Cytology Specimens Research Articles

Practical applications of three-dimensional printing for process improvement in the cytopathology laboratory.

With the increased availability of three-dimensional (3D) printers, innovative teaching and training materials have been created in medical fields. For pathology, the use of 3D printing has been largely limited to anatomic representations of disease processes or the development of supplies during the coronavirus disease 2019 pandemic. Herein, an institution's 3D printing laboratory and staff with expertise in additive manufacturing illustrate how this can address design issues in cytopathology specimen collection and processing. The authors' institutional 3D printing laboratory, along with students and trainees, used computer-aided design and 3D printers to iterate on design, create prototypes, and generate final usable materials using additive manufacturing. The program Microsoft Forms was used to solicit qualitative and quantitative feedback. The 3D-printed models were created to assist with cytopreparation, rapid on-site evaluation, and storage of materials in the preanalytical phase of processing. These parts provided better organization of materials for cytology specimen collection and staining, in addition to optimizing storage of specimens with multiple sized containers to optimize patient safety. The apparatus also allowed liquids to be stabilized in transport and removed faster at the time of rapid on-site evaluation. Rectangular boxes were also created to optimally organize all components of a specimen in cytopreparation to simplify and expedite the processes of accessioning and processing, which can minimize errors. These practical applications of 3D printing in the cytopathology laboratory demonstrate the utility of the design and printing process on improving aspects of the workflow in cytopathology laboratories to maximize efficiency, organization, and patient safety.

Comparison of immunohistochemical mesothelial biomarkers in paired biopsies and effusion cytology cell blocks from pleural mesothelioma.

Traditionally, the diagnosis of pleural mesothelioma is based on histological material. Minimally invasive effusion cytology specimens are an alternative that, like biopsies, require ancillary analyses. Validation of immunohistochemical (IHC) analyses on cytology, including the surrogate markers for molecular alterations BAP1 and MTAP, is of interest. IHC for eight different markers was performed on 59 paired formalin-fixed, paraffin-embedded pleural biopsies and pleural effusion cell blocks with mesothelioma. Immunoreactivity in ≥10% of tumour cells was considered positive/preserved. The concordance between histological and cytological materials was assessed. The overall percentage of agreement between the histological epithelioid component in 58 biopsies and paired cell blocks was 93% for calretinin, 98% for CK5, 97% for podoplanin, 90% for WT1, 86% for EMA, 100% for desmin, 91% for BAP1, and 72% for MTAP. For 11 cases with biphasic or sarcomatoid histology, the concordance between cytology and the histological sarcomatoid component was low for calretinin, CK5, and WT1 (all ≤45%). For the whole cohort, loss of both BAP1 and MTAP was seen in 40% while both markers were preserved in 11% of the biopsies for epithelioid histology. The corresponding numbers were 54% and 8%, respectively, for the paired cell blocks. Generally, a high concordance for IHC staining was seen between paired biopsies and pleural effusion cell blocks from mesotheliomas, but the somewhat lower agreement for WT1, EMA, and especially MTAP calls for further investigation and local quality assurance. The lower concordance for the sarcomatoid subtype for some markers may indicate biological differences.

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.

Malignant peritoneal cytologic contamination with robotic hysterectomy for endometrial cancer

