Urinary microbiota is associated to clinicopathological features in benign prostatic hyperplasia.

BACKGROUND: The urinary microbiota of patients with benign prostatic hyperplasia (BPH) has been associated with lower urinary tract symptoms (LUTS), however, little is known about urinary microbiota correlations with clinicopathological parameters associated with BPH. Here, we investigate associations between the urinary microbiota and clinical parameters of patients with BPH undergoing surgery. METHODS: Forty-one patients with BPH undergoing surgery were recruited from two medical centers. Catheterized urine specimens were collected and the microbiota was characterized by 16S rRNA gene sequencing. Patients were segregated into two groups according to each clinical parameter and differences in urinary microbiota diversity and composition were evaluated. RESULTS: Higher prostate weight and prostate-specific antigen (PSA) levels were associated with higher alpha diversity in the urinary microbiota of BPH patients. At the specific microbe level, we found that the greater the prostatic weight, the lower the relative abundance of Streptococcus, while the greater the PSA levels, the higher the abundance of Lactobacillus. Treatment with 5-α-reductase inhibitor was associated with overall urinary microbiota composition, in part due to a higher abundance of Corynebacterium and Anaerococcus in this group. CONCLUSIONS: We demonstrated that the urinary microbiota of BPH patients is associated with clinicopathological features, paving the way for larger studies in which causality between urinary microbiota and BPH can be appropriately explored.

The bladder microbiota is not significantly altered by intravesical BCG therapy.

INTRODUCTION: Bladder microbiota dysbiosis has been associated with several urological disorders. However, dysbiosis markers in bladder cancer have not been identified and little is known about the effect of Bacillus Calmette-Guérin (BCG) intravesical therapy on the bladder microbiota. In this study, we compared the bladder microbiota of patients with non-muscle-invasive bladder cancer (NMIBC) undergoing BCG therapy to nononcological controls. We also longitudinally analyzed the impact of BCG therapy on the bladder microbiota of NMIBC patients and addressed whether bladder microbiota is associated with BCG efficacy. METHODS: We collected catheterized urine samples from males with intermediate/high-risk NMIBC (cancer group, n = 32) or benign prostatic hyperplasia (control group, n = 41). The cancer group also provided urine samples during and after BCG induction. We used 16S rRNA gene sequencing to characterize the bladder microbiota. Bladder microbiota parameters, such as diversity and taxonomic composition, were compared between groups and associated with clinicopathological data and BCG efficacy. RESULTS: We observed no significant differences between the bladder microbiota of NMIBC patients and controls. BCG intravesical instillations did not significantly alter the bladder microbiota of NMIBC patients, and BCG was rarely detected in the bladder during and after BCG therapy. Microbiota diversity and overall composition before BCG induction did not influence disease persistence at 3 months. However, higher abundance of Lactobacillus, Streptococcus, and Cutibacterium in the pre-BCG bladder microbiota was associated with BCG effectiveness. CONCLUSION: We were unable to identify markers of bladder microbiota dysbiosis among male NMIBC patients. Moreover, we demonstrated for the first time using longitudinally collected samples that BCG cannot persist in the bladder microbiota nor significantly alter its diversity and composition. The associations found between bladder microbes and BCG efficacy highlight the potential of microbial-based therapeutic and risk-stratification strategies in the intermediate/high-risk NMIBC setting.

Detection of serum biomarkers of HPV-16 driven oropharynx and oral cavity cancer in Brazil.

