INTRODUCTION
Like the incidence rate, morbidity and mortality associated with endometrial cancer are increasing yearly with a tendency toward a younger age at diagnosis.[ 1 , 2 ] Lynch syndrome is an autosomal dominant genetic disease caused by defects in DNA mismatch repair (MMR) genes including MLH1, MSH2, MSH6, and PMS2. Endometrial cancer is the most commonly observed Lynch syndrome-related malignancy in females, with up to 40%–60% of female Lynch syndrome patients experiencing endometrial cancer as the first manifestation.[ 3 ] Recognizing the early symptoms (irregular uterine bleeding, postmenopausal vaginal bleeding, or postmenopausal bleeding) of endometrial cancer and carrying out appropriate screening is conducive to the early diagnosis of endometrial cancer with associated reductions in morbidity and mortality.[ 4 ]
The traditional gold standard for endometrial sampling is sharp dilation and curettage (D&C).[ 5 ] However, this method must be performed under anesthesia and can be associated with significant intraoperative complications.[ 6 ] More recently, suction curettage endometrial sampling has been presented as an alternative to D&C.[ 7 ] This method can be conducted on an outpatient basis and is considered less invasive. The cell and tissue samples obtained from suction curettage can be used in liquid-based cytology and histological screening with immunohistochemistry (IHC) or sequencing to detect mutations in MLH1, MSH2, MSH6, and PMS2. However, the value of screening for Lynch and Lynch-like syndromes has yet to be fully established.[ 8 , 9 ]
Despite its advantages, some authors assert a limited role for suction curettage in patients with abnormal uterine bleeding,[ 10 , 11 ] and there is little discussion available on biopsy methods for the detection of Lynch syndrome by IHC. Due to differing sampling locations and sample quality, cytological results may not be consistent with histological results.[ 12 ] Some scholars propose that the combination of liquid-based cytology and microtissue histology may be valuable to improve the clinical diagnosis of endometrial cancer .[ 13 ]
In this study, we retrospectively evaluated clinical data from patients with endometrial cancer who underwent suction curettage for liquid-based cytological and microtissue histological diagnostic screening. The diagnostic accuracy and clinical value of suction curettage were further explored in endometrial cancer patients by comparing the results with those obtained from traditional D&C. Still further, we discuss the diagnosis of endometrial cancer related to Lynch syndrome by IHC using microtissue samples.
METHODS
Patients
This retrospective study reviewed data from female patients with irregular uterine bleeding and/or abnormal uterine cavities examined from May 2018 to January 2019 in the Department of Gynecology at our Hospital. The inclusion criteria were irregular uterine bleeding, postmenopausal bleeding, and abnormal endometrium as assessed by B-mode ultrasound (endometrial thickness ≥5 mm in postmenopausal women or ≥20 mm in pre and perimenopausal women and/or a space occupying lesion in the uterine cavity/heterogeneous uterine cavity). The exclusion criteria were record of cervical disease and cervical surgery, pregnancy at diagnosis, and presence of pelvic inflammatory disease. This study was approved by the Medical Ethics Committee of our hospital. Informed consent was waived due to the retrospective nature of the study.
Data collection
Baseline patient characteristics (age, weight, BMI, blood pressure, blood sugar, and fertility status) were collected from the hospital’s electronic case system. The results of liquid-based cytology and microtissue histology from cells and tissue samples obtained by suction curettage were also collected. All diagnostic conclusions were strictly reviewed by physicians with more than 20 years of clinical work. The outcome of this study was the AUC of patients with endometrial cancer .
