Comparing triage algorithms using HPV DNA genotyping,HPV E7 mRNA detection and cytology in high-risk HPV DNA-positive women

BackgroundHigh-risk human papillomavirus (hrHPV) DNA positive women require triage testing to identify those with high-grade cervical intraepithelial neoplasia or cancer (≥CIN2).ObjectiveComparing three triage algorithms (1) E7 mRNA testing following HPV16/18/31/33/45/52/58 genotyping (E7 mRNA test), (2) HPV16/18 DNA genotyping and (3) cytology, for ≥CIN2 detection in hrHPV DNA-positive women.Study designhrHPV DNA-positive women aged 18–63 years visiting gynecology outpatient clinics were included in a prospective observational cohort study. From these women a cervical scrape and colposcopy-directed biopsies were obtained. Cervical scrapes were evaluated by cytology, HPV DNA genotyping by bead-based multiplex genotyping of GP5+6+-PCR-products, and presence of HPV16/18/31/33/45/52/58 E7 mRNA using nucleic acid sequence-based amplification (NASBA) in DNA positive women for respective HPV types. Sensitivities and specificities for ≥CIN2 were compared between E7 mRNA test and HPV16/18 DNA genotyping in the total group (n = 348), and E7 mRNA test and cytology in a subgroup of women referred for non-cervix-related gynecological complaints (n = 133).ResultsSensitivity for ≥CIN2 of the E7 mRNA test was slightly higher than that of HPV16/18 DNA genotyping (66.9% versus 60.9%; ratio 1.10, 95% CI: 1.0002–1.21), at similar specificity (54.8% versus 52.3%; ratio 1.05, 95% CI: 0.93–1.18). Neither sensitivity nor specificity of the E7 mRNA test differed significantly from that of cytology (sensitivity: 68.8% versus 75.0%; ratio 0.92, 95% CI: 0.72–1.17; specificity: 59.4% versus 65.3%; ratio 0.91, 95% CI: 0.75–1.10).ConclusionFor detection of ≥CIN2 in hrHPV DNA-positive women, an algorithm including E7 mRNA testing following HPV16/18/31/33/45/52/58 DNA genotyping performs similar to HPV16/18 DNA genotyping or cytology.     

中缅边境间日疟原虫环子孢子蛋白基因分型

根据间日疟原虫环子孢子蛋白(CSP)基因设计特异分型引物,利用巢式PCR技术(Nested-PCR)对采自中缅边境的间日疟患者血样作分型鉴定。检出的174份血样中,PV-I型热带族占54.6%(95/174)、温带族占35.6%(62/174)、PV-II型占2.9%(5/174)、混和感染占6.9%(12/174)。表明中缅边境的间日疟原虫存在4种基因型,以热带族为优势虫株,缅甸拉咱与云南腾冲间日疟原虫CSP基因型构成无差异(χ2=3.381,P0.05)。     

HLA-B27基因检定技术

用聚合酶链反应和血清学方法检测ＨＬＡ－Ｂ２７，并进行比较。方法：采用ＰＣＲ技术７８例可疑为强直性脊椎炎患者样本的ＨＬＡ－Ｂ２７基因，并与血清学比较。结果：３６例具有Ｂ２７基因者中２４例同时作血清学试验，５例血清学难以判定结果，并与ＰＣＲ结果不一致。     

Designing personalised cancer treatments

The concept of personalised medicine for cancer is not new. It arguably began with the attempts by Salmon and Hamburger to produce a viable cellular chemosensitivity assay in the 1970s, and continues to this day. While clonogenic assays soon fell out of favour due to their high failure rate, other cellular assays fared better and although they have not entered widespread clinical practice, they have proved to be very useful research tools. For instance, the ATP-based chemosensitivity assay was developed in the early 1990s and is highly standardised. It has proved useful for evaluating new drugs and combinations, and in recent years has been used to understand the molecular basis of drug resistance and sensitivity to anti-cancer drugs.     

Polymorphism of the Rat Liver Mitochondrial Aldehyde Dehydrogenase cDNA

In humans, a deficiency in mitochondrial aldehyde dehydrogenase (Class 2 ALDH) activity due to a single base-pair exchange in its structural gene serves as a deterrent to excessive alcohol consumption. Differences in Class 2 ALDH isozyme patterns on isoelectric focusing gels have been observed in the selectively bred, alcohol-preferring (P) and alcohol-nonpreferring (NP) lines of rats. To determine whether the differences are the result of sequence variation in the structural gene, we sequenced the cDNAs for Class 2 ALDH from P and NP rats. A synonymous exchange was seen in the codon for amino acid 473 in both lines, when compared with published sequences. Additionally, when the cDNA from P rats was used as reference, a substitution (G for A) was identified in the cDNA of NP rats which changes amino acid 67 from Gln (CAG codon; ALDH2Q allele) to Arg (CGG codon; ALDH2R allele). The Arg for Gln substitution makes the enzyme more basic and could account for the different electrophoretic mobilities. To determine whether the polymorphism was associated with drinking behavior, we genotyped the ALDH2 locus by amplifying rat genomic DNA encompassing the nucleotide exchange followed by probing with allele-specific oligonucleotides. There are highly significant differences in the frequencies of the two alleles in the P and NP rat lines. The frequency of the ALDH2R allele is 63% in the NP line and only 18% in the P line, whereas the frequency of the ALDH2Q allele is 82% in the P line and 37% in the NP line.     

