Fusobacterium nucleatum and Bacteroides fragilis detection in colorectal tumours: Optimal target site and correlation with total bacterial load.

BACKGROUND: Mucosal infiltration by certain bacterial species may contribute to the development and progression of colorectal cancer (CRC). There is considerable variation in reported detection rates in human CRC samples and the extent to which bacterial infiltration varies across regions of the primary tumour is unknown. This study aimed to determine if there is an optimal site for bacterial detection within CRC tumours. METHODS: Presence of target bacterial species was assessed by quantitative real-time PCR (qPCR) in 42 human CRC tumours. Abundance in primary tumour regions, normal epithelium and at metastatic sites was investigated in an expanded cohort of 51 patients. Species presence/absence was confirmed by diversity profiling in five patients. Correlation with total bacterial load and clinicopathological features was assessed. RESULTS: Fusobacterium nucleatum and Bacteroides fragilis were detected in tumours from 43% and 24% of patients, respectively (17% positive for both species). The optimal detection site was the tumour luminal surface (TLS). Patients testing positive at the TLS frequently tested negative at other sites, including central tumour and invasive margin. F. nucleatum was detected at a higher frequency in tumour versus normal epithelium (p < 0.01) and was associated with more advanced disease (p = 0.01). Detection of both species correlated with total bacterial load. However, corroboration of qPCR results via diversity profiling suggests detection of these species may indicate a specific microbial signature. CONCLUSIONS: This study supports a role for F. nucleatum in CRC development. Presence of F. nucleatum and B. fragilis varies across primary tumour regions, with the TLS representing the optimal site for bacterial detection.

Australian recommendations for EGFR T790M testing in advanced non-small cell lung cancer.

First-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are used as first-line therapy in patients with non-small cell lung cancer (NSCLC) harboring a sensitizing mutation in the EGFR gene. Unfortunately, resistance to these therapies often occurs within 10 months of commencing treatment and is mostly commonly due to the development of the EGFR T790M mutation. Treatment with the third-generation EGFR TKI, osimertinib can prolong progression free survival in patients with the T790M mutation, so it is important to determine the resistance mechanism in order to plan ongoing therapeutic strategies. Here we review the evidence and make recommendations for the timing of T790M mutation testing, the most appropriate specimens to test and the available testing methods in patients progressing during treatment with first line EGFR TKIs for NSCLC.

HSP110 T17 simplifies and improves the microsatellite instability testing in patients with colorectal cancer.

BACKGROUND: Every colorectal cancer (CRC) patient should be tested for microsatellite instability (MSI, a marker for defective DNA mismatch repair) as a first screen for Lynch syndrome (LS). In this study, we investigated whether it may be possible to improve the detection of MSI in CRC. We examined whether the HT17 DNA repeat (critical for correct splicing of the chaperone HSP110) might constitute a superior marker for diagnosis of the MSI phenotype in patients with CRC compared with the standard panel of markers (pentaplex). METHODS: The HT17 polymorphism was analysed in germline DNA from 1037 multi-ethnic individuals. We assessed its sensitivity and specificity for detecting MSI in a multicentre, population-based cohort of 685 patients with CRC and an additional series of 70 patients with CRC considered to be at-risk of LS. All cases were screened earlier for MSI using pentaplex markers. Cases showing discordant HT17/pentaplex results were further examined for the expression of mismatch repair proteins. RESULTS: HT17 status was analysed independently and blinded to previous results from pentaplex genotyping. HT17 showed no germline allelic variation outside a very narrow range. Compared with the pentaplex panel, HT17 showed better sensitivity (0.984 (95% CI 0.968 to 0.995) vs 0.951 (95% CI 0.925 to 0.972)) and similar specificity (0.997 (95% CI 0.989 to 1.000) for both) for the detection of MSI. Furthermore, HT17 alone correctly classified samples judged to be uncertain with the pentaplex panel and showed excellent ability to detect MSI in patients with LS. CONCLUSIONS: HT17 simplifies and improves the current standard molecular methods for detecting MSI in CRC.