BACKGROUND: HPV-16 driven oropharynx/oral cavity squamous cell carcinomas prevalence varies globally. We evaluated the presence of HPV-16 ctDNA and HPV-16 E6 antibodies in samples obtained from participants treated at the Instituto do Cancer do Estado de Sao Paulo, ICESP, and from whom tumoral HPV DNA, HPV-16 E6*I mRNA, and p16INK4a status was also accessed. METHODS: HPV was genotyped by PCR-hybridization. All HPV DNA positive and ∼10 % HPV DNA negative cases underwent p16INK4a immunohistochemistry and E6*I RNA testing using a multiplex bead based protocol. HPV-16 ctDNA and anti-E6 antibodies were assessed by ddPCR (digital droplet PCR) and multiplex serology, respectively. RESULTS: The prevalence of HPV-16 in oropharynx carcinoma (OPC) cases was low (8.7 %) when considering solely HPV-16 DNA detection, and even lower (5.2 %) when taken into consideration the concomitant detection of HPV-16 E6*I RNA and/or p16INK4 (HPV-16 attributable fraction - AF). None of the oral cavity cancer (OCC) cases were detected with HPV-16 DNA. HPV-16 ctDNA was more commonly detected than HPV-16 E6 antibodies (29.8 % versus 10.6 %). Both serum biomarkers attained 100 % sensitivity of detecting HPV-16 AF OPC, however the specificity of the HPV-16 anti-E6 biomarker was higher compared to ctDNA (93.2 % versus 75.0 %). Finally, when both HPV-16 ctDNA and anti-E6 biomarkers were considered together, the sensitivity and specificity for HPV-16 OPC detection was 100 % and about 70 %, respectively, independently of analyzing HPV-16 DNA positive or HPV-16 AF tumors. CONCLUSIONS: Our findings corroborate that serum biomarkers are highly sensitive and specific biomarkers for detection of HPV-associated OPC.

Suspected Germline TP53 Variants and Clonal Hematopoiesis of Indeterminate Potential: Lessons Learned From a Molecular Tumor Board.

OBJECTIVE: Li-Fraumeni syndrome (LFS) is a pan-cancer predisposition syndrome caused by germline pathogenic variants in the gene TP53. The interpretation of TP53 variants in clinical scenarios outside the classic LFS criteria may be challenging. Here, we report a patient affected by 2 primary cancers at later ages, who harbored a likely pathogenic TP53 at low allele frequency detected in a blood sample. METHODS: The Molecular Tumor Board committee at our institution revisited the case of a patient who was enrolled in a research protocol for the investigation of genetic conditions associated with neuroendocrine tumors. Clinical, familial, and molecular data were reviewed. The patient received germline testing using a next generation sequencing multi-gene panel and was incidentally found to harbor a TP53 likely pathogenic variant, with 22% of variant allele fraction. Additional samples, including a second blood sample, oral swab, and saliva, were collected for DNA analysis. A new TP53 sequencing round was performed with the attempt to distinguish between a true constitutional germline variant and a somatically acquired variant due to aberrant clonal expansion of bone marrow precursors. RESULTS: Patient's personal and familial history of cancer did not meet classic nor Chompret LFS criteria. Environmental risk factors for cancer were identified, such as alcohol abuse and tobacco exposure. The TP53 variant initially found in the next-generation sequencing was confirmed by Sanger sequencing in the previous DNA sample extracted from blood for the first analysis and in a second blood sample collected 6 years later. The TP53 variant was not detected in the DNA extracted from the oral swab and saliva samples. CONCLUSION: Considering the low TP53 variant allele fraction in blood, absence of variant detection in oral swab and saliva samples, the lack of LFS clinical criteria, and history of exposure to environmental risk factors for cancer, the main hypothesis for this case was aberrant clonal expansion due to clonal hematopoiesis. Oncologists should interpret TP53 findings during germline testing with caution.

Seroprevalence of SARS-CoV-2 in Emergency Department Healthcare Workers at Sírio-Libanês Hospital, Brazil.

COVID-19 has spread rapidly worldwide. Information on its prevalence and factors associated with infection are important for protecting both professionals and patients in healthcare centers. This study evaluated the seroprevalence of antibodies against SARS-CoV-2 and its association with the degree of exposure and use of personal protective equipment by healthcare professionals dedicated to the treatment of patients with flu-like illnesses in the emergency room. The research team included an analysis of healthcare professionals who underwent enzyme-linked immunosorbent assay serological testing for SARS-CoV-2 between May 28 and June 26, 2020, in the emergency room of Sírio-Libanês Hospital in São Paulo, Brazil. Participants answered individual questionnaires on occupational information, medical health history, and factors associated with exposure to the novel coronavirus. The questionnaire variables were compared based on the serological results. Of the 164 study participants, 96 (58.54%) reported at least 1 flu-like symptom and 42 (25.61%) presented serology results that were compatible with SARS-CoV-2 infection. The asymptomatic declared group accounted for 62 participants; of these, 8 (12.90%) had positive serology results (neutralizing antibody and IgG) for SARS-CoV-2. Data analysis showed a positive correlation with duration of work, safety in wearing and reusing personal protective equipment, and presence of anosmia, and showed a negative relationship with duration of mask use. Our findings suggest that the perception of symptoms by healthcare professionals is not a good screening parameter for the diagnosis of an infectious disease with respiratory symptoms, such as COVID-19. The main influencing factor for the control of infection is the elaboration of workflows and safety protocols based on simple and clear rules as well as investments in team training.

