Platelet Aggregation Assays Do Not Reliably Diagnose Platelet Delta Granule Storage Pool Deficiency.

Background: Patients with platelet dysfunction disorders present with a variety of mucocutaneous bleeding symptoms including easy bruising, frequent epistaxis, bleeding gums upon tooth brushing and for women, heavy menstrual bleeding. Available laboratory assays to evaluate platelet function include the platelet function analyzer (PFA) and in larger centers with coagulation laboratories, light transmission platelet aggregometry (LTA) analyses. Both assays are known to have a number of limitations, especially in the diagnosis of platelet delta granule storage pool deficiency (δ-SPD). δ-SPD is an underdiagnosed condition caused by decreased numbers of platelet dense granules (DGs) and is best diagnosed by electron microscopy (EM). Patients with platelet δ-SPD have a decreased response to low levels of the agonist adenosine diphosphate (ADP) in the second wave of light transmittance with LTA or decreased ADP secretion by fluorescence lumiaggregometry. There are few reports that have evaluated patients with δ-SPD and their respective LTA results. One report published in 1987 described normal LTA assays in 23% of patients with δ-SPD; a more recent report described LTA as having the sensitivity to detect only about 52% of patients with δ-SPD. The purpose of our study was intended to review the LTA and EM results of patients suspected of having a platelet function disorder at our institution for comparison with previously published studies. Methods: Our study included 344 patients who had been evaluated by both LTA and whole mount EM. Aggregometry utilized five agonists: ADP, epinephrine, collagen, arachidonic acid, and ristocetin. DGs were enumerated in 100 whole-mounted platelets to determine a mean number of dense granules per platelet (DGs/PL). Results: Seventy-seven percent of our patients were found to have δ-SPD (264/344); 68% (179/264) of these subjects had an abnormal platelet LTA. Thirty-two percent (85/264) of our patients had normal LTA results but were found to have δ-SPD with a mean of 2.54 ± 0.15 DG/PL (normal = 4 - 6 DG/PL). Conclusion: These data confirm previous reports suggesting the utilization of LTA alone in patients with histories of unexplained bleeding may miss the diagnosis of platelet δ-SPD. It is, therefore, prudent to assess platelet DG number by EM, especially if platelet LTA assessment is normal.

Histology-specific FGFR2 alterations and FGFR2-TACC2 fusion in mixed adenoid cystic and neuroendocrine small cell carcinoma of the uterine cervix.

Neuroendocrine small cell carcinoma of the uterine cervix portends a dismal prognosis with limited treatment options. Rarely, tumors of mixed-lineage appear in gynecologic malignancies. Here, we report a 77-year-old woman who presented with complete uterine prolapse and 4-month history of vaginal bleeding. Histopathologic evaluation revealed a mixed adenoid cystic carcinoma and neuroendocrine small cell carcinoma of the uterine cervix. The tumor was PD-L1 and HPV 35 positive. The patient was treated with up-front surgery and adjuvant radiation. Independent, histology-specific alterations in FGFR2 and a FGFR2-TACC2 fusion were identified. Progression of disease occurred within 6 months for which she received chemotherapy and immunotherapy. However, the patient expired within a year. We comprehensively review how screening for and targeting of FGFR alterations in recurrent and metastatic cervical cancer might serve as a touchstone for future treatment regimens.

Preanalytic variables in quality and quantity of nucleic acids extracted from FNA specimens of thyroid gland nodules collected in CytoLyt: Cellularity and storage time.

BACKGROUND: Morphologic and genetic analysis of thyroid nodules may be performed from a single vial. Preanalytic variables that affect nucleic acid extracted from a single vial are evaluated. METHODS: Thyroid fine-needle aspiration (FNA) specimens collected in CytoLyt were evaluated. A ThinPrep slide was prepared. Extracted nucleic acids were analyzed using Oncomine Comprehensive Panel, version 2, after Ion AmpliSeq library preparation. A pathologist and a cytotechnologist enumerated specimen cellularity. RESULTS: Fifty-six samples were collected from 55 nodules in 53 patients. Bethesda category correlated with cellularity (P = .01), and storage time (median, 43 days; range, 7-77 days) was longer for specimens in categories II and III than for those in categories IV and VI (P = .01). The mean specimen DNA concentration was 4.5 ng/µL (range, 0-23.8 ng/µL), and 25 (45%) had concentrations >3.3 ng/µL. The mean specimen RNA concentration was 4.8 ng/µL (range, 0-42.4 ng/µL), and 31 (55%) had concentrations >1.4 ng/µL. Nucleic acid quantity increased with epithelial cellularity. Storage time weakly correlated with the quantity of extracted DNA, independent of cellularity, but not extracted RNA. Greater proportions of cell-free DNA and lesser proportions of long, intact RNA fragments were extracted from a subset of samples with longer storage time. Among 15 single nucleotide variants, the median mutant allelic fraction was 15.1%. One false-negative result was identified. Five specimens subsequently determined to harbor a genetic alteration failed quality metrics. CONCLUSIONS: Cellularity and storage time affect the quantity and quality of nucleic acid extracted from thyroid FNA specimens collected in CytoLyt. Further investigation will serve to quantify the magnitude of such effects and to elucidate other contributing factors.

