Prelude to malignancy: A gene expression signature in normal mammary gland from breast cancer patients suggests pre-tumorous alterations and is associated with adverse outcomes.

Despite advances in early detection and treatment strategies, breast cancer recurrence and mortality remain a significant health issue. Recent insights suggest the prognostic potential of microscopically healthy mammary gland, in the vicinity of the breast lesion. Nonetheless, a comprehensive understanding of the gene expression profiles in these tissues and their relationship to patient outcomes remain missing. Furthermore, the increasing trend towards breast-conserving surgery may inadvertently lead to the retention of existing cancer-predisposing mutations within the normal mammary gland. This study assessed the transcriptomic profiles of 242 samples from 83 breast cancer patients with unfavorable outcomes, including paired uninvolved mammary gland samples collected at varying distances from primary lesions. As a reference, control samples from 53 mammoplasty individuals without cancer history were studied. A custom panel of 634 genes linked to breast cancer progression and metastasis was employed for expression profiling, followed by whole-transcriptome verification experiments and statistical analyses to discern molecular signatures and their clinical relevance. A distinct gene expression signature was identified in uninvolved mammary gland samples, featuring key cellular components encoding keratins, CDH1, CDH3, EPCAM cell adhesion proteins, matrix metallopeptidases, oncogenes, tumor suppressors, along with crucial genes (FOXA1, RAB25, NRG1, SPDEF, TRIM29, and GABRP) having dual roles in cancer. Enrichment analyses revealed disruptions in epithelial integrity, cell adhesion, and estrogen signaling. This signature, named KAOS for Keratin-Adhesion-Oncogenes-Suppressors, was significantly associated with reduced tumor size but increased mortality rates. Integrating molecular assessment of non-malignant mammary tissue into disease management could enhance survival prediction and facilitate personalized patient care.

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.

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.

High prevalence of somatic PIK3CA and TP53 pathogenic variants in the normal mammary gland tissue of sporadic breast cancer patients revealed by duplex sequencing.

The mammary gland undergoes hormonally stimulated cycles of proliferation, lactation, and involution. We hypothesized that these factors increase the mutational burden in glandular tissue and may explain high cancer incidence rate in the general population, and recurrent disease. Hence, we investigated the DNA sequence variants in the normal mammary gland, tumor, and peripheral blood from 52 reportedly sporadic breast cancer patients. Targeted resequencing of 542 cancer-associated genes revealed subclonal somatic pathogenic variants of: PIK3CA, TP53, AKT1, MAP3K1, CDH1, RB1, NCOR1, MED12, CBFB, TBX3, and TSHR in the normal mammary gland at considerable allelic frequencies (9 × 10-2- 5.2 × 10-1), indicating clonal expansion. Further evaluation of the frequently damaged PIK3CA and TP53 genes by ultra-sensitive duplex sequencing demonstrated a diversified picture of multiple low-level subclonal (in 10-2-10-4 alleles) hotspot pathogenic variants. Our results raise a question about the oncogenic potential in non-tumorous mammary gland tissue of breast-conserving surgery patients.

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.

Clinical outcomes of an intraoperative surgical margin assessment using the fresh frozen section method in patients with invasive breast cancer undergoing breast-conserving surgery - a single center analysis.

Breast conserving treatment (BCT) is a safe standard therapeutic method in patients with early invasive breast cancer. However, it is associated with an increased risk of residual neoplastic tissues in surgical margins. The aim of this study was to assess the outcome of the use of the intraoperative pathologic analysis by the frozen section (FS) method for evaluation of the extent of the primary lumpectomy. The study concerns a retrospective analysis of a group of 1102 patients who underwent BCT between Jan 2015 and Dec 2016. The assessment focused on the frequency of the intraoperative pathologic analysis of the primary lumpectomy extent (fresh frozen section method). The influence of the BCT specimen analysis method on the free margins width, as well as the rate and the cause of reoperation were evaluated. The intraoperative lumpectomy evaluation was performed in 45.8% (505/1102) of patients (Group I), while in the remaining 54.2% of the cases it was decided to abandon this procedure (Group II). Although in 72 (14.3%) patients the intraoperative analysis gave negative results, the margins contained residual tumor tissue (vs. 16.9% in Group II). In Group I, conversion from the previously planned BCT to mastectomy was necessary in 5.9% (30/505) patients (vs. 9.7% in Group II). The duration of surgery was 48.9 ± 17.3 minutes (Group I) and 42.9 ± 13.6 minutes (Group II). In patients undergoing BCT, the use of the intraoperative pathologic analysis by the FS method resulted in a reduction of the total number of reoperations performed due to residual tumor found in the margins following the primary lumpectomy. However, it statistically significantly extended the duration of the surgery.

