Establishing molecular pathology curriculum for pathology trainees and continued medical education: a collaborative work from the Molecular Pathology Study Group of the Korean Society of Pathologists

Background@#The importance of molecular pathology tests has increased during the last decade, and there is a great need for efficient training of molecular pathology for pathology trainees and as continued medical education. @*Methods@#The Molecular Pathology Study Group of the Korean Society of Pathologists appointed a task force composed of experienced molecular pathologists to develop a refined educational curriculum of molecular pathology. A 3-day online educational session was held based on the newly established structure of learning objectives; the audience were asked to score their understanding of 22 selected learning objectives before and after the session to assess the effect of structured education. @*Results@#The structured objectives and goals of molecular pathology was established and posted as a web-based interface which can serve as a knowledge bank of molecular pathology. A total of 201 pathologists participated in the educational session. For all 22 learning objectives, the scores of self-reported understanding increased after educational session by 9.9 points on average (range, 6.6 to 17.0). The most effectively improved items were objectives from next-generation sequencing (NGS) section: ‘NGS library preparation and quality control’ (score increased from 51.8 to 68.8), ‘NGS interpretation of variants and reference database’ (score increased from 54.1 to 68.0), and ‘whole genome, whole exome, and targeted gene sequencing’ (score increased from 58.2 to 71.2). Qualitative responses regarding the adequacy of refined educational curriculum were collected, where favorable comments dominated. @*Conclusions@#Approach toward the education of molecular pathology was refined, which would greatly benefit the future trainees.

The prognostic significance of p16 expression pattern in diffuse gliomas

Background@#CDKN2A is a tumor suppressor gene that encodes the cell cycle inhibitor protein p16. Homozygous deletion of the CDKN2A gene has been associated with shortened survival in isocitrate dehydrogenase (IDH)–mutant gliomas. This study aimed to analyze the prognostic value of p16 and to evaluate whether p16 immunohistochemical staining could be used as a prognostic marker to replace CDKN2A genotyping in diffuse gliomas. @*Methods@#p16 immunohistochemistry was performed on tissue microarrays of 326 diffuse gliomas with diagnoses that reflected IDH-mutations and 1p/19q codeletion status. The results were divided into three groups (negative, focal expression, overexpression) according to the presence and degree of p16 expression. Survival analysis was performed to assess the prognostic value of p16 expression. @*Results@#A loss of p16 expression predicted a significantly worse outcome in all glioma patients (n=326, p<.001), in the IDH-mutant glioma patients (n=103, p=.010), and in the IDH-mutant astrocytoma patients (n=73, p=.032). However, loss of p16 expression did not predict the outcome in the IDH-wildtype glioma patients (n=223, p=.121) or in the oligodendroglial tumor patients with the IDH-mutation and 1p/19q codeletion (n=30, p=.457). Multivariate analysis showed the association was still significant in the IDH-mutant glioma patients (p=.008; hazard ratio [HR], 2.637; 95% confidence interval [CI], 1.295 to 5.372) and in the IDH-mutant astrocytoma patients (p=.001; HR, 3.586; 95% CI, 1.649 to 7.801). Interestingly, patients who presented with tumors with p16 overexpression also had shorter survival times than did patients with tumors with p16 focal expression in the whole glioma (p< .001) and in IDH-mutant glioma groups. (p=.046). @*Conclusions@#This study suggests that detection of p16 expression by immunohistochemistry can be used as a useful surrogate test to predict prognosis, especially in IDH-mutant astrocytoma patients.

