B-cell to T-cell ratio as a novel indicator in flow cytometry in the diagnosis of thyroid lymphoma.

Preoperative flow cytometry is recommended to prove the monoclonality and confirm the diagnosis of thyroid lymphoma. However, lymphoma cases without light chain restriction may also have monoclonality. The aim of our study was to identify a novel marker for thyroid lymphomas using aspirated materials for flow cytometry. We retrospectively analyzed 26 patients with primary thyroid lymphomas and 16 patients with benign lymphoproliferative lesions. The materials for flow cytometry were obtained by fine-needle aspiration cytology using a 22-gauge needle under ultrasound guidance. Light chain restriction was defined as a κ to λ ratio of less than 0.5 or more than 3.0. According to the light chain-positive rate, 25% or less and more than 25% were classified as the low and high light chain-positive rate groups, respectively. B-cell predominance was defined as a CD19 to CD4 ratio (B- to T-cell ratio) of more than 2.0. B-cell predominance was more frequently observed in lymphomas (88.5%) than in benign lymphoproliferative lesions (25.0%; p < 0.001). Light chain restriction based on the κ/λ ratio was detected in 69.2% of lymphomas, but not in benign lymphoproliferative lesions. Among lymphomas belonging to the low light chain-positive rate group, 88.9% did not exhibit light chain restriction and B-cell predominance was present. In contrast, benign lymphoproliferative lesions with B-cell predominance were not detected in the low light chain-positive rate group. B-cell predominance was a useful indicator for diagnosing thyroid lymphoma in the low light chain-positive rate group without light chain restriction.

Potential role of mobile rapid on-site evaluation® in thyroid fine-needle aspiration cytology to reduce delayed repeated aspiration.

Rapid on-site evaluation of fine-needle aspiration cytology is time-consuming and requires specialized cytopathology staff. Mobile Rose® is a newly developed device for rapid on-site evaluation of fine-needle aspiration cytology. This study aimed to investigate the potential role of Mobile Rose® in reducing delayed repeated aspiration of the thyroid. A total of 120 cytological samples were collected and observed using Mobile Rose® after fine-needle aspiration cytology between September and October 2020, with immediate assessment of minimal or no cell clusters after conventional smear preparation. After qualifying and scoring, needle washout materials were prepared using the BD CytoRichTM method and correlated with cytology results. The average turn-around time of Mobile Rose® was found to be 1.5 minutes. Sensitivity, specificity, positive predictive value, and negative predictive value were 94.4%, 100%, 100%, and 57.1%, respectively. False-negative results were attributed to small aggregates of cells that were difficult to distinguish from the background and artifacts. Mobile Rose® may represent an important innovation for rapid on-site evaluation that is fast, has high diagnostic performance, does not require the presence of specialized cytology staff, and can reduce delayed repeated aspiration of the thyroid gland. However, further minor improvements and confirmation are required.

Impact of the modification of the diagnostic criteria in the 2017 Bethesda System for Reporting Thyroid Cytopathology: a report of a single institution in Japan.

The Bethesda System for Reporting Thyroid Cytopathology has recently been revised in 2017 (TBSRTC 2017). This study aimed to evaluate the impact of modifying the diagnostic criteria in TBSRTC 2017 at a single institute. We retrospectively reviewed cytological specimens of 10,399 thyroid nodules submitted for thyroid fine-needle aspiration cytology. Among them, 56 atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS) nodules, 16 suspicious for malignancy (SFM) nodules, and 8 malignant nodules were re-categorized into follicular neoplasm or suspicious for a follicular neoplasm (FN/SFN). The incidence of FN/SFN was increased by 0.8%, while that of AUS/FLUS, SFM, and malignant nodule was decreased by 0.5%, 0.2%, and 0.1%, respectively. In nine (60%) of the 15 nodules that were re-classified from AUS/FLUS to FN/SFN nodules and re-aspiration was performed, it was possible to judge whether they were benign or malignant. Of the 24 patients with FN/SFN nodules originally diagnosed with SFM or malignant, 16 were followed up without surgical resection. In conclusion, TBSRTC 2017 only caused minor changes in the incidence of each diagnostic category. TBSRTC 2017 was revised to avoid false positives owing to noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) that account for >10% of papillary thyroid carcinomas; however, it is not necessary in low frequency NIFTP institutes or countries. In Japan, we propose active surveillance as an accepted option for clinically managing AUS/FLUS, FN/SFN, SFM, or malignant nodules having favorable benign clinical findings or being part of the low-risk group.