The importance of the concept and histological criteria of "intraepithelial squamous cell carcinoma" of the esophagus: in comparison between Western and Japanese criteria.

BACKGROUND: There are differences in the histological diagnostic criteria for early stage gastrointestinal carcinoma between Western and Japanese pathologists. Western histological criteria of carcinoma are "presence of stromal invasion of neoplastic cells", while Japanese criteria are "the degree of cytological and structural abnormality of neoplastic cells, regardless of stromal invasion". The aim of the present study is to clarify and review the present status of the Western and Japanese histological criteria of early stage esophageal squamous cell carcinoma (SCC) and also to clarify their significance and accuracy. METHODS: Twenty-nine Polish, German, and Japanese pathologists participated in this study. A total of 18 histological slides of biopsy, endoscopic submucosal dissection (ESD), and surgical resection of esophageal squamous lesions were diagnosed using a virtual slide system. RESULTS: Most of noninvasive (intraepithelial) carcinomas diagnosed by Japanese pathologists were diagnosed as high- or low-grade dysplasia (intraepithelial neoplasia) or reactive atypia by the majority of Polish and German pathologists. Diagnoses of not only high-grade dysplasia but also low-grade dysplasia or reactive lesion by Western criteria were given for many biopsy specimens of cases in which the corresponding ESD or surgical specimens showed definite stromal invasion. CONCLUSION: There still exist differences in the histological diagnostic criteria for early stage esophageal carcinoma between Western and Japanese pathologists. The Japanese diagnostic criteria could improve agreement of diagnoses between biopsy and resected specimens of esophageal SCC. Moreover, diagnostic approaches using Western criteria may cause delay in the early diagnosis and treatment of esophageal SCC.

Comparison of FLT-PET/CT and CECT in gastric cancer diagnosis.

AIM: To date, no data are available on the use of 18-fluorothymidine positron emission tomography/computed tomography (FLT-PET/CT) for preoperative gastric cancer staging. Herein, we attempt to assess the value of FLT-PET/CT for preoperative gastric cancer staging in comparison with contrast-enhanced computed tomography (CECT). MATERIALS AND METHODS: In a group of 96 gastric cancer patients, 96 FLT-PET/CT, 56 abdominal cavity CECT, and 51 resective operations were done. All three (FLT-PET/CT, CECT, and resective operation) were done in 29 patients. The results of FLT-PET/CT, CECT, and histopathological examinations were used to assess the ability of FLT-PET/CT and CECT to identify primary tumors, regional nodal metastases, and distant abdominal metastases. Assessment of regional lymph nodes was based on SUVmax in FLT-PET/CT and SAD (short-axis diameter) in CECT. RESULTS: In the group of 56 patients examined with FLT-PET/CT and CECT, identification of the primary tumor was possible in 56 cases (100%) and in 53 cases (94.6%), respectively, (p = 0.013). Using ROC curve, the sensitivity and specificity of FLT-PET/CT in metastatic regional lymph node assessment were higher than those of CECT (p = 0.0033). FLT-PE/CT enabled identification of a greater number of extraregional abdominal metastases than CECT (n = 56; 19 vs. 15, respectively), but the difference was not statistically significant (p > 0.41). CONCLUSIONS: The ability of FLT-PET/CT to identify primary tumors is greater than that of CECT, and thus FLT-PET/CT was better in evaluating regional nodal metastases. FLT-PET/CT enabled identification of a greater number of abdominal metastases than CECT, but the difference was not statistically significant.