An Autopsy Proven Case of CSF1R-mutant Adult-onset Leukoencephalopathy with Axonal Spheroids and Pigmented Glia (ALSP) with Premature Ovarian Failure

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a progressive degenerative white matter disorder caused by mutations in the tyrosine kinase domain of the CSF1R gene. ALSP is often misdiagnosed as other diseases due to its rarity and various clinical presentations such as Parkinsonism, pyramidal signs, cognitive impairment and/or psychiatric symptoms. We describe an autopsy case of ALSP with a CSF1R mutation. A 61-year-old woman presented insidious-onset gait difficulty for 12 years since her age of 49, and premature ovarian failure since her age of 35. At initial hospital visit, brain magnetic resonance imaging revealed hydrocephalus. Initially, Parkinson's syndrome was diagnosed, and she was prescribed L-dopa/carbidopa because of spasticity and rigidity of extremities, which had worsened. Subsequently, severe neuropsychiatric symptoms and cognitive impairment developed and radiologically, features of leukoencephalopathy or leukodystrophy were detected. She showed a down-hill course and died, 12 years after initial diagnosis. At autopsy, the brain showed severe symmetric atrophy of bilateral white matter, paper-thin corpus callosum, thin internal capsule, and marked hydrocephalus. Microscopically, diffuse loss of white matter, relatively preserved subcortical U-fibers, and many eosinophilic bulbous neuroaxonal spheroids were noted, but there was no calcification. Pigmented glia with brown cytoplasmic pigmentation were readily found in the white matter, which were positive for Periodic acid-Schiff, p62, and CD163 stains, but almost negative for CD68. Whole-exome and Sanger sequencing revealed a CSF1R mutation (c.2539G>A, p.Glu847Lys) which was reported in prior one ALSP case. This example demonstrates that ALSP could be associated with premature ovarian failure.

Recent Advancement of the Molecular Diagnosis in Pediatric Brain Tumor

Recent discoveries of brain tumor-related genes and fast advances in genomic testing technologies have led to the era of molecular diagnosis of brain tumor. Molecular profiling of brain tumor became the significant step in the diagnosis, the prediction of prognosis and the treatment of brain tumor. Because traditional molecular testing methods have limitations in time and cost for multiple gene tests, next-generation sequencing technologies are rapidly introduced into clinical practice. Targeted sequencing panels using these technologies have been developed for brain tumors. In this article, focused on pediatric brain tumor, key discoveries of brain tumor-related genes are reviewed and cancer panels used in the molecular profiling of brain tumor are discussed.

Recent Advancement of the Molecular Diagnosis in Pediatric Brain Tumor

Recent discoveries of brain tumor-related genes and fast advances in genomic testing technologies have led to the era of molecular diagnosis of brain tumor. Molecular profiling of brain tumor became the significant step in the diagnosis, the prediction of prognosis and the treatment of brain tumor. Because traditional molecular testing methods have limitations in time and cost for multiple gene tests, next-generation sequencing technologies are rapidly introduced into clinical practice. Targeted sequencing panels using these technologies have been developed for brain tumors. In this article, focused on pediatric brain tumor, key discoveries of brain tumor-related genes are reviewed and cancer panels used in the molecular profiling of brain tumor are discussed.

Reclassification of Mixed Oligoastrocytic Tumors Using a Genetically Integrated Diagnostic Approach

BACKGROUND: Mixed gliomas, such as oligoastrocytomas (OA), anaplastic oligoastrocytomas, and glioblastomas (GBMs) with an oligodendroglial component (GBMO) are defined as tumors composed of a mixture of two distinct neoplastic cell types, astrocytic and oligodendroglial. Recently, mutations ATRX and TP53, and codeletion of 1p/19q are shown to be genetic hallmarks of astrocytic and oligodendroglial tumors, respectively. Subsequent molecular analyses of mixed gliomas preferred the reclassification to either oligodendroglioma or astrocytoma. This study was designed to apply genetically integrated diagnostic criteria to mixed gliomas and determine usefulness and prognostic value of new classification in Korean patients. METHODS: Fifty-eight cases of mixed OAs and GBMOs were retrieved from the pathology archives of Seoul National University Hospital from 2004 to 2015. Reclassification was performed according to genetic and immunohistochemical properties. Clinicopathological characteristics of each subgroup were evaluated. Overall survival was assessed and compared between subgroups. RESULTS: We could reclassify all mixed OAs and GBMOs into either astrocytic or oligodendroglial tumors. Notably, 29 GBMOs could be reclassified into 11 cases of GBM, IDH-mutant, 16 cases of GBM, IDH-wildtype, and two cases of anaplastic oligodendroglioma, IDH mutant. Overall survival was significantly different among these new groups (p<.001). Overall survival and progression-free survival were statistically better in gliomas with IDH mutation, ATRX mutation, no microscopic necrosis, and young patient age (cut off, 45 years old). CONCLUSIONS: Our results strongly suggest that a genetically integrated diagnosis of glioma better reflects prognosis than former morphology-based methods.

