Anti-SSA Ro52 and anti-Ro60 autoantibodies: association with clinical phenotypes.

OBJECTIVES: Anti-SSA autoantibodies can be differentiated according to their antigenic target proteins as anti-Ro60 (60 kDa) or anti-Ro52 (52 kDa). Anti-SSA(Ro60) antibodies are clearly associated with connective tissue diseases (CTD), but the clinical significance of anti-SSA(Ro52) antibodies remains unclear. The aim of the present study was to analyse the disease phenotype of patients with anti-Ro52 and/or anti-Ro60 antibodies. METHODS: A multicentre, cross-sectional study was carried out of positive anti-Ro52 and/or Ro60 antibodies patients followed at 10 Rheumatology centres from January 2018 until December 2021. Patients were categorised into 3 groups: group 1 (Ro52+/Ro60-); group 2 (Ro52-/Ro60+); group 3 (Ro52+/Ro60+). Antinuclear antibodies were evaluated by indirect immunofluorescence assay and further screened for anti-extractable nuclear antigen (ENA) antibodies. Demographicsand clinical data were compared between the 3 groups, by patients' medical chart review. Univariate analysis was performed and subsequently logistic regression was used to identify intergroup differences and calculate the odds ratio with a 95% confidence interval (95% CI). RESULTS: We included 776 patients [female: 83.1%; median age: 59 (46-71) years]. Groups 1, 2, and 3 comprised 31.1%, 32.6%, and 36.3% of the patients, respectively. Anti-Ro52 antibody alone was more frequently associated with non-rheumatic diseases, older age, and men (p<0.05). Among patients with CTD, the diagnosis of systemic lupus erythematosus is 3 and 2 times more prevalent in groups 2 and 3, respectively, than in group 1 [OR 2.8 (95% CI 1.60, 4.97), p<0.001; OR 2.2 (95% CI 1.28, 3.86), p<0.01]. In group 2, the diagnosis of undifferentiated CTD is more frequent than in the other groups. Group 1 was more frequently associated with inflammatory myositis than group 2 [OR 0.09 (95% CI 0.01, 0.33), p<0.001] or group 3 [OR 0.08 (95% CI 0.01, 0.29), p<0.001]. Group 1 was also more frequently associated with arthritis (p<0.01), interstitial lung disease (p<0.01), and myositis (p<0.01). CONCLUSIONS: Anti-Ro52+ antibody alone is frequently found in patients with non-rheumatic diseases. In addition, anti-Ro52+ antibody is also prevalent in patients with CTD and associated with clinical phenotypes that are different from anti-Ro60+ antibody.

Hepatitis B vaccination associated with low response in patients with rheumatic diseases treated with biologics.

BACKGROUND: Hepatitis B virus (HBV) vaccination is recommended for non-immunised patients with rheumatic diseases starting biological disease-modifying antirheumatic drugs (bDMARDs). There is some evidence that HBV vaccination is effective in patients under conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs), but it is currently unclear whether this also applies to bDMARDs. OBJECTIVES: To assess the efficacy and safety of HBV vaccination in patients with inflammatory arthritides treated with bDMARDs. METHODS: A prospective cohort with inflammatory arthritides treated with bDMARDs, negative for anti-HBs and anti-HBc and never vaccinated for HBV was recruited. Engerix B was administered at 0, 1 and 6 months and anti-HBs was reassessed ≥1 month after last dose. Response was defined as anti-HBs≥10 IU/L and compared against vaccinated healthy controls. Disease flare, serious adverse events and immune-related disorders not previously present were recorded. RESULTS: 62 patients, most treated with TNF inhibitors (TNFi), and 38 controls were recruited. Most patients were taking csDMARDs (67.7%) and were in remission/low disease activity (59.4%). Only 20/62 patients (32.3%) had a positive response to vaccination, in comparison to 36/38 age-matched controls (94.7%, p<0.001). Response was seen in 19/51 patients treated with TNFi (37.3%) and in 1/11 (9.1%) patients treated with non-TNFi (p=0.07), including 1/6 treated with tocilizumab (16.7%). Among TNFi, response rates ranged from 4/22 (18.2%) for infliximab to 8/14 (57.1%) for etanercept. No relevant safety issues were identified. CONCLUSIONS: HBV vaccination response in patients with rheumatic diseases treated with bDMARDs was poorer than expected. Our data reinforce the recommendation for vaccination prior to starting bDMARDs.

The idiopathic inflammatory myopathies module of the Rheumatic Diseases Portuguese Register.

