Focal nodular hyperplasia with concomitant hepatocellular carcinoma: a case report and clonal analysis

This report describes a hepatocellular carcinoma (HCC) with concomitant focal nodular hyperplasia (FNH) in a 56 years old Chinese man. There were two well circumscribed tumours measuring 3×2.5×2 cm and 2×1.5×1.5 cm. The larger mass was grey and soft with a small area of bleeding and necrosis and an intact capsule. The smaller mass was yellow and had no capsule. Clonal analysis was carried out to clarify the relation between the HCC and the adjacent FNH. The clonal analysis was based on the methylation pattern of the polymorphic X chromosome linked androgen receptor gene (HUMARA). In FNH, after Hpa Ⅱ digestion, the allelic bands showed two well defined peaks. The intensity of the two peaks in the DNA from cirrhotic tissue did not differ significantly, consistent with a random pattern of X chromosome inactivation. However, in HCC, after Hpa Ⅱ digestion, the allelic bands differed significantly in intensity. Therefore, there was a typical polyclonal pattern of inactivation in FNH but the HCC was interpreted as being monoclonal.

Clonal Analysis of Peripheral Papilloma and Cancerous Cells of the Breast

OBJECTIVE Because almost all malignancies represent monoclonal proliferations, we have studied the clonal status of peripheral papillomas （peri-PM）, ductal carcinomas in situ （DCIS）, and normal breast tissues to explore a reliable way to distinguish benign and malignant （or pre-malignant） cases previously diagnosed morphologically. METHODS Twenty-six cases of peri-PM, 25 cases of peri-PM with atypical ductal hyperplasia （ADH）, 27 cases of DCIS, 16 cases of developed canceration and 20 specimens of normal tissue were examined in the study. The clonal status of these tissues was studied using an assay based on inactivation mosaicism of the lenth-polymorphic X-chromosomes at the androgen receptor （AR） locus. RESULTS Loss of polymorphism at the AR locus was found in all DCIS cases and 10 cases （10/25, 40.0%） of peri-PM with ADH, in.dicating the monoclonality of the tumors. Twenty-four out of 26 （92.3%） cases with peri- PM and 19 specimens of normal tissue were shown to be polyclonal. In 16 cases of developed Canceration, identical X chromosome inactivation （monoclonal alterations） was observed from both the peri-PM with ADH part, and the DCIS part in each Case. CONCLUSION These results contribute to the understanding of the genetic changes of peri-PM, and confirm the peri-PM with ADH as a precancerous lesion of the breast. Clonal analysis might be a useful modality to screen high-risk cases from precancerous lesions or to distinguish between benign hyperplasia and early carcinoma.

EARLY DIAGNOSIS OF MYELODYSPLASTIC SYNDROMES USING CLONAL ANALYSES

Objective: To study the value of clonal analysis to the early diagnosis of myelodysplastic syndrome (MDS). Methods: Four types of clonal analyses were performed on the bone marrow samples from 50 patients suspected of MDS: (1) Conventional Cytogenetics (CC) for clonal chromosomal abnormalities; (2) BrdU-Sister Chromatid Differentiation (BrdU-SCD) for cell cycle kinetics; (3) Fluorescence in Situ Hybridization (FISH) for trisomy 8; (4) Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) for N-ras mutation. Results: The diagnosis of forty-three patients was compatible with the FAB criteria for MDS. The other seven cases didn’t meet the FAB criteria, with only one lineage of dyspoiesis or with no obvious dysplastic changes. Among these seven cases, two were morphologically diagnosed with suspicious refractory anemia, one with sideroblastic anemia, one with leukemoid reaction, one with hypercellular anemia and two with chronic aplastic anemia. Clonal analyses of the 7 patients showed that six cases had clonal karyotype abnormalities, four had prolonged cell cycle patterns, four had trisomy 8 of different proportions and one had mutation of the exon 1 of N-RAS. Thus, they were revaluated as MDS patients. Conclusion: The untypical MDS patients with one lineage dyspoiesis or without obvious dysplastic changes can be diagnosed early by combining multiple clonal analysis techniques such as CC, SCD, FISH and PCR-SSCR.

