Liquid-based Cytology Specimens Research Articles

Advancing Precision Oncology: Whole-Exome Sequencing in Endometrial Cancer Liquid-Based Cytology.

Diagnostic strategies for endometrial cancer have been evolving, with cytologic analysis being considered a key method in integrated oncologic diagnostics because of its less invasive nature and adaptability to various assessments. Liquid-based cytology (LBC) has emerged as a promising method for intact DNA preservation; it exhibits improved efficiency in advanced sequencing applications such as next-generation sequencing. However, despite the use of LBC in panel assays, its application in whole-exome sequencing (WES) for comprehensive genomic profiling remains underexplored. To investigate whether molecular classification is possible based on WES using DNA derived from LBC specimens. We combined WES with targeted gene panel analysis to compare genomic findings of LBC and traditional tissue samples obtained from 7 cases of endometrial cancer. We investigated pathogenic mutations, tumor mutational burden, and microsatellite instability, and achieved molecular classification with high accuracy. We found a substantial concordance between LBC and traditional tissue samples in terms of pathogenic mutation detection, with a 95% match in the LBC samples and 94% in the tissue samples. Notably, our results highlight the importance of combining WES with panel-based analysis in identifying the ultramutated status of a case that had been missed during panel analysis. Our findings emphasize the potential of LBC samples in the precise and noninvasive genomic analysis of cases of endometrial cancer and offer a new avenue for developing diagnostic and therapeutic strategies in precision oncology.

Methodological and TNM Focus-Based Comparison of EGFR Mutation Status in Non-Small-Cell Lung Carcinomas.

Epidermal growth factor receptor (EGFR) mutations in non-small-cell lung carcinomas (NSCLC) are a frequent class of driver mutations, and tyrosine kinase inhibitor (TKI) therapy provides considerable clinical benefits. Using the most effective and also easiest method for EGFR analysis is cost-effective and time-saving. In this study, we aimed to determine which method could be more effective by comparing the incidences of EGFR mutations in cytological and histological samples which were obtained by different methods also, whether there was a difference in the incidences of EGFR mutations between the primary foci, mediastinal lymph nodes, and distant metastatic foci. We retrospectively reviewed 420 cases of cytological materials, small biopsies, and surgical samples reported as NSCLC underwent EGFR analysis in our department between 2016 and 2022. We collected the data and interpreted the results from two different perspectives. We identified 36 EGFR mutations in 362 biopsies (9.94%) and 17 in 58 cytology samples (29.31%). There is a significant difference between the two methods (P = 0.01*). We observed 38 EGFR mutations in 320 primary foci (11.87%), 7 EGFR mutations in 36 mediastinal or subcarinal lymph nodes (19.44%), and 8 EGFR mutations in 64 distant metastatic foci (12.50%). A significant difference was also observed in pleural samples (P = 0.005*). We observed more successful results with cell blocks obtained from liquid-based cytological specimens than with formalin-fixed, paraffin-embedded tissues obtained from resection or otherwise in our clinical routine. Our study results highlight the benefits of cytological specimens in molecular treatments and current therapy modalities.

Liquid-based cytology specimens for next-generation sequencing in lung adenocarcinoma: challenges and evaluation of targeted therapy

BackgroundTo explore challenges of liquid-based cytology (LBC) specimens for next-generation sequencing (NGS) in lung adenocarcinoma and evaluate the efficacy of targeted therapy.MethodsA retrospective analysis was conducted on the NGS test of 357 cases of advanced lung adenocarcinoma LBC specimens and compared with results of histological specimens to assess the consistency. The impact of tumor cellularity on NGS test results was evaluated. The utility of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) was collected. Clinical efficacy evaluation was performed and survival curve analysis was conducted using the Kaplan-Meier method.ResultsThere were 275 TKI-naive and 82 TKI-treated specimens, the mutation rates of cancer-related genes detected in both groups were similar (86.2% vs. 86.6%). The EGFR mutation rate in the TKI treated group was higher than that in the TKI-naive group (69.5% > 54.9%, P = 0.019). There was no significant difference in the EGFR mutation frequency among different tumor cellularity in the TKI-naive group. However, in the TKI treated group, the frequency of EGFR sensitizing mutation and T790M resistance mutation in specimens with < 20% tumor cellularity was significantly lower than that in specimens with ≥ 20% tumor cellularity. Among 22 cases with matched histological specimens, 72.7% (16/22) of LBC specimens were completely consistent with results of histological specimens. Among 92 patients with EGFR-mutant lung adenocarcinoma treated with EGFR-TKIs in the two cohorts, 88 cases experienced progression, and the median progression-free survival (PFS) was 12.1 months.ConclusionsCytological specimens are important sources for gene detection of advanced lung adenocarcinoma. When using LBC specimens for molecular testing, it is recommended to fully evaluate the tumor cellularity of the specimens.