AIMS: To characterise the idiopathic inflammatory myopathies (IIM) module of the Rheumatic Diseases Portuguese Register (Reuma.pt/myositis) and the patients in its cohort. METHODS: Reuma.pt is a web-based system with standardised patient files gathered in a registry. This was a multicentre open cohort study, including patients registered in Reuma.pt/myositis up to January 2022. RESULTS: Reuma.pt/myositis was designed to record all relevant data in clinical practice and includes disease-specific diagnosis and classification criteria, clinical manifestations, immunological data, and disease activity scores. Two hundred eighty patients were included, 71.4% female, 89.4% Caucasian, with a median age at diagnosis and disease duration of 48.9 (33.6-59.3) and 5.3 (3.0-9.8) years. Patients were classified as having definite (N=57/118, 48.3%), likely (N=23/118, 19.5%), or possible (N=2/118, 1.7%) IIM by 2017 EULAR/ACR criteria. The most common disease subtypes were dermatomyositis (DM, N=122/280, 43.6%), polymyositis (N=59/280, 21.1%), and myositis in overlap syndromes (N=41/280, 14.6%). The most common symptoms were proximal muscle weakness (N=180/215, 83.7%) and arthralgia (N=127/249, 52.9%), and the most common clinical signs were Gottron's sign (N=75/184, 40.8%) and heliotrope rash (N=101/252, 40.1%). Organ involvement included lung (N=78/230, 33.9%) and heart (N=11/229, 4.8%) involvements. Most patients expressed myositis-specific (MSA, N=158/242, 65.3%) or myositis-associated (MAA, 112/242, 46.3%) antibodies. The most frequent were anti-SSA/SSB (N=70/231, 30.3%), anti-Jo1 (N=56/236, 23.7%), and anti-Mi2 (N=31/212, 14.6%). Most patients had a myopathic pattern on electromyogram (N=101/138, 73.2%), muscle oedema in magnetic resonance (N=33/62, 53.2%), and high CK (N=154/200, 55.0%) and aldolase levels (N=74/135, 54.8%). Cancer was found in 11/127 patients (8.7%), most commonly breast cancer (N=3/11, 27.3%). Most patients with cancer-associated myositis had DM (N=8/11, 72.7%) and expressed MSA (N=6/11) and/or MAA (N=3/11). The most used drugs were glucocorticoids (N=201/280, 71.8%), methotrexate (N=117/280, 41.8%), hydroxychloroquine (N=87/280, 31.1%), azathioprine (N=85/280, 30.4%), and mycophenolate mofetil (N=56/280, 20.0%). At the last follow-up, there was a median MMT8 of 150 (142-150), modified DAS skin of 0 (0-1), global VAS of 10 (0-50) mm, and HAQ of 0.125 (0.000-1.125). CONCLUSIONS: Reuma.pt/myositis adequately captures the main features of inflammatory myopathies' patients, depicting, in this first report, a heterogeneous population with frequent muscle, joint, skin, and lung involvements.

Predictors of cardiac involvement in idiopathic inflammatory myopathies.

Objectives: Idiopathic inflammatory myopathies (IIM) are a group of rare disorders that can affect the heart. This work aimed to find predictors of cardiac involvement in IIM. Methods: Multicenter, open cohort study, including patients registered in the IIM module of the Rheumatic Diseases Portuguese Register (Reuma.pt/Myositis) until January 2022. Patients without cardiac involvement information were excluded. Myo(peri)carditis, dilated cardiomyopathy, conduction abnormalities, and/or premature coronary artery disease were considered. Results: 230 patients were included, 163 (70.9%) of whom were females. Thirteen patients (5.7%) had cardiac involvement. Compared with IIM patients without cardiac involvement, these patients had a lower bilateral manual muscle testing score (MMT) at the peak of muscle weakness [108.0 ± 55.0 vs 147.5 ± 22.0, p=0.008] and more frequently had oesophageal [6/12 (50.0%) vs 33/207 (15.9%), p=0.009] and lung [10/13 (76.9%) vs 68/216 (31.5%), p=0.001] involvements. Anti-SRP antibodies were more commonly identified in patients with cardiac involvement [3/11 (27.3%) vs 9/174 (5.2%), p=0.026]. In the multivariate analysis, positivity for anti-SRP antibodies (OR 104.3, 95% CI: 2.5-4277.8, p=0.014) was a predictor of cardiac involvement, regardless of sex, ethnicity, age at diagnosis, and lung involvement. Sensitivity analysis confirmed these results. Conclusion: Anti-SRP antibodies were predictors of cardiac involvement in our cohort of IIM patients, irrespective of demographical characteristics and lung involvement. We suggest considering frequent screening for heart involvement in anti-SRP-positive IIM patients.

