Adjuvant combined therapy with trastuzumab in patients with HER2­ positive breast cancer and cardiac alterations: implications for optimal cardio­oncology care.

INTRODUCTION: Recently, the prognosis of patients with HER2­positive breast cancer (BC) has improved significantly owing to the use of combined treatment modalities. However, systemic treatment is as-sociated with increased risk of cardiotoxicity. OBJECTIVES: We aimed to assess subclinical cardiac alterations during the final stage of adjuvant com-bined therapy, that is, trastuzumab therapy (TT), as potential predictors of late cardiac complications in patients with HER2­positive BC. PATIENTS AND METHODS: We enrolled 251 patients with HER2­positive BC treated with a radical local therapy, adjuvant chemotherapy (anthracyclines or anthracyclines + taxanes), and immunotherapy (trastuzumab). Patients underwent 6 echocardiographic examinations: at baseline, during TT, and after TT, with assessment of left ventricular ejection fraction (LVEF), degree of valvular regurgitation, and cardiac chamber diameters. RESULTS: Valvular fibrosis (28.4% of patients) was associated with older age, hypertension at baseline, and a higher degree of regurgitation during TT. Reduced LVEF, greater regurgitation, and larger cardiac chamber diameters were noted during TT. The patients who received higher anthracycline doses showed a greater degree of aortic insufficiency and a larger right ventricular diameter. Reduced LVEF during TT was associated with radiotherapy or chemotherapy and the degree of valvular regurgitation. Significantly larger diameters were observed in older patients and in those with comorbidities at baseline, high body mass index, and regurgitation. CONCLUSIONS: Asymptomatic subclinical cardiac alterations during TT may predict late cardiac complica-tions; however, longer follow­up is necessary to confirm this hypothesis. Patients with HER2­positive BC should be closely monitored for possible cardiac alterations during and after therapy to ensure optimal care and guide therapeutic decision­making.

Low frequency of HPV positivity in breast tumors among patients from south-central Poland.

BACKGROUND: Some studies suggest that Human Papilloma Virus (HPV) infection is important factor in carcinogenesis of breast tumors. This study' objective was to analyze HPV prevalence in breast cancers of patients from south-central Poland. MATERIALS AND METHODS: The study was performed based on archival paraffin embebbed and formalin fixed blocks in the group of 383 patients with breast cancer. HPV prevalence and its genotype were assessed, respectively by: nested PCR (with two groups of primers: PGMY09/PGMY11 and GP5+/GP6+), quantitative PCR (qPCR). Tumors were classified as HPV positive in case of at least one positive result in nested PCR and positive results in genotyping procedure. For all HPV positive tissues P16 immunostaining was applied in order to confirm active viral infection. RESULTS: In the group of 383 breast cancers, HPV positivity was found in 17 samples (4.4%) in nested PCR. All these samples were subjected to HPV genotyping. This analysis revealed presence of HPV type 16 into two tumors (0.5%). In these two cancers, P16 overexpression was reported. CONCLUSION: In breast tumors of patients from south-central Poland in Poland, HPV positivity is demonstrated in very low percentage of cases.

Intra-Tumor Heterogeneity Revealed by Mass Spectrometry Imaging Is Associated with the Prognosis of Breast Cancer.