BackgroundMalignant peritoneal cytology in endometrial cancer (EC) is not considered an independent adverse prognostic factor for uterine-confined disease and is not a determinant factor in the International Federation of Gynecology and Obstetrics (FIGO) staging system. NCCN Guidelines still recommend obtaining cytologies. The aim of this study was to determine the prevalence of peritoneal cytologic contamination following robotic hysterectomy for EC. MethodsPeritoneal cytology from the pelvis and diaphragm were obtained at the initiation of surgery, and from the pelvis only at the completion of robotic hysterectomy with sentinel lymph node mapping (SLNM). Cytology specimens were evaluated for the presence of malignant cells. Pre- and post-hysterectomy cytology results were compared, and pelvic contamination was defined as conversion from negative to positive cytology following surgery. Results244 patients underwent robotic hysterectomy with SLNM for EC. Pelvic contamination was identified in 32 (13.1%) cases. In multivariate analysis, pelvic contamination was associated with >50% myometrial invasion, tumor size >2 cm, lymphovascular space invasion (LVSI), and lymph node metastasis. There was no association with FIGO stage or histology subtypes. ConclusionsMalignant peritoneal contamination occurred during robotic surgery for EC. Large lesions (>2 cm), deep invasion (>50%), LVSI, and lymph node metastasis were each independently associated with peritoneal contamination. Whether or not peritoneal contamination increases risk for disease recurrence should be studied in larger series, including an evaluation of patterns of recurrence and the potential impact of adjuvant therapies. Until the clinical impact of peritoneal contamination during hysterectomy for EC is better understood, methods to reduce peritoneal contamination are warranted.

Remotely operated robotic microscopy for rapid diagnosis of bronchoscopic cytology specimens.

Obtaining a diagnosis and treating pulmonary malignancies during the same anesthesia requires either an on-site pathologist or a system for remotely evaluating microscopic images. Cytology specimens are challenging to remotely assess given the need to navigate through dispersed and three-dimensional cell clusters. Remote navigation is possible using robotic telepathology, but data are limited on the ease of use of current systems, particularly for pulmonary cytology. Air dried modified Wright-Giemsa stained slides from 26 touch preparations of transbronchial biopsies and 27 smears of endobronchial ultrasound guided fine needle aspirations were scored for ease of adequacy assessment and ease of diagnosis on robotic (rmtConnect Microscope) and non-robotic telecytology platforms. Diagnostic classifications were compared between glass slides and the robotic and non-robotic telecytology assessments. Compared to non-robotic telecytology, robotic telecytology had a greater ease of adequacy assessment and non-inferior ease of diagnosis. The median time to diagnosis using robotic telecytology was 85 s (range 28-190 s). Diagnostic categories were concordant for 76% of cases in robotic versus non-robotic telecytology and 78% of cases in robotic telecytology versus glass slide diagnosis. Weighted Cohen's kappa scores for agreement in these comparisons were 0.84 and 0.72, respectively. Use of a remote-controlled robotic microscope improved the ease of adequacy assessment compared to non-robotic telecytology and enabled strongly concordant diagnoses to be expediently rendered. This study provides evidence that modern robotic telecytology is a feasible and user-friendly method of remotely and potentially intraoperatively rendering adequacy assessments and diagnoses on bronchoscopic cytology specimens.

Urgent intraoperative cytological diagnosis of spherical mass lesions in the lungs

Differential diagnosis of pulmonary mass lesions is one of the most difficult tasks not only in pulmonology but also in phthisiology, because clinical manifestations, data from visual and laboratory examinations are undifferentiated in the early stages. In the case of an unclear diagnosis, morphological verification of the process played a crucial role. Urgent intraoperative examination is the most difficult option for morphologic verification of lung lesions, which requires a quick, detailed, and accurate answer.The aim of this study was to evaluate the clinical significance and efficacy of urgent intraoperative cytologic examination in the differential diagnosis between granulomatous (tuberculosis, sarcoidosis), tumor and tumor-like lesions of the lung.Methods. The results of urgent intraoperative cytologic examination of 173 tissue specimens from lung masses were retrospectively analyzed. Cytologic specimens were stained by the May – Grunwald – Giemse and Ziehl – Neelsen methods and then visualized on a Zeiss Lab A1 microscope (Carl Zeiss, Germany). The obtained data are compared with the results of histological examination. To evaluate the effectiveness of intraoperative cytologic examination, the accuracy, sensitivity, and specificity were calculated.Results. Analysis of the results of urgent cytological examinations (n = 173) shows that granulomatosis (tuberculosis/sarcoidosis) was established in 64.7% of cases, malignancy – in 31 cases (cancer – 9.2%, carcinoid tumor – 2.9%, malignant neoplasm of unclear etiology – 4%, suspected malignant lesion – 1.7%); benign process – in 9.2% of cases (hamartoma + chondroma – 5.2%, chronic inflammation – 4%). 1.2% of the cases had no diagnostic material. When comparing the obtained results, there was a discrepancy between the histological picture (2.9% of cases) and the primary clinical diagnosis (5.7% of cases). Sensitivity, specificity, and accuracy of cytologic examination of intraoperative material amounted to 95.9, 71, and 98%, respectively.Conclusion. The high sensitivity and accuracy (&gt; 90%) of urgent cytologic diagnosis of pathologic processes in the lung under intraoperative conditions were demonstrated by the correct assessment of morphologic signs of a given lesion and by their comparison with the clinical picture and the results of additional examinations. Analysis of cytological specimens showed that urgent cytological examination significantly complements the morphological picture of lung lesions and can be considered as the first stage of phenotypic diagnosis.

