Study of prognostic and diagnostic significance of P53 and PTEN mutation in proliferative lesions of endometrium.

Background: Endometrial hyperplasia essentially implies an overgrowth of the endometrium. Complex hyperplasia associated with cellular atypia, seems to be the most important predictor of malignant potential. Endometrioid Endometrial Carcinomas account for three-fourths of Endometrial Carcinomas and are thought to develop following a continuum of premalignant lesions ranging from endometrial hyperplasia without atypia, to hyperplasia with atypia, and finally to well-differentiated carcinoma. Currently the most frequently observed gene mutation in endometrioid carcinoma is located on chromosome 10 and is related with the PTEN gene (phoshatase and tensin homolog). PTEN inactivation is found to correlate with clonal growth pattern detected in endometrial hyperplasia and carcinoma. The p53 tumor suppressor gene locates to chromosome 17p13. The abnormal p53 expression has been found in 11% of grade 1 endometrioid endometrial carcinoma, while p53 mutations occur in 90% of non-endometrioid endometrial carcinoma. Aims and objectives: In this study we aim to evaluate the immuno histochemical expression of P53 and PTEN genes in endometrial hyperplasia and endometrial carcinoma and correlate their expression with prognostic outcomes like grade and stage, in cases of endometrial carcinoma. Material and methods: A prospective study of 60 patients with abnormal uterine bleed in the peri and post menopausal age was conducted, for a period of three years. Histological specimens were studied for HPE and IHC for the markers PTEN and P53. Results: The mean age for hyperplastic and carcinomatous lesions was 44.9 years and 53.2 years respectively. 35% (21 cases) were endometrial hyperplasia and 65% (39 cases) of cases were endometrial carcinoma. Among endometrial carcinoma 87% are of endometrioid type and 13% are of other types, which include serous, clear and malignant mixed Mullerian type of carcinoma. IHC study showed that PTEN expression is higher in endometrial hyperplasia than endometrial carcinoma cases. Elevated P53 expression correlated with poor differentiation of endometrial cancer. P53 was found to be more in cases with FIGO staging III &IV compared to stage I & II (100% vs 18.1% p value= 0.0016) and grade 3 compared to grade 1&2 (50% vs 0 p value= 0.0116). Conclusion: Immuno histochemical biomarkers like PTEN and P53 may contribute to better tumor characterization and thus more precisely determine its clinical behavior. Key words: endometrial hyperplasia, endometrial carcinoma, PTEN, P53.

P0075EXPERT OPINION ON THE MULTIDISCIPLINARY MANAGEMENT OF CYSTINOSIS IN ADOLESCENT AND ADULT PATIENTS

Background and Aims Cystinosis is a rare autosomal recessive lysosomal storage disorder caused by mutations in the CTNS gene on chromosome 17p13. It affects between 1:150,000–1:200,000 live births, with a prevalence of approximately 1.6 per million. It is a life-long progressive disease which results in an abnormal lysosomal accumulation of the amino acid cystine in multiple organs and tissues of the body. The most common presentation of cystinosis (95%) is the infantile nephropathic form, with renal symptoms developing in the first few months of life. Over the next 10–20 years, extra-renal manifestations of cystinosis become apparent, which require multidisciplinary care. Here we describe a consensus-based programme with the objective of creating clinical recommendations to support HCPs with the management of adolescents and adults living with the multi-organ effects of cystinosis. Method The programme was led by a Steering Committee (SC) of European clinicians with expertise in managing cystinosis. Recommendations were developed using a quasi-Delphi methodology. The SC identified and prioritised a list of key questions. An Extended Faculty (EF) of additional specialists with extensive experience managing cystinosis in patients of all ages, were invited to answer the questions via an online digital platform. The consolidated answers of expert opinion were summarised into recommendations that were supported by evidence-based guidance and additional published data where possible. The EF were invited to agree / disagree with the draft clinical recommendations. Where there was disagreement, the SC members amended the recommendations and the EF re-voted on the revisions. This process continued until consensus was achieved on all final recommendations. Results The expert-agreed clinical recommendations reflect the multi-organ effects of cystinosis. Thus, advice on factors relating to the nervous system, muscle involvement, ophthalmology, cardio-respiratory system, dental care and nutrition, dermatology, endocrine system, and gastrointestinal and hepatological involvement, are given, along with renal considerations. Guidance on fertility and family planning that reflect some of the major advances in recent years, are also provided. Ideas on improving psychological well-being and adherence includes recommendations on the use of validated screening tests, increasing access to occupational therapy, and interacting with patient groups. The programme has also produced an online checklist to support HCPs in their daily clinical practice by providing a focus to guide regular consultations with the patient. Conclusion These expert recommendations offer HCPs relevant advice that support them in the management of adolescents and adults living with the multi-organ effects of cystinosis. The recommendations complement existing international and local guidance and aim to improve patient outcomes.

