Is detection of intraperitoneal exfoliated tumor cells after surgical resection of rectal cancer a prognostic factor of survival?

BACKGROUND: The prognostic significance of free cancer cells detected in peritoneal fluid at the time of rectal surgery remains unclear. A substantial number of patients will develop metastatic disease even with successful local treatment. This prospective non-randomized study investigated the prognostic value of intraperitoneal free cancer cells harvested in peritoneal lavage after surgery for rectal cancer. Mutational hotspots in mitochondrial DNA were examined as potential molecular signatures to detect circulating intraperitoneal free cancer cells when present in primary tumor and in lavage. METHODS: Point mutations in mitochondrial DNA amplifications were determined in primary tumors and corresponding exfoliated intraperitoneal free cancer cells in lavage from 191 patients with locally advanced rectal cancer scheduled for radical treatment. Mitochondrial DNA target sequences were amplified by polymerase chain reaction and base substitutions were detected by denaturant, cycling temperature capillary electrophoresis. Detection of intraperitoneal free cancer cells was correlated to survival. RESULTS: Of 191patients analyzed, 138 (72%) were identified with somatic mitochondrial point mutations in rectal cancer tumors. From this fraction, 45 patients (33%) had positive lavage fluid with corresponding somatic mtDNA point mutations in lavage representing circulating intraperitoneal free cancer cells. There was no significant survival difference between patients identified with or without somatic mitochondrial DNA point mutations in the corresponding lavage. CONCLUSION: Somatic mitochondrial DNA point mutations identified in primary rectal tumors enable detection of circulating intraperitoneal free cancer cells in lavage fluid. Intraperitoneal free cancer cells harvested from lavage immediately after surgery for rectal cancer does not represent an independent prognostic factor on survival.

Extraction of cell-free DNA from urine, using polylysine-coated silica particles.

DNA analysis is used for a variety of purposes, including disease diagnosis and DNA profiling; this involves extracting DNA from living organisms. In this study, we prepared polycationic silica particles to extract DNA that has the negatively charged phosphate backbone from solution. The coated particles were prepared by mixing conventional silica gel particles and poly-Lys; these particles could efficiently extract 1.3 µg of cell-free DNA from 50 mL of (male) urine. It is expected that these easily prepared particles (just a mixture of two commercially available chemicals) can be used as a noninvasive diagnostic tool for genetic disorders such as cancer, diabetes, and hypertension. Graphical abstract Effective extraction method of cfDNA from urine was developed that used commercially available silica gel particles and poly-Lys.

Mapping mitochondrial heteroplasmy in a Leydig tumor by laser capture micro-dissection and cycling temperature capillary electrophoresis.

BACKGROUND: The growth of tumor cells is accompanied by mutations in nuclear and mitochondrial genomes creating marked genetic heterogeneity. Tumors also contain non-tumor cells of various origins. An observed somatic mitochondrial mutation would have occurred in a founding cell and spread through cell division. Micro-anatomical dissection of a tumor coupled with assays for mitochondrial point mutations permits new insights into this growth process. More generally, the ability to detect and trace, at a histological level, somatic mitochondrial mutations in human tissues and tumors, makes these mutations into markers for lineage tracing. METHOD: A tumor was first sampled by a large punch biopsy and scanned for any significant degree of heteroplasmy in a set of sequences containing known mutational hotspots of the mitochondrial genome. A heteroplasmic tumor was sliced at a 12 µm thickness and placed on membranes. Laser capture micro-dissection was used to take 25000 µm2 subsamples or spots. After DNA amplification, cycling temperature capillary electrophoresis (CTCE) was used on the laser captured samples to quantify mitochondrial mutant fractions. RESULTS: Of six testicular tumors studied, one, a Leydig tumor, was discovered to carry a detectable degree of heteroplasmy for two separate point mutations: a C → T mutation at bp 64 and a T → C mutation found at bp 152. From this tumor, 381 spots were sampled with laser capture micro-dissection. The ordered distribution of spots exhibited a wide range of fractions of the mutant sequences from 0 to 100% mutant copies. The two mutations co-distributed in the growing tumor indicating they were present on the same genome copies in the founding cell. CONCLUSION: Laser capture microdissection of sliced tumor samples coupled with CTCE-based point mutation assays provides an effective and practical means to obtain maps of mitochondrial mutational heteroplasmy within human tumors.

