Sentinel lymph node biopsy in medullary thyroid microcarcinomas.

The aim of this prospective study was to analyze accuracy of sentinel lymph node biopsy with methylene blue dye for intraoperative detection of lateral metastases in clinically N0M0 medullary microcarcinomas with calcitonin <1,000 pg/mL and selection of true-positive patients for one-time therapeutic lateral dissection. In addition to total thyroidectomy and central neck dissection, all patients had bilateral sentinel biopsy of jugulo-carotid regions after methylene blue injection to decide upon necessity for lateral dissection. If sentinels were benign on frozen section, additional non-sentinels were extirpated, with no further lateral dissection. If sentinels were malignant, one-time lateral dissection was performed. 20 patients were included in this study. Hereditary disease form was observed in 3/20 (15%) of patients with RET proto-oncogene mutation C634F; remaining 17/20 (85%) were negative for germline mutations. There were no allergic reactions to methylene blue and identification rate of sentinels was 100%. In total, 2/20 (10%) cN0 patients had lymphonodal metastases, thus were reclassified as pN1b. Remaining 18/20 (90%) were classified pN0 based on standard pathohistology. Frozen section findings on sentinels were 100% match with standard pathohistology, and there were no skip metastases in lateral compartments. Sensitivity, specificity and accuracy of sentinel biopsy method with methylene dye and frozen section were 100%. Dzodic's sentinel lymph node biopsy method can be used for intraoperative assessment of lateral compartments and optimization of initial surgery of medullary microcarcinomas with calcitonin <1,000 pg/mL. This way, cN0 patients with sentinel metastases can receive one-time lateral dissection, and those without benefit from less extensive surgery.

Successfully Resected Cardiac Metastatic Melanoma in a Lifesaving Cardiac Surgery: A Case Report.

INTRODUCTION: Metastatic tumors are the most frequent tumors of the heart with the melanoma metastasis being the most frequent. In cases of a cardiac tamponade or a low cardiac output syndrome due to the position of the tumor it could lead to an acute life-threatening condition for the patient. CASE REPORT: We present a case of a successfully treated metastatic cardiac melanoma in a lifesaving cardiac surgery. A 42-year-old woman was admitted to the emergency department of the Clinical Center of Serbia in a critical state with signs of tricuspid orifice obstruction with a tumor mass. Her previous medical history showed that she had an adequate surgical excision of the melanoma in the right lumbar region at the age of 39. An emergency cardiac surgery was performed with the resection of the tumor and the atrial wall. The postoperative course was uneventful and a HP exam once again confirmed the same type of melanoma as previously diagnosed. The patient was alive and well on follow-up exams for 4 months when she was diagnosed with metastatic tumor masses in the pelvis with ascites and melanosis of the entire skin followed by lethal outcome 5 months after the cardiac surgery. CONCLUSION: Surgical resection of metastatic cardiac melanoma can be safe and effective in an emergency scenario, especially in the case of solitary metastasis. It can be performed with excellent results and very few postoperative complications. However, due to the nature of the principal disease, the long-term survival rate remains low, giving the surgery a place in palliative treatment.

Pre-operative and intra-operative detection of axillary lymph node metastases in 108 patients with invasive lobular breast cancer undergoing mastectomy.

BACKGROUND: Despite the recent changes in the treatment of the axilla in selected breast cancer patient, positive sentinel lymph node (SLN) in patients undergoing mastectomy still necessitates axillary lymph node dissection (ALND). In invasive lobular carcinoma (ILC), pre-operative detection of the lymph node metastasis may be demanding due to its unique morphology. The aim of this study was to examine the benefit of preoperative axillary ultrasound (AUS), ultrasound-guided fine-needle aspiration biopsy (US-FNAB), and intra-operative imprint cytology (IIC), in order to avoid two-stage axillary surgery in patients with ILC undergoing mastectomy. METHODS: The object of this study were 102 patients (median age 52, range 34-73 years) with clinically non-suspicious axilla in whom 108 mastectomies were performed after a pre-operative AUS investigation. Whenever a metastasis was detected in a sentinel lymph node, ALND was done. Reports of the pre-operative AUS investigation, US-FNAB, and IIC were compared with definitive histopathological reports of surgical specimens. RESULTS: In 46 cases lymph node metastases were diagnosed. AUS suspicious lymph nodes were found in 29/108 cases and histopathology confirmed metastases in 22/30 cases. US-FNAB was performed in 29 cases with AUS suspicious lymph nodes. Cytology proved metastases in 11/29 cases. Histopathology confirmed metastases in 10/11 cases with only isolated tumor cells found in one case. IIC investigation was performed in 63 cases and in 10/27 cases metastases were confirmed by histopathology. Pre-operative AUS, US-FNAB, and/or IIC investigation enabled ALND during a single surgical procedure in 20/46 patients with metastases in lymph nodes. CONCLUSION: Pre-operative AUS, US-FNAB, and/or IIC are/is beneficial in patients with ILC planned for mastectomy in order to decrease the number of two stage axillary procedures.

