In vivo cytometry: a spectrum of possibilities.

BACKGROUND: We investigate whether optical imaging can reliably detect abnormalities in tissue, in a range of specimens (live cells in vitro; fixed, fresh ex-vivo and in vivo tissue), without the use of added contrast agents, and review our promising spectral methods for achieving quantitative, real-time, high resolution intrasurgical optical diagnostics. METHODS: We use reflectance, fluorescence, two-photon, and Mie scattering imaging, performed with instrumentation we developed or modified, to detect intrinsic tissue signatures. Emphasis is on spectral/hyperspectral imaging approaches allowing the equivalent of in vivo pathology. RESULTS: With experimental focus on unstained specimens, we demonstrate the ability to segment tissue images for cancer detection. Spectral reflectance imaging, coupled with advanced analysis, typically yields 90% specificity and sensitivity. Autofluorescence is also shown to be diagnostically useful, with lymph nodes results highlighted here. Elastic scattering hyperspectral imaging endoscopy, using a new instrument we designed and built, shows promise in bronchoscopic detection of dysplasia and early cancer in patients. CONCLUSIONS: The results demonstrate that advanced optical imaging can detect and localize cellular signatures of cancer in real-time, in vivo, without the use of contrast agents, in animals and humans. This is an important step towards tight spatio-temporal coupling between such detection and clinical intervention.     

In vivo somatic microsatellite mutations identified in non-malignant human tissue

Microsatellite instability (MSI) has been described in cancer cells and in vitro cell lines, and meiotic changes in repeat length have also been documented. We report the novel observation of somatic microsatellite mutation (SMM) in normal human somatic cells in vivo, detected while genotyping 5,767 prenatal samples (4,640 amniotic fluid samples and 1,127 chorionic villus biopsies) as a diagnostic test for exclusion of trisomy 13, 18 or 21. Quantitative fluorescence-PCR using a multiplex of 12 primer pairs, for four loci on each of the three chromosomes, was followed by fragment analysis on a capillary-based genetic analyser. Forty-seven (4.2%) chorionic villus samples and six (0.1%) amniotic fluid samples showed allelic mosaicism, interpreted as SMM. In four cases, analysis of parental blood samples confirmed the presence of a de novo allele. SMM was detected at all but two of the 12 loci tested, and the incidence of mutation increased with repeat length. Detection of SMM in chorionic villus samples may imply less rigorous correction of replication errors in these extra-embryonic tissues, and is likely to have been facilitated by clonal expansion in the small samples of tissue tested. The presence of the same phenomenon in six amniotic fluid samples would imply that in these pregnancies, the instability event had occurred early in embryogenesis. The results suggest that defective proof reading during DNA replication may be more common in non-malignant human somatic tissue than previously recognised.     

The frequency spectrum of a mutation,and its age,in a general diffusion model

General formulae are derived for the probability density and expected age of a mutation of frequency x in a population, and similarly for a mutation with b copies in a sample of n genes. A general formula is derived for the frequency spectrum of a mutation in a sample. Variable population size models are included. Results are derived in two frameworks: diffusion process models for the frequency of the mutation; and birth and death process models. The coalescent structure within the mutant gene group and the non-mutant group is considered.     

In vivo transgenic mutation assays

Transgenic rodent gene-mutation models provide relatively quick and statistically reliable assays for gene mutations in the DNA from any tissue. This report summarizes those issues that have been agreed upon at a previous IWGT meeting [Environ. Mol. Mutagen. 35 (2000) 253], and discusses in depth those issues for which no consensus was reached before. It was previously agreed that for regulatory applications, assays should be based upon neutral genes, be generally available in several laboratories, and be readily transferable. For phage-based assays, five to ten animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of approximately 3x10(-5) mutants/locus and 125,000-300,000 plaque or colony forming units (pfu or cfu) per tissue per animal. A full set of data should be generated for a vehicle control and two dose groups. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a blocked design, where samples from negative control, positive control and each treatment group are processed together. The total number of pfus or cfus and the MF for each tissue and animal are reported. Statistical tests should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose-response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is a statistically non-significant change, with all mean MFs within two standard deviations of the control. During the current workshop, a general protocol was agreed in which animals are treated daily for 28 consecutive days and tissues sampled 3 days after the final treatment. This recommendation could be modified by reducing or increasing the number of treatments or the length of the treatment period, when scientifically justified. Normally male animals alone are sufficient and normally at least one rapidly proliferating and one slowly proliferating tissue should be sampled. Although, as agreed previously, sequencing data are not normally required, they might provide useful additional information in specific circumstances, mainly to identify and correct for clonal expansion and in some cases to determine a mechanism associated with a positive response.     

