Journal of Pathology and Translational Medicine

Review

Single-cell and spatial sequencing application in pathology: Yoon-Seob Kim, Jinyong Choi, Sug Hyung Lee; J Pathol Transl Med. 2023;57(1):43-51. Published online January 10, 2023; DOI: https://doi.org/10.4132/jptm.2022.12.12

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Traditionally, diagnostic pathology uses histology representing structural alterations in a disease’s cells and tissues. In many cases, however, it is supplemented by other morphology-based methods such as immunohistochemistry and fluorescent in situ hybridization. Single-cell RNA sequencing (scRNA-seq) is one of the strategies that may help tackle the heterogeneous cells in a disease, but it does not usually provide histologic information. Spatial sequencing is designed to assign cell types, subtypes, or states according to the mRNA expression on a histological section by RNA sequencing. It can provide mRNA expressions not only of diseased cells, such as cancer cells but also of stromal cells, such as immune cells, fibroblasts, and vascular cells. In this review, we studied current methods of spatial transcriptome sequencing based on their technical backgrounds, tissue preparation, and analytic procedures. With the pathology examples, useful recommendations for pathologists who are just getting started to use spatial sequencing analysis in research are provided here. In addition, leveraging spatial sequencing by integration with scRNA-seq is reviewed. With the advantages of simultaneous histologic and single-cell information, spatial sequencing may give a molecular basis for pathological diagnosis, improve our understanding of diseases, and have potential clinical applications in prognostics and diagnostic pathology.

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Original Articles

Copy Number Alterations of BCAS1 in Squamous Cell Carcinomas.: Yu Im Kim, Ahwon Lee, Jennifer Kim, Bum Hee Lee, Sung Hak Lee, Suk Woo Nam, Sug Hyung Lee, Won Sang Park, Nam Jin Yoo, Jung Young Lee, Sang Ho Kim, Su Young Kim; Korean J Pathol. 2011;45(3):271-275.; DOI: https://doi.org/10.4132/KoreanJPathol.2011.45.3.271

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Abstract PDF

BACKGROUND
Breast carcinoma amplified sequence 1 (BCAS1), located in 20q13, is amplified and overexpressed in breast cancers. Even though BCAS1 is expected to be an oncogene candidate, its contribution to tumorigenesis and copy number status in other malignancies is not reported. To elucidate the role of BCAS1 in squamous cell carcinomas, we investigated the copy number status and expression level of BCAS1 in several squamous cell carcinoma cell lines, normal keratinocytes and primary tumors.
METHODS
We quantitated BCAS1 gene by real-time polymerase chain reaction (PCR). Expression level of BCAS1 was measured by real-time reverse transcription-PCR and immunoblot.
RESULTS
Seven (88%) of 8 squamous cell carcinoma cell lines showed copy number gain of BCAS1 with various degrees. BCAS1 gene in primary tumors (73%) also showed copy number gain. However, expression level did not show a linear correlation with copy number changes.
CONCLUSIONS
We identified copy number gain of BCAS1 in squamous cell carcinomas. Due to lack of linear correlation between copy numbers of BCAS1 and its expression level, we could not confirm that the overexpression of BCAS1 is a common finding in squamous cell carcinoma cell lines. However, this study shows that the copy number gain of BCAS1 is a common finding in squamous cell carcinomas.

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Electrochemical Approaches for Preparation of Tailor-Made Amino Acids
Nana Wang, Jingcheng Xu, Haibo Mei, Hiroki Moriwaki, Kunisuke Izawa, Vadim A. Soloshonok, Jianlin Han
Chinese Journal of Organic Chemistry.2021; 41(8): 3034. CrossRef

Mutation and Expression of DNA2 Gene in Gastric and Colorectal Carcinomas.: Sung Hak Lee, Yoo Ri Kim, Nam Jin Yoo, Sug Hyung Lee; Korean J Pathol. 2010;44(4):354-359.; DOI: https://doi.org/10.4132/KoreanJPathol.2010.44.4.354

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Abstract PDF

BACKGROUND
Deregulation of DNA repair and replication are involved in cancer development. DNA2 is a nuclease/helicase that plays roles in DNA repair and replication. The aim of this study was to explore DNA2 mutation and DNA2 protein expression in gastric cancers (GCs) and colorectal cancers (CRCs).
METHODS
We analyzed two mononucleotide repeats in DNA2 in 27 GCs with high microsatellite instability (MSI-H), 34 GCs with stable MSI (MSS), 29 CRCs with MSI-H and 35 CRCs with MSS by single-strand conformation polymorphism. We also analyzed DNA2 expression in GCs and CRCs either with MSI-H or MSS.
RESULTS
We found DNA2 mutations in two GCs (7.1%) and two CRCs with MSI-H (6.9%), but not in cancers with MSS. The mutations consisted of three cases of a c.2593delT and one of a c.2592_2593delTT, which would result in premature stopping of amino acid synthesis (p.Ser865Hisfsx6 and p.Ser865Thrfsx20, respectively). DNA2 expression was observed in 16 (80%) of the GCs and 15 (75%) of the CRCs with MSI-H, but all of the cancers with DNA2 frameshift mutations were weak or negative for DNA2.
CONCLUSIONS
Our data indicate that DNA2 mutation and loss of DNA2 expression occur in GCs and CRCs, and suggest that these alterations may contribute to cancer pathogenesis by deregulating DNA repair and replication.

