Circulating Tumor Cells: Overview and Opportunities in Cytology.

Circulating tumor cells (CTCs) have long been assumed to be the substrate of cancer metastasis. However, only in recent years have we begun to leverage the potential of CTCs found in minimally invasive peripheral blood specimens to improve care for cancer patients. Currently, CTC enumeration is an accepted prognostic indicator for breast, prostate, and colorectal cancer; however, CTC enumeration remains largely a research tool. More recently, the focus has shifted to CTC characterization and isolation which holds great promise for predictive testing. This review summarizes the relevant clinical, biological, and technical background necessary for pathologists and cytopathologists to appreciate the potential of CTC techniques. A summary of relevant systematic reviews of CTCs for specific cancers is then presented, as well as potential applications to precision medicine. Finally, we suggest future applications of CTC technologies that can be easily incorporated in the pathology laboratory, with the recommendation that pathologists and particularly cytopathologists apply these technologies to small specimens in the era of "doing more with less."

Circulating and Imaging Biomarkers of Radium-223 Response in Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: Radium-223 improves overall survival (OS) and reduces skeletal events in patients with bone metastatic castration-resistant prostate cancer (CRPC), but relevant biomarkers are lacking. We evaluated automated bone scan index (aBSI) and circulating tumor cell (CTC) analyses as potential biomarkers of prognosis and activity. PATIENTS AND METHODS: Patients with bone metastatic CRPC were enrolled on a prospective single-arm study of standard radium-223. 99mTc-MDP bone scan images at baseline, 2 months, and 6 months were quantitated using aBSI. CTCs at baseline, 1 month, and 2 months were enumerated and assessed for RNA expression of prostate cancer-specific genes using microfluidic enrichment followed by droplet digital polymerase chain reaction. RESULTS: The median OS was 21.3 months in 22 patients. Lower baseline aBSI and minimal change in aBSI (<+0.7) from baseline to 2 months were each associated with better OS (P = .00341 and P = .0139, respectively). The higher baseline CTC count of ≥5 CTC/7.5 mL was associated with worse OS (median, 10.1 v 32.9 months; P = .00568). CTCs declined at 2 months in four of 15 patients with detectable baseline CTCs. Among individual genes in CTCs, baseline expression of the splice variant AR-V7 was significantly associated with worse OS (hazard ratio, 5.20 [95% CI, 1.657 to 16.31]; P = .00195). Baseline detectable AR-V7, higher aBSI, and CTC count ≥5 CTC/7.5 mL continued to have a significant independent negative impact on OS after controlling for prostate-specific antigen or alkaline phosphatase. CONCLUSION: Quantitative bone scan assessment with aBSI and CTC analyses are prognostic markers in patients treated with radium-223. AR-V7 expression in CTCs is a particularly promising prognostic biomarker and warrants validation in larger cohorts.

Comprehensive epithelial biomarker analysis of malignant mesothelioma: EpCAM positivity is a potential diagnostic pitfall.

BACKGROUND: Epithelial cell adhesion molecule (EpCAM) is frequently used to distinguish carcinoma from background mesothelial cells during cytologic examination of body cavity fluids. Previously, the authors identified one malignant mesothelioma case with strong and diffuse membranous EpCAM staining, making it indistinguishable from carcinoma. METHODS: In this study, the authors evaluated all available effusion specimens from patients with malignant mesothelioma, including the above-mentioned index case, obtained at Stanford Health Care, from 2011 to 2021 (N = 17) as well as control cases (N = 5). Analyses included an immunohistochemistry (IHC) assay for EpCAM and claudin-4, a multiplexed immunofluorescent (IF) assay for EpCAM, and an RNA in situ hybridization assay targeting EpCAM. RESULTS: The authors detected EpCAM positivity of variable intensity and percentage in four malignant mesothelioma cases (23.5%; although only two showed positivity for the epithelial-specific IHC marker MOC31 in ≥40% of cells) and claudin-4 negativity in all cases, with two cases displaying focal and weak claudin-4 staining in <1% of cells. Multiplexed IF staining on the cases with EpCAM IHC positivity showed strong, membranous EpCAM staining in one of four cases. RNA in situ hybridization also was used to assess the correlation between EpCAM positivity by IHC/IF and RNA expression levels. Strong EpCAM RNA expression was detected in the three malignant mesothelioma cases. CONCLUSIONS: The current findings revealed that a subset of epithelioid malignant mesothelioma cases mimic or exhibit the immunophenotypic features of carcinoma when evaluating for EpCAM only. Additional biomarker testing, such as claudin-4, may help avoid this potential pitfall to yield accurate diagnoses.