Intra-tumor heterogeneity (ITH) results from the coexistence of genetically distinct cancer cell (sub)populations, their phenotypic plasticity, and the presence of heterotypic components of the tumor microenvironment (TME). Here we addressed the potential association between phenotypic ITH revealed by mass spectrometry imaging (MSI) and the prognosis of breast cancer. Tissue specimens resected from 59 patients treated radically due to the locally advanced HER2-positive invasive ductal carcinoma were included in the study. After the on-tissue trypsin digestion of cellular proteins, peptide maps of all cancer regions (about 380,000 spectra in total) were segmented by an unsupervised approach to reveal their intrinsic heterogeneity. A high degree of similarity between spectra was observed, which indicated the relative homogeneity of cancer regions. However, when the number and diversity of the detected clusters of spectra were analyzed, differences between patient groups were observed. It is noteworthy that a higher degree of heterogeneity was found in tumors from patients who remained disease-free during a 5-year follow-up (n = 38) compared to tumors from patients with progressive disease (distant metastases detected during the follow-up, n = 21). Interestingly, such differences were not observed between patients with a different status of regional lymph nodes, cancer grade, or expression of estrogen receptor at the time of the primary treatment. Subsequently, spectral components with different abundance in cancer regions were detected in patients with different outcomes, and their hypothetical identity was established by assignment to measured masses of tryptic peptides identified in corresponding tissue lysates. Such differentiating components were associated with proteins involved in immune regulation and hemostasis. Further, a positive correlation between the level of tumor-infiltrating lymphocytes and heterogeneity revealed by MSI was observed. We postulate that a higher heterogeneity of tumors with a better prognosis could reflect the presence of heterotypic components including infiltrating immune cells, that facilitated the response to treatment.

High Ki-67 expression is a marker of poor survival in apocrine breast carcinoma.

Apocrine carcinoma is a very rare type of breast cancer, which represents 0.5-4% of all breast cancers. The aim of the study was to analyze biological and clinical features of apocrine carcinoma and their influence on patients survival. The studied group consists of 57 patients, who underwent treatment between 1987 and 2010. Expression of ER, PgR, HER2, AR, GCDFP-15, EGFR, CK 5/6, CK 8/18 and Ki-67 was assessed immunohistochemically on formalin-fixed paraffin-embedded tissue sections. Presence of emboli and extent of lymphocyte infiltration were assessed on haematoxylin-eosin-stained slides. In the investigated group, 16 cases were ER/PgR positive and 49 were AR-positive. ER/PgR-negative tumours were often characterised by CK5/6 and EGFR positivity. The presence of AR was related to HER-2 and GCDFP-15 expression and tumours with expression of CK5/6 were more likely be EGFR positive and had higher Ki-67 LI. Higher probability of 10-years OS and DFS was observed in patients with tumours characterized by Ki-67 LI < 20% (p = 0.036 and p = 0.009, respectively). Favourable trend in OS was noted for patients with smaller tumours (p = 0.053), without lymph node metastases (p = 0.074) and without EGFR expression (p = 0.060). In apocrine breast carcinoma expression of Ki-67 is one of the most important factors influencing patients' survival.

Comparison of mutation profile between primary phyllodes tumors of the breast and their paired local recurrences.

Phyllodes tumor of the breast (PTB) is a rare neoplasm and accounts for 0.2-2.0% of breast cancer in women. Histopathological diagnosis of the tumor is difficult, and histological features do not always predict the course of the disease and the risk of progression. Pathogenesis and molecular biological characteristics as well as PTB prognostic factors are unknown. In search for genetic factors affecting PTB progression, 10 patients were analyzed for whom material from the primary tumor and local recurrence was available. DNA isolated from paraffin blocks was sequenced using the next-generation sequencing method (NGS). In 4 pairs, consisting of primary tumor and local recurrence, probably pathogenic/pathogenic variants were detected, and in three pairs they were observed in the CDKN2A gene, while other variants were found in PTEN and TP53 genes. NGS results indicate that the above-mentioned variants are hereditary, which suggests that the CDKN2A gene might be involved in cancerogenesis of PTB. Additionally, the selected pathogenic variant of EGFR gene was exclusively detected in one recuurent tumor, which might suggest the involvement of this gene in the mechanism of progression. In order to determine if this variant is associated with progression, the frequency of this mutation should be examined in larger group of malignant and borderline tumors.

Correlation between quantitative assessment of contrast enhancement in contrast-enhanced spectral mammography (CESM) and histopathology-preliminary results.