Use of nongynecologic cytologic-histologic correlation to identify patterns of error: An institutional experience.

Quality management practices empower cytology laboratories to deliver consistent, high-quality patient care. Monitoring of key performance indicators is one way by which laboratories can identify patterns of error and focus their improvement activities. Cytologic-histologic correlation (CHC) identifies error by retrospectively reviewing cytology cases when discordant surgical pathology diagnoses are reported. Analysis of CHC data can elucidate patterns of error and direct quality improvement initiatives. CHC data of nongynecologic cytology specimens were reviewed over a 3-year period (2018-2021). Errors were separated by anatomic site and classified as either sampling or interpretive errors. A total of 364 discordant cases were identified out of 4422 cytologic-histologic pairs (a discordant rate of 8%). The majority (272; 75%) were sampling errors, with fewer interpretive errors (92; 25%). Sampling errors were found to occur most commonly in lower urinary tract and lung. Interpretive errors were most commonly found in lower urinary tract and thyroid. Nongynecologic CHC data can be a valuable resource for cytology laboratories. By studying the types of errors, quality improvement activities can be targeted toward problem areas.

Diagnosis of thoracic SMARCA4-deficient undifferentiated tumor in cytology.

Although alterations in SMARCA4-deficient occur in non-small cell lung carcinoma (SD-NSCLC), thoracic SMARCA4-deficient undifferentiated tumor (TSDUT) is recognized as a distinct entity in the 2021 World Health Organization Classification of Thoracic Tumors because of unique morphologic, immunophenotypic and molecular features, and worse survival compared with SD-NSCLC. Cytologic diagnosis of TSDUT is clinically important because of its aggressive behavior and because it is often diagnosed by fine-needle aspiration because TSDUTs are usually unresectable at presentation. Here, we identify cytologic features that can be used for recognition of TSDUT and distinction from SD-NSCLC. Cytomorphologic features were investigated in cytology specimens from patients with TSDUT (n=11) and compared with a control group of patients with SD-NSCLC (n=20). The presence of classic rhabdoid morphology, at least focally, was entirely specific for TSDUT (n=6, 55%) compared with SD-NSCLC (n=0) in this study. TSDUT more frequently showed tumor necrosis (n=11, 100% vs. n=8, 40%; p=.001), dominant single-cell pattern on aspirate smears or touch preparation slides (n=8 [of 9], 80% vs. n=3, 15%; p=.010), nuclear molding (n=5, 45% vs. n=1, 5%; p=.013), and indistinct cell borders (n=11, 100% vs. n=5, 25%; P<.001) compared with SD-NSCLC, respectively. Cytomorphologic features occurring more frequently in TSDUT include tumor necrosis, dominant single-cell pattern, nuclear molding indistinct cell borders, and focal rhabdoid cells. Presence of these features in a cytology specimen of an undifferentiated tumor, particularly in a patient with a thoracic mass, should raise suspicion for TSDUT and prompt appropriate ancillary workup.