Clonality and allelotype analyses of focal nodular hyperplasia compared with hepatocellular adenoma and carcinoma

AIM： To identify clonality and genetic alterations in focal nodular hyperplasia （FNH） and the nodules derived from it. METHODS： Twelve FNH lesions were examined. Twelve hepatocellular adenomas （HCAs） and 22 hepatocellular carcinomas （HCCs） were used as references. Nodules of different types were identified and isolated from FNH by microdissection. An X-chromosome inactivation assay was employed to describe their clonality status. Loss of heterozygosity （LOH） was detected, using 57 markers, for genetic alterations.RESULTS： Nodules of altered hepatocytes （NAH）, the putative precursors of HCA and HCC, were found in all the FNH lesions. Polyclonality was revealed in 10 FNH lesions from female patients, and LOH was not detected in any of the six FNH lesions examined, the results apparently showing their polyclonal nature. In contrast, monoclonality was demonstrated in all the eight HCAs and in four of the HCCs from females, and allelic imbalances were found in the HCAs （9/9） and HCCs （15/18）, with chromosomal arms 11p, 13q and 17p affected in the former, and 6q, 8p, 11p, 16q and 17p affected in the latter lesions in high frequencies （≥ 30%）. Monodonality was revealed in 21 （40%） of the 52 microdissected NAH, but was not found in any of the five ordinary nodules. LOH was found in all of the 13 NAH tested, being highly frequent at six loci on 8p, 11p, 13q and 17p. CONCLUSION： FNH, as a whole, is polyclonal, but some of the NAH lesions derived from it are already neoplastic and harbor similar allelic imbalances as HCAs.

Dissection of gene function at clonal level using mosaic analysis with double markers

MADM （Mosaic Analysis with Double Markers） technology offers a genetic approach in mice to visualize and concomitantly manipulate genetically defined cells at clonal level and single cell resolution. MADM employs Cre recombinase/loxP-dependent interchromosomal mitotic recombination to reconstitute two split marker genes--green GFP and red tdTomato -- and can label sparse clones of homozygous mutant cells in one color and wild-type cells in the other color in an otherwise unlabeled background. At present, major MADM applications include lineage tracing, single cell labeling, conditional knockouts in small populations of cells and induction of uniparental chromosome disomy to assess effects of genomic imprinting. MADM can be applied universally in the mouse with the sole limitation being the specificity of the promoter controlling Cre recombinase expression. Here I review recent developments and extensions of the MADM technique and give an overview of the major discoveries and progresses enabled by the implementation of the novel genetic MADM tools.

Tbr2-expressing intermediate progenitor cells in the adult mouse hippocampus are unipotent neuronal precursors with limited amplification capacity under homeostasis

Neurogenesis persists in two locations of the adult mammalian brain, the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus in the hippocampus. In the adult subgranular zone, radial glial- like cells （RGLs） are multipotent stem cells that can give rise to both astrocytes and neurons. In the process of generating neurons, RGLs divide asymmetrically to give rise to one RGL and one intermediate progenitor cell （IPC）. IPCs are considered to be a population of transit amplifying cells that proliferate and eventually give rise to mature granule neurons. The properties of individual IPCs at the clonai level are not well understood. Furthermore, it is not clear whether IPCs can generate astrocytes or revert back to RGLs, besides generating neurons. Here we developed a genetic marking strategy for clonal analysis and lineage-tracing of individual Tbr2-expressing IPCs in the adult hippocampus in vivo using Tbr2-CreERT2 mice. Using this technique we identified Tbr2-CreERT2 labeled IPCs as unipotent neuronal precursors that do not generate astrocytes or RGLs under homeostasis. Additionally, we showed that these labeled IPCs rapidly generate immature neurons in a synchronous manner and do not undergo a significant amount of amplification under homeostasis, in animals subjected to an enriched environment/running, or in animals with different age. In summary, our study suggests that Tbr2-expressing IPCs in the adult mouse hippocampus are unipotent precursors and rapidly give rise to immature neurons without major amplification.

Recent updates on the biological basis of heterogeneity in bone marrow stromal cells/ skeletal stem cells

Based on studies over the last several decades,the self-renewing skeletal lineages derived from bone marrow stroma could be an ideal source for skeletal tissue engineering.However,the markers for osteogenic precursors;i.e.,bone marrow-derived skeletal stem cells(SSCs),in association with other cells of the marrow stroma(bone marrow stromal cells,BMSCs)and their heterogeneous nature both in vivo and in vitro remain to be clarified.This review aims to highlight:i)the importance of distinguishing BMSCs/SSCs from other“mesenchymal stem/stromal cells”,and ii)factors that are responsible for their heterogeneity,and how these factors impact on the differentiation potential of SSCs towards bone.The prospective role of SSC enrichment,their expansion and its impact on SSC phenotype is explored.Emphasis has also been given to emerging single cell RNA sequencing approaches in scrutinizing the unique population of SSCs within the BMSC population,along with their committed progeny.Understanding the factors involved in heterogeneity may help researchers to improvise their strategies to isolate,characterize and adopt best culture practices and source identification to develop standard operating protocols for developing reproducible stem cells grafts.However,more scientific understanding of the molecular basis of heterogeneity is warranted that may be obtained from the robust high-throughput functional transcriptomics of single cells or clonal populations.