Overcoming resistance of targeted EGFR monotherapy by inhibition of STAT3 escape pathway in soft tissue sarcoma.

Although epidermal growth factor receptor (EGFR) is often over-expressed in soft tissue sarcoma (STS), a phase II trial using an EGFR inhibitor gefitinib showed a low response rate. This study identified a new secondary resistance mechanism of gefitinib in STS, and developed new strategies to improve the effectiveness of EGFR inhibition particularly by blocking the STAT3 pathway.We demonstrated that seven STS cell lines of diverse histological origin showed resistance to gefitinib despite blockade of phosphorylated EGFR (pEGFR) and downstream signal transducers (pAKT and pERK) in PI3K/AKT and RAS/ERK pathways. Gefitinib exposure was not associated with decrease in the ratio of pSTAT3/pSTAT1. The relative STAT3 abundance and activation may be responsible for the drug resistance. We therefore hypothesized that the addition of a STAT3 inhibitor could overcome the STAT3 escape pathway.We found that the addition of STAT3 inhibitor S3I-201 to gefitinib achieved synergistic anti-proliferative and pro-apoptotic effects in all three STS cell lines examined. This was confirmed in a fibrosarcoma xenografted mouse model, where the tumours from the combination group (418mm3) were significantly smaller than those from untreated (1032mm3) or single drug (912 and 798mm3) groups.Our findings may have clinical implications for optimising EGFR-targeted therapy in STS.

KRAS mutation testing of metastatic colorectal cancer in Australia: where are we at?

AIM: To carry out a nationwide study of KRAS testing in metastatic colorectal cancer as reported by nine major molecular pathology service providers in Australia, including mutation frequencies and turnaround times that might impact on patient care. METHODS: Participating laboratories contributed information on KRAS mutation frequencies, including the G13D mutation type, as well as turnaround times for tumor block retrieval and testing. RESULTS: The KRAS mutation frequency observed by nine different test sites for a total of 3688 metastatic colorectal cancers ranged from 34.4% to 40.7%, with an average across all sites of 38.8%. The average frequency of the G13D mutation type among all cases was 8.0%. The median turnaround time was 17 days (range 0-191), with 20% of cases requiring more than 4 weeks for a KRAS test result. The major contributor to long turnaround times was the time taken to retrieve archived blocks of primary tumor, particularly from sources external to the test site. CONCLUSION: The frequency of KRAS mutations in metastatic colorectal cancer reported by the major Australian test sites is very similar to that reported by other large overseas studies. More widespread introduction of routine testing at the time of initial diagnosis should eliminate the long turnaround times currently being experienced in a significant proportion of cases. Future expansion of testing to include other KRAS and NRAS mutation hotspots may spur the introduction of next-generation sequencing platforms.

Influence of 16S rDNA primer sequence mismatches on the spectrum of bacterial genera detected in prostate tissue by universal eubacterial PCR.

BACKGROUND: Propionibacterium sp. and Staphylococcus spp. are the most frequent bacteria cultured from prostatectomy specimens but are seldom detected by universal eubacterial PCR. MATERIALS AND METHODS: We obtained from GenBank representative 16S rRNA gene sequences from Propionibacterium sp., Staphylococcus spp. and from 34 bacterial genera that were recently detected in prostate tissues using universal eubacterial PCR. We compared these 16S rDNA sequences with the universal eubacterial 16S PCR primer sets chosen for detection of bacterial DNA in prostate tissues. RESULTS: We show that failure to detect DNA from Propionibacterium sp. and Staphylococcus spp. in prostate tissues is strongly associated with the presence of mismatches near the 3' termini of the 16S rDNA primer sets used. CONCLUSIONS: The choice of 16S PCR primers may play an important role in determining the spectrum of bacterial genera detected in prostate tissue by universal eubacterial PCR.