Good Laboratory Standards for Clinical Next-Generation Sequencing Cancer Panel Tests

Next-generation sequencing (NGS) has recently emerged as an essential component of personalized cancer medicine due to its high throughput and low per-base cost. However, no sufficient guidelines for implementing NGS as a clinical molecular pathology test are established in Korea. To ensure clinical grade quality without inhibiting adoption of NGS, a taskforce team assembled by the Korean Society of Pathologists developed laboratory guidelines for NGS cancer panel testing procedures and requirements for clinical implementation of NGS. This consensus standard proposal consists of two parts: laboratory guidelines and requirements for clinical NGS laboratories. The laboratory guidelines part addressed several important issues across multistep NGS cancer panel tests including choice of gene panel and platform, sample handling, nucleic acid management, sample identity tracking, library preparation, sequencing, analysis and reporting. Requirements for clinical NGS tests were summarized in terms of documentation, validation, quality management, and other required written policies. Together with appropriate pathologist training and international laboratory standards, these laboratory standards would help molecular pathology laboratories to successfully implement NGS cancer panel tests in clinic. In this way, the oncology community would be able to help patients to benefit more from personalized cancer medicine.

An Autopsy Proven Child Onset Chronic Traumatic Encephalopathy

Here we present an autopsy case of chronic traumatic encephalopathy (CTE) in a 36-year-old man. He had a history of febrile seizures at the age of four and was severely demented at age 10 when he was admitted to a mental hospital. He had suffered repetitive self-harm, such as frequent banging of the head on the wall in his hospital record, but he had no clear history between the ages of four and ten. Autopsy revealed global cerebral atrophy, including the basal ganglia, thalamus, hippocampus, amygdala, mammilary bodies and lateral geniculate bodies. This case showed typical pathological features of CTE. Phosphorylated tau (p-tau)-positive neurofibrillary tangles (NFTs) and neuropil threads (NT) we are widely distributed in the brain, especially in the depth of the cerebral sulci. NFT and NT were also found in the basal ganglia, thalamus, amygdala and brainstem. Scanty β-amyloid deposits were found in the motor and sensory cortices, but α-synuclein was completely negative in the brain. This example showed that CTE can occur in young ages and that even children can experience CTE dementia.

Rare Manifestations of Anaplastic Thyroid Carcinoma: the Role of BRAF Mutation Analysis

Anaplastic thyroid carcinoma (ATC) is difficult to distinguish from other cancers, especially when its pathological features are atypical for ATC or when the tumor is totally undifferentiated and occurs after a considerable lapse of time, in an area remote from the original site of the tumor. Here, we present two patients (68-year-old man and 56-year-old woman) with rare manifestations of ATC, which were initially thought to be other malignancies. Immunohistochemical tests, using various markers, failed to provide information about the origin of these tumors. However, both patients had a history of papillary thyroid carcinoma (PTC) from several years ago and BRAF mutations were observed in the undifferentiated tumors, as well as in the previous PTCs. Therefore, we could make a diagnosis of ATC derived from PTC. As such, BRAF mutation analysis may serve as a useful tool for ATC diagnosis in challenging ATC cases.

