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.

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.

Assessment of HPV16 infection in patients with laryngeal cancer.

The purpose of the study was to investigate HPV16 infection in laryngeal cancer patients treated with surgery and adjuvant radiotherapy as well as to analyze treatment results in relation to HPV16 infection and selected clinical, histopathological, and radiotherapy parameters. A retrospective analysis was performed in a group of 60 patients with squamous cell carcinoma of the larynx treated surgically and qualified for adjuvant radiotherapy at the Oncology Center in Cracow between 1995 and 2001. The studied group consisted of 57 men (95%) and 3 women (5%) of mean age of 56 years. In 13 patients (22%) underweight was noted. In the analyzed material, locally advanced laryngeal cancer prevailed (pT3-pT4) - 52 cases (87%), with the involvement of cervical lymph nodes (pN+) - 32 cases (53%). Histopathological examination revealed that microscopic radicality was not obtained in 18 patients (30%). Human papillomavirus 16 infection status as well as infection type (integrated, episomal, or mixed) were assessed in each patient by means of quantitative polymerase chain reaction (qPCR) using real-time detection. The 5-year OS, DFS, and LC rates were 45%, 61%, and 69%, respectively. Multivariate analysis revealed that local relapse risk and local failure risk were statistically significantly influenced by underweight and positive surgical margin. Underweight had also a statistically significant impact on death risk. The HPV16 infection was noticed in 4 cancers (6.8%). In all cases it was the same episomal type. On the basis of our observations it can be assumed that HPV infection does not play an important role in etiology of laryngeal cancer. Although, further study is needed in larger patient populations; optimal methodology for detecting HPV infection should also be determined. Positive surgical margin has a significant effect on worse treatment outcomes. Underweight before radiotherapy diminishes the probability of treatment success and survival of laryngeal cancer patients.

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.

Active HPV infection and its influence on survival in head and neck squamous-cell cancer.

PURPOSE: HPV is involved in the development of some head and neck squamous-cell carcinomas (HNSCC). It was suggested that only transcriptionally active virus can induce carcinogenesis, therefore, the aim of our study was to analyze the frequency of active HPV infection, virus type, and its prognostic role in HNSCC patients. METHODS: Status of active HPV infection was assessed for 155 HNSCC patients based on p16 expression and HPV DNA presence. Univariate and multivariate analyses with Cox proportional regression model were performed to select independent prognostic factors. RESULTS: Active HPV infection was detected in 20.65% of patients. We identified 16.0, 40.9 and 1.7% of HPV positive oral cavity, oropharyngeal, and laryngeal cancer cases, respectively. HPV16 was dominant (81.25%) followed by HPV35 (9.38%) and double infections with HPV16 and 35 (6.25%) or HPV35 and 18 (3.12%). Patients with active HPV infection demonstrated significantly higher survival than HPV negative ones (OS 80.89% vs. 37.08%, p = 0.000; DFS 93.0% vs. 53.35%, p = 0.000, respectively). Longer OS and DFS were maintained for infected patients when oropharyngeal and non-oropharyngeal cases were analyzed separately. Interestingly, all patients infected with other than HPV16 types survived 5 years without cancer progression. In the analyzed group of 155 patients the strongest independent favourable prognostic factor for both OS and DFS was HPV presence. CONCLUSIONS: High prevalence of HPV-driven HNSCC (mostly within oropharynx) was detected, with HPV16 type the most frequent, followed by HPV35 and HPV18. The presence of active HPV infection improved survival of both oropharyngeal and non-oropharyngeal cancer patients and should be taken into account in treatment planning.

Differences in the prognosis of HPV16-positive patients with squamous cell carcinoma of head and neck according to viral load and expression of P16.