The influence of breast cancer subtypes on the response to anthracycline neoadjuvant chemotherapy in locally advanced breast cancer patients.

PURPOSE: The objective of neoadjuvant chemotherapy (NACT) for locally advanced breast cancer (LABC) is downstaging to achieve resectability. According to the protocol for the treatment of LABC more than 10 years ago, the routine NACT for LABC in Serbia consisted of 4 cycles of FAC (fluorouracil, doxorubicin, cyclophosphamide). The aim of this analysis was to assess the influence of biologic subtypes of BC on the response to NACT and on the disease outcome in these patients. METHODS: We analyzed 190 patients with median age of 52 years (range 26-74), diagnosed with LABC between Jun/2002 and Dec/2005 and treated with 4 cycles of FAC. Patients with clinical response to NACT (162/192;85.26%) were subjected to radical mastectomy after which the majority of them received 3 cycles of adjuvant FAC, adjuvant tamoxifen if HR-positive disease, and postoperative radiotherapy. We retrospectively determined by immunohistochemistry estrogen receptor (ER)/ progesterone receptor (PgR)/HER2 status from BC biopsies in all patients who were divided in 4 subgroups. Pathological complete remission (pCR) was defined as ypT0N0. The main end points were disease-free survival (DFS) and overall survival (OS). Statistics included Fisher's exact test, KaplanMeier product-limit method and Log-rank test. RESULTS: After a median follow up of 76 months (range 3-128) 104/190 patients (54.74%) experienced disease relapse, while 78/190 (41.05%) died. Of 157 patients with known receptor status the numbers of 4 subtypes were as follows: 31/190 (16.32%) triple negative (TN) BC, 22/190 (11.58%) HR-/HER2+, 97/190 (51%) HR+/HER2- and 17/190 (8.95%) HR+/HER2+. Ten out of 190 patients (6.17%) achieved pCR and had significantly longer DFS (Log-rank test, p=0.042), and a trend to prolonged OS (Log-rank test, p=0.092). There was a significant difference (Fisher exact test, p=7.7 × 10-6) between pCR rates among 4 BC subtypes: 3/31 (9.68%) in TNBC, 6/22 (27.27%) in HR-/HER2+, 0/97 in HR+/HER2- and 1/17 (5.88%) in HR+/HER2+ patients. This difference was achieved on the account of the difference between TNBC and HR-/HER2+ BC subtypes (Fisher's exact test, p=6.85×10-6, Bonferroni correction: 0.05/6=0.0083). There were no differences in DFS and OS between the 4 BC subtypes. CONCLUSION: Although there was a significantly higher number of patients achieving pCR among HR-/HER2+ subtype compared to other BC subtypes, this did not translate into improvement in long-term disease outcome of these patients.

Promoter hypermethylation of p16, BRCA1 and RASSF1A genes in triple-negative breast cancer patients from Serbia.

PURPOSE: In order to investigate if aberrant promoter methylation of p16, BRCA1 and RASSF1A genes contributes to biological behavior of triple-negative breast cancer (TNBC), marked as the most aggressive phenotype of breast cancer, we compared the hypermethylation pattern between TNBC and ER+PR+Her2- breast cancer. METHODS: 131 patients with histologically confirmed breast cancers were included - 61 TNBC and 70 ER+PR+Her2- cases. The patients were followed up for 1-87 months (median 78). DNA from tumor tissues was isolated by the salting out procedure. The methylation status was assessed by nested methylation-specific PCR after bisulfite modification of DNA. RESULTS: The frequency of p16 hypermethylated breast cancer cases was significantly higher in TNBC than in ER+PR+Her2- group (33; 54.1% vs. 20; 28.6%, p=0.00298). Co-methylated p16 and RASSF1A genes were more frequent in the TNBC than in ER+PR+Her2- group (20; 32.8% vs. 10; 14.3%, p=0.0225). The same result was observed when hypermethylated BRCA1 gene was added in the analysis: 12; 19.7% vs. 3; 4.3%, p=0.00791. Although there was significant difference in disease-free survival (DFS) and overall survival (OS) between TNBC and ER+PR+Her2- group, further analysis of co-methylation of p16 and RASSF1A (p16+RASSF1A+) showed that DFS was significantly shorter in the patients with both genes co-methylated in TNBC than in ER+PR+Her-2- group (8/20; 40% vs. 2/10; 20%, p=0.03272). CONCLUSIONS: The obtained data indicate that hypermethylated p16 and RASSF1A cell-cycle inhibitor genes might be considered as biomarkers for bad prognosis in breast cancer. Hypermethylation of these genes may influence the clinical disease course, distinguishing a particular group of TNBC patients with even more aggressive phenotype.