中国汉人HLA—DQA1基因对类风湿关节炎的遗传易感性研究

用ＰＣＲ－ＰＡＧＥ方法，结合高灵敏的银染色作ＨＬＡ－ＤＱＡ１等位基因分型，研究ＤＱＡ１基因对类风湿关节炎（ＲＡ）的遗传易感性。选择无亲缘关系的广东籍汉族健康者１０６例和５０例ＲＡ患者。发现该方法测的６种ＨＬＡ－ＤＱＡ１等位基因中，ＲＡ组ＤＱＡ１＊０１０１（２７％，ＲＲ＝２．３３４，Ｐ＜０．００５，ＥＦ＝０．１５４）等位基因明显增高；而ＤＱＡ１＊０１０２（１％，ＲＲ＝０．０６８，Ｐ＜０．０１，ＰＦ＝０．５７７）明显下降；ＤＱＡ１的２种纯合子基因型（０１０１／０１０１和０３０１／０３０１）在ＲＡ组明显增高（Ｐ值分别小于０．０２５和０．００５）。上述结果显示：ＨＬＡ－ＤＱＡ１＊０１０１对ＲＡ有遗传易感作用，ＤＱＡ１＊０１０２等位基因有遗传抵抗作用；ＤＱＡ１基因型的检测对预测ＲＡ易感者和判断预后及疗效可能提供理论依据。     

Determination of ABO genotypes with DNA extracted from formalin-fixed,paraffin-embedded tissues

Summary The gene encoding the specific glycosyltransferases which catalyze the conversion of the H antigen to A or B antigens shows a slight but distinct variation in its allelic nucleotide sequence and can be divided into 6 genotypes when digested with specific restriction enzymes. We extracted DNA from formalin-fixed, paraffin-embedded tissues using SDS/proteinase K treatment followed by phenol/chloroform extraction. The sequence of nucleotides for the A, B and O genes was amplified by the polymerase chain reaction (PCR). DNA fragments of 128 by and 200 by could be amplified in the second round of PCR, using an aliquot of the first round PCR product as template. Degraded DNA from paraffin blocks stored for up to 10.7 years could be successfully typed. The ABO genotype was deduced from the digestion patterns with an appropriate combination of restriction enzymes and was compatible with the phenotype obtained from the blood sample.     

Genotyping of Cerebrospinal Fluid Associated With Osimertinib Response and Resistance for Leptomeningeal Metastases in EGFR-Mutated NSCLC

IntroductionPatients with NSCLC with leptomeningeal metastases (LM) presented dismal prognosis. Cerebrospinal fluid (CSF) is suggested as a medium of liquid biopsy of LM. However, the clinical implications of CSF genotyping on treatment outcomes remained elusive.MethodsPatients with EGFR-mutated advanced NSCLC with LM were included: cohort 1, patients with LM who were treated with osimertinib with CSF and plasma genotyping performed before the first dosing of osimertinib (baseline, n = 45); cohort 2, CSF genotyping on progression on osimertinib and development of LM (the progression event on osimertinib is the diagnosis of LM, n = 35). Circulating tumor DNA in CSF underwent next-generation sequencing.ResultsSensitivity of CSF genotyping for EGFR-sensitizing mutations was 93.3% (42 of 45) and 97.1% (34 of 35) in cohorts 1 and 2, respectively. In cohort 1, patients with EGFR exon 19 deletion had higher median intracranial progression free survival (iPFS) than those with EGFR exon 21 L858R mutation (11.9 versus 2.8 mo; p = 0.02). Median iPFS was significantly longer in patients with T790M-positive CSF genotyping (15.6 mo) than T790M-negative CSF (7.0 mo, p = 0.04). Concurrent CDK4 (2.8 versus 11.6 mo, p = 0.002) and CDKN2A (2.5 versus 9.6 mo, p = 0.04) mutation with EGFR-sensitizing mutations indicated lower median iPFS. Patients with T790M-negative CSF, EGFR exon 21 L858R mutation, concurrent FGF3 alteration, and over first-line osimertinib had shortened iPFS. In cohort 2, possible EGFR-related and EGFR-independent resistance mechanisms were found including C797S mutation, MET dysregulation, and TP53 plus RB1 co-occurrence. Patients with loss of T790M in CSF had a shorter median iPFS (7.4 mo) compared with those with reserved T790M (13.6 mo, p = 0.01).ConclusionsGenotyping of CSF indicated heterogeneous response to osimertinib and revealed the genetic characteristic of LM on osimertinib failure in patients with EGFR-mutated NSCLC diagnosed with LM.     

广西巴马县壮族长寿老人HLA-DRB1基因的多态性

目的探讨人类白细胞抗原DRB1(HLA DRB1)基因与长寿的相关性。方法应用聚合酶链式反应序列特异引物(PCR SSP)对广西巴马县109名90岁以上壮族长寿老人(90～103岁)、56名80～89岁和71名当地健康、无血缘关系、生活习性相似、随机抽样的壮族成年人(23～70岁)进行HLA DRB1基因分型及相应等位基因频率的比较。结果在该研究群体中共检出13个HLA DRB1等位基因,最常见的等位基因是HLA DRB11501(长寿组、80岁组和对照组分别为26.61%、26.79%和26.06%),其次是HLA DRB11601/2(以上3组分别为14.22%、14.29%和16.20%)和HLA DRB11401/4(以上3组分别为11.93%、10.71%和11.97%);长寿组HLA DRB11201/2的频率与对照组比较有升高趋势,但无统计学意义;3组间其他相应等位基因频率的比较均无显著差异(P<0.05)。结论在巴马县壮族长寿人群的HLA DRB1座位上,没发现与其长寿明显相关的等位基因。