The diagnosis of cystinosis in patients reveals new CTNS gene mutations in the Chinese population

Background Cystinosis is a rare autosomal-recessive disorder caused by a defective transport of cystine across the lysosomal membrane. Previous studies have mapped cystinosis to the CTNS gene which is located on chromosome 17p13, and various CTNS mutations have been identified to correlate them with this disease. Methods We analyzed six patients from five unrelated families who were diagnosed with cystinosis in our hospital. We described the diagnostic procedures for all the patients and proposed alternative therapies for cystinosis patients instead of using cysteamine, an orphan drug which was commercially unavailable in China. Moreover, genetic analysis of all patients’ samples was carried out to identify novel CTNS gene mutations. Results and conclusions The patients in this study were followed up from 1 to more than 10 years to monitor their growth and development, which indicated that the alternative therapies we used were helpful to ameliorate the complications of the cystinosis patients without cysteamine. Furthermore, by sequencing the patients’ genome, we identified novel mutations in the CTNS gene including: c.477C > G (p.S159R), c.274C > T (p.Q92X) and c.680A > T (p.E227V); these mutations were only observed in cystinosis patients and had never been reported in any other populations, suggesting they might be specific to Chinese cystinosis patients.

The SLC16A11 risk haplotype is associated with decreased insulin action, higher transaminases and large-size adipocytes

Objective A haplotype at chromosome 17p13 that reduces expression and function of the solute carrier transporter SLC16A11 is associated with increased risk for type 2 diabetes in Mexicans. We aim to investigate the detailed metabolic profile of SLC16A11 risk haplotype carriers to identify potential physiological mechanisms explaining the increased type 2 diabetes risk. Design Cross-sectional study. Methods We evaluated carriers (n = 72) and non-carriers (n = 75) of the SLC16A11 risk haplotype, with or without type 2 diabetes. An independent sample of 1069 subjects was used to replicate biochemical findings. The evaluation included euglycemic–hyperinsulinemic clamp, frequently sampled intravenous glucose tolerance test (FSIVGTT), dual-energy X-ray absorptiometry (DXA), MRI and spectroscopy and subcutaneous abdominal adipose tissue biopsies. Results Fat-free mass (FFM)-adjusted M value was lower in carriers of the SLC16A11 risk haplotype after adjusting for age and type 2 diabetes status (β = −0.164, P = 0.04). Subjects with type 2 diabetes and the risk haplotype demonstrated an increase of 8.76 U/L in alanine aminotransferase (ALT) (P = 0.02) and of 7.34 U/L in gamma-glutamyltransferase (GGT) (P = 0.05) compared with non-carriers and after adjusting for gender, age and ancestry. Among women with the risk haplotype and normal BMI, the adipocyte size was higher (P < 0.001). Conclusions Individuals carrying the SLC16A11 risk haplotype exhibited decreased insulin action. Higher serum ALT and GGT levels were found in carriers with type 2 diabetes, and larger adipocytes in subcutaneous fat in the size distribution in carrier women with normal weight.