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Nature Communications.2016;[Epub] CrossRef

The Role of gadd and p53 Genes in Apoptosis and Cell Cycle Delay by Genotoxic Agents.: Jung Young Lee, Jung Duk Lee, Seung Myung Dong, Eun Young Na, Min Sun Shin, Su Young Kim, Sug Hyung Lee, Won Sang Park, Nam Jin Yoo; Korean J Pathol. 1998;32(4):239-247.

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Abstract PDF: The aim of this study was to investigate the relationships between the gadd genes expression and an apoptosis induction in two different growing cell types after treatments with cisplatin and methylmethan sulfonate (MMS). We have examined the kinetics and specificity of gadd45 and gadd153 expression following cisplatin and MMS treatments to HL-60 cells and primary cultured human kidney (HKN) cells. We have also determined an induction time of apoptosis by DNA fragmentation analysis and the presence of the cell cycle arrest by a flow cytometric measurement. The results were as follows. In non-adherent HL-60 cells, a typical ladder pattern was observed within 4 hours after treatments of 20 micrometer of cisplatin and 100 microgram/ml of MMS. At the same time while adherent HKN cells failed to exhibit a ladder pattern at even higher doses of genotoxic agents. Since HL-60 cells do not have p53 gene, these findings suggest the presence of a p53-independent apoptotic pathway. The increasing patterns of the mRNA levels of gadd45 and gadd153 varied with the type of genotoxic agents. In the case of MMS treatment, the induction was rapid and transient, regardless of the cell types. The mRNA level peaked at 4 hours after MMS treatment and markedly decreased after 12 hours. On the other hand, cisplatin-induced transcriptions of gadd45 and gadd153 continued to increase for at least 24 hours and reached a peak level at 48 hours after cisplatin treatment, regardless of the cell types. HL-60 cells revealed G2 arrest following 24 hours after cisplatin and MMS treatments. These findings suggest that the regulation mechanism of apoptosis between adherent and non-adherent cells, might be different and that gadd45 and gadd153 might have an important role in DNA repair rather than apoptosis. Also, the findings suggest that an expression pattern of gadd45 and gadd153 might be different according to the type of genotoxic agents.

Identification of Differentially Expressed Genes Using RNA Fingerprinting in Cell after DNA Damage.: Jung Young Lee, Min Sun Shin, Seung Myung Dong, Eun Young Na, Su Young Kim, Sug Hyung Lee, Won Sang Park, Nam Jin Yoo; Korean J Pathol. 1998;32(5):321-327.

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Abstract: RNA fingerprinting using on arbitrary primed polymerase chain reaction (RAP-PCR) was carried out to identify differentially expressed genes in HL-60 cell after treatment of methylmethane sulfonate (MMS). Twenty differentially expressed PCR products were cloned and analyzed. We have successfully obtained eight partial cDNA sequences by TA cloning method. Among these, six cDNAs were up-regulated and two cDNAs were down-regulated after the MMS treatment. Of these six up-regulated cDNAs, 3 cDNAs were equivalent to known genes in the GenBank/EMBL databases with 98~100% homology searched by BLAST program: genomic DNA fragment containing CpGg island (clone 26h8), Human Rev interacting protein-1 (RIP-1), and human zinc finger protein-4 (HZF4). The sequences of the three remaining cDNA were entirely new genes, but we didn't try to identify a full cDNA sequence. Two clones called KIAA0060 and KIAA0065, were down-regulated in HL-60 cells after the MMS treatment. These findings suggest that the RNA fingerprinting method using RAP-PCR is an effective method which can identify and separate the differentially expressed cDNAs and that the isolated cDNAs might involve in regulation mechanism of apoptosis and/or cell cycle delay, especially a p53-independent pathway, in the cells after DNA damage. But the nature of cDNAs that we have isolated remains to be elucidated.

Identification of Differentially Expressed Genes from Serum Deprived p388D1 Cells.: Su Young Kim, Sang Ho Kim, Sug Hyung Lee, Nam Jin Yoo, Jung Young Lee, Choo Soung Kim; Korean J Pathol. 1998;32(7):488-493.

