PRACTICAL HISTOLOGICAL MICRODISSECTION FOR PCR ANALYSIS

Recovery of cells by histological microdissection is increasingly used for analysis by polymerase chain reaction (PCR) or microchemical techniques. This paper describes techniques of histological microdissection. Sections of archival formalin-fixed, paraffin-embedded tissue up to 15 years old were mounted on plain glass slides. Sections 6–7 μm in thickness stained with toluidine blue were dissected under proteinase K buffer solution, using an electrolytically sharpened tungsten needle in a bacteriological loop-holder and a Leitz mechanical micromanipulator (model M). Detached cell groups were recovered in a silicone-coated pipette tip for PCR analysis after digestion in 25–50 μl of proteinase K (500/ml) in TRIS–HCl buffer (pH 8·3). Consistent amplification and analysis of microsatellite loci were obtained from 2 μl of crude lysate using 28–30 cycles of PCR incorporating a ³²P 5′-end-labelled primer, electrophoresis under denaturing conditions on 6 per cent polyacrylamide gels, and autoradiographic detection.     

原发性胃癌中19p部分微卫星多态位点杂合性缺失分析

目的 筛选胃癌19p部分微卫星多态位点的杂合性缺失（loss of heterozygosite,LOH)频率,以初步确定19p上与胃癌相关基因连锁最密切的微卫星多态位点。方法 采用聚合酶链反应-单链长度多态（polymerase chain reaction-single strand length polymorophism,PCR-SSLP)-银染法选取19p上9对微卫星多态标记（D19S424,D19S216,D19S406,D19S413。D19S221,D19S226,D19S411,D19S883,D19S886）,对43例原发性胃癌的杂合性缺失情况进行了分析。结果 43例中22例至少在1个位点发生LOH,总缺失率为48.88%,这9个位点的LOH频率分别为29.63%,11.53%,33.33%,8.57%,13.15%,8.00%,6.45%,6.89%,10.71%,在D19S886也同时出现微卫星不稳定性（microsatellite instability,MSI)17.85%。结论 提示19p上的LOH缺失频发区域可能涉及与人类原发性胃癌发生发展相关基因的存在。     

The Clostridium difficile PCR ribotype 027 lineage: a pathogen on the move

Clostridium difficile is a Gram-positive, spore-forming, human and animal pathogen that is the major cause of antibiotic-associated diarrhoea worldwide. The past decade has seen the rapid emergence of the hypervirulent PCR ribotype (RT) 027 complex, which has been associated with increases in the incidence and severity of disease and mortality. In this review, we describe the potential virulence factors that have been reported in strains from the RT 027 complex. We review the emergence, population structure, dissemination and evolution of this lineage.     

Criteria to define HLA haplotype loss in human solid tumors

Short tandem repeat (STR) markers are currently used to define loss of heterozygosity (LOH) of genes and chromosomes in tumors. Chromosome 6 and chromosome 15 STR markers are applied to define loss of HLA and related genes (e.g. TAP and beta2m). The number of STR identified in the HLA region is still increasing. In this study, seven representative STR markers covering the 6p/6q arms of chromosome 6 including the HLA region and two for chromosome 15 flanking the beta2m gene, were selected as minimally required for reliable LOH studies. A multiplex polymerase chain reaction (PCR) strategy is proposed when small number of cells are available in microdissected tumor samples.     

REVIEW ARTICLE. MUCUS GLYCOPROTEINS AND THEIR ROLE IN COLORECTAL DISEASE

In this review, the nature and impact of progress in the study of mucins is outlined, emphasizing the current understanding of the structure and physiological function of these molecules in the colorectum. The use of new methods for preparation and separation has led to improvements in the analysis of mucins; these are detailed, as are their difficulties and pitfalls. Results obtained with these methods are correlated with long-established histochemical techniques and the use of chemical, lectin, and antibody reagents for general and specific detection of mucins in all procedures is described. Improvements in the detection and analysis of mucins in biopsy-size tissue samples and in larger numbers of individual clinical cases have now permitted a much wider approach to the pathological evaluation of mucin biology and progress with these techniques is outlined. The significance of the discovery of a family of mucin genes is presented and new concepts of mucin structure resulting from these studies are described. Bacterial degradation of the mucus layer at the surface of the colorectal mucosa is considered in line with the homeostatic relationship with mucosal mucin synthesis. Finally, the implications of abnormal mucins in colorectal disease are considered in the light of recent methodological advances.     

