The application of molecular techniques to solid tumors

Recent developments in the field of molecular biology including the sequencing of the human genome and related high throughput methodologies are presenting the diagnostic pathologist with new opportunities to expand our understanding of human disease. These techniques enable the comprehensive assessment of molecular alterations with cell populations of interest, including cancer. It will be necessary for the diagnostic pathologist to become familiar with these techniques to effectively translate their potential into the clinical environment.     

Detection of YMDD motif mutants by oligonucleotide chips in lamivudine-untreated patients with chronic hepatitis B virus infection

Lamivudine, a nucleoside analogue, has been used widely as an effective antiviral agent for the treatment of patients with chronic hepatitis B virus (HBV) infection. However, the YMDD motif mutation of HBV polymerase resistant to lamivudine occurs very frequently after long term therapy. We developed an oligonucleotide chip for the detection of YMDD motif mutants resistant to lamivudine and investigated the prevalence of the mutants in patients with chronic HBV infection who had not been treated by lamivudine before. Forty patients who had not been treated with lamivudine were included in this study. Serum samples were tested by the oligonucleotide chips designed for detection of wild-type YMDD motif, M552V and M552I. Samples were confirmed by restriction fragment length polymorphism (RFLP) and direct sequencing. M552I mutants were detected by the oligonucleotide chips in 7.5% (3/40) of chronic HBV infected patients (2 chronic hepatitis and 1 cirrhosis). The results were in accordance with those of RFLP. YMDD motif mutants occur as natural genome variabilities in patients with chronic HBV infection who had not been treated with lamivudine before. Oligonucleotide chip technology is a reliable and useful diagnostic tool for the detection of mutants resistant to antiviral therapy in chronic HBV infection.     

Comparison of deltaFVC between patients with allergic rhinitis with airway hypersensitivity and patients with mild asthma.

BACKGROUND: In asthmatic individuals, airway sensitivity and maximal airway response are increased. Airway sensitivity is usually evaluated by measuring the provocation concentration of inhaled methacholine or histamine that causes a decrease in forced expiratory volume in 1 second of 20% (PC20). The percentage decrease in forced vital capacity at the PC20 (deltaFVC) has been proposed as a surrogate marker for maximal airway response. Individuals with allergic rhinitis and no clinical evidence of asthma frequently exhibit airway hypersensitivity. OBJECTIVE: To compare the deltaFVC between patients with allergic rhinitis and mild asthmatic patients with a similar degree of airway hypersensitivity. METHODS: A retrospective analysis of methacholine challenge test data from 72 children with allergic rhinitis and airway hypersensitivity (methacholine PC20 < 16 mg/mL) (rhinitis group) and from 72 children with mild atopic asthma matched to the rhinitis group regarding the methacholine PC20 (asthma group). The deltaFVC was calculated on the concentration-response curve to methacholine. RESULTS: The mean +/- SD deltaFVC was significantly lower in the rhinitis group (15.0% +/- 3.6%) vs the asthma group (17.4% +/- 5.3%) (P = .002). There was no significant correlation between the deltaFVC and PC20 in the rhinitis (r = -0.101; P = .41) and asthma (r = -0.023; P = .85) groups when 2 patients with PC20 less than 1 mg/mL were excluded from each group. CONCLUSIONS: Patients with allergic rhinitis and airway hypersensitivity had a significantly lower deltaFVC than methacholine PC20-matched mild asthmatic patients, suggesting that the level of maximal airway response in patients with allergic rhinitis is lower than that in mild asthmatic patients with a similar degree of airway hypersensitivity.     

Complex mosaic structural variations in human fetal brains

Somatic mosaicism, manifesting as single nucleotide variants (SNVs), mobile element insertions, and structural changes in the DNA, is a common phenomenon in human brain cells, with potential functional consequences. Using a clonal approach, we previously detected 200–400 mosaic SNVs per cell in three human fetal brains (15–21 wk postconception). However, structural variation in the human fetal brain has not yet been investigated. Here, we discover and validate four mosaic structural variants (SVs) in the same brains and resolve their precise breakpoints. The SVs were of kilobase scale and complex, consisting of deletion(s) and rearranged genomic fragments, which sometimes originated from different chromosomes. Sequences at the breakpoints of these rearrangements had microhomologies, suggesting their origin from replication errors. One SV was found in two clones, and we timed its origin to ∼14 wk postconception. No large scale mosaic copy number variants (CNVs) were detectable in normal fetal human brains, suggesting that previously reported megabase-scale CNVs in neurons arise at later stages of development. By reanalysis of public single nuclei data from adult brain neurons, we detected an extrachromosomal circular DNA event. Our study reveals the existence of mosaic SVs in the developing human brain, likely arising from cell proliferation during mid-neurogenesis. Although relatively rare compared to SNVs and present in ∼10% of neurons, SVs in developing human brain affect a comparable number of bases in the genome (∼6200 vs. ∼4000 bp), implying that they may have similar functional consequences.