Analysis of Fifty Hotspot Mutations of Lung Squamous Cell Carcinoma in Never-smokers

Smoking is the major risk factor for lung squamous cell carcinoma (SCC), although a small number of lung SCCs occurs in never-smokers. The purpose of this study was to compare 50 hotspot mutations of lung SCCs between never-smokers and smokers. We retrospectively reviewed the medical records of patients newly diagnosed with lung SCC between January 1, 2011 and December 31, 2013 in the Seoul National University Hospital. Formalin-fixed, paraffin-embedded tumor samples were used for analysis of hotspot mutations. Fifty cancer-related genes in never-smokers were compared to those in ever-smokers. Of 379 lung SCC patients, 19 (5.0%) were never-smokers. The median age of these 19 patients was 67 years (interquartile range 57–73 years), and 10 of these patients were women (52.5%). The incidence rates of stage I, II, III, and IV disease in this group were 26.4%, 5.3%, 31.6%, and 36.8%, respectively, and sequencing was performed successfully in 14 cases. In the 26 lung SCC tumor samples (12 from never-smokers and 14 from ever-smokers) sequenced using personal genome machine, the most common mutations were in TP53 (75.0%), RAS (66.7%), and STK11 (33.3%), but mutations were also found in EGFR, KIT, and PTEN. The distribution of hotspot mutations in never-smokers was similar to that in ever-smokers. There was no significant difference in overall survival between the 2 groups. The 50 hotspot mutations of lung SCC in never-smokers were similar to those of ever-smokers.

Graves' Patient with Thymic Expression of Thyrotropin Receptors and Dynamic Changes in Thymic Hyperplasia Proportional to Graves' Disease Activity

Thymic hyperplasia is frequently observed in Graves' disease. However, detectable massive enlargement of the thymus is rare, and the mechanism of its formation has remained elusive. This case showed dynamic changes in thymic hyperplasia on serial computed tomography images consistent with changes in serum thyrotropin receptor (TSH-R) antibodies and thyroid hormone levels. Furthermore, the patient's thymic tissues underwent immunohistochemical staining for TSH-R, which demonstrated the presence of thymic TSH-R. The correlation between serum TSH-R antibody levels and thymic hyperplasia sizes and the presence of TSH-R in her thymus suggest that TSH-R antibodies could have a pathogenic role in thymic hyperplasia.

The Frequency and Clinical Implications of the BRAF(V600E) Mutation in Papillary Thyroid Cancer Patients in Korea Over the Past Two Decades

BACKGROUND: Over the past several decades, there has been a rapid worldwide increase in the prevalence of papillary thyroid cancer (PTC) as well as a number of changes in the clinicopathological characteristics of this disease. BRAF(V600E), which is a mutation of the proto-oncogene BRAF, has become the most frequent genetic mutation associated with PTC, particularly in Korea. Thus, the present study investigated whether the prevalence of the BRAF(V600E) mutation has increased over the past two decades in the Korean population and whether various PTC-related clinicopathological characteristics have changed. METHODS: The present study included 2,624 patients who underwent a thyroidectomy for PTC during two preselected periods; 1995 to 2003 and 2009 to 2012. The BRAF(V600E) mutation status of each patient was confirmed using the polymerase chain reaction-restriction fragment length polymorphism method or by the direct sequencing of DNA. RESULTS: The prevalence of the BRAF(V600E) mutation in Korean PTC patients increased from 62.2% to 73.7% (P=0.001) over the last two decades. Additionally, there was a greater degree of extrathyroidal extension (ETE) and lymph node metastasis in 2009 to 2012 patients with the BRAF(V600E) mutation and a higher frequency of thyroiditis and follicular variant-PTC in 2009 to 2012 patients with wild-type BRAF. However, only the frequency of ETE was significantly higher in 1995 to 2003 patients with the BRAF(V600E) mutation (P=0.047). Long-term recurrence rates during a 10-year median follow-up did not differ based on BRAF(V600E) mutation status. CONCLUSION: The BRAF(V600E) mutation rate in Korean PTC patients has been persistently high (approximately 70%) over the past two decades and continues to increase. The present findings demonstrate that BRAF(V600E)-positive PTC was associated with more aggressive clinicopathological features, especially in patients who were recently diagnosed, suggesting that BRAF(V600E) mutation status may be a useful prognostic factor for PTC in patients recently diagnosed with this disease.