Innovative Biomarkers for Obesity and Type 1 Diabetes Based on Bifidobacterium and Metabolomic Profiling.

The role of Bifidobacterium species and microbial metabolites such as short-chain fatty acids (SCFAs) and human milk oligosaccharides in controlling intestinal inflammation and the pathogenesis of obesity and type 1 diabetes (T1D) has been largely studied in recent years. This paper discusses the discovery of signature biomarkers for obesity and T1D based on data from a novel test for profiling several Bifidobacterium species, combined with metabolomic analysis. Through the NUTRISHIELD clinical study, a total of 98 children were recruited: 40 healthy controls, 40 type 1 diabetics, and 18 obese children. Bifidobacterium profiles were assessed in stool samples through an innovative test allowing high taxonomic resolution and precise quantification, while SCFAs and branched amino acids were measured in urine samples through gas chromatography-mass spectrometry (GC-MS). KIDMED questionnaires were used to evaluate the children's dietary habits and correlate them with the Bifidobacterium and metabolomic profiles. We found that B. longum subs. infantis and B. breve were higher in individuals with obesity, while B. bifidum and B. longum subs. longum were lower compared to healthy individuals. In individuals with T1D, alterations were found at the metabolic level, with an overall increase in the level of the most measured metabolites. The high taxonomic resolution of the Bifidobacterium test used meant strong correlations between the concentrations of valine and isoleucine, and the relative abundance of some Bifidobacterium species such as B. longum subs. infantis, B. breve, and B. bifidum could be observed.

Fact-based nutrition for infants and lactating mothers-The NUTRISHIELD study.

Background: Human milk (HM) is the ideal source of nutrients for infants. Its composition is highly variable according to the infant's needs. When not enough own mother's milk (OMM) is available, the administration of pasteurized donor human milk (DHM) is considered a suitable alternative for preterm infants. This study protocol describes the NUTRISHIELD clinical study. The main objective of this study is to compare the % weight gain/month in preterm and term infants exclusively receiving either OMM or DHM. Other secondary aims comprise the evaluation of the influence of diet, lifestyle habits, psychological stress, and pasteurization on the milk composition, and how it modulates infant's growth, health, and development. Methods and design: NUTRISHIELD is a prospective mother-infant birth cohort in the Spanish-Mediterranean area including three groups: preterm infants <32 weeks of gestation (i) exclusively receiving (i.e., >80% of total intake) OMM, and (ii) exclusively receiving DHM, and (iii) term infants exclusively receiving OMM, as well as their mothers. Biological samples and nutritional, clinical, and anthropometric characteristics are collected at six time points covering the period from birth and until six months of infant's age. The genotype, metabolome, and microbiota as well as the HM composition are characterized. Portable sensor prototypes for the analysis of HM and urine are benchmarked. Additionally, maternal psychosocial status is measured at the beginning of the study and at month six. Mother-infant postpartum bonding and parental stress are also examined. At six months, infant neurodevelopment scales are applied. Mother's concerns and attitudes to breastfeeding are registered through a specific questionnaire. Discussion: NUTRISHIELD provides an in-depth longitudinal study of the mother-infant-microbiota triad combining multiple biological matrices, newly developed analytical methods, and ad-hoc designed sensor prototypes with a wide range of clinical outcome measures. Data obtained from this study will be used to train a machine-learning algorithm for providing dietary advice to lactating mothers and will be implemented in a user-friendly platform based on a combination of user-provided information and biomarker analysis. A better understanding of the factors affecting milk's composition, together with the health implications for infants plays an important role in developing improved strategies of nutraceutical management in infant care. Clinical trial registration: https://register.clinicaltrials.gov, identifier: NCT05646940.

Applications of Probiotic-Based Multi-Components to Human, Animal and Ecosystem Health: Concepts, Methodologies, and Action Mechanisms.