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.

Problems concerning patients' qualification for surgical procedures allowing for evaluation of the condition of axillary fossa lymph nodes in the radical treatment of breast cancer.

UNLABELLED: The presence of metastases in the lymph nodes of the axillary fossa is the most important prognostic factor in patients with breast cancer. The surgical treatment option required for evaluation of the condition of the axillary lymph nodes depends on the results of a preoperative physical examination of the patients. The aim of the study was to evaluate the correctness of breast cancer patients' qualification to surgical procedures allowing for evaluation of the condition of the axillary lymph nodes. MATERIAL AND METHODS: A retrospective analysis of a group of 963 patients with a diagnosed malignancy of the breast, treated surgically in the period from 01 Jan 2011 to 29 Feb 2012. Depending on the result of evaluation of the axillary lymph node clinical condition, the patients underwent sentinel lymph node biopsy or elective axillary lymphadenectomy. RESULTS: In 27.4% of patients subjected to excision of the sentinel lymph node, metastatic lesions were found in the lymph nodes removed during the procedure. In most cases (98.1%) that concerned the lymph nodes of the lower part of the axilla. In 17.4% of patients, metastases were located also in the middle or upper part (9%). In the group of patients primarily qualified to lymphadenectomy, the metastatic lesions in the axillary lymph nodes were diagnosed in 67.2% of patients. They were most commonly located in the lower part of the axillary fossa (in 96.7% of cases), and in 68.8% of patients in the middle or upper part (35.8%). CONCLUSIONS: Biopsy of the sentinel lymph node in patients with clinically advanced breast cancer is an effective and safe method of evaluation of the condition of the axillary fossa lymph nodes. A high rate of false positive results concerning the clinical stage of the disease requires changing the rules of patients' qualification to elective axillary lymphadenectomy.

Application of immunohistochemistry for detection of metastases in sentinel lymph nodes of non-advanced breast cancer patients.

Any diagnostic method for detection of occult metastases in sentinel lymph node (SLN) could change postoperative therapeutic management. In this study, we attempted to evaluate the usefulness of immunohistochemical (IHC) studies for histopathological SLN examination and their impact on the diagnosis of metastatic lesions. Analyzed data concerned 1358 breast cancer patients referred for sentinel lymph node biopsy (SLNB) between 2004 and 2012 with particular emphasis on broadening the scope of postoperative SLN histopathological assessment to include IHC. Sentinel lymph node involvement was diagnosed in 22.2% of patients. Use of the IHC method facilitated detection of 21.4% of all diagnosed SLN lesions (9.9% of macrometastases, 60.0% of micrometastases, 100% of isolated tumor cells). 61.6% sensitivity and 100% specificity were obtained in the group of patients who underwent intraoperative SLN histopathological examination. Use of immunohistochemistry for diagnostics of sentinel lymph node metastases in patients with breast cancer enables detection of a greater proportion of metastases, thus modifying both surgical (SLN macrometastases) and adjuvant treatment. As compared to pN0 patients and those with a metastasis found in HE staining, in patients with a metastasis in the sentinel lymph node diagnosed in IHC studies, no statistically significant differences were found concerning the long-term results of the implemented treatment.

Core-needle biopsy under CT fluoroscopy guidance and fine-needle aspiration cytology: Comparison of diagnostic yield in the diagnosis of lung and mediastinum tumors. Analysis of frequency and types of complications.

