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.

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.

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.

Prevention of hip fracture by external hip protectors: an intervention in 17 nursing homes in two municipalities in Norway.

AIMS: This study was undertaken to estimate the effect of hip protectors on the incidence of hip fracture when introduced into nursing homes as a regular part of the healthcare for all residents. METHODS: A pre-test/test design was used. The pre-intervention period lasted 18 months from May 1996. The intervention period lasted 18 months from May 1998. During the intervention period all residents (965 beds) in nursing homes in two municipalities in Norway were offered free use of hip protectors. The project manager provided motivational activities in the nursing homes during the whole period, aimed at increasing the participation rate. RESULTS: The intervention period showed a 39% reduction in the hip fracture incidence when compared with the pre-intervention period (p = 0.003). The percentage of daily users of the protector varied from 35% during the first months to 22% at the end of the period. Among the 61 persons who suffered a hip fracture 31 were registered as daily users. Fourteen of the 31 users were not wearing the protector when the hip fracture occurred, while five of the 31 had the protector on their knees. Twelve of the 31 suffered a hip fracture while properly wearing the protector. CONCLUSIONS: This non-randomized study showed that hip protectors introduced to all residents in nursing homes considerably reduced the incidence of hip fracture. It may be possible to achieve higher compliance and a further reduction in the incidence of hip fractures if the producers of hip protectors increase the comfort of the protector without reducing its effect. In addition, it is important that health workers encourage more individuals at high risk to use the protector.