Reliability of intraoperative ultrasound in margin status assessment in women undergoing breast conserving surgery

Background: Effective treatment for breast cancer requires accurate preoperative planning, developing and implementing a consistent definition of margin clearance, and using tools that provide detailed real-time intraoperative information on margin status. Intraoperative ultrasound (IOUS) may fulfil these requirements and may offer few advantages that other preoperative localization and intraoperative margin assessment techniques may notPurpose: The goal of the present work is to determine how accurate the intraoperative ultrasound should be to acquire complete surgical excision with negative histological margins in patients undergoing Breast Conservative SurgeryDesign: A diagnostic test study with a cross-sectional design carried out in a tertiary referral hospital in Girona within a Breast Pathology UnitParticipants: Women diagnosed with breast cancer undergoing a Breast Conservative Surgery in the Breast Pathology Unit at Hospital Universitari de Girona Dr. Josep Trueta

Patson Approach versus the Standard Approach for increasing breast reconstruction rates in women undergoing mastectomy. A randomized, controlled, open-label clinical trial

Breast cancer is the most prevalent neoplasm among women in the majority of countries worldwide. Breast cancer treatment include mastectomy which is associated to strong impact in women. Breast reconstruction is an option for many women to re-establish their body image and also to decrease psychological impact. However, breast reconstruction rates are low and many factors are involved in not undergoing breast reconstruction. Patient involvement in the decision-making process increases breast reconstruction rates and is associated to higher satisfaction and less anxiety and depression symptoms. More physician-patient relation and more education in terms of breast reconstruction are needed to achieve our objective. A new approach of medical care, called Patson Approach, is created in order to meet our goal with more patient involvement, as well as, physician and psychological counsellingObjective: to increase breast reconstruction rates in women who are candidates for breast reconstruction after mastectomy and are included in the Patson Approach compared to women included in the Standard ApproachMethods: the study design will be a randomized, controlled, open-label clinical trial. 62 patients will be recruited during two years and randomly divided in two groups, 31 will be included in the Standard Approach and 31 will be included in the Patson Approach. Preoperative and postoperative appointments are established in order to do a follow-up of the patients and collect all the data

Development and validation of a high performance liquid chromatographic method for determination of cyclosporine-A from biodegradable intraocular implants

An HPLC method was developed and validated aiming to quantify the cyclosporine-A incorporated into intraocular implants, released from them; and in direct contact with the degradation products of PLGA. The separation was carried out in isocratic mode using acetonitrile/water (70:30) as mobile phase, a C18 column at 80 ºC and UV detection at 210 nm. The method provided selectivity based on resolution among peaks. It was linear over the range of 2.5-40.0 µg/mL. The quantitation and detection limits were 0.8 and 1.2 µg/mL, respectively. The recovery was 101.8% and intra-day and inter-day precision was close to 2%.

Characterization and in vitro release of cyclosporine-A from poly(D,L-lactide-co-glycolide implants obtained by solvent/extraction evaporation

Cyclosporine-A-loaded PLGA implants were developed intended for ocular route. Implants were prepared using solvent extraction/evaporation technique followed by casting of the cake into rods in a heated surface. XRD patterns showed that cyclosporine-A was completely incorporated into PLGA. FTIR and DSC results indicated alterations on drug molecular conformation aiming to reach the most stable thermodynamic conformation at polymer/drug interface. Implants provided controlled/sustained in vitro release of the drug. During the first 7 weeks, the drug release was controlled by the diffusion of the cyclosporine-A; and between 7-23 week period, the drug diffusion and degradation of PLGA controlled the drug release.

Development and validation of a High Performance Liquid Chromatographic method for determination of etoposide in biodegradable polymeric implants

A method using HPLC-UV was developed and validated for the determination of etoposide incorporated into polycaprolactone implants. The method was carried out in isocratic mode using a C18 column (250 x 4.6 mm; 5 µm), at 25 ºC, with acetonitrile and acetic acid 4% (70:30) as mobile phase, a flow rate of 2 mL/min, and UV detection at 285 nm. The method was linear (r² > 0.99) over the range of 5 to 65 µg/mL, precise (RSD < 5%), accurate (recovery of 98.7%), robust, selective regarding excipient of the sample, and had a quantitation limit equal to 1.76 µg/mL. The validated method can be successfully employed for routine quality control analyses.