Fine-needle aspiration cytology for medullary thyroid carcinoma: a single institutional experience in Japan.

Many cytological studies on medullary thyroid carcinoma (MTC) have been reported; however, such studies in large series of patients with MTC have not been performed. We investigated MTC at a single institution in Japan using fine-needle aspiration cytology (FNAC), and aimed to establish a preoperative diagnostic algorithm for MTC. FNAC was performed in 119 of 149 patients with MTC (79.9%) who ultimately underwent surgical resection. Moreover, 22 of 56 hereditary MTC (39.3%) were diagnosed preoperatively without FNAC by their high serum calcitonin levels or increased response to calcium stimulation (11 cases each), as well as RET mutation analysis. On FNAC, 76.5% of nodules were categorized as 'malignancy' or 'suspicious for malignancy'. The sensitivity and specificity of calcitonin measurement in aspiration needle wash-out fluid and in immunocytochemical staining for calcitonin were 96.3% and 92.3% respectively. We proposed an algorithm for preoperative diagnosis of MTC utilizing FNAC: When thyroid nodules are highly suspicious for MTC by their clinical and ultrasonographic features, serum calcitonin measurement with or without a calcium stimulation test is required. Furthermore, FNAC should be performed for patients who do not have those findings. When there is a possibility of MTC at the time of FNAC, calcitonin measurement using needle wash-out fluid is a reliable diagnostic tool. When MTC is suspected on cytological examination, immunocytochemical staining for calcitonin is useful for confirming MTC diagnosis.

Diagnostic significance of PAX8 in thyroid squamous cell carcinoma.

Most types of thyroid carcinomas express PAX8 transcription factor; however, whether thyroid squamous cell carcinoma (SCC) also expresses PAX8, currently remains unknown. We herein examined the immunoreactivity of PAX8 in SCC of thyroidal and extrathyroidal origin, and discussed the diagnostic significance of PAX8. We immunohistochemically examined specimens from 11 SCC, 22 papillary thyroid carcinoma (PTC), 8 anaplastic thyroid carcinoma (ATC), and 2 mucoepidermoid carcinoma (MEC) cases as well as 5 uterine cervical SCC, 5 esophageal SCC, and 5 pulmonary SCC cases. The rates of PAX8-positive SCC, PTC, ATC, and MEC were 90.9%, 90.9%, 75.0%, and 100%, respectively. Two PAX8-negative PTC cases were cribriform variants. No uterine cervical, esophageal, or pulmonary SCC specimen reacted with PAX8 antibody. Thyroid transcription factor-1 (TTF-1) was positive in 9.1% and 95.5% of SCC and PTC cases, respectively, but negative in all ATC and MEC cases. These results demonstrate that PAX8 staining is useful for distinguishing between primary thyroid SCC and invasion or metastasis from extrathyroidal SCC. We recommend using an immunohistochemical panel of antibodies to PAX8 and TTF-1 to confirm a diagnosis of primary thyroid carcinoma.

Fine-needle aspiration of parathyroid adenomas: Indications as a diagnostic approach.