Metastases to the parotid glands: An institutional experience.

Metastatic lesions to the salivary gland are rare and mostly affect the parotids. Metastases represent 8% of all malignant lesions of the parotid gland. Around 80% originate from squamous cell carcinomas (SCC) of the head and neck region. Fine needle aspiration (FNA) plays a crucial role in distinguishing primary salivary gland lesions from metastases. Herein we describe our series of metastases to the parotid glands. We analysed 630 parotid gland FNAs over a decade including conventional and liquid-based cytology specimens. Ancillary techniques such as immunocytochemistry (ICC) were conducted on cell blocks. Eighty (12.4%) cases were malignant lesions, of which 53 (63.75%) were metastases including 24% melanoma, 22.6% SCC, 19% renal carcinomas, 7.5% breast carcinomas, 11.3% lung, 9% intestinal and 1.8% testicular, malignant solitary fibrous tumour and Merkel cell carcinoma. The 53 cases, classified according to the Milan system for salivary cytopathology, belonged to 5 Suspicious for malignancy (SFM) and 48 malignant (M) categories. Forty had a known history of primary malignancy (75.4%), while 13 were suspicious to be a metastatic localisation (24.5%), distributed as 5SFM (2SCC and 3Melanoma) and 8 M. A combination of clinical history, cytomorphology and ICC identified 100% of them. Fine needle aspiration plays a central role in the diagnostic workup of patients with metastatic lesions to their parotid glands, thereby defining the correct management. Diagnostic accuracy may be enhanced by applying ICC. Although melanoma and SCC are the most common histological types, several other malignancies may also metastasize to the parotid glands and should be kept into consideration.

Evaluation of dual p16/Ki-67 immunostaining on anal cytology specimens.

Dual immunostaining for p16/Ki67 is FDA-approved for use on liquid-based cervical cytology specimens; however, the utility of dual staining in anal cytology especially for ASCUS risk stratification is not well established. We investigated dual staining performance on anal cytology specimens and correlated with subsequent cytologic interpretation, high-risk HPV status, and anal biopsy results. Dual staining for p16/Ki-67 was performed on all liquid-based anal cytology specimens from December 2021 to June 2022 (n = 43). Three patients had high grade squamous intraepithelial lesion (HSIL/AIN2-3) on biopsy; dual staining was positive in all three cases. All HR-HPV negative cases were negative for dual staining. Among the 12 ASCUS samples with subsequent anal biopsy results all also had HR-HPV testing. Due to small sample size of cases with squamous intraepithelial lesion (SIL) diagnosed on biopsy, the sensitivity and positive predictive value was not calculated. However, the specificity and negative predictive value of p16/Ki-67 dual staining for SIL of any grade on biopsy were 1 (95% CI: 0.66-1) and 0.9 (95% CI: 0.65-0.97) respectively, whereas the specificity and negative predictive value of HR-HPV testing for SIL of any grade on biopsy were 0.44 (95% CI: 0.14-0.79) and 0.8 (95% CI: 0.41-0.96) respectively. Dual p16/Ki-67 staining indicates transforming HPV infection and could help serve as an ancillary test for risk stratification for atypical anal cytology specimens. Among ASCUS samples, dual staining was specific for SIL of any grade with a high negative predictive value and therefore could be useful in clinical practices with limited availability for follow-up care.