PURPOSE: To evaluate the impact of HPV16 load (VL-the number of virus genome copies per cell) and P16 expression on prognosis of patients with squamous cell carcinomas (SCCs) of head and neck (HN). MATERIALS AND METHODS: HPV16 presence was assessed in the group of 109 patients with HNSCCs by quantitative polymerase chain reaction (qPCR). VL (assessed by qPCR) and P16 expression (evaluated by immunohistochemistry) were analysed only in the subgroup of HPV16-positive tumours. These features were correlated with 5-year overall survival (OS) and disease-free survival (DFS). RESULTS: HPV16 infection was found in 36 tumours (33.0%). Virus-positive patients had better OS and DFS than those without infection (P = 0.041 and 0.005). Among HPV16-positive HNSCCs, 18 (50.0%) had higher VL (median value > 6764.3 copies/cell) and 25 (73.5%) P16 over expression. The significant differences in OS and DFS (P = 0.008 and 0.004) were noticed according to VL, wherein 100% DFS was found for patients with higher VL. According to P16 expression, significant difference was found only for OS (P = 0.020). In multivariate analysis, VL (P = 0.045; HR = 2.795; CI 0.121-1.060) and the level of smoking (P = 0.023, HR = 2.253; CI 1.124-4.514) were independent factors affecting DFS of HPV16-positive patients. CONCLUSION: On the basis of viral load, it is possible to differentiate prognosis of patients with HPV16-positive HNSCCs. In this subgroup, viral load has stronger prognostic potential than P16 expression.

The role of histology, grading, location of tumour and ploidy in evaluation of outcome in patients with liposarcoma.

Department of Tumour Pathology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Kraków, Poland The review of literature indicates that several clinico-morphological factors such as location of the primary tumour as well as its size, histologic subtype, and grade or even selected molecular changes may significantly affect survival of liposarcoma (LPS) patients. Data concerning prognostic importance of DNA ploidy status in LPS cells are extremely limited and results of flow cytometry (FCM) studies have never been compiled with the current classification of malignant adipocytic tumours. Based on evaluation of material from 54 liposarcomas which was available for both histological and FCM analysis, we distinguished four prognostic groups of patients. The best prognosis was noticed for diploid and grade G1 well-differentiated or myxoid liposarcomas localised on extremities. None of the patients with lipoma-like WDLPS and myxoid liposarcoma grade 1 metastasised, while metastases were observed among patients with dedifferentiated LPS (70% of 5-year MFS) and cellular myxoid or round cell liposarcoma (20% of 5-year MFS, only). The metastasis-free survival curves for the above mentioned groups of patients differed significantly (p = 0.00001).

HPV16 detection by qPCR method in relation to quantity and quality of DNA extracted from archival formalin fixed and paraffin embedded head and neck cancer tissues by three commercially available kits.

The aim of the present study was to compare HPV16 detection by quantitative polymerase chain reaction (qPCR) in relation to the quantity and quality of DNA isolated from 21 formalin fixed and paraffin embedded (FFPE) head and neck cancer tissues by three commercially available kits: EX-WAX™ DNA Extraction Kit (M) (Merck Millipore, Darmstadt, Germany), QIAamp(®) DNA FFPE Tissue (Q) (Qiagen, Hilden, Germany) and ReliaPrep™ FFPE gDNA Miniprep System (P) (Promega, Madison, USA). Quantity of extracted DNA was assessed spectrophometrically and fluorometrically. Its quality was analyzed using A260/280 and A260/230 ratios and the ß-actin fragment amplifiability in qPCR. HPV16 presence was detected by qPCR, using specific primers and TaqMan probe. HPV infection was found in 8 DNA samples extracted with M kit (38.1%) and in 7 (33.3%) isolated with Q and P kits. Three samples from M and Q kits were characterized by HPV16 positivity and lack of ß-actin amplifiability. They had significantly lower A260/280 ratio (M: 1.6±0.0, p=0.044 and Q: 1.7±0.0, p=0.016) compared to samples with both fragments amplification (M: 1.7±0.0 and Q: 1.9±0.0). Therefore, for HPV detection by qPCR in FFPE tissues we recommend ReliaPrep™ FFPE gDNA Miniprep System.

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.

Clinico-morphological parameters affecting survival of patients with advanced cervical cancer.

The aim of the study was to investigate the prognostic significance of selected clinico-morphological parameters including Ki-67 antigen expression and microvessel density. The data of 122 patients with squamous cell carcinoma, FIGO stages IB-IIIB and treated with radiochemotherapy and brachytherapy were studied. Significant prognostic factors for disease-free survival in univariate analysis were the FIGO stage and the presence of atypical mitoses in carcinoma cells. Multivariate Cox analysis confirmed prognostic significance of the FIGO stage and Ki-67 expression with regard to disease-free survival. With regard to overall survival, the most important prognostic factor was Ki-67 antigen expression. The data concerning the pretreatment status of these parameters may be helpful in clinical practice.