液体活检在子宫内膜癌中的应用

子宫内膜癌是常见的女性生殖系统恶性肿瘤之一.大多数子宫内膜癌可以早期诊断,预后良好,但是晚期和复发性子宫内膜癌预后不佳.液体活检作为无侵袭性的肿瘤标本获取方式,具有无创和可重复获取等优点,目前已经批准应用于多种恶性肿瘤的诊治过程.目前液体活检在子宫内膜癌的早期诊断、风险分层、治疗选择和实时疾病监测等方面也有相关的研究和...     

低中危子宫内膜癌术中腹腔冲洗液细胞学阳性患者的预后相关因素分析

目的:探讨影响腹腔细胞学阳性的低中危组子宫内膜癌患者复发及预后的相关因素。方法:选择腹腔细胞学阳性的低中危组子宫内膜癌患者,其中18例患者术后未行化学治疗,28例患者术后行全身化疗,分析与患者复发有关的临床病理因素,分析影响子宫内膜癌腹腔细胞学阳性复发及预后因素。结果:患者的复发与年龄（P=0.044）、病理分级（P=0.035）、脉管癌栓（P=0.009）及治疗方法（P=0.015）有关。单因素分析显示患者年龄、病理分级、浸润肌层深度、脉管癌栓、化疗与患者的预后有关,多因素分析表明影响患者预后的独立危险因素是全身化疗和病理分级。结论:全身化疗及病理分级是影响腹腔冲洗液阳性的低中危组子宫内膜癌患者预后的独立危险因素,全身化疗可以提高患者的5年生存率,改善患者预后。     

Impact of peritoneal cytology on survival of endometrial cancer patients treated with surgery and radiotherapy

Stage IIIA endometrial cancer includes patients with serosal or adnexal invasion and patients with positive peritoneal cytology only. In this study, we assessed the impact of peritoneal cytology on endometrial cancer survival. All endometrial cancer patients receiving surgery and radiotherapy at the Geneva University Hospitals between 1980 and 1993 were included. Stage IIIA cancers were categorised into 'cytological' stage IIIA (only positive peritoneal cytology) and 'histological' stage IIIA (serosal or adnexal infiltration). Survival rates were analysed by Kaplan-Meier method and compared using log-rank test. The prognostic importance of peritoneal cytology was analysed by multivariate regression analysis. This study included 170 endometrial cancers (112 stage I, 17 cytological stage IIIA, 18 histological stage IIIA, 9 stage IIIB+). Disease-specific survival of cytological stage IIIA was not different from stage I (94 vs 88% respectively, P=0.5) but better than histological stage IIIA (94 vs 51% respectively, P<0.01). Histological stage IIIA patients were at increased risk to die from cancer compared to stage I patients (HR 2.7, 95% CI 1.0-7.7), while cytological stage IIIA patients were not (HR 0.3, 95% CI 0.3-2.0). Cytological stage IIIA endometrial cancer has similar prognosis as stage l and better prognosis than histological stage IIIA. Additional research, definitively separating stage and cytology is warranted.     

Positive peritoneal cytology in early-stage endometrial cancer does not influence prognosis

The aim of this study was to assess the prognostic importance of positive peritoneal cytology in early-stage endometrial cancer. All 278 stage I and 53 stage IIIA (without cervical involvement) endometrial cancer patients operated between 1980 and 1996, recorded at the Geneva Cancer registry, were included. Stage IIIA cancers were recategorised into 'cytological' stage IIIA (positive peritoneal cytology alone, n=33) and 'histological' stage IIIA (serosal or adnexal infiltration, n=20). Survival rates were analysed by Kaplan-Meier method and compared using log-rank test. The prognostic importance of cytology was analysed using a Cox model, accounting for other prognostic factors. The 5-year disease-specific survival of cytological stage IIIA cancer was similar to stage I (91 vs 92%) and better than histological stage IIIA cancer (50%, P<0.001). After adjustment for age, myometrial invasion, differentiation and radiotherapy, cytological stage IIIA patients were still at similar risk to die from endometrial cancer compared to stage I patients (hazard ratio (HR) 0.7, 95% confidence interval (CI): 0.18-2.3), while histological stage IIIA patients were at a four-fold increased risk to die from their disease (HR 4.2, 95% CI: 1.7-10.3). This population-based study shows that positive peritoneal cytology in itself has no impact on survival of patients with localised endometrial cancer. Based on the present and previous studies, FIGO (Fédération Internationale de Gynécologie et d'Obstétrique) might consider reviewing its classification system.     