OBJECTIVES: Contrast-enhanced spectral mammography (CESM) is a novel method for breast cancer detection. The aim of this study is to check if there is a possibility of quantitative assessment of contrast enhancement in CESM and if there is any correlation between quantitative assessment of contrast enhancement in CESM and histopathology. METHODS: A total of 167 female patients underwent CESM. All subjects previously had suspicious lesions found on mammography, breast ultrasound, or both. After imaging, the following parameters were evaluated: number of enhancing lesions in each breast and size and degree of enhancement of each lesion. Based on the collected data, the percentage signal difference between enhancing lesion and background (%RS) and signal-difference-to-noise ratio (SDNR) were measured for each lesion. RESULTS: The number of lesions detected in the study population was 195. Among all diagnosed lesions, 120 (62%) were assessed to be infiltrating cancers, 16 (8%) non-infiltrating cancers, and 59 (30%) were benign. Thirteen (7%) lesions did not enhance in CESM; all non-enhancing lesions were confirmed to be benign under histopathological examination. Analysis of enhancement indices showed that signal values within lesions and signal values within background ROIs (regions of interest) were similar in CC (craniocaudal) and MLO (mediolateral) projections. Mean %RS values were correlated with the type of enhancing lesion, infiltrating cancers having the highest values, benign lesions the lowest. CONCLUSIONS: This work has demonstrated a significant correlation between the degree of lesion enhancement in CESM and malignancy. Quantitative analysis of enhancement levels in CESM can distinguish between invasive cancers and benign or in situ lesions. KEY POINTS: • There is a possibility of quantitative assessment of contrast enhancement in CESM. • Correlation between quantitative assessment of contrast enhancement in CESM and histopathology was observed.

Relationship between HER2 gene status and selected potential biological features related to trastuzumab resistance and its influence on survival of breast cancer patients undergoing trastuzumab adjuvant treatment.

BACKGROUND: The aim of the study was to investigate if parameters associated with human epidermal growth factor receptor type 2 (HER2) status (HER2 gene copy number, HER2/CEP17 ratio or polysomy of chromosome 17) are related to various biological features potentially responsible for trastuzumab resistance (PTEN, IGF-1R, MUC4, EGFR, HER3, HER4, and mutation status of PIK3CA) as well as their influence on survival of HER2-positive breast cancer patients treated with adjuvant chemotherapy and trastuzumab. PATIENTS AND METHODS: The investigated group consisted of 117 patients with invasive ductal breast cancer (T≥1, N≥0, M0) with overexpression of HER2, who underwent radical surgery between 2007 and 2014. Status of ER, PR, and HER2 expression was retrieved from patients' files. HER2 gene copy number was investigated by fluorescence in situ hybridization using PathVysion HER-2 DNA Probe Kit II. Expression of PTEN, IGF-1R, MUC4, EGFR, HER3, and HER4 was assessed immunohistochemically on formalin-fixed paraffin-embedded tissue sections. PIK3C mutation status was determined by qPCR analysis. RESULTS: Overexpression of HER2 protein (IHC 3+) and ER negativity corresponded to higher HER22 copy number and HER2/CEP17 ratio (.<0.001). Tumors with polysomy were characterized by higher HER22 gene copy number but lower HER2/CEP17p ratio (p<0.026, p<0.001). Patients with tumors featuring HER3 immunonegativity or low HER2/CEP17 ratio (#4) were characterized by 100% metastasis-free survival (.=0.018, p=0.062). CONCLUSION: Presence of both unfavorable factors, ie, HER3 expression and high HER2/CEP17 ratio, allowed to distinguish a group of patients with worse prognosis (.=0.001).

Podoplanin-positive Cancer-associated Stromal Fibroblasts in Primary Tumor and Synchronous Lymph Node Metastases of HER2-overexpressing Breast Carcinomas.