Washing solution of tissue biopsy compared with paraffin-embedded tissue for NSCLC gene test.

e21104 Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer related death, whose targeted therapy is precisely based on gene type. Gene diagnosis is as important as pathological diagnosis and driver gene mutations should be detected as soon as possible. There are generally a few sources to obtain patients’ deoxyribonucleic acid (DNA), such as paraffin tissue of tumor, cytological specimens from bronchoalveolar fluid or pleural effusion, free DNA from blood, hydrothorax, ascite, or cerebrospinal fluid. Our research aims to find out whether washing solution of lung mass biopsy in double distilled water is feasible for detecting gene mutation, and whether its gene type is consistent with that from Next Generation Sequencing (NGS) of paraffin tissue. Methods: 25 clinically diagnosed NSCLC patients from Department of Oncology, Second Xiangya Hospital, Central South University were collected. We used double distilled water to wash the CT-guided percutaneous pulmonary mass biopsy, which was processed to extract DNA. DNA samples were tested for epidermal growth factor receptor (EGFR) gene mutation by amplification refractory mutation system (ARMS)-PCR. 6 of them as well as all 25 paraffin tissue samples were tested for large panel gene mutations by NGS. The gene type consistency between washing solution and paraffin tissue was analyzed by GraphPad Prism V9.4.1. Results: 16 of 25 washing solution DNA samples tested by ARMS-PCR were EGFR mutation positive, including 9 cases of EGFR exon19 deletion, 5 cases of EGFR exon21 L858R, and 2 cases of EGFR exon21 L858R and T790M. Other 9 of 25 samples were EGFR mutation negative. 17 of 25 paraffin tissue samples tested by NGS were EGFR mutation positive, including 9 cases of EGFR exon19 deletion, 7 cases of EGFR exon21 L858R, and 1 case of EGFR exon21 L858R with T790M. There was one washing solution sample showed no EGFR mutation while its tissue sample showed EGFR exon21 L858R. Compared to NGS on paraffin tissue, the sensibility of ARMS-PCR on washing solution was 94.1%. The specificity was 100%, positive predictive value was 100%, negative predictive value was 88.9%, accuracy rate was 96.0%, misdiagnosis rate was 0, and omission diagnostic rate was 5.9%. EGFR types of 6 washing solution samples tested by NGS were exactly the same as that by ARMS-PCR and that of paraffin tissue samples by NGS. The time to obtain gene result was significantly different. ARMS-PCR of washing solution took 1 day, NGS of washing solution took around 7 days, while pathological examination and NGS of paraffin-embedded tissue took around 14 days. Conclusions: The washing solution of CT-guided percutaneous lung mass biopsy in double distilled water is feasible to obtain EGFR type rapidly and efficiently for NSCLC. It is the fastest method to get patients’ gene type at present. Thus, the washing solution of biopsy could be a new DNA source for NSCLC gene test and making decision on targeted therapy ahead of time.

KRAS Mutation Analysis Using Cell-free DNA of Pancreatic Cancer.

Detection of pancreatic cancer using small samples of the pancreas obtained by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) remains a challenge. The purpose of this study was to investigate whether the detection of KRAS mutations in cell-free DNA (cfDNA) extracted from supernatants of liquid-based fixed cytology (LBC) specimens obtained using EUS-FNA in solid pancreatic cancer can be an auxiliary test for differential diagnosis. The purpose of this study was to investigate whether the detection of KRAS mutations in cell-free DNA (cfDNA) extracted from supernatants of liquid-based fixed cytology (LBC) specimens obtained using EUS-FNA in solid pancreatic cancer can be an auxiliary test for differential diagnosis. This was a single-institution cohort study that included 50 patients with pancreatic lesions. cfDNA was isolated from the supernatant of fixed LBC samples, and KRAS mutation status was compared between cfDNA samples and FFPE small fragment tissues. Of the 50 cfDNA samples, 84% (42/50) were valid. KRAS mutations were detected in 57.1% (24/42) of the 42 valid samples. The sensitivity, specificity, and accuracy of KRAS mutation detection using cfDNA samples in the pancreatic lesions were 63.2% (24/38), 100.0% (4/4), and 66.7% (28/42), respectively. KRAS mutation status between FFPE small tissues and cfDNA samples were comparable. Gene mutation analysis using cfDNA from the supernatant of fixed LBC samples is an effective ancillary diagnostic tool for pancreatic cancer.

