Mass Spectrometry Proteomics Characterization of Plasma Biomarkers for Colorectal Cancer Associated With Inflammation.

Background: Colorectal cancer (CRC) prognosis is determined by the disease stage with low survival rates for advanced stages. Current CRC screening programs are mainly using colonoscopy, limited by its invasiveness and high cost. Therefore, non-invasive, cost-effective, and accurate alternatives are urgently needed. Objective and design: This retrospective multi-center plasma proteomics study was performed to identify potential blood-based biomarkers in 36 CRC patients and 26 healthy volunteers by high-resolution mass spectrometry proteomics followed by the validation in an independent CRC cohort (60 CRC patients and 44 healthy subjects) of identified selected biomarkers. Results: Among the 322 identified plasma proteins, 37 were changed between CRC patients and healthy volunteers and were associated with the complement cascade, cholesterol metabolism, and SERPIN family members. Increased levels in CRC patients of the complement proteins C1QB, C4B, and C5 as well as pro-inflammatory proteins, lipopolysaccharide-binding protein (LBP) and serum amyloid A4, constitutive (SAA4) were revealed for first time. Importantly, increased level of C5 was verified in an independent validation CRC cohort. Increased C4B and C8A levels were correlated with cancer-associated inflammation and CRC progression, while cancer-associated inflammation was linked to the acute-phase reactant leucine-rich alpha-2-glycoprotein 1 (LRG1) and ceruloplasmin. Moreover, a 4-protein signature including C4B, C8A, apolipoprotein C2 (APO) C2, and immunoglobulin heavy constant gamma 2 was changed between early and late CRC stages. Conclusion: Our results suggest that C5 could be a potential biomarker for CRC diagnosis. Further validation studies will aid the application of these new potential biomarkers to improve CRC diagnosis and patient care.

Non-metastatic primary neuroendocrine neoplasms of the breast: a reference cancer center's experience of a heterogenous entity.

Background: Primary neuroendocrine neoplasms of the breast (Br-NENs) are rare. The classification has been updated in recent years making interpretation of the data published challenging. It is unclear whether neuroendocrine differentiation is associated with poorer prognosis and what treatment approaches should be applied. Methods: The database for breast cancer patients treated between 2009 and 2022 at the Maria Sklodowska-Curie National Research Institute of Oncology Branch Krakow was explored to search for Br-NENs. Patients' medical and pathological data were collected and analyzed. Results: We included 22 females with Br-NEN without metastases at the time of diagnosis. The median age was 64 years (range: 28-88), Of the cases, 18 were hormone receptor positive, all were HER-2 negative, the median Ki67 was 27% (10-100%). The median tumor size at the time of diagnosis was 29.5mm (7-75mm), 9 patients were N-positive. DCIS was present in 5 cases. Only one case was negative for chromogranin and synaptophysin staining, but data were missing for 4 cases. Nine patients received adjuvant chemotherapy, mainly based on anthracyclines and taxanes, while 16 received adjuvant hormonal therapy and 15 received postoperative radiotherapy. Radical surgery was performed in all patients, but two underwent suboptimal tumorectomy. One patient had local recurrence, three experienced metastatic disease, all involving the lungs, but these patients are still alive. The median follow-up was 96 months (8-153). Two patients died, with a follow up time of no recurrence >4 years. Our results were compared to twelve case series collecting clinical data on Br-NENs, with median patient number of 10.5 (range: 3-142). Conclusion: Br-NENs represent a heterogenous group of diseases, lacking data from prospective studies or clinical trials. There are no established treatment standards tailored for Br-NENs. Our patients' cohort exhibited a favorable prognosis, potentially attributed to lower tumor stage and Ki67 index compared to other reported case series. We suggest that radical surgery and postoperative radiotherapy be administered akin to standard treatment for breast cancer of no special type. ESMO also advocates for this approach in systemic treatment, although we recommend considering platinum-based chemotherapy for patients with poorly differentiated Br-NENs exhibiting high Ki67.

Tumor Predisposing Post-Zygotic Chromosomal Alterations in Bladder Cancer-Insights from Histologically Normal Urothelium.

Bladder urothelial carcinoma (BLCA) is the 10th most common cancer with a low survival rate and strong male bias. We studied the field cancerization in BLCA using multi-sample- and multi-tissue-per-patient protocol for sensitive detection of autosomal post-zygotic chromosomal alterations and loss of chromosome Y (LOY). We analysed 277 samples of histologically normal urothelium, 145 tumors and 63 blood samples from 52 males and 15 females, using the in-house adapted Mosaic Chromosomal Alterations (MoChA) pipeline. This approach allows identification of the early aberrations in urothelium from BLCA patients. Overall, 45% of patients exhibited at least one alteration in at least one normal urothelium sample. Recurrence analysis resulted in 16 hotspots composed of either gains and copy number neutral loss of heterozygosity (CN-LOH) or deletions and CN-LOH, encompassing well-known and new BLCA cancer driver genes. Conservative assessment of LOY showed 29%, 27% and 18% of LOY-cells in tumors, blood and normal urothelium, respectively. We provide a proof of principle that our approach can characterize the earliest alterations preconditioning normal urothelium to BLCA development. Frequent LOY in blood and urothelium-derived tissues suggest its involvement in BLCA.