We compared the status of stromal podoplanin-positive cancer-associated fibroblasts (ppCAFs) between primary tumors and paired synchronous lymph node metastases (LNMs) and analyzed the prognostic significance of tumoral ppCAFs in 203 patients with human epidermal growth factor receptor 2-positive breast carcinoma. ppCAFs were found in 167/203 and in 35/87 tumors and LNM, respectively. ppCAFs were most frequently found in tumors and corresponding LNM (n=52, 59.8%; p=0.001). However, for all LNMs (n=12) without ppCAFs, their paired tumors also lacked ppCAFs. In both tumors and LNMs, ppCAFs were α-smooth muscle actin-positive and cluster of differentiation 21 protein-negative, suggesting them not to be resident lymph node cells. Moreover, in our series, the presence of ppCAFs in tumors was borderline related to poor disease-free survival (p=0.058). These results speak in favor of a hypothesis suggesting ppCAFs accompany metastatic cancer cells migrating from tumor to LNMs.

Intensity and Pattern of Enhancement on CESM: Prognostic Significance and its Relation to Expression of Podoplanin in Tumor Stroma - A Preliminary Report.

BACKGROUND/AIM: It is possible that the degree of enhancement on contrast-enhanced spectral mammography (CESM), a new diagnostic method, might provide prognostic information for breast cancer patients. Therefore, in a group of 82 breast cancer patients, we analyzed the prognostic significance of degree and pattern of enhancement on CESM as well as its relation to: (a) breast cancer immunophenotype (based on ER/PR/HER2 status) (b) podoplanin expression in cancer stroma (lymphatic vessel density plus podoplanin-positivity of cancer-associated fibroblasts), and (c) other histological parameters. MATERIALS AND METHODS: For each tumor the intensity of enhancement on CESM was qualitatively assessed as strong or weak/medium, while the pattern - as homogenous and heterogenous. RESULTS: Herein we report, for the first time, that strong and heterogenous enhancement on CESM was related to unfavorable disease-free survival of breast cancer patients (p=0.005). Moreover, the strong enhancement was more frequent in large and node-positive tumors (pT>1, pN>0) (p=0.002), as well as in carcinomas with podoplanin-sparse stroma (p=0.008). CONCLUSION: Intensity and pattern of enhancement on CESM might provide (together with the results of other diagnostic imaging methods) not only the confirmation of presence or absence of tumor, but also prognostic information.

Gender-related prognostic significance of clinical and biological tumor features in rectal cancer patients receiving short-course preoperative radiotherapy.

AIM: To study the prognostic value of clinical and biological features of rectal cancer and potential gender differences in patients' overall survival (OS), local recurrence-free survival (RFS) and metastasis-free survival (MFS) after short-course preoperative radiotherapy (SCRT) with short or long interval between RT and surgery (break). BACKGROUND: The length of the interval between RT and surgery in SCRT is debatable and gender-related differences in patients survival are not established yet. MATERIALS AND METHODS: 126 patients received SCRT with 5 Gy dose per fraction during 5 days, followed by radical surgery after short break ≤17 days, and a long break >17 days. Pretreatment tumor proliferation (bromodeoxyuridine labeling index, BrdUrdLI and S-phase fraction) was evaluated by flow cytometry and proteins: CD34, Ki-67, GLUT-1, Ku70, BCL-2, P53 expression was studied immunohistochemically. RESULTS: The studied group included 84 men and 42 women. There were 33, 76, and 17 cTNM (AJCC) tumor stages I, II, III, respectively. The median follow-up time was 53.3 months (range 2-142 months). For the whole group Cox multivariate analysis revealed that tumor grade (G > 1), interval between RT and surgery >17 days, pTNM stage >1 and P53 positivity + BrdUrdLI > 7.9% were negative prognostic factors for OS. Tumor aneuploidy and MVD > 140.8 vessels/mm2 were important for RFS. pTNM stage > 1 and P53 positivity combined with BrdUrdLI > 7.9% were risk predictors for MFS. Based on tumor biological features, gender-related difference in OS, RFS, and MFS were observed. In multivariate analysis, male patients age > 62 years and break >17 days only appeared to be significant for OS. CONCLUSIONS: In male rectal patients treated with SCRT, breaks between RT and surgery >17 days should be avoided because they negatively influence patients' survival.