Immunofluorescent and molecular characterization of effusion tumor cells reveal cancer site-of-origin and disease-driving mutations.

BACKGROUND: Effective cancer treatment relies on precision diagnostics. In cytology, an accurate diagnosis facilitates the determination of proper therapeutics for patients with cancer. Previously, the authors developed a multiplexed immunofluorescent panel to detect epithelial malignancies from pleural effusion specimens. Their assay reliably distinguished effusion tumor cells (ETCs) from nonmalignant cells; however, it lacked the capacity to reveal specific cancer origin information. Furthermore, DNA profiling of ETCs revealed some, but not all, cancer-driver mutations. METHODS: The authors developed a new multiplex immunofluorescent panel that detected both malignancy and pulmonary origin by incorporating the thyroid transcription factor-1 (TTF-1) biomarker. Evaluation for TTF-1-positive ETCs (T-ETCs) was performed on 12 patient samples. T-ETCs and parallel ETCs from selected patients were collected and subjected to DNA profiling to identify pathogenic mutations. All samples were obtained with Institutional Review Board approval. RESULTS: Malignancy was detected in all samples. T-ETCs were identified in 9 of 10 patients who had clinically reported TTF-1 positivity (90% sensitivity and 100% specificity). Furthermore, DNA profiling of as few as five T-ETCs identified pathogenic mutations with equal or greater sensitivity compared with profiling of ETCs, both of which showed high concordance with clinical findings. CONCLUSIONS: The findings suggest that the immunofluorescent and molecular characterization of tumor cells from pleural effusion specimens can provide reliable diagnostic information, even with very few cells. The integration of site-specific biomarkers like TTF-1 into ETC analysis may facilitate better refined diagnosis and improve patient care.

Identification and characterization of effusion tumor cells (ETCs) from remnant pleural effusion specimens.

BACKGROUND: Cancer is a leading cause of death worldwide, and patients may have advanced disease when diagnosed. Targeted therapies guided by molecular subtyping of cancer can benefit patients significantly. Pleural effusions are frequently observed in patients with metastatic cancer and are routinely removed for therapeutic purposes; however, effusion specimens have not been recognized as typical substrates for clinical molecular testing because of frequent low tumor cellularity. METHODS: Excess remnant pleural effusion samples (N = 25) from 21 patients with and without suspected malignancy were collected at Stanford Health Care between December 2019 and November 2020. Samples were processed into ThinPrep slides and underwent novel effusion tumor cell (ETC) analysis. The ETC results were compared with the original clinical diagnoses for accuracy. A subset of confirmed ETCs was further isolated and processed for molecular profiling to identify cancer driver mutations. All samples were obtained with Institutional Review Board approval. RESULTS: The authors established novel quantitative standards to identify ETCs and detected epithelial malignancy with 89.5% sensitivity and 100% specificity in the pleural effusion samples. Molecular profiling of confirmed ETCs (pools of 5 cells evaluated) revealed key pathogenic mutations consistent with clinical molecular findings. CONCLUSIONS: In this study, the authors developed a novel ETC-testing assay that detected epithelial malignancies in pleural effusions with high sensitivity and specificity. Molecular profiling of 5 ETCs showed promising concordance with the clinical molecular findings. To promote cancer subtyping and guide treatment, this ETC-testing assay will need to be validated in larger patient cohorts to facilitate integration into cytologic workflow.