Central zone carcinoma of the prostate gland: a distinct tumor type with poor prognostic features.

PURPOSE: The central zone of the prostate gland is a region rarely associated with carcinoma. To our knowledge central zone tumors have not previously been compared to carcinoma originating in the peripheral or transition zone of the prostate gland. MATERIALS AND METHODS: All 2,010 radical prostatectomy cases seen at our institution from October 1998 to December 2006 were reviewed to identify tumor zonal origin. Central zone carcinoma was characterized and compared with tumors of other zones. RESULTS: Zonal origin was determined in a total of 2,494 tumors in 1,703 cases. Of the tumors 63 (2.5%) were of central zone origin with 59 of the 63 representing the index or main tumor. Comparative analysis of a defined subset of 726 cases showed that central zone cancers were significantly more aggressive than peripheral or transition zone cancers with a far greater risk of extracapsular extension, seminal vesicle invasion and positive surgical margins. Escape from the gland was often via the ejaculatory ducts and seminal vesicles. Kaplan-Meier analysis confirmed that the probability of post-prostatectomy biochemical failure was double that of tumors of the other zones with a far more rapid rate of failure. Multivariate Cox regression analysis identified Gleason grade, positive margins, extracapsular extension, tumor volume and preoperative serum prostate specific antigen as the major contributors to this poor prognosis, rather than specific zonal origin. CONCLUSIONS: To our knowledge this study provides the first characterization and comparative analysis of central zone carcinoma, identifying these tumors as a rare but highly aggressive form of prostate carcinoma with a distinct route of spread from the gland that contrasts with tumors of other zones. Preoperative identification is currently hampered by the avoidance of biopsy targeting the central zone. However, if recognized preoperatively, aggressive intervention may possibly improve the currently bleak outlook.

The antibody response to Propionibacterium acnes is an independent predictor of serum prostate-specific antigen levels in biopsy-negative men.

OBJECTIVE: To investigate whether the serum titres of Propionibacterium acnes antibodies in patients undergoing prostate biopsy are associated with prostate cancer or markers of prostate disease, including serum prostate-specific antigen (PSA) levels. PATIENTS AND METHODS: The cell wall-associated proteins from P. acnes types IA, IB and II were extracted and characterized by Western blotting and immunoblotting. We developed an enzyme-linked immunosorbent assay (ELISA) based on extracted proteins to determine the anti-P. acnes antibody titres in the sera of 68 patients undergoing prostate biopsy. Correlations between these titres and multiple markers of prostate disease were investigated. RESULTS: In patients with biopsies negative for cancer, a high anti-P. acnes antibody titre was associated with high serum PSA levels (>or=10.0 ng/mL, P = 0.04), and multiple linear regression analysis identified antibody titre as the predominant independent predictor of serum PSA level (P = 0.03). The titre was positively correlated with patient age, prostate volume and aggressive inflammation, suggesting an involvement with benign prostatic hyperplasia (BPH). However in patients with histologically detected cancer, the volume of cancer in the biopsy cores was the predominant independent predictor of serum PSA (P = 0.01). CONCLUSIONS: These results support our hypothesis that P. acnes might be involved in the development of inflammation-related prostate diseases, in particular with BPH. Our ELISA might be valuable for identifying P. acnes infection of the prostate gland in patients with elevated serum PSA levels but a negative biopsy, and might identify men at risk of developing clinical BPH. However, an investigation with more patients is needed to confirm these preliminary findings.

Polymerase chain reaction-based identification of Propionibacterium acnes types isolated from the male urinary tract: evaluation of adolescents, normal adults and men with prostatic pathology.

