Additional confirmation of successful cell suspension fractionation of metastatic axillary node tissue.

We present herein an extension to our recently developed and published method termed "Fractionation of Nodal Cell Suspension" (FNCS). The method enables efficient subcellular fractionation into nuclear (N) and cytosolic (C) compartments of extremely fibrous and problematic metastatic axillary lymph node (mALN) tissue, using the entire nodule. For the purpose of the present study, a case of invasive lobular breast cancer (BC) patient with pT2N3aMx status and defined primary tumor markers (ERα 8, PR-B 8, and HER2 score 0) was available. Initially, the mALN tissue of this patient was analyzed by immunohistochemistry (IHC), and a positive correlation of nodal ERα, PR-B and HER2 biomarkers to those of the primary tumor was obtained. Subsequently, the mALN was FNCS fractionated into N and C, and Western blot (WB) analysis demonstrated a single band for ERα, PR-B and nuclear loading control (HDAC1) in nuclear, but not in the cytosolic compartments, confirming the efficiency of our fractionation protocol. At the same time, HER2 bands were not observed in either compartment, in accordance with HER2 negativity determined by IHC in both primary tumor and mALN tissue. In conclusion, by confirming the nuclear expression of ERα and PR-B biomarkers in metastatic loci, we demonstrate the purity of the FNCS-generated compartments - the protocol that offers a reliable tool for further analysis of nuclear versus cytosolic content in downstream analysis of novel biomarkers in the whole mALN of BC patients.

Establishment and Fractionation of Metastatic Axillary Lymph Node Cell Suspension for Determination of Protein Expression Levels of Nuclear cFOS and Cytosolic TGFß1 from Breast Cancer Patients.

BACKGROUND: Metastatic Axillary Lymph Node (mALN) status is currently the most important prognostic factor in the management of primary breast cancer (BC). Thus, development of specimens which enable identification of new mALN markers, involved in the progression of the disease, are of considerable interest. The specific aim of this work was to describe the method of establishment of Metastatic Axillary Nodal Cell Suspension and its fractionation, termed Fractionated Nodal Cell Suspension (FNCS), into nuclear and cytosolic extracts to enable determination of protein expression levels of nuclear cFOS and cytosolic Transforming Growth Factor ß1 (TGFß1) in BC patients. RESULTS: To standardize the procedure, HeLa cells were successfully fractionated into nuclear/cytosolic extracts with confirmed presence of nuclear cFOS and cytosolic TGFß1 proteins. Subsequently, the ALN Cell Suspension specimens were obtained and further fractionated from a pilot sample of six ALN tissue pairs, mALN versus autologous normal ALN (nALN), dissected from invasive BC patients. The mALN/nALN results revealed overexpression of both nuclear cFOS and cytosolic TGFß1 protein levels. However, only the TGFß1 data exhibited statistically significant overexpression, which was proportional to the respective values of mALN diameter of tumor deposits. CONCLUSIONS: Detailed protocol for establishment and fractionation of mALN cell suspension specimens, termed FNCS, into nuclear and cytosolic extracts is here described for the first time. This approach might be a convenient ex vivo model for simultaneous analysis of protein, RNA and DNA biomarkers from nuclear/cytosolic extracts of the same mALN tissue sample. It might have potential to enable, in the age of genomics and personalized medicine, an identification of novel mALN biomarkers and thus improve the screening, diagnosis and prognosis of invasive BC.

Case with triple-negative breast cancer shows overexpression of both cFOS and TGF-ß1 in node-positive tissue.

We present herein a case report style article on a rare advanced triple-negative breast cancer (TNBC) patient with 6-month disease-free interval, and 10-month overall survival. Our results demonstrate that the poor clinical outcome of this patient was associated with pronounced, more than fivefold higher, overexpression of both cFOS and TGF-ß1 proteins in its metastatic nodal tissue extracts, when compared with the values of the two non-TNBC controls (with 'zero' disease-free interval and overall survival). This original observation suggests, for the first time, that both the cFOS and TGF-ß1 may be considered as a pair of biomarkers for an early assessment of poor prognosis for TNBC patients. The possible clinical implication of this observation is discussed.