Somatic mosaicism, the presence of more than one genotype in the somatic cells of an individual, is a prominent phenomenon in the human central nervous system. Forms of mosaicism include aneuploidies and smaller copy number variants (CNVs), structural variants (SVs), mobile element insertions, indels, and single nucleotide variants (SNVs). The developing human brain exhibits high levels of aneuploidy compared to other tissues, generating genetic diversity in neurons (Pack et al. 2005; Yurov et al. 2007; Bushman and Chun 2013). Such aneuploidy was suggested to be a natural feature of neurons, rather than a distinctive feature of neurodegeneration. However, the frequency of aneuploidy in neurons has been debated, with a separate study suggesting that aneuploidies occur in only about 2.2% of mature adult neurons (Knouse et al. 2014). They hence infer that such aneuploidy could have adverse effects at the cellular and organismal levels. Additionally, analysis of single cells from normal and pathological human brains identified large, private, and likely clonal somatic CNVs in both normal and diseased brains (Gole et al. 2013; McConnell et al. 2013; Cai et al. 2014; Knouse et al. 2016; Chronister et al. 2019; Perez-Rodriguez et al. 2019), with 3%–25% of human cerebral cortical nuclei carrying megabase-scale CNVs (Chronister et al. 2019) and deletions being twice as common as duplications (McConnell et al. 2013). Given that CNVs often arise from nonhomologous recombination and replication errors, their likely time of origin is during brain development. However, when CNVs first arise in human brain development has not yet been investigated. The present work is the first to examine this question using clonal populations of neuronal progenitor cells (NPCs) obtained from fetal human brains.Detection of CNVs in single neurons is challenging, given the need to amplify DNA. Such amplification may introduce artifacts that could, in turn, be misinterpreted as CNVs. In order to address this technical limitation, Hazen et al. reprogrammed adult postmitotic neurons using somatic cell nuclear transfer (SCNT) of neuronal nuclei into enucleated oocytes (Hazen et al. 2016). These oocytes then made sufficient copies of the neuronal genome allowing for whole-genome sequencing (WGS), thus eliminating the need for amplification in vitro. Using this method, they identified a total of nine structural variants in six neurons from mice, three of which were complex rearrangements. However, it is not possible to extend such studies to humans, given the ethical issues involved, besides the technical challenges in obtaining and cloning adult neurons. To circumvent the need of single-cell DNA amplification or nuclear cloning, we examined clonal cell populations obtained from neural progenitor cells from the frontal region of the cerebral cortex (FR), parietal cortex (PA) and basal ganglia (BG) and describe here the discovery and analysis of mosaic SVs in these NPCs (Bae et al. 2018). These clones were sequenced at 30× coverage (much higher than most previous single-cell studies), allowing identification of SVs other than large deletions and duplications as well as precise breakpoint resolution.     

The effects of total sleep deprivation on brain functional organization: mutual information analysis of waking human EEG.

Sleep deprivation can affect the waking electroencephalogram (EEG) that may reflect functional organization of the brain. We examined the effect of total sleep deprivation (TSD) on functional organization between different cortical areas from the waking EEG. Waking EEG data were recorded from 18 healthy male volunteers with eyes closed after 8-h night's sleep and after 24 h of TSD. The averaged cross mutual information (A-CMI) after 24 h of TSD were compared to before TSD. 24 h of TSD yielded the decreased A-CMIs in the inter-hemispheric C3-F4, C3-F8, and C3-C4 pairs: therefore, the electrodes that contribute to pairs with significant decrease of A-CMI were C3, F4, F8, and C4. The decreased A-CMIs between C3 and right frontal and central brain areas after 24 h of TSD may reflect the changes of cortico-cortical functional organization by homeostatic process during TSD. Our results of the frontal-area-related A-CMI decreases may support that the frontal brain regions are related to the homeostatic deterioration of brain function due to TSD.     