Clinical Impact of Clonal and Subclonal TP53 Mutations in Chronic Lymphocytic Leukemia

Introduction. The clinical course of patients with chronic lymphocytic leukemia (CLL) is highly heterogeneous. The deletions/mutations of 17p/TP53 are predictors of chemorefractoriness, and for this reason, in the management algorithm of CLL patients, when present, indicate treatment with with chemo-free regimens also in the context of first-line therapy. Recent studies based on ultra-deep-next generation sequencing (NGS) have shown that TP53 mutations can be present at very low clonal abundance in tumor cell populations, although whether these mutations may have a detrimental clinical impact on disease course is still to be established. Aim. To investigate the presence of clonal and subclonal mutations of TP53 in a large cohort of CLL cases using an ultra-deep NGS strategy, and determined their clinical relevance for patients outcome. Methods. The study includes 590 CLL patients characterized for the deletion at chromosome 17p13 (FISH analysis) and TP53 mutations in samples before treatment. In all cases, analyses were carried out on DNA extracted from nearly pure (>90%) tumor cells. TP53 mutational status was investigated by NGS with an amplicon based strategy. Sequencing reads analysis was made by the Burrows-Wheeler Aligner-MEM algorithm and by SAMtools. Variant calling was performed using the entire pipeline established on the MiSeq Reporter software. Results were expressed as percentage of mutated DNA. The minimal allelic fraction for mutation calling was set at 1%. Synonymous variants and polymorphisms described in the Single Nucleotide Polymorphism Database (dbSNP138) were removed. Outcome variable was overall survival (OS). Clinical correlations were made using Kaplan-Meier plots and log-rank test. Results. FISH and mutational analyses were performed in samples within 2 years from diagnosis in 92% of the cases (Figure 1A). A total of 125 TP53 mutations (Figure 1B) were found in 96 patients (11.7%). Subclonal mutations have similar molecular characteristics as their respective high frequency allele mutations supporting a comparable pathogenic effect (Figure 1B). According to a 15% cutoff of variant allele frequency (VAF), 78 cases were considered clonal and 18 subclonal (Figure 1C) for TP53 mutations (1% < VAF < 15%). In this context, cases with subclonal and clonal TP53 mutations experienced significant shorter OS than TP53 wild-type (wt) cases, without differences between clonal and subclonal cases (Figure 1E). Accordingly, ROC analysis on the same cohort identified a cutoff of >0% for the clinical impact of TP53 mutations (Figure 1E inset). Deletion of chromosome 17p was found in 180 out of 574 patients (31.3%), and using a 10% cutoff, 61 patients presented a percentage of deleted nuclei above the cutoff (Figure 1D). Using only 17p deletion data and considering the above mentioned cutoff, patients with 17p13 deletion ≥10% experienced shorter OS than wt cases, while patients with 17p13 deletion <10% experienced OS superimposable to wt cases (Figure 1F). These data were confirmed by ROC analysis that selected >9% of deleted nuclei as optimal cutoff for OS discrimination (Figure 1F inset). Given the frequent co-occurrence of TP53 mutations with 17p deletions, we also evaluated the impact of isolated TP53 mutations and 17p deletions. By using the ROC cutoffs for the definition of mutated/deleted cases, 466 cases (81.1%) presented no TP53 disruption (TP53 mutations and deletion), 47 cases (8.2%) were TP53 mutated only, 15 cases (2.6%) were 17p deleted only and 46 cases (8.1%) presented a concomitant TP53 mutation and 17p deletion. Kaplan-Meier curves demonstrated comparable significant shorter OS intervals for TP53 mutated and/or deleted CLL cases respect to wt cases, while no differences were observed between these three groups (Figure 1G). Conclusion. By using a highly sensitive NGS approach, we have detected small subclones of TP53 in a relative high proportion of patients. TP53 mutations conferred a significant shorter OS irrespectively of VAF percent, while deletion of chromosome 17p impacted on OS only when detectable in more than 10% of nuclei. These cutoffs, once validated by prospective studies, may be employed in daily practice for the clinical management of CLL patients. Disclosures Zaja: Novartis: Honoraria, Research Funding; Abbvie: Honoraria; Celgene: Honoraria, Research Funding; Amgen: Honoraria; Janssen: Honoraria; Takeda: Honoraria; Sandoz: Honoraria.