The Contribution of Cell Blocks in the Diagnosis of Mediastinal and Hilar Lymphadenopathy Samples From Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration (EBUS-TBNA).

Background Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a diagnostic procedure that allows clinicians to stage lung cancer by sampling lymph nodes in the mediastinum. EBUS-TBNA is recommended as a first step prior to mediastinoscopy for lung cancer mediastinal staging. This procedure has greatly aided pulmonologists in diagnosing mediastinal pathologies with substantial progress. In this study, our aim is to analyze how cell blocks affect the diagnostic yield of mediastinal and hilar lymphadenopathy using an EBUS cytology needle. Methods This retrospective study was conducted at King Abdulaziz University Hospital between May 2021 and September 2021. Patients with mediastinal and hilar lymphadenopathy in the absence of known or suspected primary lung cancer were included. The EBUS procedure was performed using a flexible bronchoscope equipped with a working channel suitable for transbronchial needle aspirationunder direct ultrasound guidance. Data were recorded using Microsoft Excel and analyzed using Statistical Package for the Social Sciences (SPSS) v. 26.0 (IBM Corp., Armonk, NY). Diagnostic accuracy measures were determined, and a p-value of 0.05 was established as the final threshold for statistical significance. Results The total number of patients in our study was 151. The sensitivity for cytology specimens, histology specimens, and a combined evaluation for the full group of patients was 77.14%, 83.33%, and 87.5%, respectively, with a negative predictive value of 27.22%, 25%, and 21.42%. The diagnostic accuracy for cytology specimens, histology specimens, and a combined evaluation was 71.42%, 76.19%, and 80%, respectively. Conclusion Our study found that the combined examination of specimens for both cytology and histology in the diagnosis of lung cancer, sarcoidosis, and tuberculosis resulted in a higher diagnostic yield compared to cytological assessment alone using EBUS-TBNA.

New Opportunities for Preoperative Diagnosis of Medullary Thyroid Carcinoma.

The preoperative diagnostics of medullary thyroid carcinoma (MTC), including the measuring of the blood calcitonin level, has a number of limitations. Particular focus has recently been placed on the role of miRNAs in the development of various malignant tumors; a comparative analysis of accuracy of the existing methods for MTC diagnosis with a novel diagnosis method, evaluation of the miRNA-375 expression level, was performed in this study. The expression level of miRNA-375 in cytology samples from 555 patients with the known histological diagnosis, including 41 patients with confirmed postoperative diagnosis of MTC, was assessed. The diagnostic parameters of the basal calcitonin level, calcitonin in wash-out fluid from the FNAB needle, and miRNA-375 were compared. An assessment of the miRNA-375 expression level made it possible to detect all the MTC samples with a 100% accuracy among all the 555 cytology specimens, as well as in non-informative FNAB specimens, and specimens from the ipsilateral thyroid lobe. Parameters such as sensitivity, specificity, PPV, and NPV were 100%. The miRNA-375 level, unlike calcitonin, does not correlate with tumor volume, so it does not have the so-called "gray zone". An assessment of the miRNA-375 expression allows one to accurately distinguish MTC from other malignant and benign thyroid tumors.

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.

SOX17 is a highly sensitive and specific marker for metastatic ovarian and endometrial carcinomas in cytology cell block specimens.