Integration of rare cell capture technology into cytologic evaluation of cerebrospinal fluid specimens from patients with solid tumors and suspected leptomeningeal metastasis.

INTRODUCTION: Dissemination of tumor to the leptomeninges, subarachnoid space, and cerebrospinal fluid (CSF) is termed leptomeningeal metastasis (LM) and occurs in approximately 5% of patients with solid tumors. LM is associated with dismal clinical prognosis, and routine cytologic and radiologic methods for diagnosing LM have limited sensitivity. The CellSearch immunomagnetic rare cell capture assay is FDA-approved to detect circulating tumor cells (CTCs) in peripheral blood, but whether it may have a role in identifying CSF CTCs is still unclear. MATERIAL AND METHODS: CSF specimens from 20 patients with clinically suspected solid tumor LM collected from 2 institutions between October 2016 and January 2019 were evaluated with routine CSF cytology and underwent concurrent CTC testing with the CellSearch assay (Menarini-Silicon Biosystems, Huntingdon Valley, PA). The results of CTC testing were compared to routine CSF cytology and radiologic studies for detecting LM. RESULTS: The CellSearch assay achieved a sensitivity of 88.9% and specificity of 100% for detecting LM (using a threshold of 1 CTC/mL of CSF as the definition of a positive CTC result). One patient with negative CSF cytology but positive CTCs developed positive cytology 37 days later. CONCLUSIONS: In this proof-of-principle pilot study, we demonstrate that the CellSearch assay can be successfully integrated with the routine CSF cytologic workflow to aid in the diagnosis of solid tumor LM. Importantly, CTCs detected by this rare cell capture assay are found in a subset of patients with non-positive routine CSF cytology, which may have significant implications for patient management.

Circulating tumor cell analysis in locally advanced and metastatic squamous cell carcinoma of the head and neck.

BACKGROUND: Circulating tumors cells (CTCs) are considered an early step towards metastasis and have been linked to poor prognosis in several types of cancer. CTCs in squamous cell carcinoma of the head and neck (SCCHN) have an unclear role. METHODS: In this prospective study, patients with locally advanced or metastatic SCCHN had CTC counts assessed before starting systemic treatment using the CellSearch System. Select cases also had sequential CTC evaluation. Presence of CTCs was correlated with patient characteristics and outcomes. RESULTS: Forty-eight patients enrolled, and 36 had evaluable clinical data and baseline CTC counts. Twenty-five patients had locally advanced disease (LAD) and 11 had metastatic disease. ≥1 CTCs were detected in six patients with LAD (24%) and four with metastatic disease (36%). On univariate analysis, smoking was associated with CTCs. CONCLUSION: CTCs are not associated with prognosis in patients with LAD and metastatic disease; however, they are present in this patient population, and ≥1 CTCs is associated with a history of smoking. LEVEL OF EVIDENCE: 1b; individual prospective cohort study.

A lab-on-a-disc platform enables serial monitoring of individual CTCs associated with tumor progression during EGFR-targeted therapy for patients with NSCLC.