Microsatellite instability in young patients with colorectal cancer

Genetic instability Is closely correlated to the pathogenesis of hereditary non-palyposis colon cancer (HNPCC), which is clinicaily characterized by a family history and early onset. To investigate the role of genetic instabllity in young patients with colorectal cancer (CRC), 22 CRC patients, who were aged younger than 30 at the time of diagnosis, were studied. Patients with famllial adenomatous polyposis were excluded. Among the 22 cases, seven were identifled as microsateillte instability posltive (MI+), and more than five microsatellite markers among the 15 tested markers showed an additlonal band pattern in the tumor tissue. None of the remaining 15 cases showed instability in any microsatellite marker. Two of seven MI+ cases were classic HNPCC. While all MI+ cases had one or no metastatic lymph node, 53.3% of MI- cases showed metastasls in two or more reglonal lymph nodes. Allelic deletion of the 17p12–13 chromosome around the p53 locus occurred in 16.7% of MI+ cases, and 80.0% of MI- cases showed loss of het-erozygosity at that locus. hMSH2 Protein expression, assessed by immunohistochemistry, was absent in two cases, both of which were MI+. When we tested two to four sites of MI+ tumors, transforming growth factor β receptor type II was mutated in a homogeneous pattern in five MI+ cases. in addition, frame-shift mutations of BAX , insulin-like growth factor II receptor, hMSH3 and hMSH6 were found in three cases, five cases, five cases and one case, respectively. In contrast to the consistent mutatlon of the transforming growth factor-β receptor type II gene, mutations of other genes varied in different portions of the tumors.     

乳腺癌和导管增生病变与1p染色体不稳定的关系

目的检测乳腺普通型导管增生(usual dutal hyperplasia,UDH)、乳腺不典型增生(atypical dutal hyperplasia,ADH)、乳腺导管原位癌(dutal carcinoma in situ,DCIS)及乳腺浸润性导管癌(invasive dutal carcinoma,IDC)中染色体1p微卫星杂合性缺失(loss of heterozygosity,LOH)/微卫星不稳定(microsatellite instability,MSI)的改变规律,探讨乳腺导管增生病变进展为IDC的遗传学改变特征。方法应用PCR法检测20例UDH、7例ADH、25例DCIS、25例IDC石蜡包埋组织,8%尿素变性聚丙烯酰胺凝胶电泳后银染检测染色体1p区间微卫星位点D1S193、D1S463、D1S2881、D1S2885、D1S234、D1S252的LOH/MSI。结果染色体1p的每个位点均发生LOH/MSI,其发生的频率UDH为55.0%、ADH为42.9%、DCIS为64.0%、IDC为72.0%,各组间差异无显著性(P0.05);微卫星位点D1S193、D1S2881、D1S2885、D1S252、D1S234在各组中LOH/MSI的频率差异均无显著性(P0.05)。D1S463的频率在UDH组为5.0%、ADH组为0、DCIS组为45.0%、IDC组为40.0%,其中UDH组与ADH组明显低于DCIS组、IDC组(P0.008)。结论 1p染色体遗传学不稳定是乳腺癌发生的早期事件,D1S463位点的改变可能在乳腺导管增生性病变进展为乳腺癌中起关键作用。     

No evidence of microsatellite instability but frequent loss of heterozygosity in primary resected lung cancer