A COVID-19 Outbreak in a Rheumatology Department Upon the Early Days of the Pandemic.

Objectives: To describe our experience with a coronavirus disease 2019 (COVID-19) outbreak within a large rheumatology department early in the pandemic. Methods: Symptomatic and asymptomatic healthcare workers (HCWs) had a naso-oropharyngeal swab for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and were followed clinically. Reverse transcription polymerase-chain reaction (RT-PCR) was repeated to document cure, and serological response was assessed. Patients with risk contacts within the department in the 14 days preceding the outbreak were screened for COVID-19 symptoms. Results: 14/34 HCWs (41%; 40 ± 14 years, 71% female) tested positive for SARS-CoV-2, and 11/34 (32%) developed symptoms but were RT-PCR-negative. Half of RT-PCR-positive HCWs did not report fever, cough, or dyspnea before testing, which were absent in 3/14 cases (21%). Mild disease prevailed (79%), but 3 HCWs had moderate disease requiring further assessment, which excluded severe complications. Nevertheless, symptom duration (28 ± 18 days), viral shedding (31 ± 10 days post-symptom onset, range 15-51), and work absence (29 ± 28 days) were prolonged. 13/14 (93%) of RT-PCR-positive and none of the RT-PCR-negative HCWs had a positive humoral response Higher IgG indexes were observed in individuals over 50 years of age (14.5 ± 7.7 vs. 5.0 ± 4.4, p = 0.012). Of 617 rheumatic patients, 8 (1.3%) developed COVID-19 symptoms (1/8 hospitalization, 8/8 complete recovery), following a consultation/procedure with an asymptomatic (7/8) or mildly symptomatic (1/8) HCW. Conclusions: A COVID-19 outbreak can occur among HCWs and rheumatic patients, swiftly spreading over the presymptomatic stage. Mild disease without typical symptoms should be recognized and may evolve with delayed viral shedding, prolonged recovery, and adequate immune response in most individuals.

Tuberculosis under anti-TNF therapy: case series of a center (reporting the experience from the period 2006-2019).

Patients with inflammatory rheumatic diseases refractory to conventional disease modifying antirheumatic drugs (DMARDs)have been treated with biologics for the last two decades. It is also known that patients under biotechnological therapy present a higher risk of developing Tuberculosis (TB).Portugal has now a TB incidence classified as low. National recommendations advise on latent TB screening before the beginning of the biological therapy. This screening consists in the detection of risk factors and/or signs and symptoms of latent TB through clinical history, physical examination, chest X-ray, tuberculin skin test and Interferon Gamma Release Assay (IGRA) test. We describe five clinical cases of patients who underwent biotechnological therapy at our Hospital after 2006 and developed TB.

Efficacy, immunogenicity and cost analysis of a systematic switch from originator infliximab to biosimilar CT-P13 of all patients with inflammatory arthritis from a single center.

Biosimilar drugs are intended to be as effective as the originator product but with a lower cost to healthcare systems. In our center we promoted a switch from originator infliximab (IFXor) to biosimilar infliximab (CT-P13). We analyzed efficacy, safety, immunogenicity and cost savings of switching. Eligible patients were adults with the diagnosis of rheumatoid arthritis (RA), spondyloarthritis (SpA) and psoriatic arthritis (PsA) on therapy with IFXor for at least 6 months and with stable disease activity. Efficacy was measured considering change from baseline in Disease Activity Score in 28 joints (DAS28) for RA and PsA and in Ankylosing Spondylitis Disease Activity Score (ASDAS) for SpA. Disease worsening was considered when an increase of 1.2 from baseline in DAS28 or an increase of 1.1 in ASDAS occurred. Serum IFX levels (sIFX) were dichotomized as therapeutic (between 3-6 µg/mL), low (< 3 µg/mL), and high (> 6 µg/mL). Anti-drug antibody (ADA) levels were dichotomized into detectable (> 10 ng/ml) or non-detectable (< 10 ng/ml). A cost analysis was done based on the purchasing prices of the 2 drugs at our center. During a period of 1 year switch to CT-P13 was performed in 60 patients for non-medical reasons. We had a total of 36 patients with SpA, 16 with RA and 8 with PsA. Disease activity was stable over the observation period and similar to the values observed with IFXor. Median follow-up time was 15 months during which 5 patients stopped CT-P13. Forty two switchers had blood samples collected before and after switch. A total of 27 patients had unaltered sIFX levels and ADA status during follow up. Three patients had detectable ADA at baseline, with low sIFX levels. After switch, ADAs became negative in 2 of those patients, and the other patient kept detectable ADA levels. ADAs became positive in 5 patients after switch. The switch to CT-P13 represented a 26.4 % reduction of costs in the use of IFX therapy in these patients. The switch in routine care of a group of RA, SpA and PsA patients from IFXor to CT-P13 did not affect efficacy, safety, immunogenicity and reduced costs in 26.4%. The observed changes in blood samples were not associated with higher disease activity and did not lead to stopping IFX therapy.