Sample collection
The patients were subjected for sample collection by suction curettage using a disposable curette (Juno Corp., Beijing, CN) before traditional D&C. The disposable endometrial curette was 3.1 mm in diameter and 243 mm in length, with a flexible, inclined sampling straw, which can collect specimens from a pandemic range of the uterine cavity and guarantee a sufficient amount tissue and cell specimens for examination. Its capacity was 10 mL with a working negative pressure of about 450 mmHg. The sampling procedure was performed as follows: expose the cervix, disinfect the cervix using iodine volts, slowly insert the top of the disposable sampling tube into the bottom of the uterus through the cervix, measure the depth of the uterus, attach the suction port of the tip of the sampling tube to the endometrium, tighten the connecting knot of the sampling tube (ES disposable endometrial sampler) with the negative pressure screw port, hold the disposable negative pressure suction device, maintain the negative pressure, and rotate the sampling tube to obtain the inner membrane sample. After the collection, slowly draw the sampling tube out of the uterus, then open the cap of the preservation solution bottle (Juno Corp., Beijing, CN), and quickly inject the collected cells and tissue samples into the endometrial preservation solution bottle. This was followed by a standard D&C procedure.
Cytological analysis
The liquid-based thin-layer cell smears were prepared using the sedimentation liquid-based method of the Auto-Cyte PREP system (Tri-Path Imagining, USA) which was followed by Pap staining and microscopic observation. The technique was performed as follows: The preservation solution bottle was transferred into centrifuge tubes with a density reagent (BD Diagnostic, Burlington, NC, USA). After a two-stage centrifugation at 200 rpm for 2 min 15s and then 800 rpm for 10 min, the supernatant in the centrifuge tube is discarded. Finally, centrifuge tubes were processed using Auto-Cyte PREP system (Tri-Path Imaging, Burlington, NC, USA). The liquid-based thin-layer cell smears were stained with routine Pap staining for microscopic observation.
Histopathological analysis
The samples from the D&C endometrium scrape were fixed by neutral formalin solution for further examination. Histopathological diagnosis by traditional sharp D&C was regarded as the gold standard.
IHC staining and interpretation
Endometrial cancer microtissue paraffin specimens were continuously sliced at 4 μm of thickness and stained by IHC PV9000 two-step method. The MMR monoclonal antibodies, MLH1 (mouse monoclonal antibody 1:100), MSH2 (rabbit monoclonal antibody 1:150), MSH6 (rabbit monoclonal antibody 1:150), and PMS2 (rabbit monoclonal antibody 1:100), were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. If all four nuclear MMR proteins were positively stained, the sample was designated as MMR intact (normal endometrial cells or mesenchymal cells are positively stained). MMR protein expression was defined as MMR absent if the nucleus was not stained by the MMR antibodies. MMR was defined as MMR is absent if at least one of the four proteins was absent.
Statistics
SPSS version 18.0 (SPSS Inc., Chicago, IL, USA) software was used for statistical analysis. Continuous data conforming to the normal distribution (according to the Kolmogorov–Smirnov test) were expressed as mean ± standard deviations, and continuous data with non-normal distribution were presented as medians (range). Categorical data were presented as count (percentage) and tested using the χ2 test. ROC curves were used to analyze the diagnostic efficiency of three diagnostic methods. P <0.05 is considered as statistically significant.
RESULTS
Patient characteristics
This study enrolled 100 patients. The average age of the cohort was 49.3 (22–76) year. Taken as a whole, the patients had a normal BMIs (23.48 ± 6.01) kg/cm2 , blood pressure (126.92 ± 3.45/75.85 ± 2.8) mmHg, and blood sugar levels were (4.72 ± 2.43) mmol/L. The indications for biopsy were irregular uterine bleeding (n = 27), postmenopausal bleeding (n = 13), endometrium thickness ≥5 mm by B-ultrasound with postmenopausal uterine bleeding (n = 34), and a space occupying lesion in the uterine cavity (n = 26) [Table 1 ].
Table 1: Patient characteristics
Sampling adequacy rates
Among 100 patients, the sampling adequacy rate of uterine cavity cytology sampling was 96.0% (96/100), of which 93 (93.0%) endometrial samples had usable tissues. The sampling adequacy rate for the D&C method was 99% (99/100). There was no statistically significant difference in the sampling adequacy rate between the two methods (χ2 = 1.85, P > 0.05) [Table 2 ]. Unsatisfactory samples were excluded from this study.
