Zhi-Ping Ren

Biobanking of fresh frozen tissue : RNA is stable in nonfixed surgical specimens

Molecular tools for tissue profiling, such as expression microarrays and real-time PCR, generally require collection of fresh frozen tissues as sources of high-quality RNA. The fragile nature of RNA prompted us to examine the effects of storage time and transport conditions with regard to RNA integrity and gene expression in nonfixed surgical human specimens. At surgery, fresh normal tonsil and colon tissue was cut into pieces and snap frozen. Additional fresh tissue pieces were (i) left at room temperature, (ii) kept on ice, (iii) in normal saline or (iv) in a commercial RNA-stabilizing buffer (RNAlater) and snap frozen after 0.5, 1, 3, 6 and 16 h. Structural RNA integrity was analysed by microchip electrophoresis. Surprisingly, RNA remained stable in both tissue types under all conditions tested for up to 6–16 h. Gene expression by real-time PCR of cfos, HIF1α, Bcl2, PCNA, TGFβ1 and SMAD7 was analysed at different storage time points in tonsil tissue. Expression levels were essentially stable when samples were kept on ice, while marked regulation of single genes was observed during storage at room temperature, in normal saline and in RNAlater. Furthermore, we analysed selected tissue types from the local biobank representing 47 normal and malignant tissues transported on ice for up to 2–3 h before biobanking. RNA prepared from 45 of the 47 samples exhibited distinct ribosomal peaks indicating intact RNA. This study shows that RNA degradation is a minor problem during handling of fresh human tissue before biobanking. Our data indicate that nonfixed tissue specimens may be transported on ice for hours without any major influence on RNA quality and expression of the selected genes. However, further studies are warranted to clarify the impact of transport logistics on global gene expression.

Molecular pathology in basal cell cancer with p53 as a genetic marker.

Human basal cell cancer (BCC) has unique growth characteristics with virtual inability to metastasize. We investigated clonality and genetic progression using p53 mutations as marker. Sampling was done through microdissection of frozen immunohistochemically stained 16 μm slices of tumors. From 11 BCC tumors 78 samples were analysed. Direct DNA sequencing of exons 5 – 8 was performed, haplotypes were determined after cloning of p53 exons and loss of heterozygosity (LOH) ascertained by microsatellite analysis. All tumors had p53 mutations and in a majority both p53 alleles were affected, commonly through missense mutations. Microdissection of small parts (50 – 100 cells) of individual tumors showed BCC to be composed of a dominant cell clone and prone to genetic progression with appearance of subclones with a second and even third p53 mutation. Samples from normal immunohistochemically negative epidermis always showed wild type sequence, except for a case of previously unknown germline p53 mutation. Our analysis also included p53 immunoreactive patches i.e. morphologically normal epidermis with a compact pattern of p53 immunoreactivity. Mutations within those were never the same as in the adjacent BCC. This detailed study of only one gene thus uncovered a remarkable heterogeneity within a tumor category famous for its benign clinical behavior.

Two distinct p53 immunohistochemical patterns in human squamous‐cell skin cancer, precursors and normal epidermis

Specimens of squamous‐cell neoplasms (81 invasive cancers, 36 in situ cancers, 70 dysplasias, 5 keratoacanthomas, 19 papillomas) and normal skin were immunostained with p53 antibody. Nuclear accumulation of p53 was visualized as following 2 distinct patterns: dispersed or compact. The former is interpreted as a reversible reaction to sunlight, whereas the latter, after microdissection and sequencing of DNA, has been shown to reflect clonal multiplication of keratinocytes with mutated p53. The dispersed pattern was diffusely distributed and usually only involved a small proportion of epidermal cells. The compact pattern was characterized as a contiguous area of homogeneously stained cells sharply demarcated from its surroundings. It involved patches of normal epidermis or large areas of dysplastic or malignant squamous epithelium. Immature cells were always stained, whereas immunoreactivity was variably present in differentiating keratinocytes. Dispersed patterns occurred in 94.7% of strongly UV‐exposed skin (mainly face) and to a lesser extent in less exposed parts of the body. It showed no correlation to the age of the individual. About two‐thirds of biopsies from individuals over age 50 displayed compact patterns in sun‐exposed, otherwise normal, epidermis. About 65% of pre‐malignant and malignant squamous‐cell neoplasms had a compact pattern. The presence of p53 immunoreactivity as a compact pattern supports the idea that mutations of the p53 gene are early events in the sequence from dysplasia to invasive squamous‐cell cancer of the skin. Also, even in the absence of cellular atypia, patches of epidermal cells can accumulate p53 in a way that is indistinguishable from that of cancer and pre‐cancer.