Treatment outcome in patients with breast conserving surgery after neoadjuvant therapy for breast carcinoma - a single institution experience.

PURPOSE: The aim of this study was to analyze outcomes of breast conserving surgery (BCS) after neoadjuvant treatment (NAT) in comparison to radical mastectomy (RM) after NAT in terms of disease-free survival (DFS), overall survival (OS) and patients' satisfaction with the esthetic outcomes of surgery. METHODS: This prospective study was conducted at the National Cancer Research Center of Serbia, Belgrade, from January 1st 2011 to December 31st 2015, on breast carcinoma patients receiving NAT. Treatment outcome was assessed by MDAPI (MD Anderson Prognostic Index). Female patients (n=52) with satisfactory clinical response to NAT and MDAPI scores 0 or 1 were included into the treatment group (NAT-BCS group). The control group (NAT-RM group) consisted of patients (n=52) with poorer clinical response and MDAPI scores 2 to 4. On check-ups, local or distant relapses were noted and both groups were asked to value their satisfaction with the esthetic outcomes of surgery using the Likert scale. RESULTS: OS was 100% in both groups. DFS was 96.1% in NAT-BCS group and 100% in NAT-RM group. Local recurrences were observed in two patients from the age group ≥60 years, with initial disease stage IIIA and "clear" resection margins on frozen section study. Patients in the NAT-BCS group were more satisfied with the esthetic outcome of surgery than the control group. CONCLUSIONS: BCS after NAT provides good esthetic outcome and is oncologically safe if adequate clinical response is achieved after NAT and if established criteria for patient selection are followed.

Multifocality as independent prognostic factor in papillary thyroid cancer - A multivariate analysis.

PURPOSE: The incidence of multifocality of papillary thyroid carcinoma (PTC) ranges from 18 to 87.5% The mechanisms of multifocal spreading, correlation with tumor size, histology variants of PTC, lymph node metastases, and prognostic impact remains unclear. The purpose of this study was to clarify the prognostic significance of multifocality on relapse and survival rates of patients with PTC by analyzing the correlation of multifocality with patient age, gender, tumor size, histological variants of PTC, presence of lymph node metastases and extent of surgery. METHODS: 153 patients with PTC were included in this study. Patients with pT4 tumors or initially distant metastases were excluded from study. Total thyroidectomy was done in all 153 patients. Central and level III and IV lateral neck lymph node dissection was done in 76.5% of the patients, followed by modified radical neck dissection if positive. RESULTS: Multifocality was found in 43 (28%) whole thyroid gland specimens, and was significantly more frequent in patients older than 45 years and in tumors greater than 4 cm in diameter (p<0.01). Presence of multifocality didn't significantly correlate with gender, histology variants of PTC or lymph node metastases. In a median follow up of 84 months locoregional relapse occurred in 8.4% and 1.3% of the patients, while 7.2% patients died due to PTC. The incidence of relapse was significantly higher (p<0.01), and relapse free interval and survival were significantly shorter (p=0.0095, p=0.0004, respectively) in patients with multifocal PTC. Cox multivariate regression analysis showed that multifocality was independent prognostic factor for both disease-free interval (DFI) and cancer-specific survival (CSS) of patients with PTC. CONCLUSION: Due to high incidence of multifocality and potential prognostic impact, total thyroidectomy should be advocated in all patients with PTC, aiming to reduce relapse rate and improve DFI and CSS.

Attenuated in vitro effects of IFN-α, IL-2 and IL-12 on functional and receptor characteristics of peripheral blood lymphocytes in metastatic melanoma patients.