Detection of free-circulating tumor-associated DNA in plasma of colorectal cancer patients and its association with prognosis

Tumor cells are characterized by specific genetic alterations. When such genetic alterations are identified in body fluid including plasma, regardless of the presence of detectable tumor cells, it shows the existence of free-circulating tumor-associated DNA. The objective of our study was to assess the prognostic value of free-circulating tumor-associated DNA in colorectal cancer patients' plasma. The first step of our work was to find common genetic alterations in tumors that would subsequently be used for plasma DNA screening. We focused on KRAS2 mutations in codons 12 and 13 by the mutant allele-specific amplification (MASA) method and p16 hypermethylation by the methylation-specific polymerase chain reaction (MSP) method. Patients with a tumor presenting either alteration were selected for plasma screening; 58 tumors were analyzed for KRAS2 mutations and tested for p16 gene promoter methylation. Survival and recurrence rates were assessed in patients with and without free-circulating tumor-associated DNA alterations in plasma. Of the 58 tumors analyzed, 39 (67%) demonstrated either one or both of the studied genetic alterations. Twenty-two (38%) were mutated at KRAS2, and an identical alteration was detected in 10 (45%) of the 22 corresponding plasma samples. Thirty-one (53%) had p16 gene promoter hypermethylation that could also be detected in the plasma in 21 cases (68%). Among the 39 patients who had one or the other alteration in tumor DNA, 37 had at least one reliable plasma test. In 26 (70%) of the 37 patients, free-circulating tumor-associated DNA was detected in plasma. The 2-year overall survival rate was 48% in the group where free-circulating tumor-associated DNA was detected in plasma and 100% in the one where free-circulating tumor-associated DNA was not detected in plasma (p < 0.03). Among these 37 patients, 25 patients had a stage I, II or III disease. In this subgroup of patients, the 2-year recurrence-free survival rate for the 17 patients with free-circulating tumor-associated DNA detected in plasma was 66%, compared to 100% for the 8 patients without free-circulating tumor-associated DNA detected in plasma (p = 0.044). The presence of free-circulating tumor-associated DNA in plasma seems to be a relevant prognostic marker for patients with colorectal cancer and may be used to identify patients with a high risk of recurrence.     

Analysis of colorectal cancer‐related mutations by liquid biopsy: Utility of circulating cell‐free DNA and circulating tumor cells

We recruited 56 colorectal cancer patients and compared the mutational spectrum of tumor tissue DNA, circulating cell‐free DNA (ccfDNA) and circulating tumor cell (CTC) DNA (ctcDNA) to evaluate the potential of liquid biopsy to detect heterogeneity of cancer. Tumor tissue DNA, ccfDNA, and ctcDNA were extracted from each patient and analyzed using next‐generation sequencing (NGS) and digital PCR. To maximize yields of CTC, three antibodies were used in the capture process. From 34 untreated patients, 53 mutations were detected in tumor tissue DNA using NGS. Forty‐seven mutations were detected in ccfDNA, including 20 not detected in tissues. Sixteen mutations were detected in ctcDNA, including five not detected in tissues. In 12 patients (35.3%), mutations not found in tumor tissues were detected by liquid biopsy: nine (26.5%) in ccfDNA only and three (8.8%) in ctcDNA only. Combination analysis of the two liquid biopsy samples increased the sensitivity to detect heterogeneity. From 22 stage IV patients with RAS mutations in their primary tumors, RAS mutations were detected in 14 (63.6%) ccfDNA and in eight (36.4%) ctcDNA using digital PCR. Mutations not detected in primary tumors can be identified in ccfDNA and in ctcDNA, indicating the potential of liquid biopsy in complementing gene analysis. Combination analysis improves sensitivity. Sensitivity to detect cancer‐specific mutations is higher in ccfDNA compared with ctcDNA.     