OBJECTIVE: To assess whether colonization of the male urinary tract with Propionibacterium acnes, in particular types IB and II (which are associated with inflammation in radical prostatectomy specimens and might be involved in the development of prostate cancer), is associated with prostate disease, and thus to develop a urine test to detect men at risk of prostate disease. PATIENTS, SUBJECTS AND METHODS: We developed the first polymerase chain reaction (PCR)-based technique for identifying P. acnes types IA, IB and II, and used this in combination with selective culture medium to compare the prevalence of these subtypes in the urinary tract of adolescent males, healthy adult men and patients with confirmed prostate pathology. RESULTS: P. acnes types IB and II were no more prevalent in the urinary tract of patients with prostate pathology than in normal control men. However, the prevalence of types IB and II appeared to be higher in adult men (at 11 of 15 and six of 15, respectively) than in adolescents (two of six and one of six), suggesting an age-related increase. Comparison of urinary tract and facial skin P. acnes from three subjects showed that type IA was more often predominant on facial skin, whereas types IB or II were more often predominant in the urinary tract. CONCLUSIONS: A urine test might not be useful for detecting men with prostatic P. acnes infection and thus at greater risk of associated prostate disease. However, this work validated our technique for detecting and identifying the three P. acnes subtypes, and identified some interesting trends worth further investigation.

Links between Propionibacterium acnes and prostate cancer.

Incidental foci of prostate cancer are found at autopsy in 30% of men in their third decade, and by their eighth decade more than 75% have histological evidence of cancer. This unprecedented cancer prevalence points to a ubiquitous causative agent or perhaps an interaction between multiple common carcinogenic cofactors. We propose that one of these carcinogens is Propionibacterium acnes. Several characteristics of prostate cancer suggest the involvement of an infectious agent and we provide evidence that P. acnes is an excellent candidate. We have cultured P. acnes from a substantial proportion of prostate glands containing cancer and shown a significant positive association with prostatic inflammation. P. acnes is well suited to cause persistent, low-grade infection involving a marked inflammatory response and the P. acnes subtypes most frequently associated with prostate cancer become highly prevalent in the urinary tract of males following puberty.

Propionibacterium acnes associated with inflammation in radical prostatectomy specimens: a possible link to cancer evolution?

PURPOSE: Inflammation is commonly observed in the prostate gland and has been implicated in the development of prostate cancer. The etiology of prostatic inflammation is unknown. However, the involvement of a carcinogenic infectious agent has been suggested. MATERIALS AND METHODS: Prostatic tissue from 34 consecutive patients with prostate cancer was cultured to detect the presence of bacterial agents. Prostatic inflammation was assessed by histological examination of wholemount tissue sections. RESULTS: The predominant microorganism detected was Propionibacterium acnes, found in 35% of prostate samples. A significantly higher degree of prostatic inflammation was observed in cases culture positive for P. acnes (p =0.007). P. acnes was separated into 3 groups based on cell surface properties, phenotype and genetic grouping. All skin control isolates were classified as group 1 whereas most prostatic isolates were classified as groups 2 and 3. CONCLUSIONS: P. acnes has been isolated from prostatic tissues in men who underwent radical prostatectomy for localized cancer and has been shown to be positively associated with prostatic inflammation. This inflammation may then be linked to the evolution of carcinoma. Furthermore, organisms infecting these patients with prostate cancer differ genetically and phenotypically from the commonly identified cutaneous P. acnes isolates, suggesting that specific subtypes may be involved in development of prostatic inflammation.

Epithelial differentiation of the lower urinary tract with recognition of the minor prostatic glands.