Considering tumor-induced suppression of lymphocytes the aim of this study was to investigate in vitro effects of IFN-α, IL-2, IL-12 and IL-18 as immunomodulating agents on the functional and receptor characteristics of peripheral blood lymphocytes (PBL) in metastatic melanoma (MM) patients compared to healthy controls (HC). In HC IFN-α, IL-2 and IL-12 enhanced mRNA level of perforin by inducing pSTAT-1 and pSTAT-5 signaling molecules. Additionally, the expression of NKG2D activating receptor and its DAP10 signaling molecule was upregulated by IL-2. Contrary to this, in MM patients only IL-2 by upregulating pSTAT-5 increased perforin-mediated cytotoxicity of lymphocytes. Furthermore, there was significantly negative correlation between the percentage of CD4+CD25bright+CD27+ regulatory T (Treg) cells and NK cell cytotoxicity, as well as the expression of NKG2D receptor on PBL in HC and MM patients. Therefore, the absence of IL-2 effect on the increase of NKG2D/DAP10 level in MM patients could be the consequence of the increased percentage of immunosuppressive CD4+CD25bright+CD27+ cells after this cytokine treatment in patients. However, in MM IL-12 significantly decreases the percentage of these inhibitory cells. Although IL-2 as a single agent has numerous side effects, it remains the important cytokine for PBL activation in melanoma immunotherapy. Additionally, the removal of Treg cells from patient PBL by IL-12 before in vitro stimulation with IL-2, may lead to the generation of more potent cytotoxic lymphocytes against tumor cells. Therefore, lymphocyte based therapy for MM patients should integrate not only the choice of appropriate immunostimulatory cytokine, but also the removal of inhibitory cells from tumor microenvironment.

Small nonfunctional parathyroid cysts: single institution experience.

Parathyroid cysts (PCs) account for less than 1% of all parathyroid lesions and are most commonly located along thyroid lobes, rarely at ectopic sites. PCs are important because they can pose a differential diagnostic challenge against other cystic formations of the neck. PCs can be functional (elevated serum parathyroid hormone level) and nonfunctional. Four cases of nonfunctional PCs are presented. All four female patients underwent physical examination and ultrasonography of the neck with ultrasound-guided fine-needle aspiration biopsy (UG-FNA). The material thus obtained was stained by the standard May-Grünwald-Giemsa method. Parathyroid hormone level was determined in aspirate and serum, along with serum levels of total calcium, inorganic phosphates. In two asymptomatic patients, remission occurred after initial aspiration biopsy; one patient had compression syndrome with vocal cord paresis that required surgical treatment; and one patient had cyst recurrence that was surgically removed. Cystic neck masses can pose a major differential diagnostic problem considering different approach, treatment method, and preoperative and postoperative follow up. Surgical treatment is necessary in case of functional and large nonfunctional PCs (due to compression syndrome), whereas individualized therapeutic approach is used in case of small nonfunctional PCs. Ultrasonography with UG-FNA, cytologic analysis of the material obtained, and determination of parathyroid hormone level in aspirate and serum are crucial for making an accurate diagnosis.

In situ preservation of parathyroid glands:advanced surgical tips for prevention of permanent hypoparathyroidism in thyroid surgery.

Hypoparathyroidism (HPT) is one of the most frequentand severe complications of thyroid surgery. It is caused by intraoperative damage, devascularization or accidental removal of the parathyroid glands (PTGs). The incidence of postoperative HPT is directly proportional to surgery extent and surgeon's experience. After 40 years of experience in thyroid surgery, the first author summarizes the already known surgical steps in thyroid surgery and adds some useful practical tips for in situ preservation of PTGs. Our surgical technique focuses on meticulous capsular dissection and preservation of the middle thyroid, Kocher's vein trunk, as well as vein branches that accompany the posterior branch of the superior thyroid artery and inferior thyroid artery trunk. Ligation of all blood vessels should be as close as possible to the thyroid capsule. Identified PTGs should be de-attached from the thyroid capsule on the vascular pedicle without significant dislocation. PTGs preservation during central neck dissection (CND) can be facilitated by using methylene blue dye for sentinel lymph nodes biopsy. PTGs are not colored in blue, unlike central lymph nodes, which facilitates central neck dissection and reduces the possibility of accidental removal of PTGs. After several thousands of preserved PTGs using this original technique, a total prevalence of permanent HPT in the first author's series is less than 0.5%. Following given key points and recommendations to surgical in situ preservation of PTGs, a surgeon can provide good outcome for patients after total thyroidectomy (with or without central neck dissection), regarding HPT as one of the most severe complications of thyroid surgery.