In vivo transgenic mutation assays

Transgenic rodent gene-mutation models provide relatively quick and statistically reliable assays for gene mutations in the DNA from any tissue. This report summarizes those issues that have been agreed upon at a previous IWGT meeting [Environ. Mol. Mutagen. 35 (2000) 253], and discusses in depth those issues for which no consensus was reached before. It was previously agreed that for regulatory applications, assays should be based upon neutral genes, be generally available in several laboratories, and be readily transferable. For phage-based assays, five to ten animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of 3×10⁻⁵ mutants/locus and 125,000–300,000 plaque or colony forming units (pfu or cfu) per tissue per animal. A full set of data should be generated for a vehicle control and two dose groups. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a blocked design, where samples from negative control, positive control and each treatment group are processed together. The total number of pfus or cfus and the MF for each tissue and animal are reported. Statistical tests should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose–response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is a statistically non-significant change, with all mean MFs within two standard deviations of the control. During the current workshop, a general protocol was agreed in which animals are treated daily for 28 consecutive days and tissues sampled 3 days after the final treatment. This recommendation could be modified by reducing or increasing the number of treatments or the length of the treatment period, when scientifically justified. Normally male animals alone are sufficient and normally at least one rapidly proliferating and one slowly proliferating tissue should be sampled. Although, as agreed previously, sequencing data are not normally required, they might provide useful additional information in specific circumstances, mainly to identify and correct for clonal expansion and in some cases to determine a mechanism associated with a positive response.     

In vivo human tendon mechanical properties: effect of resistance training in old age

Recent advances in ultrasound scanning have made it possible to obtain the mechanical properties of human tendons in vivo. Application of the in vivo method in elderly individuals showed that their patellar tendons stiffened in response to a 14-week resistance training program by approximately 65% both structurally and materially. The rate of muscle torque development increased by approximately 27%, indicating faster contractile force transmission to the skeleton. The present findings suggest that strength training in old age can, at least partly, reverse the deteriorating effect of ageing on tendon properties and function.     

In defense of the somatic mutation theory of cancer

According to the somatic mutation theory (SMT), cancer begins with a genetic change in a single cell that passes it on to its progeny, thereby generating a clone of malignant cells. It is strongly supported by observations of leukemias that bear specific chromosome translocations, such as Burkitt's lymphoma, in which a translocation activates the c-myc gene, and chronic myeloid leukemia (CML), in which the Philadelphia chromosome causes production of the BCR-ABL oncoprotein. Although the SMT has been modified and extended to encompass tumor suppressor genes, epigenetic inheritance, and tumor progression through accumulation of further mutations, perhaps the strongest validation comes from the successful treatment of certain malignancies with drugs that directly target the product of the mutant gene.     

The effect of recurrent mutation on the frequency spectrum of a segregating site and the age of an allele

The sample frequency spectrum of a segregating site is the probability distribution of a sample of alleles from a genetic locus, conditional on observing the sample to be polymorphic. This distribution is widely used in population genetic inferences, including statistical tests of neutrality in which a skew in the observed frequency spectrum across independent sites is taken as a signature of departure from neutral evolution. Theoretical aspects of the frequency spectrum have been well studied and several interesting results are available, but they are usually under the assumption that a site has undergone at most one mutation event in the history of the sample. Here, we extend previous theoretical results by allowing for at most two mutation events per site, under a general finite allele model in which the mutation rate is independent of current allelic state but the transition matrix is otherwise completely arbitrary. Our results apply to both nested and nonnested mutations. Only the former has been addressed previously, whereas here we show it is the latter that is more likely to be observed except for very small sample sizes. Further, for any mutation transition matrix, we obtain the joint sample frequency spectrum of the two mutant alleles at a triallelic site, and derive a closed-form formula for the expected age of the younger of the two mutations given their frequencies in the population. Several large-scale resequencing projects for various species are presently under way and the resulting data will include some triallelic polymorphisms. The theoretical results described in this paper should prove useful in population genomic analyses of such data.     

The somatic mutation landscape of the human body

A major challenge in the analysis of DNA methylation (DNAm) data is variability introduced from intra-sample cellular heterogeneity, such as whole blood which is a convolution of DNAm profiles across a unique cell type. When this source of variability is confounded with an outcome of interest, if unaccounted for, false positives ensue. Current methods to estimate the cell type proportions in whole blood DNAm samples are only appropriate for one technology and lead to technology-specific biases if applied to data generated from other technologies. Here, we propose the technology-independent alternative: methylCC, which is available at https://github.com/stephaniehicks/methylCC.     