Choice of 16S Ribosomal RNA Primers Impacts Male Urinary Microbiota Profiling.

Accessibility to next-generation sequencing (NGS) technologies has enabled the profiling of microbial communities living in distinct habitats. 16S ribosomal RNA (rRNA) gene sequencing is widely used for microbiota profiling with NGS technologies. Since most used NGS platforms generate short reads, sequencing the full-length 16S rRNA gene is impractical. Therefore, choosing which 16S rRNA hypervariable region to sequence is critical in microbiota profiling studies. All nine 16S rRNA hypervariable regions are taxonomically informative, but due to variability in profiling performance for specific clades, choosing the ideal 16S rRNA hypervariable region will depend on the bacterial composition of the habitat under study. Recently, NGS allowed the identification of microbes in the urinary tract, and urinary microbiota has become an active research area. However, there is no current study evaluating the performance of different 16S rRNA hypervariable regions for male urinary microbiota profiling. We collected urine samples from male volunteers and profiled their urinary microbiota by sequencing a panel of six amplicons encompassing all nine 16S rRNA hypervariable regions. Systematic comparisons of their performance indicate V1V2 hypervariable regions better assess the taxa commonly present in male urine samples, suggesting V1V2 amplicon sequencing is more suitable for male urinary microbiota profiling. We believe our results will be helpful to guide this crucial methodological choice in future male urinary microbiota studies.

Monoallelic deleterious MUTYH germline variants as a driver for tumorigenesis.

MUTYH encodes a glycosylase involved in the base excision repair of DNA. Biallelic pathogenic germline variants in MUTYH cause an autosomal recessive condition known as MUTYH-associated adenomatous polyposis and consequently increase the risk of colorectal cancer. However, reports of increased cancer risk in individuals carrying only one defective MUTYH allele are controversial and based on studies involving few individuals. Here, we describe a comprehensive investigation of monoallelic pathogenic MUTYH germline variants in 10,389 cancer patients across 33 different tumour types and 117,000 healthy individuals. Our results indicate that monoallelic pathogenic MUTYH germline variants can lead to tumorigenesis through a mechanism of somatic loss of heterozygosity of the functional MUTYH allele in the tumour. We confirmed that the frequency of monoallelic pathogenic MUTYH germline variants is higher in individuals with cancer than in the general population, although this frequency is not homogeneous among tumour types. We also demonstrated that the MUTYH mutational signature is present only in tumours with loss of the functional allele and found that the characteristic MUTYH base substitution (C>A) increases stop-codon generation. We identified key genes that are affected during tumorigenesis. In conclusion, we propose that carriers of the monoallelic pathogenic MUTYH germline variant are at a higher risk of developing tumours, especially those with frequent loss of heterozygosity events, such as adrenal adenocarcinoma, although the overall risk is still low. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Impressive response to dabrafenib, trametinib, and osimertinib in a metastatic EGFR-mutant/BRAF V600E lung adenocarcinoma patient.

The survival outcomes of the FLAURA trial support osimertinib as the new standard of care for untreated patients harboring activating mutations in the epidermal growth factor receptor (EGFR). Despite the initial response, disease progression invariably occurs. Although uncommon, BRAF V600E mutation arises as a unique mechanism of resistance, and thus far, no prospective studies are available to support concurrent EGFR/BRAF blockade. We report a case of impressive radiological and ctDNA response under dabrafenib, trametinib, and osimertinib in an advanced EGFR-mutant lung adenocarcinoma patient who developed BRAF V600E as one of the acquired resistance mechanisms to second-line osimertinib. Moreover, the patient experienced remarkable clinical improvement and good tolerance to combination therapy. The present case suggests the importance of prospective studies evaluating both efficacy and safety of the combination in later line settings and points towards the potential of ctDNA to monitor resistance mechanisms and treatment benefit in clinical practice.