Prognostic role of lymphatic vessel density and lymphovascular invasion in chemotherapy-naive and chemotherapy-treated patients with invasive breast cancer.

It is assumed that the spread of breast cancer cells via the lymphatic system might be influenced by inflammatory reactions and/or the application of chemotherapy or molecularly targeted therapy. Therefore, we analysed survival according to lymphatic vessel density (LVD), lymphovascular invasion (LVI) (both assessed using podoplanin as immunohistochemical marker of lymphatic endothelium) and well-established clinico-pathological features in a group of 358 patients with invasive ductal breast cancer: 139 chemotherapy-naïve (pT1-2/pN0/M0) and 219 treated with chemotherapy (pT1-4/pN1-3/M0). Univariate analysis revealed that high LVD was related to unfavourable disease-free survival (DFS) in pN0/chemotherapy/trastuzumab-naïve patients (P = 0.028). Conversely, in pN+/chemotherapy-treated individuals high LVD was related to favourable DFS (P = 0.019). LVI was a significant indicator of survival (P = 0.005) only in pN0/chemotherapy/trastuzumab-naïve patients. The following parameters were significant independent adverse prognostic factors for DFS: (i) in pN0/chemotherapy/trastuzumab-naïve patients: high LVD (LVD > 7 vessels/mm2; RR = 2.7, P = 0.039), LVI (RR = 3.3, P = 0.046) and high tumor grade (G3 vs. G1 + G2; RR = 2.6, P = 0.030); (ii) in pN+/chemotherapy/trastuzumab-treated patients: low LVD (RR = 1.8, P = 0.042), the number of involved lymph nodes (pN3 vs. pN1-2; RR = 2.3, P = 0.012) and the breast cancer subtype (expression of steroid receptors together with HER2 immunonegativity and high proliferation index vs. other breast cancer immunophenotypes; RR = 3.0, P < 0.001). High LVD may identify high progression risk in pN0/chemotherapy/trastuzumab-naïve patients, and low progression risk in pN+/chemotherapy-treated patients. This phenomenon might be explained by potential involvement of lymphangiogenesis in two processes related to cancer eradication: a chemotherapy-stimulated activity of the immune system against cancer cells, or increased tumour drainage influencing the efficacy of cytotoxic drugs.

Proteins Involved in HER2 Signalling Pathway, Their Relations and Influence on Metastasis-Free Survival in HER2-Positive Breast Cancer Patients Treated with Trastuzumab in Adjuvant Setting.

AIM: Resistance to trastuzumab (which is a standard therapy for breast cancer patients with HER2 overexpression) is associated with higher risk of progression or cancer death, and might be related to activation of signalling cascades (PI3K/AKT/mTOR, Ras/Raf/MAPK) and decreased level of their inhibitors. MATERIAL AND METHODS: Formalin-fixed paraffin-embedded tumour specimens from 118 HER2-overexpressing breast cancer patients treated with radical local therapy and trastuzumab in adjuvant setting were used for the assessment of: (1) PIK3CA gene mutations (p.H1047R and p.E545K) by qPCR, and (2) expression of Ki-67, EGFR, MUC4, HER3 and PTEN by immunohistochemistry. RESULTS: Lower Ki-67LI was observed in EGFR-immunonegative and in PTEN-immunopositive tumours. MUC4-immunonegative tumours more frequently were PTEN- and HER3-immunonegative. Favourable metastasis-free survival was observed in patients with tumours characterized by Ki-67LI≤50% (p=0.027), HER3 immunonegativity or PTEN immunopositivity (vs. tumours with HER3 expression and lack of PTEN expression, p=0.043), additionally, the trend was observed for patients with pN0+pN1 pathological tumour stage (vs. pN2+pN3) (p=0.086). Cox model revealed that independent negative prognostic factors were: (i) Ki-67LI>50% (p=0.014, RR=4.6, 95% CI 1.4-15.4), (ii) HER3 immunopositivity together with PTEN immunonegativity (p=0.034, RR=3.7, 95% CI 1.1-12.5). CONCLUSION: The results of our study suggest that combined analysis of HER3 and PTEN expression might bring information on trastuzumab sensitivity in the group of HER2-positive breast cancer patients treated with trastuzumab in adjuvant setting.