A novel prognostic factor for hepatocellular carcinoma: protein disulfide isomerase

BACKGROUND/AIMS: Protein disulfide isomerase (PDI) has been implicated in the survival and progression of some cancer cells, by compensating for endoplasmic reticulum stress by upregulating the protein-folding capacity. However, its prognostic role in patients with hepatocellular carcinoma (HCC) has not been investigated. METHODS: We collected HCC tissues from 83 HCC patients who underwent surgical resection for an immunohistochemical study of PDI. Overall survival (OS) was measured from the date of surgical resection until the date of death from any cause. Radiological progression was evaluated using the modified Response Evaluation Criteria in Solid Tumors in an independent radiological assessment. RESULTS: PDI expression was found to be increased in human HCC compared to adjacent nontumor tissues. Increased immunopositivity for PDI was associated with a high Edmondson-Steiner grade (p = 0.028). Univariate analysis of patients who had undergone surgical resection for HCC showed that tumor PDI upregulation is a significant risk factor for poor OS (p = 0.016; hazard ratio [HR], 1.980) and time to progression (TTP; p = 0.007; HR, 1.971). Multivariate analyses revealed that high PDI expression was an independent predictor of a shorter TTP (p = 0.015; HR, 1.865) and poor OS (p = 0.012; HR, 2.069). CONCLUSIONS: Upregulated PDI expression is associated with aggressive clinicopathological features of HCC; thus, PDI might serve as an independent prognostic factor and a potential therapeutic target for HCC patients.

Pharmacological Unmasking Microarray Approach-Based Discovery of Novel DNA Methylation Markers for Hepatocellular Carcinoma

DNA methylation is one of the main epigenetic mechanisms and hypermethylation of CpG islands at tumor suppressor genes switches off these genes. To find novel DNA methylation markers in hepatocellular carcinoma (HCC), we performed pharmacological unmasking (treatment with 5-aza-2'-deoxycytidine or trichostatin A) followed by microarray analysis in HCC cell lines. Of the 239 promoter CpG island loci hypermethylated in HCC cell lines (as revealed by methylation-specific PCR), 221 loci were found to be hypermethylated in HCC or nonneoplastic liver tissues. Thirty-three loci showed a 20% higher methylation frequency in tumors than in adjacent nonneoplastic tissues. Correlation of individual cancer-related methylation markers with clinicopathological features of HCC patients (n = 95) revealed that the number of hypermethylated genes in HCC tumors was higher in older than in younger patients. Univariate and multivariate survival analysis revealed that the HIST1H2AE methylation status is closely correlated with the patient's overall survival (P = 0.022 and P = 0.010, respectively). In conclusion, we identified 221 novel DNA methylation markers for HCC. One promising prognostic marker, HIST1H2AE, should be further validated in the prognostication of HCC patients.

Sequential evolution of IL-17 responses in the early period of allograft rejection

In addition to CD4+CD25+Foxp3+ regulatory T (T(reg)) cells which protect against autoimmune tissue injury, IL-17-producing CD4+ T (Th17) cells have been recently described and shown to play a crucial role in autoimmune injury. It appears that there is a reciprocal developmental pathway between Th17 and T(reg) cells. Although IL-17 is known to be associated with allograft rejection, the cellular source of IL-17 and the nature of Th17 in the context of allograft rejection remain unknown. In the current study, the dynamics of T(reg) and IL-17-producing cells after syngeneic and allogeneic transplantation were examined using a wild-type murine cardiac transplantation model. Ly6G+ cells were found to produce IL-17 during the early postoperative period and CD8+ as well as CD4+ T cells were also found to produce IL-17 during alloimmune response. Graft-infiltrating Ly6G+, CD4+, and even CD8+ cells were found to express IL-17 highly compared to those in spleen. Although the frequencies of Th17 and T(reg) were found to gradually increase in both syngeneic and allogeneic recipients, Th17/T(reg) ratios were significantly higher in recipients with allograft rejection than in syngeneic recipients. In conclusion, IL-17 is produced by neutrophils during the early postoperative period and subsequently by Th17 and CD8+ T cells during allograft rejection. Th17/T(reg) imbalance is associated with the development of allograft rejection. This study would provide basic information on Th17 biology for future investigation in the field of transplantation.