Retraction Notice to: VMA21 Deficiency Causes an Autophagic Myopathy by Compromising V-ATPase Activity and Lysosomal Acidification

X-linked myopathy with excessive autophagy (XMEA) is a childhood-onset disease characterized by progressive vacuolation and atrophy of skeletal muscle. We show that XMEA is caused by hypomorphic alleles of the VMA21 gene, that VMA21 is the diverged human ortholog of the yeast Vma21p protein, and that like Vma21p it is an essential assembly chaperone of the V-ATPase, the principal mammalian proton pump complex. Decreased VMA21 raises lysosomal pH, which reduces lysosomal degradative ability and blocks autophagy. This reduces cellular free amino acids, which upregulates the mTOR pathway and mTOR-dependent macroautophagy, resulting in proliferation of large and ineffective autolysosomes that engulf sections of cytoplasm, merge together, and vacuolate the cell. Our results uncover macroautophagic overcompensation leading to cell vacuolation and tissue atrophy as a mechanism of disease.

Genetic instability in the 9q22.3 region is a late event in the development of squamous cell carcinoma.

Squamous cell carcinoma (SCC) of the skin represents a group of neoplasms which is associated with exposure to UV light. Recently, we obtained data suggesting that invasive skin cancer and its precursors derive from one original neoplastic clone. Here, the analysis were extended by loss of heterozygosity (LOH) analysis in the chromosome 9q22.3 region. A total of 85 samples, taken from twenty-two sections of sun-exposed sites, corresponding to normal epidermis, morphological normal cells with positive immuno-staining for the p53 protein (p53 patches), dysplasias, cancer in situ (CIS) and squamous cell carcinomas (SCC) of the skin were analysed. Overall, about 70% of p53 patches had mutations in the p53 gene but not LOH in the p53 gene or 9q22.3 region. Approximately 70% of the dysplasias showed p53 mutations of which about 40% had LOH in the p53 region but not in the 9q22.3 region. In contrast, about 65% of SCC and CIS displayed LOH in the 9q22.3 region, as well as frequent (80%) mutations and/or LOH in the p53 gene. These findings strongly suggest that alterations in the p53 gene is an early event in the progression towards SCC, whereas malignant development involves LOH and alterations in at least one (or several) tumor suppressor genes located in chromosome 9q22.3.

Recovering DNA and Optimizing PCR Conditions from Microdissected Formalin-Fixed and Paraffin-Embedded Materials

Microdissection is a powerful technique in molecular pathology and genetic investigations. To detect genetic alterations such as gene mutation or deletion from tumor specimen, the purity of target cells is extremely critical. Unwanted cell contamination will dramatically dilute the detectable level of the abnormality. The major obstacle in clinical research is to obtain sufficient and qualified DNA from a small amount of formalin-fixed and paraffin-embedded materials. We have successfully modified our previous protocols and overcome the difficulties of recovery of DNA. After these modifications, almost every single formalin-fixed and paraffin-embedded specimen has been successfully amplified in the required DNA region.