BACKGROUND: We aimed to determine the indication of fine-needle aspiration (FNA) for parathyroid adenoma (PA)-suspected nodules and the cytological features of PA, and to discuss the ancillary techniques for diagnostic confirmation. METHOD: Clinical, cytological, and histological examinations of 15 PA patients (4.0% of all PA resected patients) were conducted through FNA on 16 nodules. We also examined the cytological preparations of 10 follicular neoplasms (FNs) and 10 poorly differentiated thyroid carcinomas (PDTCs). RESULTS: FNA was performed to detect PA in nine (56.3%) nodules. The remaining seven (43.8%) nodules underwent FNA for lesions considered as thyroid nodules or lymph nodes. The levels of parathyroid hormone (PTH) in the aspiration needle washout fluid were observably high, except for that from one nodule with unsatisfactory FNA. Cytologically, the incidences of wedge pattern (86.7%) and salt and pepper chromatin (86.7%) in PAs were significantly higher than in FNs and PDTCs. In contrast, the appearance of colloid globules and nuclear grooves was less frequent than that of FNs and PDTCs. GATA-3 expression was intense in all PAs that immunocytochemistry were performed. Histologically, capsular invasion and/or laceration, tumor seeding, granulation tissue, and fibrosis were observed. CONCLUSIONS: When PA localization is unusual or inconclusive despite extensive imaging, FNA may be performed. We asserted that wedge pattern, salt and pepper chromatin, and the absence of colloid globules and nuclear grooves are diagnostic cytological indicators of PA rather than of FN or PDTC. We recommend PTH measurements using needle washout fluid for PA-suspected nodules, and immunocytochemistry with the GATA-3 antibody for cytologically PA-suspected nodules.

Clinical significance and cytological detection of tracheal puncture following thyroid fine-needle aspiration: A retrospective study.

BACKGROUND: There have been no detailed reports on tracheal puncture after thyroid fine-needle aspiration (FNA). This study aimed to discuss the cytological differential diagnoses of tracheal puncture after thyroid FNA and clarify its clinical significance. METHODS: Tracheal puncture was defined as aspiration of tracheal components, including ciliated cells, chondrocytes, and goblet cells. A history of air suction or cough during aspiration was also considered tracheal puncture. Among the 18,480 nodules from 13,813 patients that underwent thyroid FNA, 70 (0.38%) nodules with tracheal puncture were retrospectively examined. Eleven thyroglossal duct cysts (TGDCs) and seven bronchial cysts that could exhibit ciliated cells were included in the study to compare the cytological findings. RESULTS: Sixty-six (94.3%) nodules had no clinical evidence of complications during and after FNA. Of the nodules with tracheal puncture, 64.3%, 48.6%, and 51.4% nodules were <1.0 cm in size, located in the isthmus, and associated with calcification. Cytological examination showed that chondrocytes and ciliated cells were present in 94.3% and 32.9% nodules, respectively. Ciliated cells seen in nodules with tracheal puncture and TGDCs were non-degenerative, whereas those in bronchial cysts were degenerative. CONCLUSION: Tracheal puncture after thyroid FNA is rarely noticed clinically, does not cause serious conditions, and spontaneously resolves. This complication more likely occurs in small-calcified nodules in the isthmus. Chondrocytes are more reliable diagnostic clues than ciliated cells to indicate tracheal puncture cytologically.

Risk of malignancy and clinical outcomes of cyst fluid only nodules in the thyroid based on ultrasound and aspiration cytology.

BACKGROUND: The number of extensive studies focusing on cyst fluid only (CFO) thyroid nodules is limited, and the risk of malignancy (ROM) in CFO nodules has not been well-established. Thus, the purpose of this study was to investigate CFO nodules using cytology and ultrasound. In addition, we sought to define the ROM and determine the recommended clinical management of CFO nodules. METHODS: We retrospectively reviewed cytological preparations of 678 nodules that were originally identified as CFO nodules, including conventional specimens in 209 nodules, liquid based cytology (LBC) specimens in 221 nodules, and both conventional and LBC specimens in 248 nodules. Ultrasound reports with representative photographs were also reviewed. RESULTS: Of the 678 CFO nodules, 214 (31.6%) were reclassified into other categories, including non-diagnostic/unsatisfactory (ND/UNS) except for CFO (n = 15), benign (n = 198), and malignant (n = 1). Conventional preparations (33.5%) were more frequently reclassified than LBC preparations (13.6%; P < .0001). Re-aspiration for diagnosis was performed for only one calcified nodule. The rates of surgical resection and malignancy were 3.0% and 0.2%, respectively. Based on American Thyroid Association guidelines and the Kuma Hospital ultrasound classification, worrisome sonographic features were identified in 5.8% and 0% of CFO nodules, respectively. CONCLUSION: We propose that CFO nodules should be classified as separate from ND/UNS nodules; they should be categorized as a subtype of benign nodules. However, it is essential that fine-needle aspiration cytology be performed under ultrasound-guided real-time visualization of needle placement in the target nodule in all cases.