Identification of driver genes and somatic mutations in cell-free DNA of patients with pulmonary lymphangioleiomyomatosis

Next-generation sequencing of cell-free circulating DNA (cfDNA) has emerged as promising technique for identifying minimally invasive genomic profiling of tumor cells recently. However, it remains relatively unknown in LAM disease. In our study, paired cfDNA and genomic DNA (gDNA) in blood samples were obtained from 23 LAM patients and seven healthy controls to explore mutations profiles of targeted 70 cancer-related genes. As results, log2-based allele frequencies of mutations in cfDNA were significantly different from those of gDNA. By comparing the mutual mutations identified both in cfDNA and gDNA, a significant correlation was also observed. After removing mutations in gDNA, distinct somatic mutation profiles of cfDNA were observed in LAM patients. Forty of 70 targeted genes had recurrent mutations, of which ATM, BRCA2 and APC showed the highest frequency. Based on the mutation, correlation network constructed of 40 mutated genes, 11 hub genes bearing intensive interactions were highlighted, including BRCA1, BRCA2, RAD50, RB1, NF1, APC, MLH3, ATM, PDGFRA, PALB2 and BLM. Expression of the hub genes showed significant clusters between LAM patients and controls and that RAD50 and BRCA2 had the strongest associations with subject phenotypes. Myogenesis and estrogen response were confirmed to be positively regulated in LAM patients. Collectively, our study provided a landscape of genomic alterations in LAM and discovered several potential driver genes, that is, BRCA2 and RAD50, which shed a substantial light on the clinical application of key molecular markers and potential therapy targets for precision diagnosis and treatment in the future.     

Assessment of endometrial sampling as a predictor of final surgical pathology in endometrial cancer

Background:

The histology and grade of endometrial cancer are important predictors of disease outcome and of the likelihood of nodal involvement. In most centres, however, surgical staging decisions are based on a preoperative biopsy. The objective of this study was to assess the concordance between the preoperative histology and that of the hysterectomy specimen in endometrial cancer.

Methods:

Patients treated for endometrial cancer during a 10-year period at a tertiary cancer centre were identified from a prospectively collected pathological database. All pathology reports were reviewed to confirm centralised reporting of the original sampling or biopsy specimens; patients whose biopsies were not reviewed by a dedicated gynaecological pathologist at the treating centre were excluded. Surgical pathology data including histology, grade, depth of myometrial invasion, cervical stromal involvement and lymphovascular space invasion (LVSI) as well as preoperative histology and grade were collected. Preoperative and final tumour cell type and grade were compared and the distribution of other high-risk features was analysed.

Results:

A total of 1329 consecutive patients were identified; 653 patients had a centrally reviewed epithelial endometrial cancer on their original biopsy, and are included in this study. Of 255 patients whose biopsies were read as grade 1 (G1) adenocarcinoma, 45 (18%) were upgraded to grade 2 (G2) on final pathology, 6 (2%) were upgraded to grade 3 (G3) and 5 (2%) were read as a non-endometrioid high-grade histology. Overall, of 255 tumours classified as G1 endometrioid cancers on biopsy, 74 (29%) were either found to be low-grade (G1–2) tumours with deep myometrial invasion, or were reclassified as high-grade cancers (G3 or non-endometrioid histologies) on final surgical pathology. Despite these shifts, we calculate that omitting surgical staging in preoperatively diagnosed G1 endometrioid cancers without deep myometrial invasion would result in missing nodal involvement in only 1% of cases.

Conclusions:

Preoperative endometrial sampling is only a modest predictor of surgical pathology features in endometrial cancer and may underestimate the risk of disease spread and recurrence. In spite of frequent shifts in postoperative vs preoperative histological assessment, the predicted rate of missed nodal metastases with a selective staging policy remains low.     

Coexistence of EGFR with KRAS,or BRAF,or PIK3CA somatic mutations in lung cancer: a comprehensive mutation profiling from 5125 Chinese cohorts

Background:

Determining the somatic mutations of epidermal growth factor receptor (EGFR)-pathway networks is the key to effective treatment for non-small cell lung cancer (NSCLC) with tyrosine kinase inhibitors (TKIs).The somatic mutation frequencies and their association with gender, smoking history and histology was analysed and reported in this study.

Methods:

Five thousand one hundred and twenty-five NSCLC patients'' pathology samples were collected, and EGFR, KRAS, BRAF and PIK3CA mutations were detected by multiplex testing. The mutation status of EGFR, KRAS, BRAF and PIK3CA and their association with gender, age, smoking history and histological type were evaluated by appropriate statistical analysis.

Results:

EGFR, KRAS, BRAF and PIK3CA mutation rates revealed 36.2%, 8.4%, 0.5% and 3.3%, respectively, across the 5125 pathology samples. For the first time, evidence of KRAS mutations were detected in two female, non-smoking patients, age 5 and 14, with NSCLC. Furthermore, we identified 153 double and coexisting mutations and 7 triple mutations. Interestingly, the second drug-resistant mutations, T790M or E545K, were found in 44 samples from patients who had never received TKI treatments.