Genetically distinct astrocytic and oligodendroglial components in oligoastrocytomas

Oligoastrocytomas are glial tumours consisting of a mixture of neoplastic astrocytic and oligodendroglial cells. Genetic alterations of oligoastrocytomas include loss of heterozygosity of chromosomes 1p and/or 19q (LOH 1p/19q), typically occurring in oligodendrogliomas, and mutations of TP53, frequently occurring in astrocytomas. To investigate whether these neoplastic cell types in oligoastrocytomas have different genetic profiles, we examined the two different components of oligoastrocytomas in comparison with the histological diagnosis of the specific tumour area for LOH 1p/19q and TP53 mutations by using microdissection technique. We found a variety of lost markers for 1p and 19q, and the presence of two different TP53 mutations in the tumour samples. In the majority of cases (9/11), the oligodendroglial and astrocytic components of an individual oligoastrocytoma displayed the same genotype. We present two cases of biphasic oligoastrocytomas with aberrant findings, suggesting the coexistence of genetically and morphologically distinct tumour cell clones in these tumours. In one case, the oligodendroglial part of the tumour showed LOH19q, whereas the astrocytic part showed TP53 mutation (codon 273). In another case, we found LOH 1p/19q in the oligodendroglial component, but two retained areas on chromosome 1p in the astrocytic component of the tumour. No evidence was found for the coexistence of tumour cells with the two genotypical changes within the same morphological region of one individual tumour. The two cases of biphasic oligoastrocytomas in our sample that display a different genotype in the astrocytic and oligodendroglial part of the tumour show that different components of an oligoastrocytoma may be derived from different cell clones during neoplastic transformation.

Molecular Genetic Analysis of p53 Intratumoral Heterogeneity in Human Astrocytic Brain Tumors

We investigated genetic heterogeneity of astrocytic gliomas using p53 gene mutations as a marker. Different parts of morphologically heterogeneous astrocytic gliomas were microdissected, and direct DNA sequencing of p53 gene exons 5 through 8 was performed. Thirty-five glioma samples and tumor-adjacent normal-appearing brain tissue from 11 patients were analyzed. Sixteen different p53 gene mutations were found in 7 patients. We found that some tumors were devoid of p53 gene mutations, whereas other tumors carried 1 or often several (up to 3) different mutations. The mutations were present in grade II, III, and IV astrocytic glioma areas. Both severe functionally dead mutants and mutants with remaining transcriptional activity could be observed in the same tumor. We observed that morphologically different parts of a glioma could carry different or similar mutations in the p53 gene and could be either associated or not associated with the locus of heterozygosity at the mutant site. Coexistence of p53 gene mutations and the locus of heterozygosity was common, at least in astrocytomas grade III and in glioblastomas, and also occurred in astrocytoma grade II areas. These results support the notion that intratumoral heterogeneity in brain tumors originates from different molecular defects. Our results are of importance for a further understanding of the molecular mechanisms behind failure to treat glioma patients.

Molecular genetic and epigenetic analysis of NCX2/SLC8A2 at 19q13.3 in human gliomas

M. Qu, H. Jiao, J. Zhao, Z.‐P. Ren, A. Smits, J. Kere and M. Nistér (2010) Neuropathology and Applied Neurobiology36, 198–210
Molecular genetic and epigenetic analysis of NCX2/SLC8A2 at 19q13.3 in human gliomas

The Role of p53 Codon 72 and Human Papilloma Virus Status of Cutaneous Squamous Cell Carcinoma in the Swedish Population

The arginine variant of the p53 codon 72 polymorphism as well as anogenital and epidermodysplasia verruciformis (EV) types of human papilloma virus (HPV) are suggested to confer increased risk for developing cutaneous squamous cell carcinoma (SCC). In this pilot study, we analysed the p53 codon 72 genotype distribution in 106 microdissected samples from normal and tumour tissues of 53 cases of cutaneous SCC and 96 controls from Sweden. Both normal and tumour samples from cases of SCC were screened for anogenital and EV HPV. The p53Arg allele was not associated with the development of cutaneous SCC. Anogenital HPV (44%) was more prevalent than EV HPV (12%). Data also indicate that anogenital HPV is more common in tumour samples, but HPV infection was not identified as a significant risk factor for developing SCC. The presence of anogenital HPV, but not EV HPV might be a risk factor for development of cutaneous SCC.