Fibroblast Growth Factor 8 and its Receptors in The Growth and Angiogenesis of Experimental Breast Cancer . With Special Reference to Thrombospondin-1 as a Novel Target Gene for FGF-8

The growth of breast cancer is regulated by hormones and growth factors. Recently, aberrant fibroblast growth factor (FGF) signalling has been strongly implicated in promoting the progression of breast cancer and is thought to have a role in the development of endocrine resistant disease. FGFs mediate their auto- and paracrine signals through binding to FGF receptors 1-4 (FGFR1-4) and their isoforms. Specific targets of FGFs in breast cancer cells and the differential role of FGFRs, however, are poorly described. FGF-8 is expressed at elevated levels in breast cancer, and it has been shown to act as an angiogenic, growth promoting factor in experimental models of breast cancer. Furthermore, it plays an important role in mediating androgen effects in prostate cancer and in some breast cancer cell lines. We aimed to study testosterone (Te) and FGF-8 regulated genes in Shionogi 115 (S115) breast cancer cells, characterise FGF-8 activated intracellular signalling pathways and clarify the role of FGFR1, -2 and -3 in these cells. Thrombospondin-1 (TSP-1), an endogenous inhibitor of angiogenesis, was recognised as a Te and FGF-8 regulated gene. Te repression of TSP-1 was androgen receptor (AR)-dependent. It required de novo protein synthesis, but it was independent of FGF-8 expression. FGF-8, in turn, downregulated TSP-1 transcription by activating the ERK and PI3K pathways, and the effect could be reversed by specific kinase inhibitors. Differential FGFR1-3 action was studied by silencing each receptor by shRNA expression in S115 cells. FGFR1 expression was a prerequisite for the growth of S115 tumours, whereas FGFR2 expression alone was not able to promote tumour growth. High FGFR1 expression led to a growth advantage that was associated with strong ERK activation, increased angiogenesis and reduced apoptosis, and all of these effects could be reversed by an FGFR inhibitor. Taken together, the results of this thesis show that FGF-8 and FGFRs contribute strongly to the regulation of the growth and angiogenesis of experimental breast cancer and support the evidence for FGF-FGFR signalling as one of the major players in breast cancers.

First-Line Chemotherapy with Anthracycline and Taxane Combination in Metastatic Breast Cancer. Detection of bone metastases with TRACP 5b