Preservation of tissues in glutaraldehyde-based fixatives allows identification of prostatic glandular secretions without resorting to immunostaining. This has enabled detailed histological assessment of the entire male urethra and bladder and has confirmed prostatic epithelial cells outside the confines of the prostate gland. Male and female lower urinary tracts are also compared. Three intact bladders and penile urethras from radical surgical specimens, tissue from 10 radical prostatectomies, 12 penile urethral biopsy specimens, and 40 samples of of metaplastic bladder mucosa were evaluated after undergoing glutaraldehyde-based fixation (Solufix, Tissugen, Western Australia). All sections were immunostained for prostate-specific antigen (PSA) and high molecular-weight cytokeratin. Selected formalin-fixed samples also were assessed and stained for androgen receptor status, and 10 female control subjects also were evaluated. Prostatic epithelial cells, as recognized by their content of prostate secretory granules (PSG), were identified in almost all periurethral glands seen along the length of the penile urethra. These "minor prostatic glands" were composed entirely of prostatic cells or, more commonly, mixed prostatic and mucinous epithelium. The penile urethra was lined by transitional epithelium, whereas the prostatic urethra was lined by glandular cells with superficial androgen receptor-positive cells that had lost much of their secretory function. Foci of cystitis cystica/glandularis contained prostatic cells in more than half of the cases evaluated, and in all cases PSG secretion in extraprostatic sites was commensurate with PSA secretion. No prostatic secretion was seen in the female control cases, and the female urethra, in contrast to the male urethra, was lined entirely by glycogenated stratified squamous epithelium similar to the epithelium lining the vagina and vulva. This study defines the entity of minor prostatic glands and confirms their extensive normal distribution in the adult male subject. Minimal but persistently elevated levels of serum PSA occuring after successful radical prostatectomy may be related in part to this phenomenon. The female lower urinary tract differs considerably from the male but has similar features related to the lower genital tract.

Preclinical evaluation of a phosphorothioate oligonucleotide in the retina of rhesus monkey.

Overexpression of vascular endothelial growth factor (VEGF) has been strongly implicated in the development of choroidal neovascularization (CNV) in patients with age-related macular degeneration. In this study, a phosphorothioate oligonucleotide (PS-oligo) targeting both human and rat VEGF(165) genes upstream of the translation initiation code, named DS135 in this study, was evaluated for its uptake dynamics and retinal tolerance after intravitreal (IV) and subretinal (SR) injections in the rhesus monkey. Intravitreal and SR injections of a fluorescent-labeled DS135 (FL-DS135) resulted in both dose- and time-dependent uptake and persistence, and FL-DS135 remained detectable in the retina for at least 3 weeks after injection. Ophthalmic examination showed transient vitreous haze after IV delivery of a high dose but not with a low dose of FL-DS135. Histologic examination showed no evidence of retinal degeneration with respect to IV delivery. After SR delivery, however, dose-related cellular infiltration, transient residual fluid, and slight distortion of the neuroretina were observed. The biologic efficacy of DS135 was further assessed in a laser-induced CNV model, and development of CNV was determined by fluorescein angiography and histologic examination. Incomplete inhibition of CNV formation was observed after IV and SR injection of DS135, but no statistically significant difference was achieved when compared with dose-matched control of PS-oligo. Analysis of fluorescein angiogram and histologic examination showed less than 30% incidence of CNV development in this monkey model. Our study demonstrated that PS-oligos can be successfully introduced into the retina, although with potential limitations, after SR delivery. DS135, a PS-oligo targeting the VEGF gene upstream of the translation initiation code, partially inhibited CNV formation. An improved CNV model is necessary for further confirmation of the full therapeutic potency of DS135 before clinical application.

The use of synthetic polymers for delivery of therapeutic antisense oligodeoxynucleotides.

Developed over the past two decades, the antisense strategy has become a technology of recognised therapeutic potential, and many of the problems raised earlier in its application have been solved to varying extents. However, the adequate delivery of antisense oligodeoxynucleotides to individual cells remains an important and inordinately difficult challenge. Synthetic polymers appeared on this scene in the middle 1980s, and there is a surprisingly large variety used or proposed so far as agents for delivery of oligodeoxynucleotides. After discussing the principles of antisense strategy, certain aspects of the ingestion of macromolecules by cells, and the present situation of delivery procedures, this article analyses in detail the attempts to use synthetic polymers as carrier matrices and or cell membrane permeabilisation agents for delivery of antisense oligodeoxynucleotides. Structural aspects of various polymers, as well as the results, promises and limitations of their use are critically evaluated.