Staining Pattern of Alcian Blue in Endometrial Cytology: Utility in Distinguishing Grade 1-Endometrial Endometrioid Carcinoma from Endometrial Glandular Stromal Breakdown.

In endometrial cytology, differentiating endometrial glandular stromal breakdown (EGBD) from endometrial endometrioid carcinoma (G1-EEC) is often difficult. In this study, we provided a new focus on chondroitin sulfate (CS), a major substrate component of the endometrial stroma, and assessed the diagnostic utility of Alcian Blue (AB) staining in the differential diagnosis in liquid-based cytological (LBC) samples. LBC specimens from 19 patients with a proliferative endometrium, 36 with EGBD, and 30 with G1-EEC who underwent endometrial cytology were stained with AB (pH 1.0), and their reactivity was observed. In addition, immunocytochemical staining of CS and CD31 was performed for five cases each to evaluate their interrelationship with blood vessels. Regarding the 30 G1-EEC cases, at least one of the three representative staining patterns was observed by AB staining: dot-like, microtubular, and finely branched linear patterns. Moreover, the inner portion of the tubular material observed by AB staining expressed CD31. Conversely, in the 36 EGBD cases, only five metaplastic clusters with irregular protrusions and condensed stromal clusters (CSCs) showed a dot-like positive pattern, and background CSCs did not show reactivity to AB staining in any of the cases. Furthermore, the vascular structure expressing CD31 in cell clusters was also unclear. We demonstrated that AB staining shows different staining patterns in G1-EEC and EGBD, reflecting their different tissue structures. Our data provide new insights into endometrial cell diagnosis changes and demonstrate that AB staining is a potential new diagnostic aid tool for the differentiation of G1-EEC from EGBD.

Using the DNA Integrity Number to Analyze DNA Quality in Specimens Collected from Liquid-Based Cytology after Fine-Needle Aspiration of Breast Tumors and Lesions

Introduction: Cancer genome analysis using next-generation sequencing requires adequate and high-quality DNA samples. Genomic analyses were conventionally performed using formalin-fixed paraffin-embedded sections rather than cytology samples such as cell block or smear specimens. Specimens collected from liquid-based cytology (LBC) have the potential to be sources of high-quality DNA suitable for genetic analysis even after long-term storage. Methods: We collected breast tumor/lesion fractions from 92 residual LBC specimens using fine-needle aspiration (FNA) biopsy, including breast carcinoma (1 invasive carcinoma and 4 ductal carcinomas in situ), papillomatous lesion (5 intraductal papillomas), and fibroepithelial lesion (19 phyllodes tumors and 53 fibroadenomas) samples, and others (1 ductal adenoma, 1 hamartoma, 1 fibrocystic disease, and 7 unknown). DNA was extracted from all samples and subjected to DNA integrity number (DIN) score analysis. Results: Average DIN score collected from 92 LBC specimens was significantly higher score. In addition, high-quality DNA with high DIN values (7.39 ± 0.80) was successfully extracted more than 12 months after storage of residual LBC specimens. Conclusion: Residual LBC specimens collected from FNA of the breast were verified to carry high-quality DNA and could serve as an alternate source for genetic analysis.

The detection of PD-L1 expression on liquid-based cytology in pleural effusion of lung adenocarcinoma and its prognostic evaluation: Between paired liquid-based cytology and cell block samples.

Programmed death-ligand 1 (PD-L1) expression levels measured by immunohistochemistry have been proven to predict the outcome of immunotherapy in lung adenocarcinoma (LUAD). However, data on PD-L1 expression on liquid-based cytology (LBC) in malignant pleural effusion (MPE) is scarce. This study cohort included 60 cases with MPE suffering from LUAD. PD-L1 SP263 assay was used for immunocytochemistry (ICC) on LBC and matched cell block (CB) to validate ICC protocols on LBC slides. Clinical outcomes were analyzed based on immunotherapy and PD-L1 tumor proportion scores (TPS) on LBC slides and CBs. PD-L1 expression with TPS ≥1% was lower in LBCs than in CBs (33 of 60 [55.0%] vs. 35 of 60 [58.3%]; p = .687). Even with the TPS ≥50% threshold, PD-L1 expression was lower in LBCs (10 of 60 [16.7%] vs. 15 of 60 [25%]; p = .125). Epidermal growth factor receptor (EGFR) exon 20 mutation, tumor cell proportion, and pleural fluid neutrophil-to-lymphocyte ratio were related to PD-L1 expression on CBs (p = .013, p = 0.022, and p = .011), respectively. Patients with subsequent immune checkpoint inhibitor therapy remained a better prognostic in subgroups of PD-L1 positive expression on LBC slides (TPS ≥1%, p = .041). LBC specimens had comparable performance to CBs in PD-L1 assessment and predicting treatment response to PD-L1-defined therapy.