Background: In Finland, breast cancer (BC) is the most common cancer among women, and prostate cancer (PC) that among men. At the metastatic stage both cancers remain essentially incurable. The goals of therapy include palliation of symptoms, improvement or maintenance of quality of life (QoL), delay of disease progression, and prolongation of survival. Balancing between efficacy and toxicity is the major challenge. With increasing costs of new treatments, appropriate use of resources is paramount. When new treatment regimes are introduced into clinical practice a comprehensive assessment of clinical benefit, adverse effects and cost is necessary. Both BC and PC show a predilection to metastasize to bone. Bone metastases cause significant morbidity impairing the patients´ QoL. Diagnosis of bone metastases relies mainly on radiological methods, which however lack optimal sensitivity and specificity. New tools are needed for detection and follow-up of bone metastases. Aims: Anthracyclines and taxanes are effective chemotherapeutic agents in the treatment of metastatic breast cancer (MBC) with different mechanisms of action. Therefore, evaluation of the combination of anthracyclines with taxanes was a justifiable approach in the treatment of MBC patients. We assessed the efficacy, toxicity, cost of treatment and QoL of BC patients treated with first-line chemotherapy for metastatic disease with the combination epirubicin and docetaxel. We also evaluated the diagnostic potential of tartrate-resistant acid phosphatase 5b (TRACP 5b) and carboxyterminal telopeptides of type I collagen (ICTP) in the diagnosis of bone metastases in BC and TRACP 5b in PC patients. Results: The combination of epirubicin and docetaxel was effective in this phase II study, but required individual dose adjustment to avoid neutropenic infections, and the use of growth factors to maintain a feasible dose level. The response rate was 54 % (95 % CI 37-71) and the median overall survival (OS) was 26 months. Of the patients, 87 % were treated for infections. The treatment of adverse events required additional use of health resources mainly due to neutropenic infections, thereby raising direct treatment costs by 20 %. Despite adverse events, the global QoL was not significantly compromised during the treatment. Clinically evident acute cardiac toxicity was not observed. The combination of serum TRACP 5b and ICTP was at least equally sensitive and specific in detection of of bone metastases as commonly used total alkaline phosphatise (tALP) in BC patients. In contrast, TRACP 5b was less specific and sensitive than tALP as a marker of skeletal changes in PC patients. Conclusions: Treatment with epirubicin and docetaxel showed high efficacy in first-line chemotherapy of MBC. The relatively high incidence of neutropenic infections requiring hospitalization increased the treatment costs. Despite adverse events, the global QoL of the patients was not significantly compromised. The combination of TRACP 5b and ICTP showed similar activity as tALP in detecting bone metastases in MBC. In contrast, TRACP 5b was less specific and sensitive than tALP as a marker of skeletal changes in PC.

Dissecting the molecular mechanisms of breast cancer bone metastasis for therapeutic targeting

Breast cancer that has metastasized to bone is currently an incurable disease, causing significant morbidity and mortality. The aim of this thesis work was to elucidate molecular mechanisms of bone metastasis and thereby gain insights into novel therapeutic approaches. First, we found that L‐serine biosynthesis genes, phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1) and phosphoserine phosphatase (PSPH), were up‐regulated in highly bone metastatic MDA‐MB‐231(SA) cells as compared with the parental breast cancer cell line. Knockdown of serine biosynthesis inhibited proliferation of MDA‐MB‐231(SA) cells, and L‐serine was essential for the formation of bone resorbing osteoclasts. Clinical data demonstrated that high expression of PHGDH and PSAT1 was associated with decreased relapse‐free and overall survival and with features typical of poor outcome in breast cancer. Second, RNA interference screening pointed out heparan sulfate 6‐O‐sulfotransferase 2 (HS6ST2) as a critical gene for transforming growth factor β (TGF‐β)‐induced interleukin 11 (IL‐11) production in MDA‐MB‐231(SA) cells. Exogenous heparan sulfate glycosaminoglycans heparin and K5‐NSOS also inhibited TGF‐β‐induced IL‐11 production in MDA‐MB‐231(SA) cells. Furthermore, K5‐NSOS decreased osteolytic lesion area and tumor burden in bone in mice. Third, we discovered that the microRNAs miR‐204, ‐211 and ‐379 inhibited IL‐11 expression in MDA‐MB‐231(SA) cells through direct targeting of the IL‐11 mRNA. MiR‐379 also inhibited Smad‐mediated signaling. Gene expression profiling of miR‐204 and ‐379 transfected cells indicated that these microRNAs down‐regulate several bone metastasis‐relevant genes, including prostaglandin‐endoperoxide synthase 2 (PTGS2). Taken together, this study identified three potential treatment strategies for bone metastatic breast cancer: inhibition of serine biosynthesis, heparan sulfate glycosaminoglycans and restoration of miR‐204/‐211/‐379.