The Effects of Neoadjuvant Chemoradiation in Locally Advanced Rectal Cancer-The Impact in Intratumoral Heterogeneity.

Purpose: Intratumoral genetic heterogeneity (ITGH) is a common feature of solid tumors. However, little is known about the effect of neoadjuvant chemoradiation (nCRT) in ITGH of rectal tumors that exhibit poor response to nCRT. Here, we examined the impact of nCRT in the mutational profile and ITGH of rectal tumors and its adjacent irradiated normal mucosa in the setting of incomplete response to nCRT. Methods and Materials: To evaluate ITGH in rectal tumors, we analyzed whole-exome sequencing (WES) data from 79 tumors obtained from The Cancer Genome Atlas (TCGA). We also compared matched peripheral blood cells, irradiated normal rectal mucosa and pre and post-treatment tumor samples (PRE-T and POS-T) from one individual to examine the iatrogenic effects of nCRT. Finally, we performed WES of 7 PRE-T/POST-T matched samples to examine how nCRT affects ITGH. ITGH was assessed by quantifying subclonal mutations within individual tumors using the Mutant-Allele Tumor Heterogeneity score (MATH score). Results: Rectal tumors exhibit remarkable ITGH that is ultimately associated with disease stage (MATH score stage I/II 35.54 vs. stage III/IV 44.39, p = 0.047) and lymph node metastasis (MATH score N0 35.87 vs. N+ 45.79, p = 0.026). We also showed that nCRT does not seem to introduce detectable somatic mutations in the irradiated mucosa. Comparison of PRE-T and POST-T matched samples revealed a significant increase in ITGH in 5 out 7 patients and MATH scores were significantly higher after nCRT (median 41.7 vs. 28.8, p = 0.04). Finally, we were able to identify a subset of "enriched mutations" with significant changes in MAFs between PRE-T and POST-T samples. These "enriched mutations" were significantly more frequent in POST-T compared to PRE-T samples (92.9% vs. 7.1% p < 0.00001) and include mutations in genes associated with genetic instability and drug resistance in colorectal cancer, indicating the expansion of tumor cell subpopulations more prone to resist to nCRT. Conclusions: nCRT increases ITGH and may result in the expansion of resistant tumor cell populations in residual tumors. The risk of introducing relevant somatic mutations in the adjacent mucosa is minimal but non-responsive tumors may have potentially worse biological behavior when compared to their untreated counterparts. This was an exploratory study, and due to the limited number of samples analyzed, our results need to be validated in larger cohorts.

Circulating Tumor DNA Detection in the Management of Anti-EGFR Therapy for Advanced Colorectal Cancer.

Background: Anti-EGFR antibodies are a standard care for advanced KRAS-wild type colorectal cancers. Circulating tumor DNA (ctDNA) monitoring during therapy can detect emergence of KRAS mutant clones and early resistance to therapy. Case Presentation: We describe a 61-years-old man presenting a metastatic and recurrent rectal cancer treated with different chemotherapy regimens. His tumor was KRAS wild-type based on tissue analysis and he was treated sequentially with cetuximab-based chemotherapy, chemotherapy alone and panitumumab-based chemotherapy. We performed sequential analysis of ctDNA using droplet digital PCR (ddPCR) and a commercial assay designed for the detection of frequent KRAS mutations during his clinical follow-up. Prior to the first cetuximab-based chemotherapy ctDNA analysis demonstrated an absence of KRAS mutations. Emergence of KRAS mutations in ctDNA occurred ~3 months after treatment initiation and preceded clinical and imaging progression in about 2 months. Fractional abundance of KRAS mutation rapidly increased to 70.7% immediately before a chemotherapy alone regimen was initiated. Interestingly, KRAS mutation abundance decreased significantly during the first two months of chemotherapy, reaching a fractional abundance of 3.0%, despite minimal clinical benefit with this therapy. Re-challenge with a different anti-EGFR antibody was attempted as later line, but high levels of KRAS mutations in ctDNA before therapy correlated with an absence of clinical benefit. Conclusions: The monitoring of resistance mutations in KRAS using ctDNA during the treatment of KRAS wild-type advanced colorectal cancers can detect the emergence of resistant clones prior to clinical progression. Dynamics of resistant clones may alter during periods on and off anti-EGFR antibodies, detecting window of opportunities for a re-challenge with these therapies.