Screening of Tumor Suppressor Genes in Metastatic Colorectal Cancer

Most tumor suppressor genes are commonly inactivated in the development of colorectal cancer (CRC). The activation of tumor suppressor genes may be beneficial to suppress the development and metastasis of CRC. This study analyzed genes expression and methylation levels in different stages of CRC. Genes with downregulated mRNA expression and upregulated methylation level in advanced CRC were screened as the potential tumor suppressor genes. After comparing the methylation level of screened genes, we found that MBD1 gene had downregulated mRNA expression and upregulated methylation levels in advanced CRC and continuously upregulated methylation level in the progression of CRC. Enrichment analysis revealed that genes expression in accordance with the elevated expression of MBD1 mainly located on chromosomes 17p13 and 17p12 and 8 tumor suppressor genes located on chromosome 17p13. Further enrichment analysis of transcription factor binding site identified that SP1 binding site had higher enrichment and could bind with MBD1. In conclusion, MBD1 may be a tumor suppressor gene in advanced CRC and affect the development and metastasis of CRC by regulating 8 tumor suppressor genes through binding with SP1.

An Amplicon-Targeted Ultra-Deep Sequencing Approach Reveals the Presence at the Onset of Multiple Myeloma and the Selection over Time of TP53 Sub-Clonal Variants, Which Adversely Influence Patients' Overall Survival