Use of preclinical and early clinical data for dose selection of a selective estrogen receptor modulator toremifene in treatment of breast cancer

In development of human medicines, it is important to predict early and accurately enough the disease and patient population to be treated as well as the effective and safe dose range of the studied medicine. This is pursued by using preclinical research models, clinical pharmacology and early clinical studies with small sample sizes. When successful, this enables effective development of medicines and reduces unnecessary exposure of healthy subjects and patients to ineffectice or harmfull doses of experimental compounds. Toremifene is a selective estrogen receptor modulator (SERM) used for treatment of breast cancer. Its development was initiated in 1980s when selection of treatment indications and doses were based on research in cell and animal models and on noncomparative clinical studies including small number of patients. Since the early development phase, the treatment indication, the patient population and the dose range were confirmed in large comparative clinical studies in patients. Based on the currently available large and long term clinical study data the aim of this study was to investigate how the early phase studies were able to predict the treatment indication, patient population and the dose range of the SERM. As a conclusion and based on the estrogen receptor mediated mechanism of action early studies were able to predict the treatment indication, target patient population and a dose range to be studied in confirmatory clinical studies. However, comparative clinical studies are needed to optimize dose selection of the SERM in treatment of breast cancer.

Percutaneous core biopsy of palpable breast lesions: accuracy of frozen section histopathological exam in the diagnosis of breast cancer

OBJECTIVE: to evaluate the accuracy of frozen section histopathology from fragments of tissue obtained by percutaneous core needle biopsy of palpable tumors in the diagnosis of breast cancer. METHODS: a cohort study was performed on 57 patients with palpable tumors and suspected breast cancer undergoing percutaneous thick needle core biopsy. The fragments were analyzed by the same pathologist. RESULTS: frozen section diagnosed 16 benign cases (28.6%) and 40 malignant (71.4%), whereas paraffin showed that 15 were benign (26.8%) and 41 malignant (73.2%). Histopathological examinations were concordant in 55 cases and there was one false-negative (6.2%). Statistics rates were: negative predictive value of 93.8%, positive predictive value of 100%, no false-positive (0%), one false negative (6.2%), specificity of 100%, sensitivity of 97 6%; observed agreement = 98.2%; expected agreement = 59.9%, Kappa = 0.955 [ 95% CI = 0.925-0.974, p < 0.01 ]. CONCLUSIONS: frozen section histopathological findings showed excellent correlation with the findings by the technique in paraffin in the fragments of palpable breast tumors obtained by thick needle percutaneous core biopsy (98.2% accuracy). Therefore, in these patients, it was possible to anticipate the diagnosis, staging and the breast cancer treatment planning.

Identification of novel regulators for tumor progression in breast cancer

Breast cancer is the most frequent solid tumor among women and the leading cause of cancer related death in women worldwide. The prognosis of breast cancer patients is tightly correlated with the degree of spread beyond the primary tumor. In this thesis, the aim was to identify novel regulators of tumor progression in breast cancer as well as to get insights into the molecular mechanisms of breast cancer progression and metastasis. First, the role of phospholipid remodeling genes and enzymes important for breast cancer progression was studied in breast cancer samples as well as in cultured breast cancer cells. Tumor samples displayed increased de novo synthesized fatty acids especially in aggressive breast cancer. Furthermore, RNAi mediated cell based assays implicated several target genes critical for breast cancer cell proliferation and survival. Second, the role of arachidonic acid pathway members 15-hydroxyprostaglandin dehydrogenase (HPGD) and phospholipase A2 group VII (PLA2G7) in tumorigenesis associated processes was explored in metastatic breast cancer cells. Both targets were found to contribute to epithelial-mesenchymal transition related processes. Third, a high-throughput RNAi lysate microarray screen was utilized to identify novel vimentin expression regulating genes. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) was found to promote cellular features connected with metastatic disease, thus implicating MTHFD2 as a potential drug target to block breast cancer cell migration and invasion. Taken together, this study identified several putative targets for breast cancer therapy. In addition, these results provide novel information about the mechanisms and factors underlying breast cancer progression.