Tissue processing of endoscopic ultrasound-guided fine-needle aspiration specimens from solid pancreatic lesions.

Now that tissue cores can be obtained using fine-needle biopsy (FNB) needles, the ways tissues are handled for endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) are changing. Direct smear, touch smear of core tissues, and centrifugation have been used for cytological examinations, and liquid-based cytology (LBC), which allows immunostaining and genetic tests that use residual samples, is emerging as an alternative. We emphasize that liquid cytology (Cytospin™ cytology and LBC) is still important, because it enables the diagnosis of pancreatic ductal adenocarcinoma (PDAC) when cancerous cells are scarce in specimens. Cell blocks are being replaced by core tissues obtained via FNB needles. Recent reports indicate that rapid on-site evaluation (ROSE) is not necessary when FNB needles are used, and macroscopic on-site evaluation is used to evaluate specimen adequacy. Macroscopic findings of specimens are helpful in the diagnostic workup and for clarifying specimen-handling methods. In addition to the red strings and white cores observed in PDAC, mixed red and white strings, gray tissues, and gelatinous tissues are observed. Gray (necrotic) tissues and gelatinous (mucus) tissues are more suitable than histology for cell block or cytological processing. Tumor cells in neuroendocrine tumors (NETs) are numerous in red strings but cannot be observed macroscopically. ROSE might thus be necessary for lesions that may be NETs. Core tissues can be used for genetic tests, such as those used for KRAS mutations and comprehensive genomic profiling. Cytological materials, including slides and LBC specimens, can also be genetic test materials.

Comparing the performance of 2 human papillomavirus assays for a new use indication: a real-world evidence-based evaluation in the United States