Rapid development and use of patient-specific ctDNA biomarkers to avoid a "rash decision" in an ovarian cancer patient.

Epithelial ovarian cancer (OvCa) is the most lethal female reproductive tract malignancy. A major clinical hurdle in patient management and treatment is that when using current surveillance technologies 80% of patients will be clinically diagnosed as having had a complete clinical response to primary therapy. In fact, the majority of women nonetheless develop disease recurrence within 18 mo. Thus, without more accurate surveillance protocols, the diagnostic question regarding OvCa recurrence remains framed as "when" rather than "if." With this background, we describe the case of a 61-yr-old female who presented with a 3-mo history of unexplained whole-body rash, which unexpectedly led to a diagnosis of and her treatment for OvCa. The rash resolved immediately following debulking surgery. Nearly 1 yr later, however, the rash reappeared, prompting the prospect of tumor recurrence and requirement for additional chemotherapy. To investigate this possibility, we undertook a genomics-based tumor surveillance approach using a targeted 56-gene NGS panel and biobanked tumor samples to develop personalized ctDNA biomarkers. Although tumor-specific TP53 and PTEN mutations were detectable in all originally collected tumor samples, pelvic washes, and blood samples, they were not detectable in any biosample collected beyond the first month of treatment. No additional chemotherapy was given. The rash spontaneously resolved. Now, 2 yr beyond the patient's original surgery, and in the face of continued negative ctDNA findings, the patient remains with no evidence of disease. As this single case report suggests, we believe for the first time that ctDNA can provide an additional layer of information to avoid overtreatment.

Immunohistochemical analysis of OTP and NKX6.1 in neuroendocrine tumors of the lung and pancreas.

BACKGROUND: Homeobox transcription factors have demonstrated utility in diagnosing neuroendocrine tumors. Orthopedia homeobox protein (OTP) has a well-defined role in embryonic neurodevelopment and has also been described as a prognostic marker in lung neuroendocrine tumors (NET). Additionally, NK6 homeobox-1 (NKX6.1) has been described to be necessary for the development of neuroendocrine cells in the pancreas. We evaluated immunohistochemical (IHC) expression of OTP and NKX6.1 to determine their utility in the diagnosis of NETs from lung and pancreas fine-needle aspirations (FNA). METHODS: Our study examined 50 FNA specimens, including 30 primary pulmonary NETs (8 carcinoid tumors (CT), 6 atypical carcinoids (AC), 11 small-cell neuroendocrine carcinomas (SCNEC), 5 large-cell neuroendocrine carcinomas (LCNEC)) and 20 primary pancreatic NETs (17 well-differentiated pancreatic neuroendocrine tumors (PanNET) and 3 poorly differentiated pancreatic neuroendocrine carcinomas (PanNEC)). IHC expression of OTP, NKX6.1, and Ki-67 was evaluated on FNA cell blocks. RESULTS: Half of the pulmonary TC tumors expressed OTP, while only 17% of AC and 20% of LCNEC expressed OTP. Neither SCNECs nor any pancreatic NET expressed OTP. In contrast, intermediate and high-grade tumors expressed NKX6.1 (LCNEC-80%, SCNEC-82%, and AC-83%) more often than low-grade tumors (TC-63%, PanNET-71%). All three PanNECs expressed NKX6.1. CONCLUSIONS: OTP may be useful in diagnosing well-differentiated NETs of pulmonary origin. NKX6.1 may have utility in segregating high from low-grade NETs of both pulmonary and pancreatic origin, although other methods will be required to determine site of origin.

The role of peritoneal washings in the diagnosis of endometriosis.

Endometriosis, the presence of endometrial tissue outside the uterine corpus, is a common finding in reproductive age women. It is classically diagnosed based on the presence of at least two of the following elements: endometrial glands, endometrial stroma, and hemosiderin-laden macrophages (HLMs). Although a common finding in surgical pathology specimens at the time of gynecologic surgery, there is little literature on the role of pelvic washings in diagnosing endometriosis. Our study aimed to examine the characteristics of endometriosis in pelvic washings at the time of gynecologic surgery. We report nine cases of endometriosis diagnosed on pelvic washing. Two had a reported history of endometriosis. Four had endometriosis on the concurrent surgical pathology specimen. Liquid-based cytology was diagnostic of endometriosis in seven patients, including five with glandular cells and HLMs and two with glandular cells, HLMs, and endometrial stromal cells. Cell block was diagnostic of endometriosis in eight patients, including four cases with intact fragments of endometrial glands and stroma. Three cases showed glandular cells and HLMs, while one showed separate fragments of glandular cells and stromal cells. Pelvic washings increased the diagnostic yield for endometriosis at the time of gynecologic surgery, as only four out of nine cases had endometriosis diagnosed on surgical pathology. Cell block in particular aids in the diagnosis, since intact glandular and stromal fragments frequently can be identified.