Table 2: Sampling adequacy of suction curettage and sharp dilation and curettage (D&C)
Comparison of diagnostic capabilities
Thirteen patients had endometrial cancer diagnosed by the final D&C. Among them, ten patients were correctly diagnosed at the same time by cytology screening. The remaining three patients had a false-negative result, of which two patients had local simple hyperplasia and one had complicated hyperplasia consistent with histopathological diagnosis [Figure 1 ]. Among the 96 cytology results, there were two false positives. The concordance rate, sensitivity, and specificity of the 96 patients with adequate sampling for liquid-based cytology were 94.8%, 76.9%, and 97.5%, respectively. The coincidence rate, sensitivity, and specificity of diagnosis from 93 microtissue histology specimens with satisfactory sampling were 96.8%, 84.6%, and 98.8%, respectively. The coincidence rate, sensitivity, and specificity of suction curettage cytology combined with micro-histology for the diagnosis of endometrial cancer were 99.0%, 92.3%, and 100.0%, respectively [Table 3 ].
Figure 1: Cytological and histological morphology of normal endometrium and endometrial cancer . (a) Cytology of proliferative endometrium (Pap staining; X400 400X magnification), (b) microtissue histology of proliferative endometrium (H&E staining; X400400X magnification), (c) cytology of endometrial cancer (Pap staining; X400400X magnification), and (d) microtissue histology of endometrial cancer (H and E staining; X400 400X magnification)
Table 3: Comparison of cytology, histology, and cytology combined with histology and diagnostic curettage
Further, ROC analysis was performed to evaluate the diagnostic accuracy of cytology, micro-histology, and cytology and micro-histology combined for endometrial cancer patients. The combined strategy was stronger (area under the curve (AUC) = 0.962, 95% CI: 0.000–1.000, P = 0.000) than cytologic (AUC = 0.873, 95% CI: 0.718–1.000, P = 0.000) or micro-histologic (AUC = 0.917, 95% CI: 0.000–1.000, P = 0.000) evaluation alone [Figure 2 ].
Figure 2: ROC curves for the assessment of the diagnostic accuracy of (a) liquid-based cytology, (b) microtissue histology, and (c) the two methods combined
MMR protein
Among the 13 cases of endometrial cancer , three were MMR absent and ten were MMR intact. Among them, 15.3% (2/13), 0% (0/13), 7.7% (1/13), and 15.3% (2/13) lacked MLH1, MSH2, MSH6, and PMS2, respectively. Two cases were negative for both MLH l and PMS2, while one was only MSH6 negative [Figure 3 ].
Figure 3: IHC staining in endometrial cancer (a) MLH1 negative, (b) MSH2 positive, (c) MSH6 positive, and (d) PMS2 negative in microtissue of endometrial cancer (X200)
DISCUSSION
This study verified the clinical utility of suction curettage to obtain usable cytological and histological samples for endometrial cancer screening. The method we examined is advantageous due to its high diagnostic accuracy and limited invasiveness. It can be used to confirm whether there is hemorrhage caused by endometrial cancer to promote its early identification, and further protein detection by immunohistochemistry (IHC) can confirm the cause of cancer for prompt precision treatment.
Presently, the most common endometrial collection method in China utilizes endometrial brushing, with early screening and diagnosis of endometrial lesions made according to sampled cell morphology.[ 16 , 17 ] The sensitivity of cytological diagnosis from this technique ranges from 73% to 97%.[ 18 , 19 ] In our study, the sensitivity and specificity for cytological diagnoses were 76.9% and 97.5%, respectively, and the concordance rate with sharp curettage was 94.8%. For comparison, Pipelle® suction curettage has reported sensitivity and specificity rates of 75% and 100%, respectively, for cytological specimens.[ 20 ] Compared with the operative D&C diagnosis, the concordance of cytological diagnosis was 96.4% in a further study using the Pipelle® technique,[ 21 ] which is similar to our results. However, by combing cytological diagnosis and microtissue histological techniques, we reached a concordance rate of 99%.