The US Food and Drug Administration supports innovations to facilitate new indications for high-risk human papillomavirus testing. This report describes the retrospective testing of stored specimens and analysis of existing data to efficiently and cost-effectively support a new indication for the Onclarity human papillomavirus assay (Becton, Dickinson and Company, BD Life Sciences - Integrated Diagnostic Solutions, Sparks, MD). The performance of this index test was compared with that of a predicate test, the cobas human papillomavirus assay (Roche Diagnostics, Indianapolis, IN). Both human papillomavirus assays are based on real-time polymerase chain reaction platforms that detect the presence of 14 high-risk human papillomavirus genotypes. The predicate assay reports human papillomavirus types 16 and 18 as individual results and the other 12 human papillomavirus genotypes as 1 pooled result. The index assay reports 9 independent results (human papillomavirus types 16, 18, 31, 33/58, 35/39/68, 45, 51, 52, and 56/59/66). Both the index and predicate assays are approved by the Food and Drug Administration for cervical cancer screening, but at the time that this study was initiated, the index human papillomavirus assay was not approved for use with cervical specimens collected in PreservCyt (Hologic, Inc, San Diego, CA) liquid-based cytology media. The performance of the index human papillomavirus assay was compared with that of the predicate human papillomavirus assay for the detection of cervical intraepithelial neoplasia grades 2 or greater and 3 or greater (≥CIN2 or ≥CIN3) using PreservCyt liquid-based cytology specimens collected from women aged 21 to 65 years. In addition, the ability of the index test's extended genotyping to stratify ≥CIN2 and ≥CIN3 risks, using these specimens, was evaluated. The New Mexico HPV Pap Registry was used to select an age- and cytology-stratified random sample of 19,879 women undergoing opportunistic cervical screening and follow-up in routine clinical practice across New Mexico. A subset (n = 4820) of PreservCyt specimens was selected from 19,879 women for paired testing by the index and predicate human papillomavirus assays within age and cytology strata and included women with or without cervical biopsy follow-up. Point estimate differences and ratios were calculated for cervical disease detection and positivity rates, respectively, with 95% confidence intervals to determine statistical significance. The cumulative risk of ≥CIN2 or ≥CIN3, with up to 5-year follow-up, was estimated for the index assay using Kaplan-Meier methods. The 5-year cumulative ≥CIN3 detection rates were 5.6% for the index assay and 4.6% for the predicate assay (difference, 1.0%; 95% confidence interval, 0.5%-1.5%). The ≥CIN3 positivity rates within <1 year were 95.3% for the index assay and 94.5% for the predicate assay (ratio, 1.01; 95% confidence interval, 0.98-1.06). The ≥CIN3 cumulative positivity rates for the index and predicate assays were also similar at 5 years. Among cases of ≥CIN3, the positive agreement rates between the index and predicate assays for human papillomavirus types 16 and 18 were 100.0% (95% confidence interval, 95.0%-100.0%) and 90.9% (95% confidence interval, 62.3%-98.4%), respectively. Human papillomavirus type 16 carried the highest ≥CIN2 or ≥CIN3 risk, followed by human papillomavirus types 18/31/33/58/52/45 and human papillomavirus types 35/56/59/51/56/59/66. The index and predicate human papillomavirus assays demonstrated equivalent performance, and extended human papillomavirus genotyping, using the index assay, provided effective ≥CIN2 and ≥CIN3 risk stratification, supporting a new indication for use of the index assay with PreservCyt.

Don't SUMP it! Utility of PLAG1 immunocytochemistry in basaloid SUMP subcategory.

Basaloid salivary gland neoplasm of uncertain malignant potential (B-SUMP) is an indeterminate diagnostic subcategory, with pleomorphic adenoma (PA) representing the most common benign neoplasm. Pleomorphic adenoma gene 1 (PLAG1) staining is frequently seen in PAs and could aid in distinguishing them from other basaloid neoplasms. The authors evaluated the utility of PLAG1 immunocytochemistry (ICC) in differentiating PAs from other basaloid neoplasms in smears and liquid-based cytology (LBC) specimens. In total, 45 B-SUMP cytology aspirates and corresponding surgical excision specimens were identified. PLAG1 immunostaining was performed in all aspirates and surgical excision specimens and was scored as positive (strong/diffuse), equivocal (focal/weak), or negative. PLAG1 ICC was performed directly on 38 smears and seven LBC specimens. PLAG1 was positive in 29 of 45 cases (64%), whereas six of 45 (13%) were equivocal, and 10 of 45 (22%) were negative. PLAG1-positive aspirates included 26 (90%) PAs, two (7%) basal cell adenomas (BCAs), and one (3%) carcinoma ex-PA. PLAG1-equivocal aspirates included four (67%) PAs and two (33%) BCAs, whereas negative aspirates included five (50%) BCAs, four (40%) adenoid cystic carcinomas, and one (10%) metastatic adenosquamous carcinoma. The sensitivity, specificity, positive, and negative predictive values were 87%, 86%, 93%, and 75%, respectively. Diagnostic accuracy was 87%. PLAG1 ICC is useful when positive (strong/diffuse) and can be reliably performed on smears and LBC specimens. PLAG1 was positive in most PAs and in a small subset of BCAs. Therefore, in the absence of atypical cytologic features, PLAG1-positive tumors could be diagnosed as benign, with a note favoring PA versus BCA. In contrast, PLAG1-negative/equivocal tumors should remain in the B-SUMP category.