Survival benefit of the late percutaneous coronary intervention in the patients after acute myocardial infarction who are or who are not treated with thrombolysis.

The impact of late percutaneous coronary intervention (PCI) in the patients after acute myocardial infarction (AMI) on long term mortality remains to be established. At currently, thrombolysis is accepted as standard therapy when PCI is not immediately available. However, PCI is often performed in stable patients with AMI who are/are not received thrombolysis . We performed the trial that enrolled myocardial infarction patients treated with thrombolysis, late PCI and medically to assess the potential benefits of delayed PCI. We follow up 164 consecutive patients after AMI one year. The patients are divided in two groups; first group-66 patients who received reperfusion (37 patients received only thrombolysis, 10 patients received thrombolysis and PCI 7-9 days after thrombolysis and 19 patients underwent only PCI after 7-9 days) and second group-98 patients medically treated. One year mortality was 3% in the reperfusion group (2/66) and 14,3% in the medical group (14/98) (p=0,016). There were not significant differences between groups about other end points-reinfarctus, coronary artery bypass surgery and PCI performed later after discharge. The major predictors of one year mortality were ages (p<0,001) and ejection fraction (p=0,003). Also, therapy with beta-blockers (p=0,002), statins (p=0,001) and ACE-inhibitors (p=0,024) was associated with better survival. Delayed PCI performed 7-9 days after AMI in the patients who underwent thrombolysis or those did not improves outcome at long-term follow-up.

Quantification of transforming growth factor beta 1 levels in metastatic axillary lymph node tissue extracts from breast cancer patients: a new specimen source.

OBJECTIVE: To use cytoplasmic tissue extract as a new specimen source to quantify transforming growth factor beta 1 (TGFbeta1) protein in metastatic axillary lymph node tissue (ALNT) of breast cancer (BC) patients and to confirm the feasibility of this approach in a prospective pilot study on a subgroup of patients with invasive BC. STUDY DESIGN: The 6 selected malignant and autologous nonmalignant pairs of ALNT were fractionated, under special preanalytical, nonaggressive/nondenaturing conditions, to obtain respective cytoplasmic extracts for TGFbeta1 detection by the Quantikine (R&D Systems Inc., Minneapolis, Minnesota, U.S.A.) enzyme-linked immunosorbent assay kit. RESULTS: The data indicated a highly significant (r = 0.973054) positive linear correlation between the TGFbeta1 concentration and total protein concentration in cytoplasmic extract of metastatic ALNT. The subsequent patients' pilot study, performed strictly before any clinicopathologic factors were accessible, revealed significantly (p < 0.01) elevated TGFbeta1 in malignant ALNT (median value: 1.05 ng/mg protein, range: 0.67-3.6 ng/mg protein, n = 6) vs. autologous nonmalignant ALNT controls (median value: 0.48 ng/mg protein, range: 0.29-0.90 ng/mg protein, n = 6). This elevation was correlated with the number of metastatic axillary lymph nodes with respect to the total and was consistent with an increase in size of tumor deposits in axillary lymph nodes. CONCLUSION: Our data provide for the first time suggestive evidence that the TGFbeta1 level in cytoplasmic extracts of metastatic ALNTs may be a promising biomarker of invasiveness for BC patients. Confirmatory, large-scale studies are needed to evaluate possible implications of this putative biomarker in BC diagnosis and treatment.

[Isolated aneurysm of the internal iliac artery and disorders of the pelvic organs functions].