Diagnostic clues indicating tall cell variants of papillary thyroid carcinoma in fine needle aspiration.

BACKGROUND: The definition of tall cell variant of papillary thyroid carcinoma (TCV-PTC) depends on the articles, and the defined cytological findings characteristic of TCV-PTC have not yet been fully analyzed. This study aimed to establish the cytological characteristics of TCV-PTC. METHODS: We retrospectively analyzed the smears of 19 TCV-PTC and 50 conventional PTC (C-PTC) cases. RESULTS: Palisaded pattern with the nuclei locating at the base of tall columnar carcinoma cells was seen in 94.7% of TCV-PTCs, and the incidence was significantly higher than that of C-PTCs (P < .0001). The palisaded pattern tended to appear at the periphery of the cell clusters. Isolated tall columnar carcinoma cells were present in 89.5% of TCV-PTCs. The incidence was significantly higher than that of C-PTC (P = .0001). Tombstone appearance was identified in 78.9% of TCV-PTCs, but not in C-PTCs. Spindle-like carcinoma cells with tapering cytoplasmic end appeared in 68.4% and 12.0% of TCV-PTC and C-PTC, respectively (P < .0001). The cytoplasm of TCV-PTC was densely stained and its cell border was distinct. Cytoplasmic elongation toward an outside of the cell clusters was observed in 89.5% of TCV-PTCs. CONCLUSION: It is the most important to identify the presence of the tall columnar carcinoma cells on the cytological preparations, in order to distinguish TCV-PTC from C-PTC. We propose five cytological findings indicating TCV-PTC, (1) palisaded pattern, (2) tall columnar cells with the heights of at least three times their widths, (3) tombstone appearance, (4) spindle-like carcinoma cells, and (5) cytoplasmic elongation.

Expression of chromosomal regional maintenance protein-1 may be associated with subcellular survivin expression in human gastric and colorectal carcinoma.

Survivin, a member of the inhibitor of apoptosis protein family, is a potential prognostic marker and molecular target for anticancer therapies. Chromosomal regional maintenance protein-1 (CRM-1) mediates the nuclear export of proteins such as survivin. The aims of the present study were to compare the expression and subcellular localization of CRM-1 in human gastric and colorectal carcinomas and to assess the association between CRM-1 and survivin expression in these tumor types. The nuclear and cytoplasmic CRM-1 expression rates in gastric carcinoma were 61% (42/69) and 29% (20/69), respectively, while the nuclear and cytoplasmic CRM-1 expression rates in colorectal carcinoma were 55% (43/78) and 37% (29/78), respectively. Nuclear and cytoplasmic CRM-1 expression was found to be significantly correlated with nuclear and cytoplasmic survivin expression in colorectal carcinoma, but not gastric carcinoma. These results indicate that CRM-1 expression patterns differ between gastric and colorectal carcinomas and thus, we hypothesize that CRM-1-mediated nuclear export of survivin may be deregulated in gastric carcinoma. Therefore, CRM-1 may exhibit different functions in gastric and colorectal carcinoma.

Differentiating between benign follicular nodules and follicular neoplasms in thyroid liquid-based cytology preparations.

BACKGROUND: The cytological morphology observed in liquid-based cytology (LBC) preparations is dissimilar to that of conventional preparations. The aim of this report is to clarify the cytological differences between benign follicular nodules (BFNs) and follicular neoplasms (FNs) in LBC preparations and identify novel diagnostic criteria for LBC preparations. METHODS: A retrospective review of LBC preparations from 38 BFN and 74 FN (57 follicular adenomas and 17 follicular carcinomas) cases confirmed by histological examination was conducted. LBC samples were obtained from the washout fluid in aspiration needles, fixed with CytoRich-RED(TM) , and prepared using the SurePath(TM) method. RESULTS: Fibrin was observed in 43.2% of FNs and in 23.7% of BFNs. The incidences of intercellular spaces, distinct outer margin, and cytoplasmic process were significantly higher in BFNs (P < 0.01, P < 0.01, and P < 0.05, respectively). Elongated microfollicles were seen in 55.4% of FNs and 10.5% of BFNs (P < 0.01). Membranous materials encircling the outer edge of the follicles were observed only in BFN cases (5.8%). There were no significant differences between follicular adenoma and carcinoma. CONCLUSION: Intercellular spaces, distinct outer margins of the follicular clusters, cytoplasmic process, and membranous materials constitute indicators of BFN in LBC preparations. Fibrin and elongated microfollicles point to FN. We believe that these findings will improve the diagnostic accuracy of thyroid LBC preparations. Diagn. Cytopathol. 2016;44:659-664. © 2016 Wiley Periodicals, Inc.