Tocilizumab and rituximab have similar effectiveness and are both superior to a second tumour necrosis factor inhibitor in rheumatoid arthritis patients who discontinued a first TNF inhibitor.

OBJECTIVES: To compare the effectiveness of a 2nd TNF inhibitor (TNFi), Tocilizumab (TCZ) and Rituximab (RTX), measured by drug retention and by response rates, in RA patients after discontinuing a first-line TNFi and to clarify the reasons and predictors for discontinuation of a second-line biologic. MATERIAL AND METHODS: Non-interventional prospective study of RA patients exposed to a 2nd TNFi, TCZ or RTX after previous TNFi discontinuation using real-world data from Reuma.pt database. Drug retention was estimated using Kaplan-Meier analysis and Cox models. Crude and LUNDEX adjusted response rates were evaluated at 6 months, 1 and 2 years and reasons for discontinuation were compared according to biologic class. RESULTS: In total, 643 patients were included, 88.8% females, with a mean age of 59.4±12.8 years. Of those, 390 (60.7%) initiated a 2nd TNFi, 147 (22.9%) TCZ and 106 (16.5%) RTX. Drug retention was significantly greater among patients who initiated TCZ (76.4±4.3 months) or RTX (80.8±4.8 months), compared with those who initiated a 2nd TNFi (52.7±2.6 months) (log rank test, p < 0.001). In the adjusted Cox model, hazards of discontinuation were significantly lower for TCZ (HR 0.39, 95% CI 0.23-0.64, p < 0.001) and RTX (HR 0.42, 95% CI 0.25-0.72, p=0.001). Smokers had a significantly higher risk for discontinuation (HR 2.43, 95%CI 1.50-3.95, p < 0.001) as well as patients with higher HAQ at baseline (HR 1.51, 95%CI 1.14-2.00, p=0.004). The proportion of patients in remission or low disease activity according to Clinical Disease Activity Index (CDAI) at 6 months, 1 and 2 years was, respectively, 46.5%/50.0%/61.2% for TNFi, 52.9%/53.6%/ 69.2% for TCZ and 37.7%/48.0%/50.0% for RTX. After LUNDEX adjustment, response rates were, respectively, 33.0%/31.0%/31.8% for 2nd TNFi, 42.8%/41.8%/53.3% for TCZ and 32.0%/39.4%/39.0% for RTX. The main reasons for discontinuation were inefficacy for 2nd TNFi and RTX and adverse events for TCZ (p < 0.001). CONCLUSIONS: Our findings showed a significantly higher drug retention for TCZ and RTX, compared with 2nd TNFi, and similar persistence among TCZ and RTX, in patients who discontinued a first-line TNFi. These data corroborate the notion that switching to a biologic with a different mode of action is more effective than to a second TNFi.

Loss to follow-up in registries of rheumatic patients treated with biologics: a potential information bias in assessing pharmacovigilance and efficacy outcomes.