Internet-based breast cancer patient’s pathway as an empowering patient educational tool

This three-phase study was conducted to examine the effect of the Breast Cancer Patient’s Pathway program (BCPP) on breast cancer patients’ empowering process from the viewpoint of the difference between knowledge expectations and perceptions of received knowledge, knowledge level, quality of life, anxiety and treatment-related side effects during the breast cancer treatment process. The BCPP is an Internet-based patient education tool describing a flow chart of the patient pathway during the breast treatment process, from breast cancer diagnostic tests to the follow-up after treatments. The ultimate goal of this study was to evaluate the effect of the BCPP to the breast cancer patient’s empowerment by using the patient pathway as a patient education tool. In phase I, a systematic literature review was carried out to chart the solutions and outcomes of Internet-based educational programs for breast cancer patients. In phase II, a Delphi study was conducted to evaluate the usability of web pages and adequacy of their content. In phase III, the BCPP program was piloted with 10 patients and patients were randomised to an intervention group (n=50) and control group (n=48). According to the results of this study, the Internet is an effective patient education tool for increasing knowledge, and BCPP can be used as a patient education method supporting other education methods. However, breast cancer patients’ perceptions of received knowledge were not fulfilled; their knowledge expectations exceed the perceived amount of received knowledge. Although control group patients’ knowledge expectations were met better with the knowledge they received in hospital compared to the patients in the intervention group, no statistical differences were found between the groups in terms of quality of life, anxiety and treatment-related side effects. However, anxiety decreased faster in the intervention group when looking at internal differences between the groups at different measurement times. In the intervention group the relationship between the difference between knowledge expectations and perceptions of received knowledge correlated significantly with quality of life and anxiety. Their knowledge level was also significant higher than in the control group. These results support the theory that the empowering process requires patient’s awareness of knowledge expectations and perceptions of received knowledge. There is a need to develop patient education to meet patients’ perceptions of received knowledge, including oral and written education and BCPP, to fulfil patient’s knowledge expectations and facilitate the empowering process. Further research is needed on the process of cognitive empowerment with breast cancer patients. There is a need for new patient education methods to increase breast cancer patients’ awareness of knowing.

Application of morphometry, static DNA ploidy analysis, and steroid receptor expression in diagnosis and prognosis of Libyan breast cancer

The aim of this study was to describe the demographic, clinicopathological, biological and morphometric features of Libyan breast cancer patients. The supporting value of nuclear morphometry and static image cytometry in the sensitivity for detecting breast cancer in conventional fine-needle aspiration biopsies were estimated. The findings were compared with findings in breast cancer in Finland and Nigeria. In addation, the value of ER and PR were evaluated. There were 131 histological samples, 41 cytological samples, and demographic and clinicopathological data from 234 Libyan patients. The Libyan breast cancer is dominantly premenopausal and in this feature it is similar to breast cancer in sub-Saharan Africans, but clearly different from breast cancer in Europeans, whose cancers are dominantly postmenopausal in character. At presention most Libyan patients have locally advanced disease, which is associated with poor survival rates. Nuclear morphometry and image DNA cytometry agree with earlier published data in the Finnish population and indicate that nuclear size and DNA analysis of nuclear content can be used to increase the cytological sensitivity and specificity in doubtful breast lesions, particularly when free cell sampling method is used. Combination of the morphometric data with earlier free cell data gave the following diagnostic guidelines: Range of overlap in free cell samples: 55 μm2 -71 μm2. Cut-off values for diagnostic purposes: Mean nuclear area (MNA) >54 μm2 for 100% detection of malignant cases (specificity 84 %), MNA < 72 μm2 for 100% detection of benign cases (sensitivity 91%). Histomorphometry showed a significant correlation between the MNA and most clinicopathological features, with the strongest association observed for histological grade (p <0.0001). MNA seems to be a prognosticator in Libyan breast cancer (Pearson’s test r = - 0.29, p = 0.019), but at lower level of significance than in the European material. A corresponding relationship was not found in shape-related morphometric features. ER and PR staining scores were in correlation with the clinical stage (p= 0.017, and 0.015, respectively), and also associated with lymph node negative patients (p=0.03, p=0.05, respectively). Receptor-positive (HR+) patients had a better survival. The fraction of HR+ cases among Libyan breast cancers is about the same as the fraction of positive cases in European breast cancer. The study suggests that also weak staining (corresponding to as few as 1% positive cells) has prognostic value. The prognostic significance may be associated with the practice to use antihormonal therapy in HR+ cases. The low survival and advanced presentation is associated with active cell proliferation, atypical nuclear morphology and aneuploid nuclear DNA content in Libyan breast cancer patients. The findings support the idea that breast cancer is not one type of disease, but should probably be classified into premenopausal and post menopausal types.