We examined microsatellite instability and loss of heterozygosity (LOH) in primary lung tumors from 93 cancer patients, using 16 microsatellite markers. The cases studied included 87 non-small-cell lung cancers (NSCLC) and six small-cell lung cancers (SCLC). All the patients except two were current or former smokers. The microsatellite markers were all dinucleotide repeat sequences from chromosomal locations 1p, 3p, 5q, 8p, 9p, 10p, 11p, 13q, and 17q. None of the tumors showed microsatellite instability (0/93). In NSCLC, 28% (24/87) of the cases showed LOH in at least one locus, whereas, in SCLC, 67% (4/6) had allelic losses. The frequency of LOH differed between the various cell types of NSCLC. The highest frequency was seen in large cell carcinoma (3/6, 50%) followed by squamous cell carcinoma (16/43, 37%) and adenocarcinoma (5/35, 14%). The most common site of LOH was 3p, where markers D3S1284, D3S659, D3S1289, D3S966, D3S647, and D3S1038 were studied. LOH, studied with 9p markers (D9S126, D9S171, D9S162), was less common. The present results, together with earlier reports, suggest that smoking-related primary lung cancers seldom show microsatellite instability but are characterized by frequent LOH. Environ. Mol. Mutagen. 30:217–223, 1997. © 1997 Wiley-Liss, Inc.     

Analysis of microsatellite alterations in gastric carcinoma using the crypt isolation technique

The crypt isolation technique was used to analyse loss of heterozygosity (LOH) and microsatellite instability (MSI) in gastric carcinomas (36 intestinal type, 17 solid type, and 23 diffuse type) using a polymerase chain reaction assay. Increased LOH frequencies and fractional allelic losses (FAL) were observed in samples prepared using the crypt isolation technique compared with those isolated by the conventional method. A significant increase in LOH was found at several chromosomal loci, and significant differences in FAL were found in patients with intestinal- and solid-type tumours. There was no difference in the frequency of MSI using either technique. In samples prepared by the crypt isolation technique, significant allelic losses (> or =50%) were observed at most loci tested in intestinal- and solid-type tumours, but not in diffuse-type tumours. Significant losses of some of these loci are novel findings for gastric cancer. FAL values were significantly higher in intestinal- and solid-type tumours than in diffuse-type tumours. MSI-high was observed in intestinal- (17%) and solid-type (12%) tumours. The results suggest that the crypt isolation technique is useful for accurate allelic loss analysis in gastric carcinoma and that LOH and MSI are more common in intestinal- and solid-type tumours than in diffuse-type tumors.     

Loss of heterozygosity during the development and progression of differentiated adenocarcinoma of the stomach

In a recent allelotypic analysis of differentiated adenocarcinoma of the stomach, loss of heterozygosity (LOH) was found frequently on chromosomes 2q, 4p, 5q, 6p, 7q, 11q, 14q, 17p, 18q, and 21q. To clarify the sequence of these chromosomal losses during gastric carcinogenesis, microsatellite analysis of the chromosome arms described above was performed in 25 early and 29 advanced differentiated adenocarcinomas of the stomach. LOH on these chromosome arms fell within a range of 20–50 per cent. On 4p, 7q, 14q, 17p, and 21q, LOH was detected at a similar frequency in both early and advanced carcinomas, while LOH on 2q, 5q, 6p, 11q, and 18q was observed more than twice as frequently in advanced than in early lesions. Mean fractional allelic losses (FALs) were 0·221 in early and 0·413 in advanced carcinomas, representing a significant difference (P<0·05). These results suggest that LOH on 4p, 7q, 14q, 17p, and 21q is a relatively early event, while LOH on 2q, 5q, 6p, 11q, and 18q typically accumulates during the progression of gastric carcinogenesis. © 1998 John Wiley & Sons, Ltd.     