SOX17 (SRY-box transcription factor 17) was recently identified as a highly sensitive and specific marker for ovarian and endometrial carcinomas in surgical specimens. In this study, validation of the utility of SOX17 immunohistochemistry (IHC) in diagnosing metastatic gynecologic carcinomas in cytology specimens was sought. The study cohort included 84 cases of metastatic carcinomas that included 29 metastatic gynecologic carcinomas (24 ovarian high-grade serous carcinomas, two endometrial serous carcinomas, one low-grade serous carcinoma, one ovarian clear cell carcinoma, and one endometrial endometrioid carcinoma) and 55 cases of metastatic nongynecologic carcinomas (10 clear cell renal cell carcinomas, 10 papillary thyroid carcinomas, 11 gastrointestinal adenocarcinomas, 10 breast carcinomas, 10 lung adenocarcinomas, and four urothelial carcinomas). Cytology specimen types included peritoneal fluid (n=44), pleural fluid (n=25), and fine-needle aspiration (n=15). SOX17 IHC was performed on the cell block sections. The intensity of staining and percent positivity of the tumor cells were evaluated. SOX17 was highly expressed in all tested metastatic gynecologic carcinomas with diffuse and strong nuclear expression (29 of 29; 100%). SOX17 was negative in other metastatic nongynecologic carcinomas (54 of 55; 98.18%) except for one papillary thyroid carcinoma that showed low positivity (<10%). SOX17 is a highly sensitive (100%) and specific (98.2%) marker for the differential diagnosis of metastatic gynecologic carcinomas in cytology specimens. Therefore, SOX17 IHC should be included in the workup of differential diagnosis of metastatic gynecologic carcinomas in cytology specimens.

Nucleic Acid Quality Assessment is Critical to the Success of the Oncomine Dx Target Test for Lung Cancer.

The Oncomine Dx Target Test (ODxTT) has been used as a companion diagnostic test for lung cancer. Here, we evaluated whether the amount of nucleic acid and the degree of RNA degradation are related to the success of the ODxTT. This study included 223 samples from 218 patients with lung cancer. For all samples, DNA and RNA concentrations were quantified using Qubit, and the degree of RNA degradation was evaluated using the Bioanalyzer. Of the 223 samples, 219 samples were successfully analyzed in the ODxTT and four were not. DNA analysis failed in two samples, which were attributed to low DNA concentrations and both were cytology specimens. Meanwhile, RNA analysis failed in the other two samples. These samples had sufficient amounts of RNA, but it was highly degraded with DV200 (the percentage of RNA fragments >200 base pairs) less than 30. Compared with RNA samples with DV200 ≥30, analysis of RNA with DV200 <30 yielded significantly fewer reads for the internal control genes. This test showed actionable mutations were identified in 38% (83/218) of all patients and in 46.6% (76/163) of patients with lung adenocarcinoma. DNA concentration and degree of RNA degradation are key factors determining the success of diagnostic testing by the ODxTT.

The utilisation of digital droplet PCR to enhance the diagnosis of bladder and pancreaticobiliary tumours in cytology specimens.

Digital droplet PCR (ddPCR) is a relatively new technique used to detect molecular alterations with unprecedented precision and accuracy. It is particularly useful for detecting point mutations and copy number variation (CNV) in samples with small amounts of target DNA. This proof of principle study was conducted to see if ddPCR technology could be applied to cytology specimens to detect molecular alterations which may influence diagnostic decision making. A range of cytological specimens derived from bladder or pancreaticobiliary origin, with varying diagnoses but with an emphasis on abnormality, underwent ddPCR testing. DNA was manually extracted from Thinprep vials, cell blocks or direct fine needle aspiration smears. ddPCR was performed using two somatic point mutations TP53 R248W and TP53 R273H assays for both urine and pancreaticobiliary cytology specimens. Three CNV assays; CDKN2A, E2F3 and YWHAZ were applied to urine samples. SMAD4 and CDKN2A CNVs were applied to the pancreaticobiliary samples. 16 of 21 urine specimens showed molecular alterations using ddPCR testing. 12 of those 16 cases were associated with malignant outcomes. The pancreaticobiliary specimens showed 14 of 37 specimens with molecular alterations, all of which were associated with carcinoma. We demonstrated an increasing percentage of molecular aberrations associated with increasing severity of cytological results. Our results have shown that ddPCR can identify both mutations and CNVs in urine and pancreaticobiliary cytology derived samples. Being able to detect these molecular alterations may reduce the number of equivocal results leading to more timely and informed patient management decisions.