Cytological characteristics of papillary thyroid carcinoma on LBC specimens, compared with conventional specimens.

BACKGROUND: The cytological findings in conventional specimens (C-S) and liquid-based cytology specimens (LBC-S) are not quite same. The aim of this article is to clarify the cytological findings of papillary thyroid carcinoma (PTC) characteristic of LBC-S. METHODS: Out of 5,971 cases diagnosed in a single institution from March to September in 2012, 161 PTC cases with both C-S and LBC-S were reviewed. Additionally, we compared the findings with those in LBC-S of 55 adenomatous or hyperplastic nodule (AN) and 21 follicular neoplasm (FN) cases. RESULTS: Compared to C-S, the incidences of trabecular and hobnail patterns, collagenous stroma, naked capillaries, intercellular spaces, convoluted nuclei, eosinophilic nucleoli, and perinucleolar halo were increased. Pale nuclei were observed in only one of 161 PTC cases. Specificity of convoluted nuclei and perinucleolar halo were 97.4% and 96.1%, respectively. CONCLUSION: Convoluted nuclei and perinucleolar halo might become a new indicator of PTC in LBC-S. Contrarily, we should be aware that pale nuclei are rarely observed in LBC-S.

Smac/DIABLO expression in human gastrointestinal carcinoma: Association with clinicopathological parameters and survivin expression.

Lack of apoptosis is a key factor in carcinogenesis and tumor progression. Survivin is a member of the inhibitor of apoptosis protein (IAP) family. Second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) is an antagonist of IAPs. Recently, Smac/DIABLO was identified as a potent therapeutic target. However, the clinical significance of Smac/DIABLO in gastrointestinal carcinomas remains unclear. In the present study, Smac/DIABLO expression was analyzed by immunohistochemistry in 72 gastric adenocarcinomas and 78 colorectal adenocarcinomas. The expression of Smac/DIABLO was significantly higher in colorectal carcinoma than in gastric carcinoma. Additionally, a correlation was found between the expression of Smac/DIABLO and nuclear survivin in well- to moderately-differentiated colorectal adenocarcinomas (r=0.245; P<0.01). Based on these results, it was hypothesized that gastric and colorectal carcinomas differ in the level of Smac/DIABLO expression. Our previous studies revealed that the expression of cleaved caspase-9 was significantly lower in colorectal carcinoma than in gastric carcinoma (P<0.0001). Conversely, the expression levels of microtubule-associated protein 1 light chain 3 (LC3), an autophagy marker, and survivin were significantly higher in colon cancer than in gastric cancer (P<0.0001 and P<0.01, respectively). Taken together, these results indicate that not only LC3 and survivin expression, but also Smac/DIABLO expression, are significantly higher in colorectal carcinoma than in gastric carcinoma. We hypothesize that the analysis of Smac/DIABLO, survivin and LC3 expression in colorectal carcinoma is likely to aid cancer therapy due to the involvement of these markers in apoptosis and/or autophagy.

Phosphorylation status of Akt and caspase-9 in gastric and colorectal carcinomas.