Pathologic Features of Korean Prostate Adenocarcinoma: Mapping Analysis of 83 Cases

BACKGROUND :Prostatic adenocarcinoma makes up about 2% of the total cancer incidence and cancer death in Korean men, but the incidence of this malady is continuously increasing. So far, there have been only a few studies describing the pathologic characteristics of the prostatic adenocarcinoma in Korean patients. In this study, we analyzed 83 radical prostatectomy specimens by using mapping analysis to discover the clinico pathologic characteristics of Korean prostatic adenocarcinoma. METHODS: The resected prostates were serially sectioned and embedded for histologic mapping. The clinico pathologic findings, including the Gleason score, tumor size, prostate intraepithelial neoplasia (PIN) and tumor invasion to the surrounding tissues, were examined. RESULTS: The mean values were as follows: age, 64.1+/-6.6 years; serum prostate specific antigen (sPSA), 16.6+/-16.2 ng/mL; tumor volume, 22.3+/-22.4%; tumor size, 2.2+/-1.2 cm; and Gleason score, 6.9+/-0.9. The rate of high grade PIN was 79.7%. The Gleason score, tumor extent and T stage were statistically correlated (p<0.05). CONCLUSIONS: Some prognostic factors such as sPSA and the Gleason scores showed significantly lower levels compared with those of the previous studies on Korean prostate adenocarcinoma (16-36 ng/mL vs 16.6 ng/mL and 7.3-7.7 vs 6.9, respectively). Although these values are still higher than those of the western studies, this study implies that the early detection of prostate adenocarcinoma is increasing in Korea.

Epithelial Cysts in the Intrapancreatic Accessory Spleen that Clinically Mimic Pancreatic Cystic Tumor: A Report of Two Cases

Cystic lesions in the accessory spleen are extremely rare and they present a challenging clinical differential diagnosis. We report here on two cases of epithelial cyst of intrapancreatic accessory spleen that mimicked pancreatic cystic tumor. In both cases, the patients underwent distal pancreatectomy under the impression of a benign cystic tumor of the pancreas. Unilocular or multilocular cysts in the pancreas tail were observed, and these were later shown to be epithelial cysts in the accessory spleen located within the pancreatic tail. The cysts were lined by columnar, cuboidal or stratified squamous epithelium.

Dedifferentiated Chondrosarcoma with Giant Cell-rich Sarcomatous Component Resembling Giant Cell Tumor: A Case Report

Dedifferentiated chondrosarcoma is an uncommon bone tumor, defined as a tumor in which two components -a low-grade chondrosarcoma and a high-grade non-cartilaginous sarcoma-coexist with abrupt interface. We report a rare case of giant-cell rich dedifferentiated chondrosarcoma occurred in the right distal femur shaft of a 60 year-old female. The plain X-ray film showed an irregular radiolucent mass. The T2-weighted MRI revealed a heterogeneous high signal intensity. It was an irregular mass composed of bluish-white, translucent chondroid elements and yellowish solid components with extraosseous invasion. Microscopically, a low-grade chondrosarcoma and a giant-cell rich spindle cell sarcoma with areas resembling giant cell tumor were recognized with abrupt transition. Immunohistochemical staining revealed a S100 protein positivity in chondroid cells and a few spindle cells. CD68 was strongly positive in giant cells. Vimentin was positive in both components and smooth muscle actin was positive in some spindle cells. There was no cytokeratin, desmin and myogenin immunopositivity. It is important to be aware of this rare variant of dedifferentiated chondrosarcoma to avoid the misdiagnosis of more common bone tumors including giant cell tumors.