A straightforward assay to evaluate DNA integrity and optimize next-generation sequencing for clinical diagnosis in oncology.

Next generation sequencing (NGS) has become an informative tool to guide cancer treatment and conduce a personalized approach in oncology. The biopsy collected for pathologic analysis is usually stored as formalin-fixed paraffin-embedded (FFPE) blocks and then availed for molecular diagnostic, resulting in DNA molecules that are invariably fragmented and chemically modified. In an attempt to improve NGS based diagnostics in oncology we developed a straightforward DNA integrity assessment assay based on qPCR, defining clear parameters to whether NGS sequencing results is accurate or when it should be analyzed with caution. We performed DNA extraction from 12 tumor samples from diverse tissues and accessed DNA integrity by straightforward qPCR assays. In order to perform a cancer panel NGS sequencing, DNA library preparation was performed using RNA capture baits. Reads were aligned to the reference human genome and mutation calls were further validated by Sanger sequencing. Results obtained by the DNA integrity assays correlated to the efficiency of the pre-capture library preparation in up to 0.94 (Pearson's test). Moreover, sequencing results showed that poor integrity DNA leads to high rates of false positive mutation calls, specially C:G>T:A and C:G>A:T. Poor quality FFPE DNA samples are prone to generating false positive mutation calls. These are especially perilous in cases in which subclonal populations are expected, such as in advance disease, since it could lead clinicians to erroneous conclusions and equivocated conduct.

Sequential liquid biopsies reveal dynamic alterations of EGFR driver mutations and indicate EGFR amplification as a new mechanism of resistance to osimertinib in NSCLC.

Osimertinib is an EGFR-T790M-specific TKI, which has demonstrated impressive response rates in NSCLC, after failure to first-line anti-EGFR TKIs. However, acquired resistance to osimertinib is also observed and the molecular mechanisms of resistance are not yet fully understood. Monitoring and managing NSCLC patients who progressed on osimertinib is, therefore, emerging as an important clinical challenge. Sequential liquid biopsies were used to monitor a patient with EGFR-exon19del positive NSCLC, who received erlotinib and progressed through the acquisition of the EGFR-T790M mutation. Erlotinib was discontinued and osimertinib was initiated. Blood samples were collected at erlotinib progression and during osimertinib treatment for the detection of the activating (EGFR-exon19del) and resistance mutations (EGFR-T790M, EGFR-C797S, BRAF-V600E, METamp and ERBB2amp) in the plasma DNA using digital droplet PCR. Plasma levels of the activating EGFR-exon19del accurately paralleled the clinical and radiological progression of disease and allowed early detection of AR to osimertinib. Resistance to osimertinib coincided with the emergence of a small tumor cell subpopulation carrying the known EGFR-C797S resistance mutation and an additional subpopulation carrying amplified copies of EGFR-exon19del. Given the existence of multiple AR mechanisms, quantification of the original EGFR activation mutation, instead of the resistance mutations, can be efficiently used to monitor response to osimertinib, allowing early detection of AR. Absolute quantification of both activation and resistance mutations can provide important information on tumor clonal evolution upon progression to osimertinib. Selective amplification of the EGFR-exon19del allele may represent a novel resistance mechanism to osimertinib.

Intratumoral Genetic Heterogeneity in Rectal Cancer: Are Single Biopsies representative of the entirety of the tumor?