Cytomorphologic observations on the sclerosing variant of well-differentiated pancreatic neuroendocrine tumors: Towards making specific diagnoses early.

The recently described sclerosing variant of pancreatic neuroendocrine tumor (spNET) shows prominent stromal fibrosis and decreased tumor cellularity in surgical pathology specimens. Although prognostic data are currently ambivalent, some studies have reported metastatic disease in small primary tumors, highlighting the need for early diagnosis. The aim of our study is to evaluate cytology specimens of spNET to determine its characteristic cytomorphologic features to expedite an early diagnosis. Twenty-five cytology cases of spNET from 23 patients and 29 cytology cases of typical pancreatic neuroendocrine tumor (tpNET) from 29 patients diagnosed as such by surgical pathology evaluation were reviewed by two pathologists to assess adequacy of diagnostic material, cellularity and fibrosis. Radiographic findings and outcome data were collected. With only 13 of 25 specimens deemed as diagnostic, spNET specimens were more often non-diagnostic (p < .01) and less often hypercellular (p = .03) compared to tpNET counterparts. While at least focal fibrosis was observed in both groups, a subset of spNET cases showed small tumor cell groups entrapped in large fibrotic fragments. Importantly, spNETs tended to be metastatic at diagnosis (both regionally and distant), with a smaller average tumor size. The hypocellular nature of spNET cytology samples makes this variant difficult to diagnose. However, when adequate sample is available, a subset of spNET show characteristic cytomorphology that enables us to consider this specific diagnosis at first diagnostic sampling. It is crucial to diagnose this variant early given the propensity of small tumors with regional lymph node involvement and liver metastases.

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.

One procedure—one report: the Re-Imagine Cytopathology Task Force position paper on small tissue biopsy triage in anatomic pathology

Endoscopic biopsy procedures increasingly generate multiple tissue samples from multiple sites, and frequently retrieve concurrent cytologic specimens and small core needle biopsies. There is currently lack of consensus in subspecialized practices as to whether cytopathologists or surgical pathologists should review such samples, and whether the pathology findings should be reported together or separately. In December 2021, the American Society of Cytopathology convened the Re-Imagine Cytopathology Task Force to examine various workflows that would facilitate unified pathology reporting of concurrently obtained biopsies and improve clinical care. This position paper summarizes the key points and highlights the advantages, challenges, and resources available to support the implementation of such workflows that result in "one procedure-one report".

Detection of multiple druggable mutations of lung cancer from cytology specimens by MINtS: An advanced medicine A trial.

Most multigene mutation tests require tissue specimens. However, cytological specimens are easily obtained in the clinical practice and provide high-quality DNA and RNA. We aimed to establish a test that utilizes cytological specimens and performed a multi-institutional study to investigate the performance of MINtS, a test based on next-generation sequencing. A standard procedure for specimen isolation was defined. The specimens were considered suitable for the test if >100 ng DNA and >50 ng RNA could be extracted from them. In total, 500 specimens from 19 institutions were investigated. MINtS detected druggable mutations in 63% (136 of 222) of adenocarcinomas. Discordant results between MINtS and the companion diagnostics were observed in 14 of 310 specimens for the EGFR gene, and 6 of 339 specimens for the ALK fusion genes. Confirmation by other companion diagnostics for the EGFR mutations or the clinical response to an ALK inhibitor all supported the results obtained by MINtS. MINtS along with the isolation procedure presented in the current study will be a platform to establish multigene mutation tests that utilize cytological specimens. UMIN000040415.