Evaluation of DNA and RNA quality from archival formalin‐fixed paraffin‐embedded tissue for next‐generation sequencing – Retrospective study in Japanese single institution

Genetic analysis on formalin‐fixed paraffin‐embedded (FFPE) tissue specimens has become a mainstream method, from conventional direct sequencing to comprehensive analysis using next‐generation sequencing (NGS). In this study, we evaluated the quality of DNA and RNA extracted from FFPE sections, derived from surgical specimens of different tumor types. Electrophoresis was performed using a 4200 TapeStation to evaluate DNA and RNA fragmentation. DNA Ct values were higher and significantly increased over a period of 4 years compared with those from cell lines or frozen tissues. The RNA integrity number equivalent (RIN) ranged from 1 to 4.1 and DV200 ranged from 7.3 to 81%. Twelve of the 108 cases were analyzed by NGS using the AmpliSeq Cancer HotSpot Panel v2 on a Miniseq system. A sufficient number of reads and coverage were obtained in all cases. Our results revealed that NGS analysis was sufficient for FFPE‐derived DNA within 4 years of preservation. Conversely, approximately 20% of the RNA derived from FFPE within 4 years from the collection could be inappropriate for gene analysis based on RIN and DV200. It was suggested that FFPE would be adequate for genetic analysis, although it is desirable to store frozen specimens for the tumor tissues to be subjected to genetic analysis.     

Early magnetic resonance detection of cortical necrosis and acute network injury associated with neonatal and infantile cerebral infarction

Background

Knowledge of MRI findings in pediatric cerebral infarction is limited.

Objective

To determine whether cortical necrosis and network injury appear in the acute phase in post-stroke children and to identify anatomical location of acute network injury and the ages at which these phenomena are seen.

Materials and methods

Images from 12 children (age range: 0–9 years; neonates [<1 month], n=5; infants [1 month–12 months], n=3; others [≥1 year], n=4) with acute middle cerebral artery (MCA) cortical infarction were retrospectively analyzed. Cortical necrosis was defined as hyperintense cortical lesions on T1-weighted imaging that lacked evidence of hemorrhage. Acute network injury was defined as hyperintense lesions on diffusion-weighted imaging that were not in the MCA territory and had fiber connections with the affected cerebral cortex. MRI was performed within the first week after disease onset.

Results

Cortical necrosis was only found in three neonates. Acute network injury was seen in the corticospinal tract (CST), thalamus and corpus callosum. Acute network injury along the CST was found in five neonates and one 7-month-old infant. Acute network injury was evident in the thalamus of four neonates and two infants (ages 4 and 7 months) and in the corpus callosum of five neonates and two infants (ages 4 and 7 months). The entire thalamus was involved in three children when infarction of MCA was complete.

Conclusion

In acute MCA cortical infarction, MRI findings indicating cortical necrosis or acute network injury was frequently found in neonates and early infants. Response to injury in a developing brain may be faster than that in a mature one.     

Clear cell adenocarcinoma present exclusively within endometrial polyp: report of two cases

Endometrial polyp is a common benign lesion that protrudes into the endometrial surface. The incidence of carcinoma within endometrial polyp is thought to be low, however, postmenopausal women with endometrial polyps are at an increased risk. Endometrial clear cell adenocarcinoma is a distinct and relatively rare subtype of endometrial carcinoma, and recent studies have proposed putative precursor lesions of clear cell adenocarcinoma, namely clear cell endometrial glandular dysplasia (EmGD) and clear cell endometrial intraepithelial carcinoma (EIC). Herein, we describe two cases of clear cell adenocarcinoma present exclusively within endometrial polyp and discuss the association of its precursor. Two postmenopausal Japanese females, 66-year-old (Case 1) and 54-year-old (Case 2) presented with abnormal genital bleeding. Cytological examination of both cases revealed adenocarcinoma, thus, hysterectomy was performed. Histopathological studies demonstrated clear cell adenocarcinoma within exclusively endometrial polyp in both cases. The peculiar finding in Case 1 was presence of atypical glandular cells with large round to oval nuclei and clear cytoplasm within the atrophic endometrial glands in the surrounding endometrial tissue, which corresponded to clear cell EIC. A recent study showed that 33% of uteri had at least one focus of clear cell EmGD in endometrial polyps. Accordingly, clear cell adenocarcinoma and clear cell EmGD can occur in association with endometrial polyps more frequently than previously thought. Therefore, detailed histopathological examination is important in diagnosis of endometrial polyps, especially in the postmenopausal women, moreover cytological examination is a useful tool in the postmenopausal women with endometrial polyps.     