In vivo somatic mutations in Werner’s syndrome

The frequencies of mutant erythrocytes with loss of heterozygosity at the glycophorin A (GPA) locus and mutant CD4⁺ T cells lacking surface expression of the T-cell receptor αβ (TCR)/CD3 complex were measured by flow cytometry for Japanese Werner’s syndrome (WRN) patients. The hemizygous and homozygous GPA mutant frequencies (GPA Mfs) and the TCR/CD3-defective mutant frequency (TCR Mf) in WRN patients were found to be significantly higher than those in normal controls in the same age range. However, because these Mfs in the patients are only about twice those in controls, it is difficult to conclude that the WRN gene mutations cause instability of somatic genes. This contrasts markedly with Bloom’s syndrome (BLM) patients, whose GPA and TCR Mfs were previously reported to increase about 50- and 15-fold, respectively. The difference in Mfs is one aspect of the large variation in the phenotype observed between WRN and BLM patients, suggesting a different role of the responsible genes, both of which belong to the RecQ DNA helicase gene family, in the control of somatic mutagenesis. Received: 8 June 1998 / Accepted: 21 August 1998     

Clastogenic effects in human lymphocytes of power frequency electric fields: In vivo and in vitro studies

Summary In vivo and in vitro studies of the clastogenic effects of power frequency electric fields and transient electric currents have been performed. For the in vivo investigation peripheral lymphocytes from twenty switchyard workers were screened for chromosome anomalies. The rates of chromatid and chromosome breaks were found to be significantly increased compared to the rates in 17 controls.Exposure of human peripheral lymphocytes, in vitro, to a 50-Hz current with 1 mA/cm² current density did not induce any chromosome damage. Exposure to ten 3 µs-long spark discharge pulses with a peak field strength in the samples of 3.5 kV/cm, however, resulted in chromosome breaks at a frequency similar to that induced in lymphocytes in vitro by ionizing radiation at 0.75 Gy.The biological significance of chromosomal damage induced in somatic cells is discussed.     

In vivo tracking of the human patella.

The purpose of this study was to describe the dynamic, in vivo, three-dimensional tracking pattern of the patella for one normal male subject. Intracortical pins were inserted into the patella, tibia, and femur. The subject performed seated and squatting knee flexion/extension, and maximum voluntary quadriceps contractions. In addition, the vastus medialis oblique was subjected to maximal electrical stimulation. Motions of the markers attached to the intracortical pins were analyzed using an automated video system. Patellar and tibial motions were determined relative to a femoral reference system. While the tibia flexed 50 degrees from full extension (seated condition), the patella flexed 30.3 degrees, tilted laterally 10.3 degrees, and shifted laterally 8.6 mm. In general, these results show qualitative agreement with the data collected from cadaveric specimens [van Kampen and Huiskes, J. orthop. Res. 8, 372-382 (1990)]. The differences present may reflect different passive constraints to patellar motions, and different relative loading of the individual quadriceps components, in our study compared to the cadaveric study. Only small differences were found between patellar motions in the seated and squatting conditions. Differences in patellar displacements produced by (1) maximal electrical stimulation of the vastus medialis oblique, and (2) maximum voluntary quadriceps contraction, at 30 degrees knee flexion and full extension, may reflect the dominant influence of passive constraints, and the vastus lateralis, on normal patellar motions. Further in vivo study of patellar tracking seems warranted to evaluate surgical and conservative interventions for patellofemoral disorders.     

In vivo and in vitro studies of immunoglobulin gene somatic hypermutation

Following antigen encounter, two distinct processes modify immunoglobulin genes. The variable region is diversified by somatic hypermutation while the constant region may be changed by class-switch recombination. Although both genetic events can occur concurrently within germinal centre B cells, there are examples of each occurring independently of the other. Here we compare the contributions of class-switch recombination and somatic hypermutation to the diversification of the serum immunoglobulin repertoire and review evidence that suggests that, despite clear differences, the two processes may share some aspects of their mechanism in common.     

Evidence for an elevated frequency of in vivo somatic cell mutations in ataxia telangiectasia.