BACKGROUND: The information associated with loss to follow-up (LFU) patients may affect real-world data evaluation of the use of biologics that is not being adequately captured in registries. METHODS: We identified all patients (Pts) treated with biologics in our center who had no visits registered for more than 6 months, in the Rheumatic Diseases Portuguese Register, Reuma.pt. We retrieved baseline information from Reuma.pt and from the hospital electronic clinical record. We then performed a telephonic interview to characterize the reasons for LFU at our day care unit. For Pts unable to be contacted by telephone a letter of invitation to an appointment at the hospital was sent. RESULTS: From a total of 794 Pts registered in Reuma.pt at our center with active biologic therapy 227 did not have any information registered in the last 6 months. Of this, 36 Pts were on biologic therapy prescribed by other departments and maintained follow-up in these departments. 102 Pts had suspended biologic administration by medical indication and this information was registered in the hospital electronic clinical records but not updated in Reuma.pt. For 89 Pts no information could be retrieved from either the hospital electronic clinical record or Reuma.pt and we classified these Pts as true LFU. 26 of these LFU Pts were being followed up in another Rheumatology center. 26 of the LFU Pts died. 11 Pts had an adverse effect. 4 Pts of the LFU were considering to be in remission. We were not able to contact 15 of the LFU pts. CONCLUSION: Identifying LFU Pts and clarifying the reason for the loss of data in a register contributes to a better knowledge on strategies to discontinue biologics in stable pts, to a better pharmacovigilance of adverse effects and to more efficiency in data capture by registries. Due to data protection reasons it was impossible to have access to the Pts's death certificates.

Instability of mRNA expression signatures of drug transporters in chronic myeloid leukemia patients resistant to imatinib.

Despite the success of imatinib mesylate (IM) in the treatment of chronic myeloid leukemia (CML), approximately 30% of patients are resistant to therapy, mostly due to unknown causes. To profile the expression signatures of drug transporters throughout IM therapy and correlate them with resistance, we quantified mRNA expression levels of the SLC22A12, ABCB1, ABCC1, ABCG2 and MVP genes in consecutive samples from peripheral blood or bone marrow of CML patients who were either responsive or resistant to IM. Additionally we identified and quantified BCR-ABL1 transcript variants and analyzed 1236T>C ABCB1 and 480G>C SLC22A1 polymorphisms. A relationship between the type of BCR-ABL1 transcript or ABCB1 or SLC22A1 genotype and response to treatment was not discovered. However, the studied genes had higher expression levels in follow-up compared to the diagnostic samples, demonstrating a possible induction in expression. IM-sensitive patients presented significantly higher values of SLC22A1 expression, suggesting higher drug influx. Most importantly, while responding patients demonstrated stable expression signatures in consecutive samples, there was considerable variation in IM-resistant patients, indicating that single point sampling expression signatures are not reliable in predicting clinical outcomes or prognostic features in these patients. Studies that assessed consecutive samples from CML patients in order to evaluate the variation in expression levels of transporter genes are limited yet our study emphasizes the importance of such approaches.

DNA damage response in imatinib resistant chronic myeloid leukemia K562 cells.

Resistance to imatinib in patients with chronic myeloid leukemia can lead to advanced disease and blast crisis. Conventional chemotherapy with DNA damaging agents is then used, alone or in combination with other tyrosine kinase inhibitors (TKIs). Our aim was to assess whether imatinib resistant K562 cells were also resistant to DNA damaging agents. After treatment with H(2)O(2) and doxorubicin, but not camptothecin, cell survival was higher in imatinib resistant cells compared to parental cells. DNA damage, measured by comet and γ-H2AX assays, was lower in imatinib resistant cells. mRNA expression levels of 50 genes of the DNA damage response pathway showed increased expression of the base excision repair (BER) genes MBD4 and NTHL1. Knockdown of MBD4 and NTHL1 expression in resistant cells using siRNA decreased cell survival after treatment with H(2)O(2) and doxorubicin. Our results indicate that imatinib resistant cells display cross-resistance to oxidative agents, partly through up-regulation of BER genes. Expression of these genes in imatinib resistant patients was not significantly different compared to sensitive patients. However, the strategy followed in this study could help identify chemotherapeutic agents that are more effective as alternative agents in cases of resistance to TKIs.

Genomics and cancer drug resistance.

Cellular drug resistance is a major obstacle in cancer therapy. Mechanisms of resistance can be associated with altered expression of ATP-binding cassette (ABC) family of transporters on cell membrane transporters, the most common cause of multi-drug resistance (MDR), but can also include alterations of DNA repair pathways, resistance to apoptosis and target modifications. Anti-cancer treatments may be divided into different categories based on their purpose and action: chemotherapeutic agents damage and kill dividing cells; hormonal treatments prevent cancer cells from receiving signals essential for their growth; targeted drugs are a relatively new cancer treatment that targets specific proteins and pathways that are limited primarily to cancer cells or that are much more prevalent in cancer cells; and antibodies function by either depriving the cancer cells of necessary signals or by causing their direct death. In any case, resistance to anticancer therapies leads to poor prognosis of patients. Thus, identification of novel molecular targets is critical in development of new, efficient and specific cancer drugs. The aim of this review is to describe the impact of genomics in studying some of the most critical pathways involved in cancer drug resistance and in improving drug development. We shall also focus on the emerging role of microRNAs, as key gene expression regulators, in drug resistance. Finally, we shall address the specific mechanisms involved in resistance to tyrosine kinase inhibitors in chronic myeloid leukemia.