Duchenne型肌营养不良症家系的多重PCR和STR—PCR基因诊断

目的该研究率先开展温州地区Duchenne型肌营养不良症（DMD）家系的缺失基因诊断特别是STR单体型连锁基因诊断,为基于DMD症状前、携带者基因诊断结果的遗传咨询和生育指导提供依据。方法针对4例DMD先证者,采用多重PCR检测常见18个外显子缺失,进行直接基因诊断。针对未能发现常见外显子缺失的DMD先证者及其有关家系成员,采用短串联重复序列（STR）PCR检测5个位点（3’CA、44CA、45CA、49CA和50CA）STR多态性,进行间接单体型连锁基因诊断。结果家系二的先证者缺失外显子3、4和6。其余3个家系的先证者的异常x染色体均肯定来源于其母亲。家系一先证者外婆肯定是携带者。家系三先证者年幼（4周岁）弟弟肯定为正常人,将来年龄大了也不会发病,先证者外婆肯定是携带者。家系四先证者刚出生的妹妹肯定是遗传携带者,将来其生育儿子有遗传患病风险。结论该研究的DMD家系的缺失和STR单体型连锁基因诊断,特别是对症状前男孩的诊断、对无患病后代的女性携带者的检出,具有非常重要的实际意义,可以为遗传咨询和生育指导提供可靠依据。     

THE FREQUENCY AND MECHANISM OF LOSS OF HETEROZYGOSITY ON CHROMOSOME 11q IN BREAST CANCER

Loss of heterozygosity (LOH, allele loss) occurs frequently on the long arm of chromosome 11 in breast cancer. Seventy-one paired tumour/normal DNA samples from breast cancer patients under 50 years old were studied for allele loss at four microsatellite loci on 11q: D11S29 (11q23.3), NCAM (11q22–q23), D11S968 (11qtel), and D11S1313 (11qcen). The maximum frequency of LOH (≈35 per cent) was found at the D11S29 and NCAM loci. This result is consistent with previous studies and the frequency of allele loss is moderate to high compared with the usual baseline of 0–20 per cent. In most of the cases studied, LOH on chromosome 11q could be accounted for by one of two mechanisms. Either chromosomal non-disjunction had occurred, or sequences stretching from the telomere at least as far as NCAM had undergone deletion or mitotic recombination. These results suggest that a putative tumour suppressor gene is most likely to exist near 11q22–q23. There was a very low frequency of microsatellite instability in the tumours. An association was found between lack of progesterone receptor (PgR) expression and LOH at NCAM , suggesting that deletion of sequences on 11q may prevent high levels of PgR expression in some cases.     

Involvement of genetic instability in the downregulation of sFRP1 in Chinese patients with hepatocellular carcinoma

Secreted frizzled‐related protein 1 (sFRP1) is a new tumor suppressor based on recent researches, but the correlation of the genetic instability of sFRP1 gene with the clinicopathologic features of the hepatocellular carcinoma (HCC) has not been studied in Chinese people. In this study, 42 pairs of paraffin‐embedded HCC and adjacent non‐carcinoma tissues were examined for the loss of heterozygosity (LOH) and microsatellite instability (MSI) of two microsatellite markers D8S532 and D8S1722 located in the vicinity of the sFRP1 gene. Envision immunohistochemistry was used to assess the expression of sFRP1. We found that the reduced expression of the sFRP1 protein was frequently observed in Chinese patients with HCC, which may at least partially result from the genetic instability, especially LOH. The LOH‐associated sFRP1 downregulation may play an important role in the development of HCC. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

肝细胞癌17p13.3位点CpG岛甲基化状态的分析及其意义

为探讨ＤＮＡ甲基化异常在肝细胞癌中的作用，采用Ｓｏｕｔｈｅｒｎ杂交技术对１２例肝细胞癌１７ｐ１３．３位点ＣｐＧ岛甲基化状态进行了检测，发现６例正常肝组织ＣｐＧ岛均未被甲基修饰，而４１．７％（５／１２）的肝细胞癌ＣｐＧ岛被不同程度的重新甲基化；甲基化程度显著升高者多伴１７ｐ１３．３位点的杂合缺失。结果表明：ＣｐＧ岛重新甲基化可能是１７ｐ１３．３位点基因失活的重要机理之一，过度的高甲基化状态与杂合缺失有关。为肝癌的病因学研究提供了资料。     

Microsatellite alterations and target gene mutations in the early stages of multiple gastric cancer.