Rationale: Unlike traditional biopsy, liquid biopsy, which is a largely non-invasive diagnostic and monitoring tool, can be performed more frequently to better track tumors and mutations over time and to validate the efficiency of a cancer treatment. Circulating tumor cells (CTCs) are considered promising liquid biopsy biomarkers; however, their use in clinical settings is limited by high costs and a low throughput of standard platforms for CTC enumeration and analysis. In this study, we used a label-free, high-throughput method for CTC isolation directly from whole blood of patients using a standalone, clinical setting-friendly platform. Methods: A CTC-based liquid biopsy approach was used to examine the efficacy of therapy and emergent drug resistance via longitudinal monitoring of CTC counts, DNA mutations, and single-cell-level gene expression in a prospective cohort of 40 patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer. Results: The change ratio of the CTC counts was associated with tumor response, detected by CT scan, while the baseline CTC counts did not show association with progression-free survival or overall survival. We achieved a 100% concordance rate for the detection of EGFR mutation, including emergence of T790M, between tumor tissue and CTCs. More importantly, our data revealed the importance of the analysis of the epithelial/mesenchymal signature of individual pretreatment CTCs to predict drug responsiveness in patients. Conclusion: The fluid-assisted separation technology disc platform enables serial monitoring of CTC counts, DNA mutations, as well as unbiased molecular characterization of individual CTCs associated with tumor progression during targeted therapy.

Malignancy risk for solitary and multiple nodules in Hürthle cell-predominant thyroid fine-needle aspirations: A multi-institutional study.

BACKGROUND: Hürthle cell metaplasia is common in hyperplastic nodules, particularly within the setting of lymphocytic thyroiditis (LT). The Bethesda System for Reporting Thyroid Cytopathology indicates that it is acceptable to classify Hürthle cell-predominant fine-needle aspiration (HC FNA) specimens as atypia of undetermined significance (AUS) rather than suspicious for a Hürthle cell neoplasm (HUR) within the setting of multiple nodules or known LT. The goal of the current study was to address whether this approach is justified. METHODS: HC FNA specimens were identified and correlated with ultrasound and surgical pathology reports if available. Multinodularity was determined based on findings on macroscopic examination if imaging results were unavailable. RESULTS: A total of 698 HC FNA specimens were identified, including 576 resected nodules, 455 of which (79%) were benign. The overall risk of malignancy for HUR was 27%, whereas the risk of malignancy for AUS was 10%. The mean size of the benign nodules was 2.1 cm on surgical resection specimens, with multiple nodules noted in 293 cases (64%) and histologic LT noted in 116 cases (25%). The mean size of the malignant nodules was 2.8 cm, with multiple nodules and histologic LT noted in 74 cases (61%) and 22 cases (18%), respectively. The malignancy rate did not differ between solitary or multiple nodules (P = .52) or in the presence or absence of LT (P = .12). However, size did significantly differ between malignant and benign nodules (P < 0.01). CONCLUSIONS: The malignancy rate did not differ significantly in the presence of multiple nodules or LT, although the latter demonstrated a statistical trend. A diagnosis of AUS over HUR based solely on the presence of multinodularity is not warranted.

Comparison of fluorescent in situ hybridization HER-2/neu results on core needle biopsy and excisional biopsy in primary breast cancer.

HER-2/neu status is critical for the therapy for breast carcinoma. Fluorescent in situ hybridization for gene amplification and immunohistochemical stains for protein expression are widely used methods to detect HER-2/neu status. Multiple studies have shown fluorescent in situ hybridization and immunohistochemical stain results to have high concordance rates. To our knowledge, a comparison between fluorescent in situ hybridization results for core needle biopsy and the subsequent excisional biopsy specimens has not yet been studied. We retrospectively evaluated the fluorescence in situ hybridization and immunohistochemical results in both the breast core needle and the excisional biopsy of 125 patients with invasive breast carcinoma from 2002 to 2005. There was complete concordance with respect to both immunohistochemical and fluorescence in situ hybridization results for core needle biopsy and excisional biopsy specimens in 87% of the patients evaluated. Comparison of fluorescent in situ hybridization results of the 129 core needle biopsies to the 131 excisional biopsies of all 125 patients showed a concordance rate of 92%. The immunohistochemical stain results of the same core needle and excisional biopsies showed a concordance rate of 98%. Comparison of the immunohistochemical stain results with the fluorescent in situ hybridization results for all 260 cases examined showed 95% concordance. On the basis of our study, we observed that repeating HER-2/neu testing by immunohistochemical stain and/or fluorescent in situ hybridization methods on excisional biopsy is not unreasonable, in particular in cases of intratumoral heterogeneity, indeterminate/borderline HER-2/neu results and after neoadjuvant chemotherapy.