OBJECTIVE: Demonstrate intratumoral genetic heterogeneity in rectal cancer. BACKGROUND: Several clinical management decisions in rectal cancer may be influenced by pretreatment biopsy information. However, in the setting of significant intratumoral heterogeneity, biopsies may not be representative of the entirety of the tumor and limit the reliability of the information provided from them for clinical decision management. METHODS: Three fragments from a single rectal adenocarcinoma were chosen for whole-exome sequencing followed by mutation detection analysis. About 25 Gb of unambiguously mapped sequences were generated for each sample resulting in a median fold-coverage of 35x. Captured sequences mapped to the reference human genome were then used for the detection of somatic point mutations. RESULTS: Overall, 193 unique somatic point mutations were identified. Only 53 (27%) of these were shared by all three fragments, including known genes involved in early phases of the adenoma-carcinoma sequence (such as, APC). Approximately, 115 (59%) mutations were exclusively present in only one of the fragments, including mutations in "driver" genes (DNAH12). Jaccard distances showed a median distance of 0.603 for pair-wise comparison of fragments indicating significant heterogeneity between them. CONCLUSIONS: Considerable intratumoral heterogeneity is present among naive rectal cancers. The majority of point mutations detected in different fragments from rectal cancers are frequently unique to a single fragment. These findings support that gene mutations found on single pretreatment biopsies will not necessarily be representative of mutations present in the entirety of the tumor and therefore may limit the utility of the biological information provided by single biopsy fragments for clinical management decisions.

The use of personalized biomarkers and liquid biopsies to monitor treatment response and disease recurrence in locally advanced rectal cancer after neoadjuvant chemoradiation.

Neoadjuvant chemoradiotherapy (nCRT) followed by surgery is the mainstay treatment for locally advanced rectal cancer. Variable degrees of tumor regression are observed after nCRT and alternative treatment strategies, including close surveillance without immediate surgery, have been investigated to spare patients with complete tumor regression from potentially adverse outcomes of radical surgery. However, clinical and radiological assessment of response does not allow accurate identification of patients with complete response. In addition, surveillance for recurrence is similarly important for these patients, as early detection of recurrence allows salvage resections and adjuvant interventions. We report the use of liquid biopsies and personalized biomarkers for monitoring treatment response to nCRT and detecting residual disease and recurrence in patients with rectal cancer. We sequenced the whole-genome of four rectal tumors to identify patient-specific chromosomal rearrangements that were used to monitor circulating tumor DNA (ctDNA) in liquid biopsies collected at diagnosis and during nCRT and follow-up. We compared ctDNA levels to clinical, radiological and pathological response to nCRT. Our results indicate that personalized biomarkers and liquid biopsies may not be sensitive for the detection of microscopic residual disease. However, it can be efficiently used to monitor treatment response to nCRT and detect disease recurrence, preceding increases in CEA levels and radiological diagnosis. Similar good results were observed when assessing tumor response to systemic therapy and disease progression. Our study supports the use of personalized biomarkers and liquid biopsies to tailor the management of rectal cancer patients, however, replication in a larger cohort is necessary to introduce this strategy into clinical practice.

Comprehensive evaluation of the effectiveness of gene expression signatures to predict complete response to neoadjuvant chemoradiotherapy and guide surgical intervention in rectal cancer.

Neoadjuvant chemoradiotherapy (nCRT) may lead to complete tumor regression in rectal cancer patients. Prediction of complete response to nCRT may allow a personalized management of rectal cancer and spare patients from unnecessary radical total mesorectal excision with or without sphincter preservation. To identify a gene expression signature capable of predicting complete pathological response (pCR) to nCRT, we performed a gene expression analysis in 25 pretreatment biopsies from patients who underwent 5FU-based nCRT using RNA-Seq. A supervised learning algorithm was used to identify expression signatures capable of predicting pCR, and the predictive value of these signatures was validated using independent samples. We also evaluated the utility of previously published signatures in predicting complete response in our cohort. We identified 27 differentially expressed genes between patients with pCR and patients with incomplete responses to nCRT. Predictive gene signatures using subsets of these 27 differentially expressed genes peaked at 81.8% accuracy. However, signatures with the highest sensitivity showed poor specificity, and vice-versa, when applied in an independent set of patients. Testing previously published signatures on our cohort also showed poor predictive value. Our results indicate that currently available predictive signatures are highly dependent on the sample set from which they are derived, and their accuracy is not superior to current imaging and clinical parameters used to assess response to nCRT and guide surgical intervention.

Overexpression of miR-21-5p as a predictive marker for complete tumor regression to neoadjuvant chemoradiotherapy in rectal cancer patients.