Multiple gastric cancers may develop through the same genetic background: the mutator pathway due to defects in DNA mismatch repair genes, or the suppressor pathway due to defects in tumour suppressor genes. To clarify the critical genetic events in the early stages of multiple gastric cancer development, 29 early and four advanced gastric cancers were examined from 12 patients. Microsatellite alterations were studied involving microsatellite instability (MSI) and loss of heterozygosity (LOH) at tumour suppressor loci, representative of the mutator pathway and the suppressor pathway, respectively, as well as mutations of target genes (TGF-beta RII, BAX, hMSH3, and E2F-4). MSI was determined in ten cancers (10/33; 30.3%) from seven patients (7/12; 58.3%). LOH was detected in six cancers (6/33; 18.2%) from five patients (5/12; 41.7%), most frequently at TP53, in four cancers (4/33; 12.1%) from four patients (4/12; 33.3%). In cases with multiple gastric cancers in the same stomach, the MSI status was generally the same, but in two patients (2/12; 16.8%) a tumour with MSI-H and another with LOH were found to co-exist in the same stomach. As for mutations of the target genes, it was found that E2F-4 was mutated in six cancers (6/33; 18.2%) from four patients (4/12; 33.3%). Furthermore, identical E2F-4 mutations were detected in four of the six intestinal metaplastic mucosae adjacent to each cancer carrying an E2F-4 mutation. No mutations were detected in the other target genes. In conclusion, the present results indicate that the majority of multiple gastric cancers develop from the same genetic background, with the mutator pathway playing a more important role than the suppressor pathway. Mutations of E2F-4 are early events in multiple gastric cancer development, occurring even in the intestinal metaplastic mucosa, with mutations of other target genes to follow during cancer progression.     

ALLELOTYPE OF ADENOMA AND DIFFERENTIATED ADENOCARCINOMA OF THE STOMACH

The molecular mechanism of gastric tumourigenesis has not yet been clarified, although investigators have postulated that differentiated adenocarcinoma may arise from pre-existing adenoma, similarly to the colorectal adenoma–carcinoma sequence. An allelotype analysis has been performed to identify chromosomal regions which are frequently deleted in gastric tumours and to examine the significance of the adenoma–carcinoma sequence in gastric tumourigenesis. Forty-five gastric tumours, 20 adenomas, and 25 differentiated adenocarcinomas were examined for loss of heterozygosity (LOH) using 39 microsatellite markers covering each non-acrocentric chromosome arm. Frequent LOH in the adenocarcinomas was observed on chromosomes 2q (33 per cent), 4p (33 per cent), 5q (50 per cent), 6p (33 per cent), 7q (43 per cent), 11q (36 per cent), 14q (38 per cent), 17p (45 per cent), 18q (36 per cent), and 21q (40 per cent). In contrast, the incidence of LOH in adenomas did not exceed 10 per cent at any of the loci examined. In addition to the p53 gene on 17p and the DCC gene on 18q, which are known to be frequently deleted in differentiated adenocarcinomas of the stomach, other unknown tumour suppressor genes on the above-mentioned chromosomes may also be inactivated. These observations suggest that the adenoma–carcinoma sequence is not a major pathway in gastric tumourigenesis.     

3′HVR的结构多态性及其PCR指纹分析

设计了一对与３＇ＨＶＲ（Ｄ１６Ｓ８５）中重复单位ａ以及旁侧单拷贝序列互补的寡核苷酸引物，建立了直接反映重复单位变异体排序多态性的ＰＣＲ技术，即小卫星变异重复单位ＰＣＲ（ｍｉｎｉｓａｔｅｌｌｉｔｅｖａｒｉａｂｌｅｒｅｐｅａｔＰＣＲ，ＭＶＲＰＣＲ）。对１０名无血缘关系正常个体外周血白细胞ＤＮＡ样本以及１例死胎蜡块组织ＤＮＡ进行了分析。初步证实３＇ＨＶＲ内部结构具有高度的多态性，其带型类似经典的ＤＮＡ指纹图谱。结果提示，该方法有可能代替经典的ＤＮＡ指纹技术，并具有快速、简单、不需ＤＮＡ印迹杂交而易于推广，以及可以分析微量或部分降解ＤＮＡ样本的优点，特别适于法医鉴定及遗传病基因连锁诊断。     