Circulating Tumor Cells: Applications in Cytopathology.

Circulating tumor cells (CTCs) are rare tumor cells found in the blood of patients with cancer that can be reliably detected by CTC technologies to provide prognostic, predictive, and diagnostic information. CTC sampling reflects intratumoral and intertumoral heterogeneity better than targeted biopsy. CTC samples are minimally invasive and amenable to repeated sampling, allowing real-time evaluation of tumor in response to therapy-related pressures and possibly early detection. Cytology is the most natural arena for integration of CTC testing. CTC technology may also be deployed to enhance and facilitate the practice of cytology and surgical pathology.

Molecular testing of different cytologic preparations in patients with advanced lung adenocarcinoma: which yields the best results?

INTRODUCTION: This study constitutes the first systematic comparison of molecular results between different cytology preparations in patients with lung adenocarcinoma undergoing testing for EGFR, KRAS, and BRAF mutations. MATERIALS AND METHODS: 115 archival cytology preparations (direct smears, ThinPrep preparations [TP], and cell blocks [CB]) from lung adenocarcinomas with known EGFR, KRAS, or BRAF mutations were tested and compared with clinical testing results. Results were compared between preparations and analyzed in relation to tumor purity and tumor cell content. RESULTS: 82 (77%) of 106 informative cases were concordant with clinical testing results. There was no significant difference in the concordance rate between CB, TP, air-dried smears, or alcohol-fixed smears (P = 0.3803), nor between preparations with <25%, 25% to 50%, or >50% tumor purity (P = 0.1147). Concordance rates were lower in preparations with ≤100 tumor cells (P = 0.0002). CONCLUSIONS: Smears, TP, and CB are all valid substrates for molecular testing. Although tumor purity did not significantly affect results, low tumor content showed poorer performance. Recording tumor purity and content is recommended.

Preoperative Cytologic Diagnosis of Noninvasive Follicular Thyroid Neoplasm with Papillary-Like Nuclear Features: A Prospective Analysis.

BACKGROUND: The term noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) has been proposed to replace noninvasive follicular variant of papillary thyroid carcinoma (FVPTC) in recognition of the indolent behavior of this tumor. The ability to differentiate NIFTP from classical papillary thyroid carcinoma (cPTC) by fine-needle aspiration (FNA) would facilitate conservative management for NIFTP. The aim of this study was to determine if NIFTP can be distinguished prospectively from cPTC. METHODS: From June 2015 to January 2016, thyroid FNAs with a diagnosis of "malignant" or "suspicious for malignancy" were prospectively scored for features associated with NIFTP/FVPTC (microfollicular architecture) or cPTC (papillae, psammomatous calcifications, sheet-like architecture, and nuclear pseudoinclusions) and categorized as NIFTP/FVPTC, cPTC, or indeterminate. Results were correlated with subsequent histologic diagnoses. RESULTS: The study included 52 patients with 56 resected nodules with a cytologic diagnosis of "malignant" (43/56) or "suspicious for malignancy" (13/56). Forty-nine patients (94%) underwent initial total thyroidectomy. Histopathologic diagnoses included 42 cPTC, 8 NIFTP, 3 invasive FVPTC, 2 follicular adenomas, and 1 poorly differentiated carcinoma. Excluding 7 indeterminate cases, 89% (8/9) of nodules classified as NIFTP/FVPTC on FNA demonstrated follicular-patterned lesions on histology (5 NIFTP, 1 invasive FVPTC, 2 follicular adenomas). Cytopathologists prospectively identified cPTC in 95% (38/40) of cases. CONCLUSIONS: In thyroid FNAs with cytologic features concerning for PTC, NIFTP/FVPTC can be distinguished from cPTC in most cases by assessing a limited number of features. Therefore, it is both feasible and appropriate to attempt to separate NIFTP/FVPTC from cPTC on FNA to promote appropriate clinical management.