Prognostic importance of steroid receptor status for disease free and overall survival after surgical resection of isolated liver metastasis in breast cancer patients.

PURPOSE: Breast cancer (BC) is the most common malignancy among women, while isolated operable liver metastases (LMs) from BC are very rare and occur in only 1-5% of the patients. Besides, positive steroid receptor (SR) status for oestrogen and/or progesterone is known as a factor which improves disease free survival (DFS) and overall survival (OS). The primary aim of this study was to examine the impact of SR status on DFS and OS after liver metastasectomy in female patients with primary BC. METHODS: We analyzed 32 medical records of female patients diagnosed and treated for primary BC with LMS as the first and only site of disease progression, at the Institute of Oncology and Radiology of Serbia (IORS), during 2006- 2009. All of them underwent primary BC surgery as well as LMs resection. RESULTS: Patients with metachronous BC and LMs and positive SR status in both BC and LM (BC+/LM+) had a median time from BC to LM occurrence (TTLM) of 36 months, compared to BC+/LM- and BC-/LM- subgroups, whose medians for TTLM were 30.5 and 14.5 months, respectively (p<0.01). For all patients, positive SR status showed high correlation with longer DFS and OS after LM resection (medians according survival analysis for DFS/OS in subgroups BC-/LM-, BC+/LM- and BC+-LM+ were 10/19, 25/45, 50/not reached months respectively; p<0.01 for DFS/ OS). Cox regression analysis confirmed that the subgroup of patients with BC-/LM- had 10.8 and 18.8 higher risk of events for DFS (disease relapse or death) and event for OS (death only), respectively, compared to BC+/LM+ subgroup of patients. CONCLUSION: Positive SR status in BC and LM has a high impact not only on time from BC to LM occurrence, but also on longer DFS and OS after LM resection.

Lymph node metastases in clinically N0 patients with papillary thyroid microcarcinomas - a single institution experience.

PURPOSE: To register the frequency of central and lateral lymph node metastases (LNMs) from papillary thyroid microcarcinomas (PTMCs), and to assess whether sentinel lymph node (SLN) biopsy of the lateral compartment is an accurate technique to select patients with true positive but clinically negative lymph nodes for one-time selective lateral neck dissection (sLND). The correlation between tumor characteristics (size, multifocality, bilaterality, capsular invasion) and LNMs was analyzed. METHODS: During a 10-year-period (2004-2013), 111 clinically N0 patients with PTMCs had total thyroidectomy, central neck dissection and SLN biopsy of the lateral neck compartment in our institution. SLN mapping was performed by subcapsular injection of 0.2 to 0.5ml of 1% methylene blue dye. If SLNs were positive on frozen section, one-time sLND was done. RESULTS: Forty per cent of PTMCs were multicentric. LNMs were detected in 25% of the patients and isolated central LNMs were found in 18% of the patients. Lateral LMNs were present in 7% of the patients, of which 4% were isolated, skip LNMs. All these patients had therapeutic sLND. Specificity and sensitivity of SLN biopsy were 100% and 57%, positive and negative predictive values were 100% and 97%, respectively. Method's accuracy was 97%. CONCLUSIONS: SLN biopsy of the lateral neck compartment is more precise than physical examination and ultrasonography for detection of lateral LNMs in clinically N0 patients with PTMCs. Intraoperative assessment of lateral lymph nodes (SLNs) provides one-time therapeutic dissection for patients with occult LNMs at initial operation, reducing the need for additional operations. This method provides appropriate disease staging and optimizes treatment.

Recurrent Laryngeal Nerve Liberations and Reconstructions: A Single Institution Experience.