BACKGROUND: Isolated aneurysm of internal iliac artery is very rare and often asymptomatic. Aneurysm itself can produce malfunction of the pelvic organs. Aneurysm rupture is followed by high mortality. CASE REPORT: A 76-year-old patient was admitted to the hospital with abdominal and left groin pain, disuric problems, obstipation and the signs of intestinal subocclusion. These problems persisted over the last few months. Digitorectal examination showed pulsatile tumor. Computed tomography and angiography revealed isolated aneurysm of the left internal iliac artery with a maximal diametar of 13.5 cm. The aneurysm was treated operatively using extraperitoneal approach in general anestesia. During the operation a Cell Saver was used. The left internal iliac artery was resected and ligated with end-aneurysmatic suture of its branches. CONCLUSION: Isolated aneurysm of the internal iliac artery should be considered by differential diagnosis in any case of the occurence of disorders of the pervic organs functions. Clinical findings, ultrasound examination, computed tomography and angiography are the diagnostic techniqnes of choice which can confirm the diagnosis. Surgical treatment with the use of retroperitoneal approach lead to complete recovery, so it could be considered the method of choice for patients with the condition permitting a radical surgical approach.

Elevated plasma TGF-beta1 levels correlate with decreased survival of metastatic breast cancer patients.

BACKGROUND: The role of circulating TGF-beta(1) in prognosis of breast cancer (BC) was investigated with an intention to define TGF-beta(1)-dependent high risk and low risk subsets of patients. METHODS: Fifty three BC patients of all clinical stages and 37 healthy donors (HD) were analyzed for plasma TGF-beta(1) by the TbetaRII receptor-based Quantikine TGF-beta(1) ELISA kit. RESULTS: The plasma TGF-beta(1) level of Stage I/II disease (median: 0.94 ng/ml; n=10)) remained close to HD (median: 1.30 ng/ml; n=37; p>0.1). In contrast, Stage III/IV disease (median: 2.34 ng/ml; n=43) exhibited highly significant TGF-beta(1) elevation (p<0.001) relative to HD. Further analysis revealed that TGF-beta(1) increase was predominantly attributed to Stage IV, metastatic disease patients (Q3=4.23 ng/ml) rather than to the group Stage III/IV (Q3=3.58 ng/ml). Using the plasma TGF-beta(1) concentration of 3.00 ng/ml as the cut-off value, two subgroups of patients were formed. Overall 2-year survival of the first subgroup, having elevated plasma TGF-beta(1) (>3.00 ng/ml; n=10), was 10%. This was significantly decreased (p<0.05) compared to 52% survival observed for the second subgroup of patients with plasma TGFbeta(1) values close to HD (<3.00 ng/ml, n=19). CONCLUSION: We have performed a pilot study to determine the relationship between overall survival and TGF-beta(1) concentration in the blood of metastatic breast cancer patients. The survival was significantly reduced in the patients with elevated plasma TGF-beta(1) levels compared to that of the patients with plasma TGF-beta(1) levels close to normal. We propose that plasma TGF-beta(1) concentration may be a new tumour marker attributed to the presence of metastatic BC cells that may be used in selection of metastatic BC patients with poor prognosis.

Elevated plasma levels of transforming growth factor-beta 1 (TGF-beta 1) in patients with advanced breast cancer: association with disease progression.

We examined the association between an elevated plasma TGF-beta 1 level and the disease progression of advanced breast cancer (BC) patients (n = 44). TGF-beta 1 levels were detected by an enzyme-linked immunosorbent assay (ELISA). Platelet carryover and in vitro platelet activation in our plasma samples was assessed and found to be insignificant. Plasma TGF-beta 1 values were significantly elevated (P < 0.05) in stage IIIB/IV patients (median value: 2.40 ng/ml, range: 0.13-8.48 ng/ml, n = 44) compared with healthy donors (median value: 1.30 ng/ml, range: 0.41-4.93 ng/ml, n = 36). Although pronounced in metastatic patients, especially those who had been newly diagnosed, TGF-beta 1 elevation was independent of tumour mass, site of distant metastases, histopathological type, steroid receptor (SR) content and age of the BC patients. Follow-up of 6 patients indicated a relationship between the plasma TGF-beta 1 and the patient's response. This suggests that TGF-beta 1, may be a promising prognostic marker for breast cancer patients with advanced disease. Confirmatory large-scale studies are needed, particularly given the overlap of values between our different subgroups analysed.