Posttransplant primary cutaneous Ki-1 (CD30)+/CD56+ anaplastic large cell lymphoma

An anaplastic large cell lymphoma that was negative for Epstein-Barr virus and positive for Ki-1 (CD30) presented as a polypoid scalp mass in a 56-year-old man 16 years after renal transplantation. The lymphoma was of the CD4+ cytotoxic T-cell lineage, and the tumor cells also expressed CD56. Despite reduction in the dose of immunosuppression and localized radiotherapy, the tumor had rapidly progressed to involve the soft tissue of the right hand. Systemic chemotherapy induced complete regression of the soft tissue lesion. This case illustrates that posttransplant primary cutaneous CD30+ anaplastic large cell lymphomas may assume an aggressive clinical course but can still be controlled by systemic chemotherapy.     

Analysis of the immune response to Mycobacterium avium subsp. paratuberculosis in experimentally infected calves

Johne's disease of cattle is widespread and causes significant economic loss to producers. Control has been hindered by limited understanding of the immune response to the causative agent, Mycobacterium avium subsp. paratuberculosis, and lack of an effective vaccine and sensitive specific diagnostic assays. The present study was conducted to gain insight into factors affecting the immune response to M. avium subsp. paratuberculosis. A persistent proliferative response to M. avium subsp. paratuberculosis purified protein derivative and soluble M. avium subsp. paratuberculosis antigens was detected in orally infected neonatal calves 6 months postinfection (p.i.) by flow cytometry (FC). CD4(+) T cells with a memory phenotype (CD45R0(+)) expressing CD25 and CD26 were the predominant cell type responding to antigens. Few CD8(+) T cells proliferated in response to antigens until 18 months p.i. gammadelta T cells did not appear to respond to antigen until 18 months p.i. The majority of WC1(+) CD2(-) and a few WC1(-) CD2(+) gammadelta T cells expressed CD25 at time zero. By 18 months, however, subsets of gammadelta T cells from both control and infected animals showed an increase in expression of CD25, ACT2, and CD26 in the presence of the antigens. Two populations of CD3(-) non-T non-B null cells, CD2(+) and CD2(-), proliferated in cell cultures from some control and infected animals during the study, with and without antigen. The studies clearly show multicolor FC offers a consistent reliable way to monitor the evolution and changes in the immune response to M. avium subsp. paratuberculosis that occur during disease progression.     

Somatic mitochondrial DNA mutations in cortex and substantia nigra in aging and Parkinson's disease

Oxidative damage to mitochondrial DNA (mtDNA) increases with age in the brain and can induce G:C to T:A and T:A to G:C point mutations. Though rare at any particular site, multiple somatic mtDNA mutations induced by oxidative damage or by other mechanisms may accumulate with age in the brain and thus could play a role in aging and neurodegenerative diseases. However, no prior study has quantified the total burden of mtDNA point mutation subtypes in the brain. Using a highly sensitive cloning and sequencing strategy, we find that the aggregate levels of G:C to T:A and T:A to G:C transversions and of all point mutations increase with age in the frontal cortex (FCtx). In the substantia nigra (SN), the aggregate levels of point mutations in young controls are similar to the levels in the SN or FCtx of elderly subjects. Extrapolation from our data suggests an average of 2.7 (FCtx) to 3.2 (SN) somatic point mutations per mitochondrial genome in elderly subjects. There were no significant differences between Parkinson's disease (PD) patients and age-matched controls in somatic mutation levels. These results indicate that individually rare mtDNA point mutations reach a high aggregate burden in FCtx and SN of elderly subjects.     

Methods for derivation of human embryonic stem cells

The expanded blastocysts, developed from 2PN-stage embryos, are generally divided into three categories: a good blastocyst containing a large and distinguishable inner cell mass (ICM), a blastocyst with a small and distinct ICM, and a blastocyst with a poorly defined ICM. In this study, we introduce methods for the derivation of human embryonic stem cells (hESCs) depending on the quality of the blastocysts. An immunosurgical method was used for the good expanded blastocysts. This method, however, raises the probability of ICM loss in cases of hESC derivation from blastocysts with smaller or indistinct ICMs. Furthermore, this method is also associated with a risk of the contamination of the hESCs with animal pathogens. To overcome these shortcomings, the partial- or whole-embryo culture method was used. For blastocysts with no visible ICM, the whole-embryo culture method was used to establish hESCs via the seeding of the entire blastocyst without its zona pellucida directly on a STO feeder layer. However, trophectodermal overgrowth tends to hinder the expansion of the ICM during the initial steps of hESC derivation. Therefore, the partial-embryo culture method was developed to establish hESCs from blastocysts with smaller ICMs. The surgical isolation of the region containing the ICM with an ultra-fine glass pipette alleviates trophectoderm overgrowth. This method is also applicable to blastocysts with large and distinct ICMs, and the efficiency of this method is comparable to that of the immunosurgical method.