BACKGROUND: Recurrent laryngeal nerve (RLN) palsy rates vary from 0.5 to 10%, even 20% in thyroid cancer surgery. The aim of this paper was to present our experience with RLN liberations and reconstructions after various mechanisms of injury. METHODS: Patients were treated in our institution from year 2000 to 2015. First group (27 patients) had large benign goiters, locally advanced thyroid/parathyroid carcinomas, or incomplete previous surgery of malignant thyroid disease. Second group (5 patients) had reoperations due to RLN paralysis on laryngoscopy. Liberations and reconstructions of injured RLNs were performed. RESULTS: Surgical exploration of central compartment enabled identification of the RLN injury mechanism. Liberations were performed in 11 patients, 2 months to 16 years after RLN injury, by removing misplaced ligations. Immediate or delayed (18 months to 23 years) RLN reconstructions were performed in 21 patients, by direct suture or ansa cervicalis-to-RLN anastomosis (ARA). RLN liberation provided complete voice recovery within 3 weeks in all patients. Patients with direct sutures had better phonation 1 month after reconstruction. Improved phonation was observed 2-6 months after ARA in 43% of patients. CONCLUSIONS: Vocal cords do not regain normal movement once being paralyzed after RLN transection, but they restore tension during phonation by reconstruction. Nerve liberation is a useful method which enables patients with RLN paresis/paralysis a significant improvement in phonation, even complete voice recovery. Reinnervation of vocal cords, using one of the mentioned techniques, should be a standard in thyroid and parathyroid surgery, with aim to improve quality of patient's life.

Acceptable adverse outcomes after delayed breast reconstruction using abdominal advancement flap - a 15-year experience.

PURPOSE: To evaluate the adverse outcomes after delayed breast reconstruction (DBR) by abdominal advancement flap (AAF) and permanent prosthesis in patients treated with mastectomy due to unilateral breast carcinoma, as well as to determine which factors are predictive for their occurrence. METHODS: The study included 155 patients operated at the Institute for Oncology and Radiology of Serbia from 1996 to 2010. All patients had total mastectomy and axillary lymph node dissection, followed by specific oncological treatment. Patients were selected for DBR after complete diagnostic evaluation. Adverse events were evaluated in regard to patient, disease and prostheses-related factors. RESULTS: During follow-up, DBR adverse events were observed in 23.237percnt; of the patients - the majority (91.67%) had only one. The most frequent was capsule contracture (47.22%), followed by asymmetry (22.22%), infection (16.67%) and prosthesis rupture (16.67%). There were isolated cases of prosthesis prolapse and local disease recurrence. Infections were treated conservatively in all but one patient. Other events were managed by additional operation. Statistical analysis showed that complications occurred significantly more often in patients ?51 years (vs 18-40, vs 41-50), disease stage IIb (vs Ia), T2 (vs T1) tumors and adjuvant radiotherapy (vs without). Prostheses-related factors were not significant for DBR complications, neither body mass index (BMI), nor smoking habits. CONCLUSIONS: DBR using AAF and permanent prosthesis is a safe technique with acceptable complication rate. It provides one-time surgery with satisfactory aesthetic results and good postoperative recovery. Most frequent complication is capsule contracture. Patients' age and irradiation of the chest wall after mastectomy are predictive factors for complications.

Favorable in vitro effects of combined IL-12 and IL-18 treatment on NK cell cytotoxicity and CD25 receptor expression in metastatic melanoma patients.

BACKGROUND: As IL-12 and IL-18 have important immunostimulatory role the aim of this study was to investigate their in vitro effects on functional and receptor characteristics of NK cells and their subsets in healthy controls (HC) and metastatic melanoma patients (MM). METHODS: Peripheral blood mononuclear cells (PBMC) of HC and MM were stimulated with culture medium alone, medium supplemented with IL-12 (10 ng/ml), IL-18 (100 ng/ml) and their combination. NK cell activity was determined using radioactive cytotoxicity assay, while perforin, CD107a and pSTAT-4 expression, IFN-γ production and the expression of NKG2D, DNAM-1, CD161, CD158a/b, CD25, IL-12R beta 1/2 receptors on CD3(-)CD56(+) NK cells and their CD3(-)CD56(dim+) and CD3(-)CD56(bright+) subsets were analyzed by flow cytometry. Cytokine induced level of DAP10 in PBMC was analyzed by reverse transcription polymerase chain reaction. RESULTS: IL-12 alone or in combination with IL-18 significantly induced NK cell activity and CD107a degranulation marker expression in MM and HC, while IL-18 alone did not have any effect in patients. The combination of IL-12 and IL-18 significantly increased mean fluorescence intensity (MFI) of IFN-γ in all NK cell subsets in HC and only in the bright subset in MM. MM that belong to M1c group with metastasis in liver and increased LDH serum values had significantly lower increase in NK cell cytotoxicity after combined IL-12 and IL-18 treatment compared to the patients in M1a and M1b categories. These results could be explained by decreased IL-12R expression and lower increase in pSTAT-4 and perforin expression in NK cells of M1c patients after IL-12 and combined IL-12 and IL-18 treatment. IL-18 alone significantly decreased NKG2D receptor expression and level of DAP10 signaling molecule in MM, while combined IL-12 and IL-18 increased the expression of CD25 on all NK cell subsets in HC and MM. Additionally, MM that belong to M1a + M1b group had significantly higher increase in CD25 receptor expression compared to the patients in M1c group. CONCLUSIONS: The novel data obtained in this study support the use of IL-12 and IL-18 in combination for developing new therapeutic strategies for metastatic melanoma especially for patients with better survival rate and prognosis.