Histopathologic Follow-Up of Women with Inadequate ThinPrep™ Cervical Pap Tests and Positive High-Risk Human Papillomavirus Test Results

Introduction: We found only a few studies that had performed high-risk human papillomavirus (hrHPV) analyses of inadequate ThinPrep™ Papanicolaou (Pap) tests. Therefore, this study aimed to analyze unsatisfactory ThinPrep Pap tests using hrHPV tests. The colposcopic biopsy results of cases with an unsatisfactory ThinPrep Pap test and positive hrHPV results were revealed. Methods: Between January 1, 2018, and October 31, 2022, 965 (3.7%) of 25,958 liquid-based cytology specimens were evaluated as unsatisfactory. Ninety-five (9.8%) of 965 patients were positive for hrHPV. The colposcopic evaluation was performed in 28 (29.4%) of 95 patients, in whom 23 tests were adequate. Results: Twenty-three colposcopy biopsy results showed that 17 (73.9%) of 23 patients had benign biopsy results. High-grade squamous intraepithelial lesions were observed in three (13%) of the 23 patients, and low-grade squamous intraepithelial lesions were observed in two (8.6%) of the 23 patients. One of the 23 (4.3%) patients had keratinized squamous cell carcinoma of the cervix diagnosed histologically, although no tumor was visible upon gynecologic examination. Conclusion: For the management of unsatisfactory Pap tests, The American Society for Colposcopy and Cervical Pathology (ASCCP) recommends repeat cytology within 2–4 months. Evaluation of such patients using hrHPV tests may triage those with squamous intraepithelial lesions, even invasive cervical cancer. More studies with a larger number of cases are needed to analyze the hrHPV status and biopsy follow-up of cases with unsatisfactory cytology.

Effect of liquid-based cytology fixing solution on immunocytochemistry: Efficacy of antigen retrieval in cytologic specimens.

Immunocytochemistry (ICC) is an indispensable technique to improve diagnostic accuracy. ICC using liquid-based cytology (LBC)-fixed specimens has been reported. However, problems may arise if the samples are not fixed appropriately. We investigated the relationship between the LBC fixing solution and ICC and the usefulness of antigen retrieval (AR) in LBC specimens. Specimens were prepared from five types of LBC-fixed samples using cell lines and the SurePath™ method. ICC was performed using 13 antibodies and analyzed by counting the number of positive cells in the immunocytochemically stained specimens. Insufficient reactivity was observed using ICC without heat-induced AR (HIAR) in nuclear antigens. The number of positive cells increased in ICC with HIAR. The percentage of positive cells was lower in CytoRich™ Blue samples for Ki-67 and in CytoRich™ Red and TACAS™ Ruby samples for estrogen receptor and p63 than in the other samples. For cytoplasmic antigens, the percentage of positive cells for no-HIAR treatment specimens was low in the three antibodies used. In cytokeratin 5/6, the number of positive cells increased in all LBC specimens with HIAR, and the percentage of positive cells in CytoRich™ Red and TACAS™ Ruby samples was significantly lower (p < .01). For cell membrane antigens, CytoRich™ Blue samples had a lower percentage of positive cells than the other LBC-fixed samples. The combination of detected antigen, used cells, and fixing solution may have different effects on immunoreactivity. ICC using LBC specimens is a useful technique, but the staining conditions should be examined before performing ICC.

Accurate deep learning model using semi-supervised learning and Noisy Student for cervical cancer screening in low magnification images.

Deep learning technology has been used in the medical field to produce devices for clinical practice. Deep learning methods in cytology offer the potential to enhance cancer screening while also providing quantitative, objective, and highly reproducible testing. However, constructing high-accuracy deep learning models necessitates a significant amount of manually labeled data, which takes time. To address this issue, we used the Noisy Student Training technique to create a binary classification deep learning model for cervical cytology screening, which reduces the quantity of labeled data necessary. We used 140 whole-slide images from liquid-based cytology specimens, 50 of which were low-grade squamous intraepithelial lesions, 50 were high-grade squamous intraepithelial lesions, and 40 were negative samples. We extracted 56,996 images from the slides and then used them to train and test the model. We trained the EfficientNet using 2,600 manually labeled images to generate additional pseudo labels for the unlabeled data and then self-trained it within a student-teacher framework. Based on the presence or absence of abnormal cells, the created model was used to classify the images as normal or abnormal. The Grad-CAM approach was used to visualize the image components that contributed to the classification. The model achieved an area under the curve of 0.908, accuracy of 0.873, and F1-score of 0.833 with our test data. We also explored the optimal confidence threshold score and optimal augmentation approaches for low-magnification images. Our model efficiently classified normal and abnormal images at low magnification with high reliability, making it a promising screening tool for cervical cytology.