Degree of Enhancement on Contrast Enhanced Spectral Mammography (CESM) and Lesion Type on Mammography (MG): Comparison Based on Histological Results.

BACKGROUND Contrast enhanced spectral mammography (CESM) is a new method of breast cancer diagnosis in which an iodinated contrast agent is injected and dual-energy mammography is obtained in multiple views of the breasts. The aim of this study was to compare the degree of enhancement on CESM with lesion characteristics on mammography (MG) and lesion histology in women with suspicious breast lesions. MATERIAL AND METHODS The degree of enhancement on CESM (absent, weak, medium, or strong) was compared to lesion characteristics on MG (mass, mass with microcalcifications, or microcalcifications alone) and histology (infiltrating carcinoma, intraductal carcinoma, or benign) to compare sensitivity of the two modalities and to establish correlations that might improve diagnostic accuracy. RESULTS Among 225 lesions identified with CESM and MG, histological evaluation revealed 143 carcinomas (127 infiltrating, 16 intraductal) and 82 benign lesions. This is the largest cohort investigated with CESM to date. The sensitivity of CESM was higher than that of MG (100% and 90%, respectively, p=0.010). Medium or strong enhancement on CESM and the presence of a mass on MG was the most likely indictor of malignancy (55.1% p=0.002). Among benign lesions, 60% presented as enhancement on CESM (were false-positive), and most frequently as medium or weak enhancement, together with a mass on MG (53%, p=0.047). Unfortunately, the study did not find combinations of MG findings and CESM enhancement patterns that would be helpful in defining false-positive lesions. We observed systematic overestimation of maximum lesion diameter on CESM compared to histology (mean difference: 2.29 mm). CONCLUSIONS Strong or medium enhancement on CESM and mass or mass with microcalcifications on MG were strong indicators of malignant transformation. However, we found no combination of MG and CESM characteristics helpful in defining false-positive lesions.

Survival of breast cancer patients according to changes in expression of selected markers between primary tumor and lymph node metastases.

BACKGROUND: The differences between primary and metastatic tumor cells might be important for treatment selection and prognostication. MATERIALS & METHODS: Expression of ER, PR, HER2, CK5/6, EGFR, Ki-67, Ep-CAM, P-cadherin, CD24, CD44, ALDH was assessed immunohistochemically in primary tumor (T) and corresponding synchronous nodal metastases (LNM) in 156 invasive ductal breast cancer patients (T ≥1, N ≥1, M0). RESULTS: Independent negative prognostic factors for disease-free survival were pN3, ALDH immunopositivity in LNM, nonluminal A subtype in LNM, reduction of Ep-CAM expression in LNM, lack of changes or enhancement of CK5/6 and ALDH expression in LNM. DISCUSSION: Our results suggest that in some cases expression of markers in lymph node metastases might bring additional prognostic information to that obtained from primary tumor.

The search for optimal cutoff points for apoptosis and proliferation rate in prognostification of early stage breast cancer patients treated with anthracyclines in adjuvant settings.