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Abstract: This experiment is designed to find differentially expressed genes in p388D1 cells that are specific for the serum deprived state. Serum starvation induces cells to enter the quiscent state in the cell cycle and is used to arrest cell growth or synchronize the cell cycle. Differential display and ribonuclease protection assay were used to identify quantitative change in gene expression. Nineteen genes that showed a differential expression in the differential display were cloned and 7 clones were verified by a ribonuclease protection assay. Among the 7 clones clone-16 showed same expression pattern in comparison with the differential display. Deduced amino acid sequences of clone-16 had N-glycosylation motif and seems to be a secretory protein. Getting a full sequence of clone-16 is critical for the characterization of it.

Identification of Zinc Finger Genes that are Differentially Expressed upon Apoptosis of Ramos B Cells.: Min Sun Shin, Su Young Kim, Seung Myung Dong, Eun Young Na, Sug Hyung Lee, Won Sang Park, Jung Young Lee, Nam Jin Yoo; Korean J Pathol. 1998;32(12):1043-1048.

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Abstract: Typical programmed cell death requires de novo macromolecular synthesis and shares common morphological changes referred to as apoptosis. To elucidate the molecular mechanism of apoptosis, we isolated 13 cDNA clones of zinc finger genes that are differentially expressed in calcium ionophore-induced apoptosis of Ramos human B cell by 'targeted RNA fingerprinting' protocol (Stone & Wharton, 1993). According to DNA sequence analysis of the 13 cDNA clones, three clones are identical with ZNF7, ZNF143 and MTB-Zf, respectively, and 8 out of the other 10 clones showed partial homology to known zinc finger genes. Differential expression was confirmed in the three known zinc finger genes by ribonuclease protection assay. ZNF7 and ZNF143 are up-regulated after induction of apoptosis, and, in contrast, MTB-Zf is down-regulated. According to the previous reports on these three genes, all of the three genes have been suspected to be tumor suppressor genes, but their functions have not been identified yet. Taken together, our results suggest that many of the novel and known zinc finger genes might play important roles in regulation of apoptosis and that these findings also provide clues as to the functions of the three putative tumor suppressor genes, ZNF7, ZNF143 and MTB-Zf in terms of apoptosis. In addition, the isolation of zinc finger genes by targeted RNA fingerprinting could be a straightforward approach for the identification of novel candidate genes associated with apoptosis.

Ethnic Differences of the p53 Genetic Alteration in Cutaneous Malignant Melanoma.: Won Sang Park, Eun Young Na, Sang Kyu Lee, Sug Hyung Lee, Su Young Kim, Seok Jin Kang, Kye Yong Song, Suk Woo Nam, Nam Jin Yoo, Jung Young Lee; Korean J Pathol. 2001;35(2):158-164.

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Abstract PDF: BACKGROUND
There are significant differences in the clincopathologic pattern including the incidence, favor site, and histopathologic type between cutaneous malignant melanomas arising from whites, asians and blacks. These differences might suggest that there is a racial difference in the molecular tumorigenesis mechanism of malignant melanoma.
METHODS
To determine the ethnic differences in tumorigenesis of malignant melanoma, we performed loss of heterozygosity (LOH) and sequencing analyses of the p53 gene in cutaneous malignant melanomas arising from 22 white American, 30 Korean and 15 black African patients.
RESULTS
The frequency of LOH of the p53 gene is only 12.5% in white American patients, but the frequency is significantly higher in Korean (42.1%) and black African (61.5%) patients. We also detected 17 mutations (nonsense: 1, missense: 16) of the p53 gene in the cutaneous malignant melanomas of Koreans and black Africans, but none in those of white Americans: among the 16 missense mutations, 10 mutations were C:G to T:A transitional mutations. Of these, we also detected one GG (CC) to AA (TT) tandem mutation at the pyrimidine sequence.
CONCLUSION
These results strongly suggest that there might be a racial difference in molecular carcinogenesis mechanisms among the cutaneous malignant melanomas occurring in white American, Korean and black African patients. But the role of the p53 genetic alteration in the genesis of melanomas in Korean and black African patients is subject to further evaluation.

Mutational Analysis of Proapoptotic bcl-2 Family genes in Colon Carcinomas.: Young Hwa Soung, Jong Woo Lee, Su Young Kim, Suk Woo Nam, Won Sang Park, Jung Young Lee, Nam Jin Yoo, Sug Hyung Lee; Korean J Pathol. 2005;39(3):168-171.