Conclusions:

EGFR exons 19, 20 and 21, and BRAF mutations tend to happen in females and non-smokers, whereas KRAS mutations were more inclined to males and smokers. Activating and resistant mutations to EGFR-TKI drugs can coexist and ‘second drug-resistant mutations'', T790M or E545K, may be primary mutations in some patients. These results will help oncologists to decide candidates for mutation testing and EGFR-TKI treatment.     

Noninvasive detection of microsatellite instability in patients with endometrial cancer

The analysis of mismatch repair proteins in solid tissue is the standard of care (SoC) for the microsatellite instability (MSI) characterization in endometrial cancer (EC). Uterine aspirates (UAs) or circulating-DNA (cfDNA) samples capture the intratumor heterogeneity and provide a more comprehensive and dynamic molecular diagnosis. Thus, MSI analysis by droplet-digital PCR (ddPCR) in UAs and cfDNA can provide a reliable tool to characterize and follow-up the disease. The UAs, paraffin-embedded tumor tissue (FFPE) and longitudinal plasma samples from a cohort of 90 EC patients were analyzed using ddPCR panel and compared to the SoC. A high concordance (96.67%) was obtained between the analysis of MSI markers in UAs and the SoC. Three discordant cases were validated as unstable by ddPCR on FFPE samples. Besides, a good overall concordance (70.27%) was obtained when comparing the performance of the ddPCR assay on UAs and cfDNA in high-risk tumors. Importantly, our results also evidenced the value of MSI analysis to monitor the disease evolution. MSI evaluation in minimally invasive samples shows great accuracy and sensitivity and provides a valuable tool for the molecular characterization and follow-up of endometrial tumors, opening new opportunities for personalized management of EC.     

转移性结直肠癌患者ctDNA基因突变检测方法的比较及影响因素分析

背景与目的：结直肠癌是常见的消化道肿瘤之一,中国的结直肠癌发病率和死亡率都位居前列。循环肿瘤DNA（circulating tumor DNA,ctDNA）作为一种非侵入性检测标志物在结直肠癌患者全病程管理中具有一定价值。通过对转移性结直肠癌（metastatic colorectal cancer,mCRC）患者血浆ctDNA中相关基因突变状态进行检测和分析,可协助制定患者个性化治疗方案。方法：本研究纳入2016年6月—2017年1月复旦大学附属中山医院收治的30例mCRC患者,利用二代测序（next generation sequencing,NGS）和MALDI-TOF检测患者血浆ctDNA中KRAS、NRAS、BRAF、PIK3CA基因的常见突变。将两个平台数据相互比较,同时结合病史、组织活检结果和微滴式数字聚合酶链反应（droplet digital polymerase chain reaction,ddPCR）得到的结果进行比较,评估本实验中NGS和MALDI-TOF检测的结果与综合获得的结果的符合率以及各自的阳性预测值和阴性预测值。结果：MALDI-TOF的符合率为76.67%,阳性预测值为86.67%,阴性预测值为66.67%。NGS的符合率为86.67%,阳性预测值为83.33%,阴性预测值为91.67%。结论：两个平台均可用于检测ctDNA中的常见突变,但NGS的阴性预测值要优于MALDI-TOF,同时突变位点丰度与原发灶是否手术切除以及患者是否接受过治疗有关。     

Droplet digital PCR measurement of HER2 in patients with gastric cancer

Background:

Although there are some new criteria for human epidermal growth factor receptor 2 (HER2) expression with immunohistochemistry/fluorescence in situ hybridisation (IHC/FISH) in gastric cancer, the method is still ambiguous and is somewhat dependent on the subjective qualities of the evaluator.

Methods:

We used droplet digital polymerase chain reaction (ddPCR) to evaluate HER2 amplification in formalin-fixed and paraffin-embedded (FFPE) samples and cell-free serum circulating tumour DNA (ctDNA) in 25 patients with gastric cancer.

Results:

The concordance rate of HER2 amplification examined in FFPE samples with ddPCR and IHC/FISH was 92% (23 out of 25). The concordance rate of FFPE with ctDNA was not high (62.5%); however, patients who were HER2-positive by ctDNA had significantly shorter survival compared with HER2-negative patients.