Plasma protein changes reflect colorectal cancer development and associated inflammation.

Introduction: Colorectal cancer (CRC) is the third most common malignancy and the second leading cause of death worldwide. Efficient non-invasive blood-based biomarkers for CRC early detection and prognosis are urgently needed. Methods: To identify novel potential plasma biomarkers, we applied a proximity extension assay (PEA), an antibody-based proteomics strategy to quantify the abundance of plasma proteins in CRC development and cancer-associated inflammation from few µL of plasma sample. Results: Among the 690 quantified proteins, levels of 202 plasma proteins were significantly changed in CRC patients compared to age-and-sex-matched healthy subjects. We identified novel protein changes involved in Th17 activity, oncogenic pathways, and cancer-related inflammation with potential implications in the CRC diagnosis. Moreover, the interferon γ (IFNG), interleukin (IL) 32, and IL17C were identified as associated with the early stages of CRC, whereas lysophosphatidic acid phosphatase type 6 (ACP6), Fms-related tyrosine kinase 4 (FLT4), and MANSC domain-containing protein 1 (MANSC1) were correlated with the late-stages of CRC. Discussion: Further study to characterize the newly identified plasma protein changes from larger cohorts will facilitate the identification of potential novel diagnostic, prognostic biomarkers for CRC.

Comprehensive cancer-oriented biobanking resource of human samples for studies of post-zygotic genetic variation involved in cancer predisposition.

The progress in translational cancer research relies on access to well-characterized samples from a representative number of patients and controls. The rationale behind our biobanking are explorations of post-zygotic pathogenic gene variants, especially in non-tumoral tissue, which might predispose to cancers. The targeted diagnoses are carcinomas of the breast (via mastectomy or breast conserving surgery), colon and rectum, prostate, and urinary bladder (via cystectomy or transurethral resection), exocrine pancreatic carcinoma as well as metastases of colorectal cancer to the liver. The choice was based on the high incidence of these cancers and/or frequent fatal outcome. We also collect age-matched normal controls. Our still ongoing collection originates from five clinical centers and after nearly 2-year cooperation reached 1711 patients and controls, yielding a total of 23226 independent samples, with an average of 74 donors and 1010 samples collected per month. The predominant diagnosis is breast carcinoma, with 933 donors, followed by colorectal carcinoma (383 donors), prostate carcinoma (221 donors), bladder carcinoma (81 donors), exocrine pancreatic carcinoma (15 donors) and metachronous colorectal cancer metastases to liver (14 donors). Forty percent of the total sample count originates from macroscopically healthy cancer-neighboring tissue, while contribution from tumors is 12%, which adds to the uniqueness of our collection for cancer predisposition studies. Moreover, we developed two program packages, enabling registration of patients, clinical data and samples at the participating hospitals as well as the central system of sample/data management at coordinating center. The approach used by us may serve as a model for dispersed biobanking from multiple satellite hospitals. Our biobanking resource ought to stimulate research into genetic mechanisms underlying the development of common cancers. It will allow all available "-omics" approaches on DNA-, RNA-, protein- and tissue levels to be applied. The collected samples can be made available to other research groups.

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.

Immune cells lacking Y chromosome show dysregulation of autosomal gene expression.

Epidemiological investigations show that mosaic loss of chromosome Y (LOY) in leukocytes is associated with earlier mortality and morbidity from many diseases in men. LOY is the most common acquired mutation and is associated with aberrant clonal expansion of cells, yet it remains unclear whether this mosaicism exerts a direct physiological effect. We studied DNA and RNA from leukocytes in sorted- and single-cells in vivo and in vitro. DNA analyses of sorted cells showed that men diagnosed with Alzheimer's disease was primarily affected with LOY in NK cells whereas prostate cancer patients more frequently displayed LOY in CD4 + T cells and granulocytes. Moreover, bulk and single-cell RNA sequencing in leukocytes allowed scoring of LOY from mRNA data and confirmed considerable variation in the rate of LOY across individuals and cell types. LOY-associated transcriptional effect (LATE) was observed in ~ 500 autosomal genes showing dysregulation in leukocytes with LOY. The fraction of LATE genes within specific cell types was substantially larger than the fraction of LATE genes shared between different subsets of leukocytes, suggesting that LOY might have pleiotropic effects. LATE genes are involved in immune functions but also encode proteins with roles in other diverse biological processes. Our findings highlight a surprisingly broad role for chromosome Y, challenging the view of it as a "genetic wasteland", and support the hypothesis that altered immune function in leukocytes could be a mechanism linking LOY to increased risk for disease.

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.

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.

Expression of metallothionein 3 in ductal breast cancer.