Microsatellite analysis of breast carcinoma and corresponding local recurrences

Local recurrence is a serious complication of breast carcinoma that reduces quality of life and influences prognosis. The aim of this study was to determine whether local recurrences of breast carcinoma are genetically related to the primary tumours. Forty cases of locally recurrent breast carcinomas (median onset: 3.6 years after primary surgery) were analysed: 22 patients had undergone breast-conserving therapy and 18 mastectomy. Eighteen microsatellites on chromosomes 2p, 3p, 5q, 10q, 11p, 11q, 13q, 17q, 17p, 18p were amplified by PCR using fluorescent-labelled primers, automatically detected after polyacrylamide gel electrophoresis and analysed for loss of heterozygosity (LOH) or microsatellite instability (MSI). Follow-up data were available for 39 cases with a median value of 89 months. All LOH and MSI found in the primary tumours were also present in the corresponding recurrences, indicating that they are genetically related to the primary tumours and not secondary malignancies in the same breast. MSI was found in three cases, of which one harboured MSI at more than two loci. The median value of LOH per case was significantly higher in the recurrent (four per case) compared to the primary tumours (two per case; p < 0.001, Mann-Whitney test), reflecting the genotype of tumour progression. Early local recurrence was associated with specific LOH for TP53.15 (p = 0.018, log-rank test) in the primary tumours. LOH on D13S1699 or D17S855 was associated with lymph node metastases (p = 0.024 and p = 0.019, respectively; chi-square test). In addition, tumour grade, lack of oestrogen or progesterone receptor expression, young patient age and early appearance of local recurrence significantly correlated with poor survival. The development of local recurrence despite clear resection margins may result from residual DCIS distant from the invasive carcinoma, homing of circulating tumour cells, or genetically altered, histologically normal breast tissue not immediately adjacent to the invasive carcinoma.     

Deletion mapping of chromosome 13q in head and neck squamous cell carcinoma in Indian patients: correlation with prognosis of the tumour

Deletions in chromosome (chr.) 13q occur frequently in head and neck squamous cell carcinoma (HNSCC). Previous studies failed to identify common deleted regions in chr.13q, though several candidate tumour suppressor genes (TSGs) loci, e.g. BRCA2, RB1 and BRCAX have been localized in this chromosome, as well as no prognostic significance of the deletion has been reported. Thus, in the present study, deletion mapping of chr. 13q has been done in 55 primary HNSCC samples of Indian patients using 11 highly polymorphic microsatellite markers of which three were intragenic to BRCA2 gene, one intragenic to RB1 gene and another from BRCAX locus. The deletion in chr.13q was significantly associated with progression of HNSCC. High frequencies (27-39%) of loss of heterozygosity were found in 13q13.1 (BRCA2), 13q14.2 (RB1), 13q21.2-22.1 (BRCAX) and 13q31.1 regions. Deletions in the BRCA2 and RB1 regions were significantly correlated. The four highly deleted regions were associated with clinical stage and histological grades of the tumour as well as poor patient outcome. Deletion in the 13q31.1 region was only found to be associated with HPV infection. High frequencies (11-23%) of microsatellite size alteration (MA) were seen to overlap with the highly deleted regions. Forty per cent of the samples showed rare biallelic alteration whereas loss of normal copy of chromosome 13q was seen in five tumours. Thus, it seems that the putative TSGs located in the BRCAX and 13q31.1 regions as well as the BRCA2 and RB1 genes may have some cumulative effect in progression and poor prognosis of HNSCC. Significant association between deletion in BRCA2 and RB1 gene loci may indicate functional relationship between the genes in this tumour progression.