Conclusions:

Our results demonstrated that this ddPCR method was as effective as IHC/FISH and therefore might become a standard method for analysing not only FFPE but also ctDNA.     

Microsatellite alterations and TP53 mutations in plasma DNA of small-cell lung cancer patients: Follow-up study and prognostic significance

Background:Small-cell lung cancer (SCLC), one of the major types of lung cancer, is associated with many different somatic molecular genetic changes. These alterations, observed in tumor DNA, have also been identified in the plasma DNA of patients. We undertook the present study to make a prospective investigation into the correlation between abnormal plasma DNA and patient survival. Patients and methods:Thirty-five patients with SCLC were selected after histological diagnosis. Polymorphic markers (ACTBP2, UT762 and AR) were chosen for their reported high rate of alterations in SCLC and analyzed in tumor tissue, normal blood cells and plasma DNA. Furthermore, we looked for mutations of the TP53 gene in tumor and plasma DNA. Results:In 25 patients (71%) at least one molecular change precisely matching that of the primary tumor was detected in the plasma DNA. No difference in survival was observed between patients with aberrant plasma DNA and patients without plasma DNA alterations. However, patients with microsatellite modifications and TP53 mutations concomitantly, showed a significant difference (P = 0.02) in survival compared with patients bearing only one of these molecular changes. In 15 cases it was possible to find a correlation either between tumor response and disappearance of abnormal plasma DNA, or tumor progression and persistence of plasma DNA alterations. Conclusions:Free plasma DNA with molecular alterations is present to a high degree in plasma DNA of SCLC patients and may have a role as a prognostic factor.     

Comparing single‐target and multitarget approaches for postoperative circulating tumour DNA detection in stage II–III colorectal cancer patients

Circulating tumour DNA (ctDNA) detection for postoperative risk stratification in cancer patients has great clinical potential. However, low ctDNA abundances complicates detection. Multitarget (MT) detection strategies have been developed to increase sensitivity. Yet, empirical evidence supporting performance gains of MT vs. single‐target (ST) strategies in a postoperative setting is limited. We compared ctDNA detection in 379 paired plasma samples from 112 stage II–III colorectal cancer patients by ST digital PCR and MT sequencing of 16 patient‐specific variants. The strategies exhibited good concordance (90%, Cohen''s Kappa 0.79), with highly correlated ctDNA quantifications (Pearson r = 0.985). A difference was observed in ctDNA detection preoperatively (ST 72/92, MT 88/92). However, no difference was observed immediately after surgery in recurrence (ST 11/22, MT 10/22) or nonrecurrence (both 2/34) patients. In serial samples, detection was similar within recurrence (ST 13/16, MT 14/16) and nonrecurrence (ST 3/49, MT 1/49) patients. Both approaches yielded similar lead times to standard‐of‐care radiology (ST 4.0 months, MT 4.1 months). Our findings do not support significant performance gains of the MT strategy over the ST strategy for postoperative ctDNA detection.     

The prognostic value of plasma fibrinogen levels in patients with endometrial cancer: a multi-centre trial

Background:

To analyse the correlation between pre-treatment plasma fibrinogen levels and clinical–pathological parameters in patients with endometrial cancer and to assess the value of plasma fibrinogen as a prognostic parameter.

Methods:

Within a retrospective multi-centre study, the records of 436 patients with endometrial cancer were reviewed and pre-treatment plasma fibrinogen levels were correlated with clinical–pathological parameters and patients'' survival.

Results:

The mean (s.d.) pre-treatment plasma fibrinogen level was 388.9 (102.4) mg per 100 ml. Higher plasma fibrinogen levels were associated with advanced tumour stage (FIGO I vs II vs III and IV, P=0.002), unfavourable histological subtype (endometrioid vs non-endometrioid histology, P=0.03), and higher patients'' age (⩽67 years vs >67 years, P=0.04), but not with higher histological grade (G1 vs G2 vs G3, P=0.2). In a multivariate analysis, tumour stage (P<0.001 and P<0.001), histological grade (P=0.009 and P=0.002), patients'' age (P=0.001 and P<0.001), and pre-treatment plasma fibrinogen levels (P=0.04 and P=0.02) were associated with disease-free and overall survival, respectively.

Conclusion:

Plasma fibrinogen levels can be used as an independent prognostic parameter for the disease-free and overall survival of patients with endometrial cancer.