Metallothionein 3 (MT-3) has the ability to regulate the growth of nerve cells, but the significance of MT-3 expression outside the central nervous system and its participation in carcinogenesis have not yet been clarified. The aim of our study was to investigate the expression of MT-3 in ductal breast cancer and to determine its relationship with well-defined clinicopathological factors in this type of tumor. The study was conducted on 134 cases of invasive ductal breast carcinoma (IDC), 42 samples of non-malignant breast tissue (NMBT), and 26 cases of mastopathy. Moreover, selected breast cancer cell lines (MCF-7, SKBR-3, MDA-MB-231, BO2) and normal human breast epithelial cells (hTERT-HME1) were used. The expression of MT-3 was examined on the protein level using immunohistochemistry and on the mRNA level using real-time PCR. It was shown that the MT-3 protein in cells of IDC and mastopathy appeared in the cytoplasm as well as in the cell nuclei. Both the cytoplasmic and nuclear expression of MT-3 was significantly lower in IDC than in the mastopathies (p<0.0001 and p<0.001). However, no significant correlation was demonstrated between the level of MT-3 protein and the studied clinicopathological factors. The mRNA expression of MT-3 in IDC was also lower than in non­malignant breast tissue (p<0.0001). Furthermore, in the cases of IDC with lymph node metastasis, the level of MT-3 mRNA was significantly lower than in the cases without metastasis (p=0.0199). The expression of MT-3 mRNA in breast cancer cell lines was significantly lower than in the normal human breast epithelial cell line (p<0.001). These results suggest that MT-3 may play a role in the malignant transformation of breast epithelial cells and in tumor progression.

Prolactin-induced protein as a potential therapy response marker of adjuvant chemotherapy in breast cancer patients.

Many studies are dedicated to exploring the molecular mechanisms of chemotherapy-resistance in breast cancer (BC). Some of them are focused on searching for candidate genes responsible for this process. The aim of this study was typing the candidate genes associated with the response to standard chemotherapy in the case of invasive ductal carcinoma. Frozen material from 28 biopsies obtained from IDC patients with different responses to chemotherapy were examined using gene expression microarray, Real-Time PCR (RT-PCR) and Western blot (WB). Based on the microarray results, further analysis of candidate gene expression was evaluated in 120 IDC cases by RT-PCR and in 224 IDC cases by immunohistochemistry (IHC). The results were correlated with clinical outcome and molecular subtype of the BC. Gene expression microarray revealed Prolactin-Induced Peptide (PIP) as a single gene differentially expressed in BC therapy responder or non-responder patients (p <0.05). The level of PIP expression was significantly higher in the BC therapy responder group than in the non-responder group at mRNA (p=0.0092) and protein level (p=0.0256). Expression of PIP mRNA was the highest in estrogen receptor positive (ER+) BC cases (p=0.0254) and it was the lowest in triple negative breast cancer (TNBC) (p=0.0336). Higher PIP mRNA expression was characterized by significantly longer disease free survival (DFS, p=0.0093), as well as metastasis free survival (MFS, p=0.0144). Additionally, PIP mRNA and PIP protein expression levels were significantly higher in luminal A than in other molecular subtypes and TNBC. Moreover significantly higher PIP expression was observed in G1, G2 vs. G3 cases (p=0.0027 and p=0.0013, respectively). Microarray analysis characterized PIP gene as a candidate for BC standard chemotherapy response marker. Analysis of clinical data suggests that PIP may be a good prognostic and predictive marker in IDC patients. Higher levels of PIP were related to longer DFS and MFS but not with OS.

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 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.

Signatures of post-zygotic structural genetic aberrations in the cells of histologically normal breast tissue that can predispose to sporadic breast cancer.

Sporadic breast cancer (SBC) is a common disease without robust means of early risk prediction in the population. We studied 282 females with SBC, focusing on copy number aberrations in cancer-free breast tissue (uninvolved margin, UM) outside the primary tumor (PT). In total, 1162 UMs (1-14 per breast) were studied. Comparative analysis between UM(s), PT(s), and blood/skin from the same patient as a control is the core of the study design. We identified 108 patients with at least one aberrant UM, representing 38.3% of cases. Gains in gene copy number were the principal type of mutations in microscopically normal breast cells, suggesting that oncogenic activation of genes via increased gene copy number is a predominant mechanism for initiation of SBC pathogenesis. The gain of ERBB2, with overexpression of HER2 protein, was the most common aberration in normal cells. Five additional growth factor receptor genes (EGFR, FGFR1, IGF1R, LIFR, and NGFR) also showed recurrent gains, and these were occasionally present in combination with the gain of ERBB2. All the aberrations found in the normal breast cells were previously described in cancer literature, suggesting their causative, driving role in pathogenesis of SBC. We demonstrate that analysis of normal cells from cancer patients leads to identification of signatures that may increase risk of SBC and our results could influence the choice of surgical intervention to remove all predisposing cells. Early detection of copy number gains suggesting a predisposition toward cancer development, long before detectable tumors are formed, is a key to the anticipated shift into a preventive paradigm of personalized medicine for breast cancer.

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.