Probiotics and related preparations, including synbiotics and postbiotics, are living and non-living microbial-based multi-components, which are now among the most popular bioactive agents. Such interests mainly arise from the wide range and numerous beneficial effects of their use for various hosts. The current minireview article attempts to provide an overview and discuss in a holistic way the concepts, methodologies, action mechanisms, and applications of probiotic-based multi-components in human, animal, plant, soil, and environment health. Probiotic-based multi-component preparations refer to a mixture of bioactive agents, containing probiotics or postbiotics as main functional ingredients, and prebiotics, protectants, stabilizers, encapsulating agents, and other compounds as additional constituents. Analyzing, characterizing, and monitoring over time the traceability, performance, and stability of such multi-component ingredients require relevant and sensitive analytical tools and methodologies. Two innovative profiling and monitoring methods, the thermophysical fingerprinting thermogravimetry-differential scanning calorimetry technique (TGA-DSC) of the whole multi-component powder preparations, and the Advanced Testing for Genetic Composition (ATGC) strain analysis up to the subspecies level, are presented, illustrated, and discussed in this review to respond to those requirements. Finally, the paper deals with some selected applications of probiotic-based multi-components to human, animal, plant, soil and environment health, while mentioning their possible action mechanisms.

Tracing of Human Tumor Cell Lineages by Mitochondrial Mutations.

BACKGROUND: Previous studies have shown the value in studying lineage tracing in slices of human tumors. However, a tumor is not a two-dimensional structure and to better understand how a tumor, and its corresponding metastasis grow, a three-dimensional (3-D) view is necessary. RESULTS: Using somatic mitochondrial mutations as a marker for lineage tracing, it is possible to identify and follow tumor specific cell lineages. Using cycling temperature capillary electrophoresis (CTCE) a total of 8 tissues from 5 patients (4 primary tumors and 4 metastasis) containing clear mitochondrial markers of tumor lineages were selected. From these 8 tissues over 9,500 laser capture microdisection (LCM) samples were taken and analyzed, in a way that allows 3-D rendering of the observations. CONCLUSION: Using CTCE combined with LCM makes it possible to study the 3-D patterns formed by tumors and metastasis as they grow. These results clearly show that the majority of the volume occupied by a tumor is not composed of tumor derived cells. These cells are most likely recruited from the neighboring tissue.

Somatic Mitochondrial DNA Point Mutations Used as Biomarkers to Demonstrate Genomic Heterogeneity in Primary Prostate Cancer.

Primary prostate tumor heterogeneity is poorly understood, leaving research efforts with challenges regarding the initiation and advancement of the disease. The growth of tumor cells is accompanied by mutations in nuclear and in mitochondrial genomes. Thus, mitochondrial DNA mutations may be used as tumor cell markers. By the use of laser capture microdissection coupled with assays for mitochondrial point mutation detection, mtDNA mutations were used to trace mutated cells at a histological level. Point mutations in mtDNA were determined in 12 primary prostate cancers. The tumors represent different pathology-prognostic grade groups. Known mutational hotspots of the mtDNA were scanned for heteroplasmy. All specimens with mtDNA heteroplasmy were subsequently subsampled by laser capture microdissection. From a total number of 1728 microsamples, mitochondrial DNA target sequences were amplified and base substitutions detected by cycling temperature capillary electrophoresis. Real-time PCR was used as a quantitative assay to determine the relative mtDNA copy number of 12 tumors studied, represented by two samples from each (N = 24); a high degree (75%) demonstrated tumor specimen heterogeneity. A grid of 96 spots isolated by laser capture microdissection demonstrated interfocal sample heterogeneity and increased the limit of detection. The spots demonstrated a wide range of mutant fractions from 0 to 100% mutant copies. The mitochondrial DNA copy number in the samples was determined by real-time PCR. No correlation between copy number and pathology-prognostic grade groups was observed. Somatic mitochondrial DNA point mutations represent traceable biomarkers demonstrating heterogeneity in primary prostate cancer. Mutations can be detected in areas before changes in tissue histopathology are evident to the pathologist.

Scanning the mitochondrial genome for mutations by cycling temperature capillary electrophoresis.