Young investigator challenge: Application of cytologic techniques to circulating tumor cell specimens: Detecting activation of the oncogenic transcription factor STAT3.

BACKGROUND: The circulating tumor cell (CTC) field is rapidly advancing with the advent of continuously improving technologies for enriching these rare neoplastic cells from blood. CTC enumeration provides prognostic information, and CTC characterization has the potential to provide more useful information for the clinical decision-making process in this era of personalized medicine and targeted therapeutics. Proof-of-principle studies have shown that CTC samples can be characterized with a variety of techniques in the research laboratory environment. The goal of the current study was to validate routine cytologic techniques and immunohistochemical markers in CTC samples in a clinical cytology laboratory, using inducible phosphorylated signal transducer and activator of transcription 3 (pSTAT3) as a clinically important example and Ki-67 as a positive control. METHODS: Whole blood from noncancer patients was spiked with breast cancer cell lines with constitutive or inducible pSTAT3 expression and underwent CTC processing in the CellSearch system. The resulting CTC samples were subjected to various cytologic/immunocytochemical techniques and were compared with non-CTC-processed cultured cell controls. RESULTS: CTC-processed samples showed a morphology comparable to that of controls in cytospin, ThinPrep, and cell block preparations. Immunocytochemistry for Ki-67 and pSTAT3 provided biological information from CTC samples, showing uniform Ki-67 staining across all samples, pSTAT3 positivity in the constitutive and induced cells, and an absence of pSTAT3 expression in the noninduced cells, as expected. CONCLUSIONS: CTC samples can be processed in the cytology laboratory with routine methods. CTC morphologic and immunophenotypic analysis can be easily integrated into the existing clinical workflow, moving the field closer to a true peripheral blood liquid biopsy for cancer patients.

Benign metastasizing pleomorphic adenoma presenting as a solitary kidney mass: imaging features.

Benign metastasizing pleomorphic adenoma is a rare condition that occurs in patients with a prior history of pleomorphic adenoma of the salivary glands. Metastases to the kidney are extremely rare, and, to the best of our knowledge, their imaging appearance on multiple cross-sectional imaging modalities has not been described. We present a solitary metastasis to the kidney in a 40-year-old woman. Computed tomography and magnetic resonance imaging demonstrated a 2.4 cm, well-marginated, enhancing mass that protruded into the renal sinus fat. Findings were indistinguishable from a primary renal malignancy. Prior history is crucial in suggesting the correct diagnosis.

The effect of delay in fixation, different fixatives, and duration of fixation in estrogen and progesterone receptor results in breast carcinoma.

Accurate determination of estrogen receptor (ER) and progesterone receptor (PR) status in breast carcinoma is essential. Preanalytic variation may contribute to discordant results. Recently, American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) made recommendations to normalize fixation for breast biomarkers. To evaluate this, a 4-cm invasive lobular carcinoma was processed according to ASCO/CAP guidelines. The remainder was stored fresh at 4°C for 4 days and cut into biopsy-sized pieces. Each was fixed in 10% formalin, Pen-Fix (Richard-Allan Scientific, Kalamazoo, MI), Bouin solution, Sakura Molecular Fixative (Sakura Tissue-Tek Xpress, Torrance, CA), zinc formalin, or 15% formaldehyde for times ranging between 1 and 168 hours. Immunohistochemical studies for ER and PR were performed and interpreted. After 4 days at 4°C, all samples showed no degradation or ER/PR staining differences, except 2 Bouin-fixed samples, in comparison with the patient's sample processed according to ASCO/CAP guidelines. In our study, the preanalytic variables of fixative type, fixation time, and 4 days of ischemic time did not affect immunohistochemical accuracy for ER/PR.