The serine/threonine protein kinase B/Akt plays a central role in the coordination of multiple signal transduction processes involved in transcriptional regulation, cell survival, and apoptosis. Activation of Akt kinase is a prognostic factor in several types of cancers; however, its role in gastrointestinal cancers is not fully understood. Caspase-9 is an Akt substrate that belongs to the caspase family of proteases, which function as initiators of the mitochondrial apoptotic pathway. Little is known about the role of caspase-9 phosphorylation, which downregulates the apoptotic activity of the enzyme. In this study, we investigated the expression of phosphorylated (p)-Akt and phosphorylated (p)-caspase-9 in gastric and colorectal carcinoma and the relationship between p-Akt and p-caspase-9 expression and clinicopathological parameters of gastric and colorectal cancer patients. In total, 75 samples of advanced gastric adenocarcinoma (37 well-to-moderately differentiated and 38 poorly differentiated) and 76 samples of advanced colorectal adenocarcinoma (69 well-to-moderately differentiated and 7 poorly differentiated) were analyzed for p-Akt and p-caspase-9 expression by immunohistochemistry. Our results reveal a correlation between p-Akt and p-caspase-9 expression in gastric and colorectal cancers. Levels of p-caspase-9 were significantly higher in colorectal cancer than in gastric cancer, indicating tumor-specific regulation. Although the biological role of p-Akt/p-caspase-9 signaling remains unclear, we suggest that phosphorylation of caspase-9 may be a useful tool to assess the state of gastrointestinal cancer and the effects of anti-cancer therapy.

Immunohistochemical expression of nuclear and cytoplasmic survivin in gastrointestinal carcinoma.

Survivin is a protein that is highly expressed in many embryonic tissues, as well as most human tumors. Prior studies have reported both positive and negative correlations between survivin expression and cancer prognosis, but these associations remain controversial. In the present study, we assessed the expression of nuclear and cytoplasmic survivin in gastrointestinal carcinomas. Using these data, we determined the correlation between nuclear and cytoplasmic survivin and, further, investigated correlations between survivin expression and clinicopathological parameters. Seventy-two advanced gastric adenocarcinomas and 78 colorectal adenocarcinomas were analyzed for survivin expression by immunohistochemistry. Expression of both nuclear and cytoplasmic survivin was significantly higher in colorectal carcinomas than in gastric carcinomas (P < 0.01). There was a positive correlation between nuclear and cytoplasmic expression of survivin (r = 0.42, P < 0.001). In gastric carcinomas, the level of survivin protein expression was associated with tumor differentiation, patient age, and lymphatic invasion (P < 0.05, 0.01, and 0.01, respectively). In colorectal carcinomas, the level of nuclear survivin expression was significantly higher in females than in males (P < 0.05). There were no significant associations between survivin expression and most of the clinicopathological parameters. Nevertheless, there was a trend towards an inverse correlation between nuclear survivin expression and tumor aggressiveness in gastric carcinoma; there was a similar trend for cytoplasmic survivin expression. In summary, our results suggest that levels of nuclear and cytoplasmic survivin expression differ between gastric carcinoma and colorectal carcinoma.

Immunohistochemical analysis of cell death pathways in gastrointestinal adenocarcinoma.

Caspase-8 and caspase-9 play crucial roles in the extrinsic and intrinsic apoptotic pathways, respectively. The nuclear translocation of apoptosis-inducing factor (AIF) is involved in caspase-independent apoptosis. Microtubule-associated protein 1 light chain 3 (LC3) plays a pivotal role in autophagy. In the present study, we analyzed the expression of cleaved caspase-8 (CC8), cleaved caspase-9 (CC9), AIF, and LC3 in 160 gastrointestinal adenocarcinomas. The nuclear expression of AIF was rare. The expression of CC8 in gastric and colorectal adenocarcinomas did not differ, whereas the percentage of CC9-positive tumors in gastric adenocarcinomas was significantly higher than in colorectal adenocarcinomas. In contrast, the percentage of LC3-positive tumors in gastric adenocarcinomas was significantly lower than in colorectal adenocarcinomas. CC8 and CC9 occasionally co-existed in the same tumor cells in gastric adenocarcinoma. However, LC3-positive tumor cells in colorectal adenocarcinomas were constantly negative for CC8. No correlation was identified between the expression of any markers and clinicopathological parameters. These results suggest that different cell death pathways are activated in a manner that depends upon the primary site and cell type. The extrinsic and intrinsic apoptotic pathways may be mutually regulated in gastric adenocarcinomas. Also, autophagy may function as a cellular guardian to avoid apoptosis in colorectal adenocarcinomas.