To bypass possible nuclear contamination and to exclusively amplify DNA from the mitochondrion, a set of 23 primers was selected. On the mitochondrial DNA selection fragments, a second set of fragments was used to amplify and identify mutant fractions with a detection limit of 1% . This mutation scanning method analyzed 76% of the mitochondrial genome and was used to examine 94 tumours from different tissues of origin. In all, 87 tumours had one or more mutations, leaving seven samples without observed mutations. Sanger sequencing verified samples carrying mutations with a mutant fraction exceeding 30%. The generated data validate that several regions of the mitochondrial DNA have more mutations than others.

Quantifying mitochondrial DNA copy number using robust regression to interpret real time PCR results.

BACKGROUND: Real time PCR (rtPCR) is a quantitative assay to determine the relative DNA copy number in a sample versus a reference. The [Formula: see text] method is the standard for the analysis of the output data generated by an rtPCR experiment. We developed an alternative based on fitting a robust regression to the rtPCR signal. This new data analysis tool reduces potential biases and does not require all of the compared DNA fragments to have the same PCR efficiency. RESULTS: Comparing the two methods when analysing 96 identical PCR preparations showed similar distributions of the estimated copy numbers. Estimating the efficiency with the [Formula: see text] method, however, required a dilution series, which is not necessary for the robust regression method. We used rtPCR to quantify mitochondrial DNA (mtDNA) copy numbers in three different tissues types: breast, colon and prostate. For each type, normal tissue and a tumor from the same three patients were analysed. This gives a total of six samples. The mitochondrial copy number is estimated to lie between 200 and 300 copies per cell. Similar results are obtained when using the robust regression or the [Formula: see text] method. Confidence ratios were slightly narrower for the robust regression. The new data analysis method has been implemented as an R package.

Cycling temperature capillary electrophoresis: A quantitative, fast and inexpensive method to detect mutations in mixed populations of human mitochondrial DNA.

Cycling temperature capillary electrophoresis has been optimised for mutation detection in 76% of the mitochondrial genome. The method was tested on a mixed sample and compared to mutation detection by next generation sequencing. Out of 152 fragments 90 were concordant, 51 discordant and in 11 were semi-concordant. Dilution experiments show that cycling capillary electrophoresis has a detection limit of 1-3%. The detection limit of routine next generation sequencing was in the ranges of 15 to 30%. Cycling temperature capillary electrophoresis detect and accurate quantify mutations at a fraction of the cost and time required to perform a next generation sequencing analysis.

Mutator/Hypermutable fetal/juvenile metakaryotic stem cells and human colorectal carcinogenesis.

Adult age-specific colorectal cancer incidence rates increase exponentially from maturity, reach a maximum, then decline in extreme old age. Armitage and Doll (1) postulated that the exponential increase resulted from "n" mutations occurring throughout adult life in normal "cells at risk" that initiated the growth of a preneoplastic colony in which subsequent "m" mutations promoted one of the preneoplastic "cells at risk" to form a lethal neoplasia. We have reported cytologic evidence that these "cells at risk" are fetal/juvenile organogenic, then preneoplastic metakaryotic stem cells. Metakaryotic cells display stem-like behaviors of both symmetric and asymmetric nuclear divisions and peculiarities such as bell shaped nuclei and amitotic nuclear fission that distinguish them from embryonic, eukaryotic stem cells. Analyses of mutant colony sizes and numbers in adult lung epithelia supported the inferences that the metakaryotic organogenic stem cells are constitutively mutator/hypermutable and that their contributions to cancer initiation are limited to the fetal/juvenile period. We have amended the two-stage model of Armitage and Doll and incorporated these several inferences in a computer program CancerFit v.5.0. We compared the expectations of the amended model to adult (15-104 years) age-specific colon cancer rates for European-American males born 1890-99 and observed remarkable concordance. When estimates of normal colonic fetal/juvenile APC and OAT gene mutation rates (∼2-5 × 10(-5) per stem cell doubling) and preneoplastic colonic gene loss rates (∼8 × 10(-3)) were applied, the model was in accordance only for the values of n = 2 and m = 4 or 5.