The disposable endometrial sampler in this study was applied to aspirate the endometrium by negative pressure. This sampling method is non-invasive, and the flexible, inclined sampling straw design can collect specimens from a pandemic range of the uterine cavity and guarantees a sufficient amount tissue and cell specimens for evaluation. In this study, the satisfaction rate of the endometrium sampler in obtaining tissue was 93%, which did not achieve statistically significant difference with the D&C method. The cytological diagnosis of five patients was not consistent with D&C samples, including two false positives and three false negatives. The two false positive cytology diagnoses are attributable to abnormal endometrial hyperplasia and tamoxifen treatment, which can lead to changes in cell morphology. The results of three false-negative cytology can likely be explained by limited size of the tumor, the localization of lesions in the corners of the uterus, or the overlap of proliferative glandular cells with cancer cells which can be difficult to differentiate.
Ideally, diagnostic screenings should be as accurate, low cost, and non-invasive, to increase patient compliance and be widely carried out. As the “gold standard” for the diagnosis of endometrial cancer , segmental diagnostic curettage has possibility to miss the diagnosis of small local lesions, and it is an invasive operation with risk of uterine perforation and infection. Moreover, patients have to endure significant postoperative pain. Therefore, for high-risk patients, long-term monitoring has poor compliance.[ 22 ] For asymptomatic women who should be screened but are unwilling to undergo an invasive and painful procedure, suction curettage as described by our methods can be recommended as a simple and effective alternative method for endometrial tissue collection. However, we recommend that cytology alone should only be used as a preliminary screening for endometrial diseases in high-risk groups, rather than as a definitive test for clinical decisions.
In Lynch syndrome-related endometrial cancer patients, endometrial cancer can be regarded as the “sentinel” tumor of the female patient and her family.[ 23 ] Screening Lynch syndrome-related endometrial cancer will guide clinical treatment decisions and follow-up plans not only for endometrial cancer patients but also for other members of the family. Thus, the current trend is advocating Lynch syndrome screening for all patients diagnosed with endometrial cancer [ 24 , 25 ] Currently, the most well-recognized screening method is IHC staining which indicates MMR protein expression in tumors. It has high sensitivity and can predict gene mutations causing the absence of MMR proteins.[ 20 ] However, protein testing is only a preliminary screening for endometrial cancer associated with Lynch syndrome, and diagnosis must be further confirmed by genetic testing. Our results show that suction curettage can provide adequate samples for Lynch syndrome screening.
This study had some limitations. First, due to the retrospective nature of the study, some confounders are possible. Second, the sample size was relatively small, Third, the sampling was blind. As previously stated, lesions located on the uterine horn (three patients confirmed by gross pathology) and other distal sites are difficult to sample. This concern may be ameliorated by the use of hysteroscopy during biopsy, so all concerning lesions can be visualized and sampled by the operator. Finally, while it is considered the “gold standard” traditional D&C can also miss the diagnosis of malignant lesions due to inadequate sampling, and the only definitive method for the diagnosis of endometrial cancer is hysterectomy.[ 26 ] Similarly, as previously mentioned, hysteroscopy is preferred over blind biopsy (Novak’s curette) where available due to improved sensitivity. Finally, we did not confirm the diagnosis of Lynch syndrome by genetic screening.[ 14 ]
In summary, the use of suction curettage, a non-invasive method, to obtain both cells and microtissues is valuable in the diagnosis of endometrial diseases such as endometrial cancer . Moreover, endometrial lesions may be the first sign of Lynch syndrome and initiate diagnostic testing before a deadly colon cancer develops.[ 15 ] Definitive diagnostic testing should be performed to validate our IHC results related to Lynch syndrome. Further clinical research should be carried out on a large-scale cohort to obtain more reliable and accurate data on the utility of suction curettage vs traditional sharp dilation and curettage in our studied demographic population.
Ethical approval
This study was approved by the Medical Ethics Committee of Affiliated Tumor Hospital of Xinjiang Medical University (K-2021041). Informed consent was waived due to the retrospective nature of the study.
Financial support and sponsorship
This work was supported by Xinjiang Science and Technology Project (2020E02125) and Natural Science Foundation of Xinjiang Uygur Autonomous Region (2020D01C204).
Conflicts of interest
There are no conflicts of interest.
Acknowledgement
We are grateful to Dr Guqun Shen for their assistance with patient recruitment and Dr. Huiqin Guo for study co-ordination.
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