Potential clinical relevance of uPA and PAI-1 levels in node-negative, postmenopausal breast cancer patients bearing histological grade II tumors with ER/PR expression, during an early follow-up.

We evaluated urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) prognostic value in postmenopausal, node-negative breast cancer patients bearing tumors with estrogen receptor (ER)/progesterone receptor (PR) expression, treated with locoregional therapy alone, within an early follow-up. We focused our analysis on tumors of histological grade II in order to improve its prognostic value and, consequently, to improve a decision-making process. The cytosol extracts of 73 tumor samples were used for assessing several biomarkers. ER and PR levels were measured by classical biochemical method. Cathepsin D was assayed by a radiometric immunoassay while both uPA and PAI-1 level determinations were performed by enzyme-linked immunosorbent assays. HER-2 gene amplification was determined by chromogenic in situ hybridization (CISH) in primary tumor tissue. Patients bearing tumors smaller than or equal to 2 cm (pT1) or those with low PAI-1 levels (PAI-1 < 6.35 pg/mg) showed favorable outcome compared to patients bearing tumors greater than 2 cm (pT2,3) or those with high PAI-1 levels, respectively. Analyses of 4 phenotypes, defined by tumor size and PAI-1 status, revealed that patients bearing either pT1 tumors, irrespective of PAI-1 levels, or pT2,3 tumors with low PAI-1 levels, had similar disease-free interval probabilities and showed favorable outcome compared to those bearing pT2,3 tumors with high PAI-1 levels. Our findings suggest that tumor size and PAI-1, used in combination as phenotypes are not only prognostic but might also be predictive in node-negative, postmenopausal breast cancer patients bearing histological grade II tumors with ER/PR expression, during an early follow-up period.

Early prognosis of metastasis risk in inflammatory breast cancer by texture analysis of tumour microscopic images.

Inflammatory breast cancer (IBC) is a rare and aggressive type of locally advanced breast cancer. The purpose of this study was to determine the value of microscopic tumour histomorphology texture for prognosis of local and systemic recurrence at the time of initial IBC diagnosis. This retrospective study included a group of 52 patients selected on the basis of non-metastatic IBC diagnosis, stage IIIB. Gray-Level-Co-Occurrence-Matrix (GLCM) texture analysis was performed on digital images of primary tumour tissue sections stained with haematoxylin/eosin. Obtained values were categorized by use of both data- and outcome-based methods. All five acquired GLCM texture features significantly associated with metastasis outcome. By accuracies of 69-81% and AUCs of 0.71-0.81, prognostic performance of GLCM parameters exceeded that of standard major IBC clinical prognosticators such as tumour grade and response to induction chemotherapy. Furthermore, a composite score consisting of tumour grade, contrast and correlation as independent features resulted in further enhancement of prognostic performance by accuracy of 89%, discrimination efficiency by AUC of 0.93 and an outstanding hazard ratio of 71.6 (95%CI, 41.7-148.4). Internal validation was successfully performed by bootstrap and split-sample cross-validation, suggesting that the model is generalizable. This study indicates for the first time the potential use of primary breast tumour histology texture as a highly accurate, simple and cost-effective prognostic indicator of metastasis risk in IBC. Clinical relevance of the obtained results rests on the role of prognosis in decisions on induction chemotherapy and the resulting impact on quality of life and survival.

Can we use frozen section analysis of sentinel lymph nodes mapped with methylene blue dye for decision making upon one-time axillary dissection in breast carcinoma surgery in developing countries?