Somatic cell mutation frequency in vivo was measured in individuals with high cancer risk who were from ataxia telangiectasia (A-T) families. The assay for somatic mutation measures the frequency of variant erythrocytes which are progeny of erythroid precursor cells with mutations that result in a loss of gene expression at the polymorphic glycophorin A (GPA) locus. Samples from 14 of 15 A-T homozygotes showed high frequencies of GPA gene expression-loss variant cells with normal expression of only one of the two alleles at the GPA locus (i.e., GPA hemizygous variant cells). The mean elevation of the frequency of hemizygous variant cells over those in normal controls and unaffected family members was 7-14-fold. A-T homozygotes also showed an increase in the frequency of cells in which one allele at the GPA locus had lost expression and in which the remaining allele was expressed at a homozygous level (i.e., GPA homozygous variant cells). Family members who are obligate A-T heterozygotes did not appear to have a significantly elevated frequency of GPA hemizygous or homozygous variant cells. These indications of elevated in vivo frequencies of variant erythrocytes in A-T homozygotes support a causal link between susceptibility to somatic mutation and susceptibility to cancer.     

Constitutional and somatic RB1 mutation spectrum in nonfamilial unilateral and bilateral retinoblastoma in India

An epidemiologic survey has indicated a comparatively high prevalence of retinoblastoma (Rb) in Asian countries. Recently, the development of preventive strategies in nonfamilial Rb has become a major goal. The present studies were designed for identification and characterization of constitutional and somatic RB1 gene mutations by conventional cytogenetics, fluorescent in situ hybridization (FISH) and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP)-DNA sequencing. Of 34 patients 32 were nonfamilial and 2 were familial Rb. Maternal inheritance of del (13q14) was common. FISH was sensitive in detecting monoallelic RB1 deletion/deletion mosaicism as a first genetic hit in 20% of cases. Somatic and germline RB1 point mutations affected exons 3, 17, 20, and 21 and these were identified as novel mutations. Involvement of exon 20 as a predisposing mutation in sporadic unilateral retinoblastoma (URB) probably suggests the susceptibility of exon 20 to unknown etiologic factors in our population. A de novo RB1 deletion along with transmitted RB1 point mutation from an asymptomatic parent was identified as a unique predisposing RB1 mutation chimerism in a URB case that later evolved to bilateral retinoblastoma (BRB). The predisposing mutations such as del (13q), RB1 mono-allelic deletion and RB1 point mutation in sporadic Rb were de novo as well as transmitted mutations from asymptomatic/symptomatic parents. The RB1 mutation incidence was comparatively higher (25%) in nonfamilial Rb with emphasis on high prevalence in sporadic URB (18% versus 0%-9% in the literature series). The present studies demonstrated the efficacy of a multitechnique approach to detect various types of constitutional RB1 mutations such as RB1 deletion, deletion mosaicism, point mutation, mutation chimerism in patients of symptomatic/asymptomatic parents.     

In vivo biomicroscopy of the skin with high-resolution magnetic resonance imaging and high frequency ultrasound.

Noninvasive imaging and characterization of the skin is of great interest in dermatology. In order to get relevant diagnostic information, high-resolution imaging techniques have to be applied. Ultrasonic imaging is a potential method for this purpose where the special requirements concerning the spatial resolution make it essential to apply high frequency ultrasound (HFUS). Alternatively, magnetic resonance imaging (MRI), being a very promising imaging modality, also shows the perspective of becoming a valuable diagnostic tool in dermatology. However, to account for the small dimensions of the structures under observation, very specialized system designs have to be developed. In this paper, a HFUS imaging system working in the 50 MHz and 100 MHz range is applied for high-resolution skin imaging. Furthermore, a commercial MRI-system was equipped with specially designed low noise rf (radio frequency) coils with minimized volume, and customized imaging sequences were applied to optimize the signal-to-noise ratio. With HFUS and high-resolution magnetic resonance (HR-MR) imaging complementary imaging techniques for in vivo biomicroscopy of the skin are available.     

LET and ion-species dependence for mutation induction and mutation spectrum on hprt locus in normal human fibroblasts.

We have been studying LET and ion species dependence of RBE in mutation frequency and mutation spectrum of deletion pattern of exons in hprt locus. Normal human skin fibroblasts were irradiated with heavy-ion beams, such as carbon- (290 MeV/u and 135 MeV/u), neon- (230 MeV/u and 400 MeV/u), silicon- (490 MeV/u) and iron- (500 MeV/u) ion beams, generated by Heavy Ion Medical Accelerator in Chiba (HIMAC) at national Institute of Radiological Sciences (NIRS). Mutation induction in hprt locus was detected to measure 6-thioguanine resistant colonies and deletion spectrum of exons was analyzed by multiplex PCR. The LET-RBE curves of mutation induction for carbon- and neon-ion beams showed a peak around 75 keV/micrometers and 155 keV/micrometers, respectively. On the other hand, there observed no clear peak for silicon-ion beams. The deletion spectrum of exons was different in induced mutants among different ion species. These results suggested that quantitative and qualitative difference in mutation occurred when using different ion species even if similar LET values.