Cancer growth is determined by the proportion of proliferating to dying cells; thus, the aim of the study was to analyze how proliferation rate and apoptosis level affect disease-free survival (DFS) of breast cancer (BC) patients treated with anthracycline-based chemotherapy. For 172 BC, proliferation rate was investigated by Ki-67 labeling index (Ki-67 LI)-assessed immunohistochemically. Apoptosis level was analyzed by apoptotic index (AI) estimated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. To stratify patients into subgroups with higher and lower DFS and to achieve optimal categorization, optimal cutoff points were searching by minimal P value method. The best separation of DFS curves (P = 0.001) was observed for three categories of AI: (i) AI >1.8 % (DFS = 100 %), (ii) AI ≤0.3 % (DFS = 84.6 %), and (iii) 1.8 % <= AI >0.3 % (DFS = 64.0 %). The highest DFS (86.1 %) was shown for the subgroup with low-proliferating BC (Ki-67 LI ≤18.0 %), intermediate (73.9 %) for patients characterized by Ki-67 LI in the range 18.0-37.0 % and the lowest (60.0 %) for women with fast-proliferating tumors (Ki-67 LI >37.0 %) (P = 0.022). Summarized, minimal P value method allows for optimal separation of survival curves. Apoptosis level and proliferation rate have some prognostic potential for early stage breast cancer patients treated with anthracyclines in adjuvant setting, however, as suggested by multivariate analysis, not as independent prognostic factors.

The tumour border on contrast-enhanced spectral mammography and its relation to histological characteristics of invasive breast cancer.

Contrast-enhanced spectral mammography (CESM) is one of the new diagnostic modalities implemented in clinical practice. In the case of these techniques, there are two major issues to be addressed: (1) their diagnostic usefulness, and (2) the relation between parameters assessed using these techniques and well-known diagnostic/prognostic/predictive markers (histological, clinical, and molecular). Therefore, we studied the relationship between the tumour margin assessed on CESM and (1) tumour borders defined on the basis of macroscopic and microscopic examination, (2) pT, (3) pN, and (4) tumour grade in a group of 82 breast cancer patients. Based on CESM, the tumour border was defined as sharp, indistinct or spiculated, whereas in the case of lesions showing weak or medium enhancement on CESM the borders were classified as unspecified. We found a statistically significant relationship between tumour margin on CESM and (1) macroscopic border (a spiculated margin on CESM was found only in carcinomas with an invasive border on histological examination; p = 0.004), (2) pT (p = 0.016), and (3) pN (nodal involvement was observed most frequently in carcinomas with a spiculated or indistinct margin on CESM; p = 0.045). Moreover, in cases with an undefined margin on CESM (cases showing weak or medium enhancement on CESM), both invasive and pushing borders were found on histological examination. The results of our preliminary study suggest that it is possible to assess macroscopic borders of examined lesions on the basis of CESM imaging. This might be useful in planning the extent of surgical excision. On the other hand, the assessment of the tumour margin on CESM might not be precise in cases showing weak enhancement.

Correlation between blood and lymphatic vessel density and results of contrast-enhanced spectral mammography.

UNLABELLED: Contrast-enhanced spectral mammography (CESM) is a novel technique used for detection of tumour vascularity by imaging the moment in which contrast, delivered to the lesion by blood vessels, leaks out of them, and flows out through lymphatic vessels. In our study, we included 174 women for whom spectral mammography was performed for diagnostic purposes. The relationship between enhancement in CESM and blood vessel density (BVD), lymphatic vessel density (LVD) or the percentage of fields with at least one lymphatic vessel (distribution of podoplanin-positive vessels - DPV) and other related parameters was assessed in 55 cases. BVD, LVD and DPV were assessed immunohistochemically, applying podoplanin and CD31/CD34 as markers of lymphatic and blood vessels, respectively. The sensitivity (in detection of malignant lesions) of CESM was 100%, while its specificity - 39%. We found a significant positive correlation between the intensity of enhancement in CESM and BVD (p = 0.007, r = 0.357) and a negative correlation between the intensity of enhancement in CESM and DPV (p = 0.003, r = -0.390). Lesions with the highest enhancement in CESM showed a high number of blood vessels and a low number of lymphatics. CONCLUSIONS: 1) CESM is a method characterized by high sensitivity and acceptable specificity; 2) the correlation between CESM results and blood/lymphatic vessel density confirms its utility in detection of tissue angiogenesis and/or lymphangiogenesis.