PURPOSE: To evaluate the accuracy of intraoperative frozen section analysis (FSA) of sentinel lymph nodes (SLNs) mapped using methylene blue dye (MBD) and its usefulness for selecting patients with breast carcinomas and positive axillary lymph nodes (ALNs) for one-time axillary dissection. METHODS: 152 female patients with T1/T2 breast carcinomas and clinically negative ALNs were selected for mapping using MBD (1%) from October 2010 to December 2011. Patients underwent FSA of mapped SLNs and ALN dissection. The accuracy of SLN-FSA was tested by comparing these findings with the definite histopathology (HP) of SLNs, as well as of other ALNs. Sensitivity, specificity, positive and negative predictive values were calculated. RESULTS: There was a 98%-match between FSA and definite HP findings of SLNs, suggesting high accuracy of FSA in this series. None of 3 patients with false-negative SLNs on FSA had additional axillary nodal metastases. One out of 20 (5%) patients with metastases in other ALNs had "clear" SLNs, both on FSA and definite HP (false-negative). Accuracy reached 94.1%. CONCLUSIONS: SLN-FSA enables adequate selection of patients for one-time axillary node dissection. MBD mapping technique is cheap, feasible and enables easy and precise detection of the first draining ALNs. Using FSA of SLNs mapped with MBD, patients with breast carcinoma benefit from complete surgical treatment during one hospitalization, the risk of undergoing anaesthesia twice is reduced, as well as the treatment cost, which is important in developing countries.

Surgical treatment of breast cancer in patients aged 80 years or older--how much is enough?

BACKGROUND: The population of elderly people is increasing and so is the population of breast cancer patients aged ≥80 years. The aim of our retrospective study was to identify independent prognostic factors for the duration of breast cancer-specific survival of surgically treated patients aged ≥80 years. The secondary aim was to determine the appropriate surgical treatment of breast cancer in patients aged ≥80 years. METHODS: We reviewed the medical records of 154 patients aged ≥80 years with early-stage breast cancer (mean age 83 years) who underwent surgery at the tertiary cancer center in the period from 2000 to 2008. Tumor stage was pT1/pT2 and pT3/pT4 in 75% and 25%, respectively. Surgical treatment comprised: quadrantectomy (in 27%), mastectomy (in 73%), axillary dissection (in 57%), and sentinel lymph node biopsy (in 18%), while 25% of patients had no axillary surgery. RESULTS: During a median follow-up of 5.3 years, 31% of patients died of breast cancer, while 28% of patients died of other causes. Half of our patients with poorly differentiated breast cancer or estrogen receptor-negative tumor died of breast cancer. Multivariate statistical analysis showed that the pathological T-stage, pathological N-stage and estrogen receptors were independent prognostic factors for the duration of breast cancer-specific survival of patients. CONCLUSION: Short breast cancer-specific survival indicates that, in patients aged ≥80 years, breast cancer with metastases in axillary lymph nodes can be an aggressive disease.

Trefoil factor 1 in early breast carcinoma: a potential indicator of clinical outcome during the first 3 years of follow-up.

BACKGROUND: A role of an estrogen-regulated, autocrine motogenic factor was assumed to be a major biological role of trefoil factor 1 (TFF1) in breast cancer. TFF1 is regarded as a predictive factor for positive response to endocrine therapy in breast cancer patients. The aim of our study was to examine TFF1 level distribution in breast carcinomas in order to distinguish estrogen-independent from estrogen-dependent TFF1 expression and to evaluate clinical usefulness of TFF1 status in early breast cancer during the first 3 years of follow-up. METHODS: The study included 226 patients with primary operable invasive early breast carcinomas for whom an equal, a 3-year follow-up was conducted. TFF1 levels as well as estrogen receptor (ER) and progesterone receptor (PR) levels were measured in cytosolic extracts of tumor samples by immunoradiometric assay or by use of classical biochemical method, respectively. Non-parametric statistical tests were applied for data analyses. RESULTS: Statistical analysis revealed that TFF1 levels were significantly higher in premenopausal patients (p=0.02), or in tumors with: lower histological grade (p<0.001), positive ER or PR status (p<0.001, in both cases). On the basis of TFF1 level distribution between ER-negative and ER-positive postmenopausal patients with tumors of different histological grade, 14 ng/mg was set as the cut-off value to distinguish estrogen-independent from estrogen-dependent TFF1 expression in breast cancer. Depending on menopausal and PR status, positive TFF1 status identified patients at opposite risk for relapse among ER-positive patients with grade II tumors. Among ER- and PR-positive premenopausal patients with grade II tumors, TFF1 status alone identified patients at opposite risk for relapse. CONCLUSIONS: Determination of TFF1 status might identify patients at different risk for relapse and help in making decision on administering adjuvant therapy for early breast cancer patients during the first 3 years of follow-up.