BACKGROUND: Neoadjuvant chemoradiotherapy (nCRT) followed by radical surgery is the preferred treatment strategy for locally advanced rectal cancer. However, complete tumor regression is observed in a significant proportion of patients after nCRT, making them ideal candidates for alternative treatment strategies to this considerably morbid procedure. Identification of such patients based on clinical findings (complete clinical response - cCR) is difficult mainly because it relies on subjective clinical and imaging studies. Our goal was to identify biomarkers capable of predicting complete response to nCRT. METHODS: We analyzed miRNA expression profile using deep sequencing in rectal tumor biopsies prior to nCRT. Differential expression was investigated by EdgeR for a training (n = 27) and a validation (n = 16) set of patients to identify miRNAs associated with treatment response (complete vs. incomplete). In vitro experiments with two cancer cell lines were also performed in order to evaluate the possible role of miRNAs on response to nCRT. RESULTS: We found 4 miRNAs differentially expressed between complete and incomplete responders to nCRT. In addition, validation was performed using an independent group of patients and miR-21-5p was confirmed as being overexpressed in complete responders. Overall sensitivity and specificity of miR-21-5p expression in predicting complete response to nCRT was 78% and 86% respectively. Interestingly, in a subset of patients with cCR followed by early local recurrence, the expression level of miR-21-5p was considerably low, similarly to incomplete responders. We also found SATB1, a miR-21-5p target gene and known multidrug resistance gene, whose expression was inversely correlated with miR-21-5p expression. Finally, we performed functional experiments and showed that miR-21-5p and SATB1 may be directly involved with poor response to nCRT in rectal cancer patients. CONCLUSIONS: This study suggests miR-21-5p as a promising predictive biomarker, which should aid in the selection of patients with cCR to nCRT that potentially could be spared from radical surgery.

Identification of FAM46D as a novel cancer/testis antigen using EST data and serological analysis.

Cancer/testis Antigens (CTAs) are immunogenic proteins with a restricted expression pattern in normal tissues and aberrant expression in different types of tumors being considered promising candidates for immunotherapy. We used the alignment between EST sequences and the human genome sequence to identify novel CT genes. By examining the EST tissue composition of known CT clusters we defined parameters for the selection of 1184 EST clusters corresponding to putative CT genes. The expression pattern of 70 CT gene candidates was evaluated by RT-PCR in 21 normal tissues, 17 tumor cell lines and 160 primary tumors. We were able to identify 4 CT genes expressed in different types of tumors. The presence of antibodies against the protein encoded by 1 of these 4 CT genes (FAM46D) was exclusively detected in plasma samples from cancer patients. Due to its restricted expression pattern and immunogenicity FAM46D represents a novel target for cancer immunotherapy.

Antibodies against the cancer-testis antigen CTSP-1 are frequently found in prostate cancer patients and are an independent prognostic factor for biochemical-recurrence.

Cancer-testis (CT) antigens are immunogenic proteins expressed in normal gametogenic tissues and in different types of tumors. CTSP-1 is a CT antigen frequently expressed in prostate tumors, and capable of eliciting humoral response in prostate cancer patients. Here, we analyzed the presence of anti-CTSP-1 antibodies in 147 patients with localized prostate cancer and determined its prognostic value for predicting biochemical-recurrence after radical prostatectomy. Anti-CTSP-1 antibodies were detected in 25% of the patients and a significant correlation (p = 0.017) between CTSP-1 protein expression and the presence of specific humoral response was observed. No association was found between the presence of antibodies and the pathological variables analyzed. On univariate analysis, patients without antibodies against CTSP-1 had a lower biochemical-recurrence free survival than did those with anti-CTSP-1 antibodies, although the difference between the groups was not statistically significant (57 vs. 75%, p = 0.075). However, the presence of antibodies against CTSP-1 was significantly associated with a better prognosis in patients with higher Gleason score (36 vs. 80%, p = 0.028). On multivariate analysis, antibodies against CTSP-1 were associated with a better prognosis (Hazard ratio = 0.41, 95% IC 0.18-0.90 p = 0.039), being the third most powerful prognostic factor among Gleason score and preoperative PSA levels. CTSP-1 should be considered a promising candidate for prostate cancer immunotherapy, since it is frequently expressed in prostate tumors and capable of eliciting humoral immune response in prostate cancer patients. Our results also suggest that humoral response against CTSP-1 could be used as a prognostic marker, especially among patients with a high Gleason score.