosis for the mere detection of disseminated tumor cells. In contrast, a significant association was seen between detection of u-PAR on these cells and shorter disease-free (P <.0001) and overall survival (P <.0001). Multivariate analysis revealed that u-PAR on disseminated tumor cells at the time of primary surgery is an independent prognostic factor for disease-free (95% confidence interval [CI], 1.72 to 3.21; P =.024) and overall survival (P =.0049; relative risk, 2.89; 95% CI, 1.92 to 4.30). CONCLUSION: This is the first large study to show that u-PAR, detected on disseminated tumor cells in the bone marrow, is an independent prognostic parameter in gastric cancer, in contrast to the mere detection of minimal residual disease (MRD). u-PAR may be a promising marker to define a critical subpopulation of disseminated tumor cells and a target to eliminate MRD. Molecular phenotyping of MRD is critical for defining its individual clinical relevance.     

Progression of diploid tumor cells in aneuploid head and neck squamous cell carcinomas

The development of aneuploid clones from diploid progenitor cells is a regular characteristic of head and neck squamous cell carcinoma progression. While the significance of aneuploidy formation for the acquisition of invasive and metastatic behavior is well documented, little is known about the contribution of diploid tumor cells after aneuploid clones have emerged. To distinguish diploid cells of epithelial origin from benign cellular components, we applied multiparameter flow cytometry of DNA content and cytokeratin (CK) expression to 36 primary tumors. Twenty-seven carcinomas accommodated aneuploid cell lines that stained positive for CK. All diploid cell populations obtained from aneuploid carcinomas contained CK-positive subpopulations as did all of nine tumors that consisted exclusively of diploid cells. The proportions of CK-positive diploid cells ranged between 6% and 80%, independent of whether they were achieved from entirely diploid or from aneuploid carcinomas. CK-gated diploid and aneuploid cell populations showed largely identical S-phase fractions. These results emphasize that diploid tumor cells regularly persist after the development of aneuploid clones and significantly contribute to local tumor progression. Despite the presence of diploid epithelial cells in aneuploid primary tumors, exclusively the aneuploid clones of eight corresponding lymph node metastases were CK-positive. This provides further evidence of a largely reduced metastatic potential of diploid tumor cells.     

Carbonic anhydrase IX expression, a novel surrogate marker of tumor hypoxia, is associated with a poor prognosis in non-small-cell lung cancer.

PURPOSE: To evaluate carbonic anhydrase (CA) IX as a surrogate marker of hypoxia and investigate the prognostic significance of different patterns of expression in non-small-cell lung cancer (NSCLC). METHODS: Standard immunohistochemical techniques were used to study CA IX expression in 175 resected NSCLC tumors. CA IX expression was determined by Western blotting in A549 cell lines grown under normoxic and hypoxic conditions. Measurements from microvessels to CA IX positivity were obtained. RESULTS: CA IX immunostaining was detected in 81.8% of patients. Membranous (m) (P =.005), cytoplasmic (c) (P =.018), and stromal (P <.001) CA IX expression correlated with the extent of tumor necrosis (TN). The mean distance from vascular endothelium to the start of tumor cell positivity was 90 micro m, which equates to an oxygen pressure of 5.77 mmHg. The distance to blood vessels from individual tumor cells or tumor cell clusters was greater if they expressed mCA IX than if they did not (P <.001). Hypoxic exposure of A549 cells for 16 hours enhanced CA IX expression in the nuclear and cytosolic extracts. Perinuclear (p) CA IX (P =.035) was associated with a poor prognosis. In multivariate analysis, pCA IX (P =.004), stage (P =.001), platelet count (P =.011), sex (P =.027), and TN (P =.035) were independent poor prognostic factors. CONCLUSION: These results add weight to the contention that mCA IX is a marker of tumor cell hypoxia. The absence of CA IX staining close to microvessels suggests that these vessels are functionally active. pCA IX expression is representative of an aggressive phenotype.     

The plasmacytoid carcinoma of the bladder--rare variant of aggressive urothelial carcinoma

The WHO 2004 classification defines new histological and molecular variants of urothelial carcinoma. However, there are limited data available on the clinicopathological characteristics or prognosis of these variants. We present histopathological, molecular and clinical data of 32 plasmacytoid carcinomas of the bladder (PUC) showing that PUC is a high-grade tumor with molecular features of aggressive urothelial carcinoma, usually diagnosed in advanced pathological stage (64% pT3, 23% pT4) showing metastases in 60% of the patients. Average survival of our cohort of PUC treated with radical cystectomy and adjuvant chemotherapy was lower than what is typically seen for comparable conventional urothelial carcinomas. Eighty-seven percent of the PUCs showed a negative or strongly reduced membranous staining of E-cadherin. β-Catenin staining was negative in 22.5%, and 16.7% of the remaining tumors showed nuclear accumulation. Aberrant CK20 expression (negative or >10% of cells stained) and negative CK7 staining was found in 100% and 22.6%, respectively. Ninety-seven percent revealed positive staining for PAN-CK. CD138 was positive in 78%, whereas MUM-1 expression was negative in all cases. Multitarget fluorescence in situ hybridization showed all PUCs to be highly aneuploid and polysomic. Deletions on chromosome 9p21 seem to play an important role in this variant. FGFR3 and PIK3CA mutation analyses yielded no mutations in any of the PUCs analyzed. TP53 mutation analysis showed mutations in 29%. In summary, PUC is an aggressive variant of bladder cancer with molecular features of advanced bladder cancer and evidence of WNT pathway activation in some of the cases.