Cancer gene analysis of liquid-based cytology specimens using next-generation sequencing: A technical report of bimodal DNA- and RNA-based panel application.

As liquid-based cytology (LBC) specimens harbor high-quality DNA, genomic analysis using LBC specimens is beneficial for integrative diagnosis. This study aimed to clarify the feasibility of LBC specimens for a bimodal application of DNA- and RNA-based next-generation sequencing (NGS) panels. LBC specimens were prepared from cultured human cancer HEC59 cells using commercially available fixatives (Cellprep, CytoRich Red, and SurePath solutions), and were subjected to NGS for a feasibility study. Clinical LBC specimens of thyroid and salivary gland tumors were prepared using CytoRich Red solution. After DNA and RNA extraction, NGS analyses were performed in a single run using combined DNA- and RNA-based custom-made cancer panels for the detection of gene mutations and fusions. High-quality DNA and RNA were obtained, and the expected gene mutations and fusions were detected in HEC59 cells using all types of LBC fixatives. Most available clinical cases (18 out of 20) exhibited pathogenic gene mutations (15 cases) and fusion genes (3 cases) using the bimodal DNA- and RNA-based panels. Overall, 18 cases (90%) showed oncogenic mutations or fusion genes of diagnostic values. Simultaneous application of bimodal DNA- and RNA-based gene panels was useful in NGS analysis using residual LBC specimens for integrative diagnosis. Residual LBC specimens for genomic analysis, including fusion gene analysis, are particularly useful for obtaining genomic information before surgical resection.

Next-Generation Sequencing Analysis of Pancreatic Cancer Using Residual Liquid Cytology Specimens from Endoscopic Ultrasound—Guided Fine-Needle Biopsy: A Prospective Comparative Study with Tissue Specimens

This study evaluated the feasibility and clinical utility of liquid-based cytology (LBC) specimens via endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) for next-generation sequencing (NGS) of pancreatic cancer (PC). We prospectively evaluated the performance of DNA extraction and NGS using EUS-FNB samples obtained from PC. Thirty-three consecutive patients with PC who underwent EUS-FNB at our hospital were enrolled. DNA samples were obtained from 96.8% of the patients. When stratified with a variant allele frequency (VAF) > 10% tumor burden, the NGS success rate was 76.7% (n = 23) in formalin-fixed paraffin-embedded (FFPE), 83.3% (n = 25) in LBC, and 76.7% (n = 23) in frozen samples. The overall NGS success rate was 86.7% (n = 26) using FFPE, LBC, or frozen samples. The detection rates for the main mutated genes were as follows: 86.7% for KRAS, 73.3% for TP53, 66.7% for CDKN2A, 36.7% for SMAD4, and 16.7% for ARID1A. LBC had the highest median value of VAF (23.5%) for KRAS and TP53. PC mutation analysis using NGS was successfully performed using LBC compared with FFPE and frozen samples. This approach provides an alternative and affordable source of molecular testing materials.

A Novel Preservation Method of Improving DNA Stability for Liquid-Based Cytology Specimens Using a Lung Adenocarcinoma Cell Line

A Novel Preservation Method of Improving DNA Stability for Liquid-Based Cytology Specimens Using a Lung Adenocarcinoma Cell Line

Cancer-associated gene analysis of cervical cytology samples and liquid-based cytology significantly improve endometrial cancer diagnosis sensitivity.