INTRODUCTION In newly diagnosed Multiple Myeloma (MM) patients (pts), Copy Number (CN) losses of chromosome 17p13, carrying the TP53 tumor-suppressor gene, are strong predictors of poor outcomes. On the contrary, the prognostic relevance of TP53 mutations at the onset of the disease is less clear, due to the very limited frequency of clonal lesions, as revealed by Sanger sequencing. To address this poorly investigated issue, we used an ultra-deep sequencing (UDS) approach to characterize the TP53 structural architecture in both newly diagnosed and relapsed MM pts and to assess the prognostic role and evolution over time of small TP53 mutated sub-clones. SAMPLES AND METHODS A cohort of 99 newly diagnosed MM pts treated up-front with bortezomib-based regimens and autologous stem cell transplantation, was included in this molecular study. In 29 cases, samples were collected both at diagnosis and at relapse(s). DNA was obtained from CD138+ highly purified plasma cells. TP53 gene mutational status was analysed by using an amplicon-targeted UDS approach (GSJ, 454 Roche Life Sciences). In order to discriminate between low frequency sub-clonal TP53 variants and sequencing errors, sequencing raw data were filtered according to cut-off values based on different ranges of sequences' coverage depth. Additional filters were also applied, based on both quality and biological cut-offs, to obtain a final confident list of variants. Analysis of CN alterations (CNAs) was performed by SNPs array and results analysed with ChAS software. RESULTS With a median coverage of 1386X, a list of 129 correctly called TP53 variants (either missense, or nonsense or splice ones), including 20 INDELs, was detected. Only deleterious and N/A variants (according to SIFT classification) were included in the list. Most newly diagnosed MM pts (55%) carried at least one TP53 sub-clonal variant (on average 1.08 variants per pts), with 45/99 (45%) carrying non-mutated TP53. Pts carrying TP53 sub-clonal variants bared also TP53 CN hemizygous losses (20%), CKS1B gains (56%) and cdkn2c losses (14%). According to TP53 sub-clonal mutational load, pts were stratified in two sub-groups, including 28 pts with ≥2 (high load) and 71 with <2 variants (low load), respectively. Eleven out of 129 variants were recurrent (RVs), as being detected in at least 3% of pts, with Variants Allele Frequencies (VAFs) ranging from 0.24 to 70.1% (median 0,53%); RVs were observed in 29 pts. The clinical impact of the TP53 sub-clonal mutational load, as well as of variants recurrence, was evaluated in 90/99 MM (median follow up = 70 months). Results of statistical analysis are summarized in Table 1. Pts carrying either high TP53 sub-clonal mutational load or RVs had significantly shorter OS and OS after relapse, as compared to the others, while no difference between these two groups was seen regarding PFS and TTP. Multivariate analysis showed that high TP53 mutational load, as well as the presence of TP53 RVs, both resulted independent factors adversely affecting OS and OS after relapse (Table 2). Of note, none of the detected genomic aberrations significantly influenced the response to front-line induction therapy. The distribution of both TP53 sub-clonal variants and genomic CNAs was overall modified in samples collected at relapse(s): 90% of relapsed pts carried at least one sub-clonal variant (on average 1.63 variants per pts) with 3/29 (10%) relapsed MM carrying non-mutated TP53. Moreover, 5 different sub-clonal lesions proved a linear increment of both TP53 VAFs (from 29.4% to 54.6%; from 7.8% to 12.4%; from 0.5% to 4.3%) and TP53 CN loss smooth signal (from 7% to 89% and from 50% to 100%), as evaluated in longitudinally collected samples. CONCLUSIONS The UDS analysis of TP53 coding sequence in newly diagnosed MM highlighted for the first time a high rate of variants, recurring with a wide range of frequencies among samples. The increased number of TP53 sub-clonal variants per pts in samples collected at relapse(s), compared to that seen at the onset of the disease, suggests a sub-clonal dynamics over time. This finding might explain the adverse impact of high TP53 sub-clonal mutational load and TP53 RVs on OS, due to a shorter OS after relapse. Acknowledgments: Roche Diagnostics for applicationsupport in the realization of this project. Disclosures Zamagni: Celgene Corporation: Honoraria, Speakers Bureau; Janssen Pharmaceuticals: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Ariad: Consultancy; AMGEN: Consultancy; ROCHE: Consultancy; Pfizer: Consultancy; MSD: Consultancy. Cavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.

The relationship between biochemical failure and TP53 genetic polymorphism in patients with prostate cancer

5133 Background: TP53 is a tumour suppressor gene, located at chromosome 17p13, referred as altered in 50–55% of cancer cases. The p53 protein, encoded by the TP53 gene, is known as the cellular gatekeeper for growth and division, as it plays an essential role in safeguarding the integrity of the genome. This protein is involved in processes as cell cycle arrest, gene transcription, DNA repair and apoptosis and may influence cancer progression. Few studies have been published regarding TP53 codon 72 genetic polymorphism and prostate cancer behaviour. Methods: We analysed the frequency of TP53 codon 72 genetic polymorphism in DNA isolated from blood samples of 265 individuals with prostate cancer using the Real-time PCR methodology. Biochemical failure was defined as two successive post- treatment rises in serum PSA, greater than 50 %. Results: From the 265 prostate cancer cases, we observed that 54.7% were found to be homozygous for the Arg allele (AA), 38.1% were heterozygous (AP) and 7.2% homozygous for the Pro allele (PP). We find that individuals carriers of PP genotype have three fold increased risk of biochemical relapse (OR=3.43, 95% CI 1.31–9.01; p= 0.008). Furthermore, we observed that this genotype was not associated to the gleason score (OR=1.66, 95%CI 0.53–5.13; p=0.372) or the presence of advanced disease (OR=1.69, 95%CI 0.62–4.6; p=0.296). Conclusions: We demonstrate that TP53 codon 72 genetic polymorphism may influence the clinical progression of patients with prostate cancer. Furthermore, our results are consistent with literature regarding other neoplasia suggesting that the Pro allele was associated to a poorer prognosis and survival. No significant financial relationships to disclose.