Avaliação de microcalcificações mamárias de acordo com as classificações do Breast Imaging Reporting and Data System (BI-RADS TM) e de Le Gal

OBJETIVO: avaliar a acurácia da mamografia para o diagnóstico de microcalcificações mamárias suspeitas, com as classificações do Breast Imaging Reporting and Data System (BI-RADS TM) e Le Gal em comparação com o resultado histopatológico utilizado como padrão-ouro. MÉTODOS: foram selecionados dos arquivos dos blocos cirúrgicos, 130 casos operados com mamografias contendo somente microcalcificações mamárias, inicialmente classificadas como suspeitas sem lesões detectáveis ao exame clínico. Estas foram reclassificadas por dois examinadores, utilizando as classificações de Le Gal e BI-RADS TM, obtendo-se diagnóstico de consenso. As biópsias foram revistas por dois patologistas e foi obtido diagnóstico de consenso. A leitura das mamografias e a revisão das lâminas foram feitas em duplo-cego. As análises estatísticas utilizadas neste estudo foram o teste do chi2, o modelo Fleiss quadrático para VPP e o programa Epi-Info 6.0. RESULTADOS: a correlação entre a análise histopatológica e mamográfica, usando BI-RADS TM e Le Gal, mostrou a mesma sensibilidade de 96,4%, especificidade de 55,9 e 30,3%, valor preditivo positivo (VPP) de 37,5% e 27,5% e acurácia de 64,6 e 44,6%, respectivamente. Quando discriminamos por categorias de BI-RADS TM, obtivemos VPPs: categoria 2, 0%; categoria 3, 1,8%; categoria 4, 31,6% e categoria 5, 60%. Os VPPs pela classificação de Le Gal foram: categoria 2, 3,1%; categoria 3, 18,1 %; categoria 4, 26,4%; categoria 5, 66,7% e não classificável, 5,2%. CONCLUSÕES: observou-se uma maior precisão com a classificação de BI-RADS TM, porém não se conseguiu reduzir a ambigüidade na avaliação das microcalcificações mamárias.

Comparison of nuclear grade and immunohistochemical features in situ and invasive components of ductal carcinoma of breast

PURPOSE:To compare the prognostic and predictive features between in situ and invasive components of ductal breast carcinomas. METHODS:We selected 146 consecutive breast samples with ductal carcinoma in situ (DCIS) associated with adjacent invasive breast carcinoma (IBC). We evaluated nuclear grade and immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), cytokeratin 5/6 (CK5/6), and epidermal growth factor receptor (EGFR) in both components, in situ and invasive, and the Ki-67 percentage of cells in the invasive part. The DCIS and IBC were classified in molecular surrogate types determined by the immunohistochemical profile as luminal (RE/PR-positive/ HER2-negative), triple-positive (RE/RP/HER2-positive), HER2-enriched (ER/PR-negative/HER2-positive), and triple-negative (RE/RP/HER2-negative). Discrimination between luminal A and luminal B was not performed due to statistical purposes. Correlations between the categories in the two groups were made using the Spearman correlation method. RESULTS:There was a significant correlation between nuclear grade (p<0.0001), expression of RE/RP (p<0.0001), overexpression of HER2 (p<0.0001), expression of EGFR (p<0.0001), and molecular profile (p<0.0001) between components in situ and IBC. CK 5/6 showed different distribution in DCIS and IBC, presenting a significant association with the triple-negative phenotype in IBC, but a negative association among DCIS. CONCLUSIONS: Our results suggest that classical prognostic and predictive features of IBC are already determined in the preinvasive stage of the disease. However the role of CK5/6 in invasive carcinoma may be different from the precursor lesions.