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Abstract PDF: BACKGROUND
Several lines of evidence have indicated that the deregulation of apoptosis is involved in the mechanisms of cancer development, and somatic mutations of the apoptosisrelated genes have been reported in human cancers. Members of the bcl-2 family proteins regulate the intrinsic apoptosis pathway mainly in the mitochondria. The aim of this study was to explore whether the somatic mutation of the proapoptotic bcl-2 family genes, one of the mechanisms that prolong the survival of cancer cells, occurred in colorectal carcinomas.
METHODS
In the current study, to detect the somatic mutations in the DNA sequences encoding the bcl-2 homology 3 (BH3) domain of the human bak, bid, bik, bim, PUMA, bcl-rambo, bcl-G, and bmf genes in 98 colon adenocarcinomas, we used polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP), and DNA sequencing.
RESULTS
The SSCP analysis detected no evidence of somatic mutations of the genes in the coding regions of the BH3 domain in the cancers.
CONCLUSIONS
The data presented here indicate that the proapoptotic bcl-2 family genes, bak, bid, bik, bim, PUMA, bcl-rambo, bcl-G and bmf may not be somatically mutated in human colorectal carcinomas, and suggest that the colorectal cancers may not utilize mutational events of these proapoptotic bcl-2 family genes in the mechanisms for evading apoptosis.

Case Report

Primary Cutaneous Meningioma arising from the Scalp: A case report.: Sug Hyung Lee, Seok Jin Gang, Sun Moo Kim; Korean J Pathol. 1993;27(2):181-183.

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Abstract PDF: Primary cutaneous meningiomas are extremely rare tumors found in the cutis or subcutis, and generally have a benign course. The tend to be located on the scalp, face, neck. or paravertebral area. The primary cutaneous meningioma bears similarities to developmental defects and probably originates from arachnoid cell rests in the skin, although diverse groups of cutaneous meningiomas seem to arise from several different sources. A case of primary cutaneous meningioma occuring in the scalp of left parietal area of a 27-year-old female is presented. Clinically the lesion appeared as indolent, slow growing cutaneous mass and has no connection with underlying brain tissue, as determined by examination of the roentgenographs. The definite diagnosis was made after pathological examination. Microscopically the tumor is composed of sheets and nests of meningothelial cells. Immunohistochemical and electron microscopic studies reveal the typical findings of meningioma.

Original Article

Mutation of Adenomatous Polyposis Coli Gene in Human Stomach Cancer.: Won Sang Park, Mun Gan Rhyu, Sug Hyung Lee, Yun Jun Chung, Gum Ryong Kim, Choo Soung Kim; Korean J Pathol. 1993;27(1):34-39.

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Abstract PDF: Recently the adenomaatous polyposis coli(APC) gene, a tumor suppressor gene, was identified and the cDNA was cloned from chromosome 5q21. Allelic deletion or point mutation of tumor suppressor genes(TSGs) has been considered as an important mechanism in development of human tumor. Point mutations affecting APC gene are seen in the hereditary syndrome, adenomatous polyposis and spordic colon cancer. However, the mutation of APC gene and other TSGs have not been described in gastric cancer. In order to identify the mutation of exon 11 of APC gene for gastric cancer, we amplified DNA extracted from paraffin-embedded tissues by polymerase chain reaction(PCR) and digested the PCR products with restriction enzyme Rsa I. We examined the DNA extracted from paraffin-embedded 44 gastric cancer tissues with lymph nodes. Eighteen(41%) among 44 were informative for the study exon 11 of the APC gene, and we found loss of heterozygosity(LOH) for APC in 6/18(33.3%). These data suggest that the point mutation or the base change of APC gene commonly occurs in gastric cancer. We conclude that the mutation of APC gene is strongly connected with development of human gastric cancer.

Case Report

Carcinosarcoma of the Esophagus: A report of case.: Sug Hyung Lee, Won Sang Park, Young Jin Choi, An Hee Lee, Sun Moo Kim; Korean J Pathol. 1992;26(2):191-196.

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Abstract PDF: Carcinosarcoma of the esophagus is a rare neoplasm composed of both carcinoma and spindle sarcomatous area. Usually the carcinoma component is a squamous cell carcinoma but rarely adenocarcinoma or undifferentiated carcinoma is found. The histogenesis of the sarcomatous component is still unknown. A case of ulcerated polypoid lesion with a stalk in esophagus was reported. Microscopically it was composed of spindle shaped cells interminled with squamous cell carcinoma and small cell carcinoma nests. No distinct transition between spindle shaped cells and carcinoma are was observed. Immunoreactivity to cytokeratin was observed in both carcinomatous and spindle cell component, but electron microscopic examination failed to demonstrated desmosome or tonofilaments in spindle cells. Undifferentiated small cell nests were reactive to neuron specific enolase and contained membrane bounded secretory granule in electron microscopy.

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