Cytological features of dedifferentiated adenoid cystic carcinoma of the trachea

Adenoid cystic carcinoma (AdCC) is a distinct type of carcinoma, and cytological examination has been recognized as a useful tool in its diagnosis. Dedifferentiation is defined as the abrupt transformation of a low‐grade tumor into a tumor with high‐grade components. Albeit extremely rare, dedifferentiated AdCC has been reported: however, the cytological features of this tumor have not been documented. We observed a case in which a 66‐year‐old Japanese male had stenosis and thickness of the lower tracheal and bronchial walls. Cytological smears of a bronchial brush specimen revealed features typical for low‐grade AdCC. However, a few cohesive epithelial cell clusters composed of large, atypical polygonal cells with large nuclei and conspicuous nucleoli also were present. This component was considered to represent dedifferentiated carcinoma. Histopathological study of the resected bronchial tumor revealed dedifferentiated AdCC. The cytological diagnosis of conventional low‐grade AdCC is straightforward in most cases, although extremely rare, dedifferentiated carcinoma can occur within the conventional AdCC, and detection of a dedifferentiated component is possible in a cytological specimen because of obvious nuclear atypia. Therefore, careful observation is needed because cytologic diagnosis of dedifferentiated AdCC can help expedite treatment of this highly aggressive tumor. Diagn. Cytopathol. 2014;42:880–883. © 2013 Wiley Periodicals, Inc.     

A novel automated assay for malate dehydrogenase isoenzymes

We have developed a new automated method for the determination of malate dehydrogenase (MDH) isoenzymes in serum employing guanidine hydrochloride. Our proposed method showed good reproducibility; within-run precision coefficient of variations (CVs) were less than 2.5 (mean 13.6–42.9 U/L) for total MDH (T-MDH) and less than 6.7% (mean 6.3–23.6 U/L) for mitochondrial MDH (m-MDH) (n = 10). The upper detection limit of the proposed method exhibited good linearity up to 1,000 U/L for both T-MDH and m-MDH. In the proposed m-MDH reagent, the presence of up to 2,000 U/L of cytosolic MDH(c-MDH) activity had no effect on the outcome of m-MDH assay. Results of our proposed method (y) correlated well with those of the electrophoretic method (x) giving the regression equation: y = 1.46 x + 6.87 (N = 30); r = 0.99. Normal concentrations of various anticoagulants and bilirubin did not affect the assay results. Both ascorbic acid and glucose exhibited a slight positive interference with the proposed assay. Clinically, we found that m-MDH activity in serum had greater diagnostic predictive value than T-MDH activity for judging successful outcome of reperfusion therapy; the prognosis was poor when the m-MDH/T-MDH ratio was greater than 20%. © 1996 Wiley-Liss, Inc.     

Small Cortical Infarcts Transformed to Lobar Cerebral Microbleeds: A Case Series

Cerebral microbleeds (MBs) have been often observed due to the development of imaging devices, and are classified to deep and lobar MBs. Lobar MBs are strongly associated with cerebral amyloid angiopathy. Here, we report 3 cases of lobar MBs that developed after small cortical ischemic stroke. One case underwent carotid artery stenting for severe carotid stenosis, one was diagnosed with artery-to-artery embolism, and the other was embolic stroke of undetermined source. New small cortical infarctions were detected with diffusion-weighted magnetic resonance imaging (MRI). Initial MRI revealed no hemorrhage around the ischemic lesion on T2*-weighted gradient-recalled echo or susceptibility-weighted imaging (SWI) at the onset of stroke. Follow-up SWI after 12-20 months revealed lobar MBs in the previously detected ischemic lesions, and high-intensity lesions remained around the MBs on fluid-attenuated inversion recovery imaging. These cases revealed that cerebral MBs developed through the transformation of small cortical infarctions. All cases showed lobar MBs, and these MBs existed in the previously detected ischemic lesions at a chronic stage. Lobar MBs present around ischemic lesions may predict embolic infarcts.     

Two region‐dependent pathways of eosinophilic neuronal death after transient cerebral ischemia

Various types of eosinophilic neurons (ENs) are found in the post‐ischemic brain. We examined the temporal profile of ENs in the core and peripheral regions of the ischemic cortex, and analyzed the relationship to the expression of various cell death‐related factors. Unilateral forebrain ischemia was induced in Mongolian gerbils by transient common carotid artery occlusions, and the brains from 3 h to 2 weeks post‐ischemia were prepared for morphometric and immunohistochemical analysis of ENs. ENs with minimally abnormal nuclei and swollen cell bodies appeared at 3 h in the ischemic core and at 12 h in the periphery. In both locations multiple cell death‐related factors including calcium, µ‐calpain, cathepsin D, 78 kDa glucose‐regulated protein (GRP78) and ubiquitin were activated. In the ischemic core, pyknosis and irregularly atrophic cytoplasm peaked at 12 h, which was associated with significant increases in staining for calcium and µ‐calpain. ENs with pyknosis and scant cytoplasm peaked at 4 days and were positive for TUNEL and calcium staining. In the ischemic periphery, ENs had slightly atrophic cytoplasm and sequentially developed pyknosis, karyorrhexis and karyolysis over 1 week. These cells were positive for TUNEL and calcium staining. All types of EN were negative for caspase 3. There may be two region‐dependent pathways of EN changes in the post‐ischemic brain: pyknosis with cytoplasmic shrinkage in the core, and nuclear disintegration with slightly atrophic cytoplasm in the periphery. This difference coordinates different activation patterns of cell death‐related factors in ENs.