Development of imatinib and dasatinib resistance: dynamics of expression of drug transporters ABCB1, ABCC1, ABCG2, MVP, and SLC22A1.

About 20% of patients with chronic myeloid leukemia (CML) do not respond to treatment with imatinib either initially or because of acquired resistance. To study the development of CML drug resistance, an in vitro experimental system comprising cell lines with different resistance levels was established by exposing K562 cells to increasing concentrations of imatinib and dasatinib anticancer agents. The mRNA levels of BCR- ABL1 and of genes involved in drug transport or redistribution (ABCB1, ABCC1, ABCC3, ABCG2, MVP, and SLC22A1) were measured and the ABL1 kinase domain sequenced. Results excluded BCR- ABL1 overexpression and mutations as relevant resistance mechanisms. Most studied transporters were overexpressed in the majority of resistant cell lines. Their expression pattern was dynamic: varying with resistance level and chronic drug exposure. Studied efflux transporters may have an important role at the initial stages of resistance, but after prolonged exposure and for higher doses of drugs other mechanisms might take place.

Comparative genomic hybridization, BRAF, RAS, RET, and oligo-array analysis in aneuploid papillary thyroid carcinomas.

Aneuploidy in papillary thyroid carcinomas (PTCs) is considered a marker of worse prognosis. Multiple genetic surveys have been performed in PTCs, however, we are not aware of any such studies in aneuploid PTCs. In order to contribute to a better comprehension of the genetic basis of this neoplasm's more aggressive behaviour in 17 aneuploid PTCs we performed a comparative genomic hybridization (CGH) analysis, studied the BRAF and RAS mutational status, searched for RET/PTC1 and RET/PTC3 rearrangements and determined their expression profile. Array results were validated by TaqMan and immunohistochemistry. CGH revealed multiple non-random chromosomal abnormalities. BRAFV600E and RAS mutations were found in 41.2% and 33% of the carcinomas respectively. None of the studied cases presented RET/PTC1 or RET/PTC3 rearrangement. When comparing array data with the chromosomal, mutational and clinical data we found that: a) loss of control of cellular transcription was of major relevance in this group of neoplasms, HMGA2 being one of the most overexpressed genes; b) gene expression correlated with the mutational status of PTCs, as in BRAF+ cases cMET and FN1 were concomitantly overexpressed; and c) death from disease and distant metastasis was associated to the overexpression of DDR2 and to the down-regulation of genes involved in immune, inflammatory response, signal transduction and cell adhesion processes. In conclusion we have identified in aneuploid PTCs a group of significantly altered molecules that may represent preferential targets for the development of new more efficient therapies in this type of cancer.

Who takes the lead in the development of ulcerative colitis-associated colorectal cancers: mutator, suppressor, or methylator pathway?

Although several genetic alterations have been identified in patients with ulcerative colitis (UC), it remains unclear whether these changes indicate an increased risk for malignancy. This paper analyzes the involvement of suppressor, mutator, and methylator pathways in malignant transformation associated with UC. A total of 60 colonic samples (47 affected non-neoplastic mucosa, 7 dysplasia, and 6 carcinoma) from 51 UC patients were analyzed for 22 microsatellite markers. p53 gene exons 5-8 were analyzed by single-strand conformational polymorphism, and APC gene by denaturing gradient gel electrophoresis (exons 1-14) and protein truncation test (exon 15). Methylation studies for MLH1 and CSPG2 genes were also performed. Microsatellite instability was absent in all samples whereas allelic imbalance (AI) and loss of heterozygosity (LOH) were detected mainly in samples with neoplastic transformation (P<0.0001). AI and/or LOH at loci located on chromosomes 5, 9, and 18 were significantly more frequent in neoplastic samples (P<0.01), as were TP53 gene mutations (P<0.007). A single mutation was detected for APC gene in a cancer sample. MLH1 gene methylation was absent in all analyzed samples, whereas CSPG2 gene methylation was detected in a single non-neoplastic sample. Our results suggest that the suppressor pathway plays the main role in UC associated tumorigenic progression. LOH at specific loci located on chromosomes 5, 9, and 18 appears to be specifically associated with malignancy risk.