However, inconsistencies in research protocol, analysis, and interpretation undermine the accuracy and clinical applications. In our review, “Current Trends and Challenges of Microbiome Research in Bladder Cancer”, we sought to characterize these constraints, to offer additional suggestions for research on urine microbiome within bladder cancer (BC).1
Studying the microbiome for BC presents multiple challenges due to the variability in specimen collection and handling, primers utilized for analysis, and differences across computational methods and their interpretation. In the process of collecting specimens, the urinary microbiome is known for its notably low biomass, which increases the likelihood of contamination by agents frequently employed in sequencing and collection methodology itself. Without the implementation of thorough decontamination measures and the use of both positive and negative controls, a significant portion of the urinary data reported in the scientific literature to date may be erroneous. This is due to potential contamination from external sources, such as the meatus, urethra, and prostate, as well as from PCR reagents. As urine specimens can be collected in a variety of ways, the range of methodologies not only allows for inconsistencies across studies but contributes to the validity of results and interpretation within context of true bladder microbiota vs other tissue contamination.
The 16S primers (V1-V9) refer to specific regions along the 16S rRNA gene, which is widely used in microbial identification and phylogeny. These primers target variable regions (labeled V1 through V9) within the gene, allowing for the differentiation and classification of bacteria based on their unique genetic sequences in these areas. The varied use of 16S primers (i.e., V1-V4, V3-V4) across studies also complicates interpretation and cross study comparison. In contrast, shotgun metagenomic sequencing allows for researchers to sequence all genomes in a sample. However, it is imperative for studies utilizing the shotgun approach to aim for genus-level identification at a minimum, preferably reaching species-level resolution to enhance accuracy and comparability across studies.
The urinary microbiome is inherently diverse, making it hard to generalize significant findings to a patient population. It is also therefore crucial to understand the microbiome findings in the wider context, integrating microbial data with others like genomic and metabolic data, while adjusting for many confounders including exposure (i.e., tobacco) and medication use including over the counter pre/probiotics. Only with the utilization of multi-mics data, and adjusting for potential confounders, can we reliably trust the data reported. Lastly, the translation of basic microbiome data into therapeutic uses is exceedingly challenging, as the lack of reliable and consolidated microbial research damages the opportunity for a comprehensive understanding to advance clinical practice. Without a strict, prospective collection in the setting of clinical trials, the use of urinary microbiome data as it stands, is flawed for therapeutic intervention.
In conclusion, while microbiome research holds immense promise in advancing care for our cancer patients, the field has significant challenges that compromise the validity and applicability of published results. Our review points out just several of the issues that plague urinary microbiome research. It is crucial that efforts are made towards standardizing methodologies, and integrating microbiome data with other omics data while adjusting for confounders in large prospective trials. Only through meticulous technique and accurate interpretation can we realistically aspire to translate our discoveries into therapeutic applications. The saying that "nothing is worse than dirty science" is particularly pertinent in the realm of microbiome research, underscoring the importance of rigor and clarity in this field.
Written by:
- Emma Helstrom BS, Department of Urology, Fox Chase Cancer Center, Philadelphia, PA
- Mohit Sindhani MS, Department of Urology, University Hospitals Cleveland Medical Center, Cleveland, OH
- Philip Abbosh MD, PhD, Department of Urology, Fox Chase Cancer Center, Philadelphia, PA
- Laura Bukavina MD, MPH, MSc, Department of Urology, University Hospitals Cleveland Medical Center, Case Western Reserve School of Medicine, Cleveland, OH
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