Prognostic value of PIK3CA mutation status, PTEN and androgen receptor expression for metastasis-free survival in HER2-positive breast cancer patients treated with trastuzumab in adjuvant setting.

Resistance to trastuzumab in patients with HER2-overexpressing breast cancer is associated with higher risk of progression or cancer death, and might be related to activation of PI3K/AKT/mTOR and Ras/Raf/MAPK signaling cascades and a decreased level of their inhibitor (PTEN). HER2-overexpressing breast cancer patients (n=75) treated with radical local therapy and trastuzumab in adjuvant setting were included into the study. Deoxyribonucleic acid isolated from paraffin sections was used to assess mutational status of the PIK3CA gene (p.H1047R and p.E545K mutations) by the quantitative polymerase chain reaction technique. Expression of selected proteins (ER, PgR, AR, Ki-67, EGFR) was assessed using immunohistochemistry. In the studied group we found significantly higher Ki-67LI in EGFR-positive carcinomas (p=0.048). Moreover, EGFR immunonegativity was observed more frequently in low-grade (G1/G2) carcinomas as well as in estrogen/progesterone and androgen receptor immunopositive tumors (p=0.042, p=0.016, p=0.044, respectively). Favorable metastasis-free survival was observed in patients with pN0 and pN1 (vs. pN2+3) stage (p=0.040) and with tumors characterized by low Ki-67LI (≤50% vs. >50%) (p=0.014). Patients with tumor androgen receptor immunonegativity (weak or lack of expression) or strong PTEN expression survived 3 years without metastases (p=0.007). The results of our study suggest that androgen receptor and PTEN status might be considered as indicators of trastuzumab sensitivity.

Prognostic Role of Nodal Status and Clinically Asymptomatic Valvular Insufficiency in Patients with HER2-positive Breast Cancer Treated with Chemotherapy, Radiotherapy and Trastuzumab in an Adjuvant Setting.

AIM: The aim of the present study was to assess metastasis-free survival of 134 patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer treated with chemotherapy, radiotherapy and trastuzumab in an adjuvant setting, according to sub-clinical cardiac side-effects (parameters not tested previously) evaluated before, during and after trastuzumab therapy, as well as selected clinicopathological parameters. RESULTS: In our series, left ventricular ejection fraction decreased significantly from 68.1% before trastuzumab treatment to 66.7% after therapy (p<0.001). Further analysis revealed that this decrease was significant only in patients who received radiotherapy and developed valve insufficiency during or after (but not before) trastuzumab therapy (p<0.001). Cox multivariate analysis revealed that both pN2a tumor stage and valve regurgitation during/after trastuzumab therapy (vs. lack of valve insufficiency or insufficiency before trastuzumab therapy) were significant independent factors for a negative prognosis. CONCLUSION: Valve insufficiency diagnosed during or after trastuzumab application might be cancer-unrelated indicator of decreased sensitivity to trasuzumab.

Lymphatic and blood vessels in male breast cancer.

BACKGROUND: It is assumed that lymphatic vessels are responsible for breast cancer dissemination. PATIENTS AND METHODS: In 32 male breast carcinomas we evaluated the correlation between: (i) lymphatic vessel density (LVD), distribution of podoplanin-immunostained vessels (DPV), blood vessel density (BVD), infiltration of immune cells and (ii) known clinicopathological parameters. RESULTS: Lymphatic and blood vessels were found in 77.8% and 100% of breast carcinomas, respectively. Double-negative estrogen and progesterone receptor tumors (ER-/PR-) presented significantly higher LVD than ER/PR positive cases, while high-grade tumors exhibited significantly higher DPV than low-grade carcinomas. We detected significantly higher frequency of vascular invasion in high-grade and double-negative carcinomas than in low-grade and ER/PR-positive ones, respectively. CONCLUSION: The relationship between high number of lymphatic vessels and high tumor grade or steroid receptor negativity might confirm the hypothesis regarding the influence of lymphangiogenesis on the formation of a more aggressive phenotype in male breast cancer.