BACKGROUND: Patients with pathological tissue mass in thoracic cage found with imaging require histopathological or cytological confirmation of malignancy before treatment. The tissue material essential for patomorphological evaluation can be acquired with fine-needle aspiration biopsies (FNAB) controlled with CT and core-needle biopsy (CNB) under real-time CT fluoroscopy guidance. The purpose of this work is to carry out a retrospective analysis of the two methods with regards to their informativity, frequency and the kind of complications. MATERIAL/METHODS: From January, 2012 to May 2013, 76 core-needle biopsies of lung and mediastinum tumors were conducted and compared with 86 fine-needle aspiration biopsies(FNAB) of lung and mediastinum tumors, including 30 patients who underwent FNAB and were referred to CNB in order to specify the diagnosis. RESULTS: Complete histopathological diagnosis was made in 91% with the use of CNB and in 37% when FNAB was the chosen method. Early complications were observed in 32% patients who underwent BG and in group of 11% who underwent FNAB. Late complications, however, appeared in 29% patients after CNB and 13% after FNAB. In 24 cases CNB specified the complete diagnosis. CONCLUSIONS: Core-needle biopsy in comparison to fine-needle aspiration biopsy has more frequent rate of negligible complications, however, it offers higher diagnostic yield for diagnostic of lung and mediastinum neoplastic disease and allows for more precise diagnosis of focal lesions.

Post-zygotic and inter-individual structural genetic variation in a presumptive enhancer element of the locus between the IL10Rß and IFNAR1 genes.

Although historically considered as junk-DNA, tandemly repeated sequence motifs can affect human phenotype. For example, variable number tandem repeats (VNTR) with embedded enhancers have been shown to regulate gene transcription. The post-zygotic variation is the presence of genetically distinct populations of cells in an individual derived from a single zygote, and this is an understudied aspect of genome biology. We report somatically variable VNTR with sequence properties of an enhancer, located upstream of IFNAR1. Initially, SNP genotyping of 63 monozygotic twin pairs and multiple tissues from 21 breast cancer patients suggested a frequent post-zygotic mosaicism. The VNTR displayed a repeated 32 bp core motif in the center of the repeat, which was flanked by similar variable motifs. A total of 14 alleles were characterized based on combinations of segments, which showed post-zygotic and inter-individual variation, with up to 6 alleles in a single subject. Somatic variation occurred in ∼24% of cases. In this hypervariable region, we found a clustering of transcription factor binding sites with strongest sequence similarity to mouse Foxg1 transcription factor binding motif. This study describes a VNTR with sequence properties of an enhancer that displays post-zygotic and inter-individual genetic variation. This element is within a locus containing four related cytokine receptors: IFNAR2, IL10Rß, IFNAR1 and IFNGR2, and we hypothesize that it might function in transcriptional regulation of several genes in this cluster. Our findings add another level of complexity to the variation among VNTR-based enhancers. Further work may unveil the normal function of this VNTR in transcriptional control and its possible involvement in diseases connected with these receptors, such as autoimmune conditions and cancer.

(18)F-FLT PET/CT in Patients with Gastric Carcinoma.

The aim of the study was to evaluate the usefulness of 18F-FLT PET/CT in the detection and differentiation of gastric cancers (GC). 104 consecutive patients (57 cases of adenocarcinoma tubulare (G2 and G3), 17 cases of mucinous adenocarcinoma, 6 cases of undifferentiated carcinoma, 14 cases of adenocarcinoma partim mucocellulare, and 10 cases of end stage gastric cancer) with newly diagnosed advanced gastric cancer were examined with FLT PET/CT. For quantitative and comparative analyses, the maximal standardized uptake value (SUVmax) was calculated for both the tumors and noninvaded gastric wall. Results. There were found, in the group of adenocarcinoma tubulare, SUVmax 1.5-23.1 (7.46 ± 4.57), in mucinous adenocarcinoma, SUVmax 2.3-10.3 (5.5 ± 2.4), in undifferentiated carcinoma, SUVmax 3.1-13.6 (7.28 ± 3.25), in adenocarcinoma partim mucocellulare, SUVmax 2-25.3 (7.7 ± 6.99), and, in normal gastric wall, SUVmax 1.01-2.55 (1.84 ± 0.35). For the level of 2.6 cut-off value between the normal wall and neoplasm FLT uptake from ROC analysis, all but five gastric cancers showed higher accumulation of FLT than noninfiltrated mucosa. Conclusion. Gastric cancer presents higher accumulation of 18F-FLT than normal, distended gastric mucosa. Significantly higher accumulation was shown in cancers better differentiated and with higher cellular density.