To the best of our knowledge, there are no useful screening methods for early detection of endometrial cancer in asymptomatic individuals. The present study evaluated the usefulness of genetic analysis of liquid-based cytology (LBC) specimens by assessing whether pathological genetic mutations detected in cancer tissue sections were detected in LBC specimens from the cervix and uterus. The primary endpoint was genetic analysis of cervical cytology specimens and LBC for the detection of endometrial cancer. Endometrial thickening (>11 mm) assessed using transvaginal ultrasonography was present in 60% of cases and adenocarcinoma assessed using cervical cytology was present in 50% of cases. In 70% of cases, pathogenic mutations detected in cancer tissue sections were also detected in cervical and/or endometrial LBC specimens. The pathogenic variants identified were PTEN in four cases, tumor protein P53, PI3K catalytic subunit α and fibroblast growth factor receptor 2 in two cases each and APC regulator of WNT signaling pathway, KRAS and catenin β1 in one case each. In the present study, a combination of endometrial thickening assessed by transvaginal ultrasonography, cervical cytology and genetic analysis resulted in a high sensitivity of 90% for detection of endometrial cancer. The combination of these tests is more expensive than conventional methods, but delayed detection of uterine cancer requires multidisciplinary treatment, which increases healthcare costs. Increased spending on early detection of uterine cancer is better economically and may improve patient quality of life.

Relationship between Liquid-Based Cytology Preservative Solutions and Artificial Intelligence: Liquid-Based Cytology Specimen Cell Detection Using YOLOv5 Deep Convolutional Neural Network

Introduction: Deep learning is a subset of machine learning that has contributed to significant changes in feature extraction and image classification and is being actively researched and developed in the field of cytopathology. Liquid-based cytology (LBC) enables standardized cytological preparation and is also applied to artificial intelligence (AI) research, but cytological features differ depending on the LBC preservative solution types. In this study, the relationship between cell detection by AI and the type of preservative solution used was examined. Methods: The specimens were prepared from five preservative solutions of LBC and stained using the Papanicolaou method. The YOLOv5 deep convolutional neural network algorithm was used to create a deep learning model for each specimen, and a BRCPT model from five specimens was also created. Each model was compared to the specimen types used for detection. Results: Among the six models, a difference in the detection rate of approximately 25% was observed depending on the detected specimen, and within specimens, a difference in the detection rate of approximately 20% was observed depending on the model. The BRCPT model had little variation in the detection rate depending on the type of the detected specimen. Conclusions: The same cells were treated with different preservative solutions, the cytologic features were different, and AI clarified the difference in cytologic features depending on the type of solution. The type of preservative solution used for training and detection had an extreme influence on cell detection using AI. Although the accuracy of the deep learning model is important, it is necessary to understand that cell morphology differs depending on the type of preservative solution, which is a factor affecting the detection rate of AI.

Sequential evaluation of MUC promoter methylation using next-generation sequencing-based custom-made panels in liquid-based cytology specimens of pancreatic cancer.

As liquid-based cytology (LBC) specimens preserve high-quality DNA, clinical sequencing of LBC specimens using next-generation sequencing (NGS) is becoming a common strategy. This study aimed to evaluate the feasibility of NGS-based custom-made panels for evaluating MUC promoter methylation in LBC specimens. Thirty-one patients with pancreatic cancer were enrolled in the study. Cancer tissue samples were obtained using endoscopic ultrasound-guided fine-needle aspiration/biopsy (EUS-FNA/FNB). LBC, formalin-fixed paraffin-embedded (FFPE), and fresh frozen specimens were prepared for DNA extraction after pathological diagnosis. These specimens were then subjected to NGS analysis using custom-made cancer gene screening and methylation panels comprising 28 cancer-related genes and 13 gene promoter regions, including MUC1, MUC2, and MUC4. The success rate of NGS using the cancer gene panel was comparable among the LBC, FFPE, and frozen specimens, and the presence of cancer cell-derived somatic mutations in each specimen was confirmed. The specimens were then tested using a methylation panel that revealed the sequential methylation status of CpG islands located in the promoter regions of MUC genes. The methylation status results obtained from LBC specimens were almost comparable with those from FFPE and frozen specimens. MUC and other gene methylation analyses using an NGS-based panel were successfully performed in residual LBC specimens obtained by EUS-FNA/FNB. Therefore, this approach provides an alternative source of molecular tests for gene mutations and methylation, especially in the pancreatic cancers, which are often unresectable and unsuitable for obtaining FFPE specimens.