Frequent genetic differences between matched primary and metastatic breast cancer provide an approach to identification of biomarkers for disease progression.

Breast cancer is a major cause of morbidity and mortality in women and its metastatic spread is the principal reason behind the fatal outcome. Metastasis-related research of breast cancer is however underdeveloped when compared with the abundant literature on primary tumors. We applied an unexplored approach comparing at high resolution the genomic profiles of primary tumors and synchronous axillary lymph node metastases from 13 patients with breast cancer. Overall, primary tumors displayed 20% higher number of aberrations than metastases. In all but two patients, we detected in total 157 statistically significant differences between primary lesions and matched metastases. We further observed differences that can be linked to metastatic disease and there was also an overlapping pattern of changes between different patients. Many of the differences described here have been previously linked to poor patient survival, suggesting that this is a viable approach toward finding biomarkers for disease progression and definition of new targets useful for development of anticancer drugs. Frequent genetic differences between primary tumors and metastases in breast cancer also question, at least to some extent, the role of primary tumors as a surrogate subject of study for the systemic disease.

Chromosome 22 array-CGH profiling of breast cancer delimited minimal common regions of genomic imbalances and revealed frequent intra-tumoral genetic heterogeneity.

Breast cancer is a common malignancy and the second most frequent cause of death among women. Our aim was to perform DNA copy number profiling of 22q in breast tumors using a methodology which is superior, as compared to the ones applied previously. We studied 83 biopsies from 63 tumors obtained from 60 female patients. A general conclusion is that multiple distinct patterns of genetic aberrations were observed, which included deletion(s) and/or gain(s), ranging in size from affecting the whole chromosome to only a few hundred kb. Overall, the analysis revealed genomic imbalances of 22q in 22% (14 out of 63) of tumors. The predominant profile (11%) was monosomy 22. The smallest identified candidate region, in the vicinity of telomere of 22q, encompasses approximately 220 kb and was involved in all but one of the tumors with aberrations on chromosome 22. This segment is dense in genes and contains 11 confirmed and one predicted gene. The availability of multiple biopsies from a single tumor provides an excellent opportunity for analysis of possible intra-tumor differences in genetic profiles. In 15 tumors we had access to two or three biopsies derived from the same lesion and these were studied independently. Four out of 15 (26.6%) tumors displayed indications of clonal intra-tumor genotypic differences, which should be viewed as a high number, considering that we studied in detail only a single human chromosome. Our results open up several avenues for continued genetic research of breast cancer.

Microarray-based survey of CpG islands identifies concurrent hyper- and hypomethylation patterns in tissues derived from patients with breast cancer.

Maintenance of CpG island methylation in the genome is crucial for cellular homeostasis and this balance is disrupted in cancer. Our rationale was to compare the methylation of CpG islands in tissues (tumor, healthy breast and blood) from patients with breast cancer. We studied 72 genes in 103 samples using microarray hybridization and bisulfite sequencing. We observed tumor specific hyper- or hypomethylation of five genes; COL9A1, MT1A, MT1J, HOXA5 and FLJ45983. A general drop of methylation in COL9A1 was apparent in tumors, when compared with blood and healthy breast tissue. Furthermore, one tumor displayed a complete loss of methylation of all five genes, suggesting overall impairment of methylation. The downstream, evolutionary conserved island of HOXA5 showed hypomethylation in 18 tumors and complete methylation in others. This CpG island also displayed a semimethylated state in the majority of normal breast samples, when compared to complete methylation in blood. Distinct methylation patterns were further seen in MT1J and MT1A, belonging to the metallothionein gene family. The CpG islands of these genes are spaced by 2 kb, which shows selective methylation of two structurally and functionally related genes. The promoters of FLJ45983 and MT1A were methylated above 25% in 18 primary and metastatic tumors. Concurrently, there was also >10% methylation of healthy breast tissue in 11 and 5 samples, respectively. This suggests that the methylation process for the latter two genes takes place already in normal breast cells. Our results also point to a considerable heterogeneity of epigenetic disturbance in breast cancer. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.