Flow cytometric analysis for Ki67 assessment in formalin-fixed paraffin-embedded breast cancer tissue.

BACKGROUND: Pathologists commonly employ the Ki67 immunohistochemistry labelling index (LI) when deciding appropriate therapeutic strategies for patients with breast cancer. However, despite several attempts at standardizing the Ki67 LI, inter-observer and inter-laboratory bias remain problematic. We developed a flow cytometric assay that employed tissue dissociation, enzymatic treatment and a gating process to analyse Ki67 in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue. RESULTS: We demonstrated that mechanical homogenizations combined with thrombin treatment can be used to recover efficiently intact single-cell nuclei from FFPE breast cancer tissue. Ki67 in the recovered cell nuclei retained reactivity against the MIB-1 antibody, which has been widely used in clinical settings. Additionally, since the method did not alter the nucleoskeletal structure of tissues, the nuclei of cancer cells can be enriched in data analysis based on differences in size and complexity of nuclei of lymphocytes and normal mammary cells. In a clinical study using the developed protocol, Ki67 positivity was correlated with the Ki67 LI obtained by hot spot analysis by a pathologist in Japan (rho = 0.756, P < 0.0001). The number of cancer cell nuclei subjected to the analysis in our assay was more than twice the number routinely checked by pathologists in clinical settings. CONCLUSIONS: The findings of this study showed the application of this new flow cytometry method could potentially be used to standardize Ki67 assessments in breast cancer.

Aggressive cribriform-morular variant of papillary thyroid carcinoma: Report of an unusual case with pulmonary metastasis displaying poorly differentiated features.

The cribriform-morular variant of papillary thyroid carcinoma (CMV-PTC) is a rare morphologic entity in which metastasis rarely occurs. Until now, only three cases of metastasis by CMV-PTC have been reported. We present a rare sporadic case of CMV-PTC with multiple lung metastases in a 28-year-old female, 3 years after total thyroidectomy. The lung tumor was not encapsulated but well-circumscribed and showed a mixture of cribriform, papillary, and solid patterns of growth with necrosis. The tall columnar carcinoma cells did not display the typical nuclear features of PTC. Carcinoma cells were positive for thyroid transcription factor 1, paired-box gene 8, estrogen receptor, progesterone receptor, and adenomatous polyposis coli, and showed positive nuclear and cytoplasmic staining for ß-catenin. Carcinoma cells were negative for thyroglobulin and CDX-2, and the Ki-67 labeling index was 22.1%. This immunoprofile suggests a pathological diagnosis of metastasis by a CMV-PTC displaying poorly differentiated features. To the best of our knowledge, our case is the first report of CMV-PTC with pulmonary metastasis that was confirmed by histological and immunohistochemical examinations. The present case suggests that CMV-PTC with a high Ki-67 labeling index may cause visceral metastasis.

Intraductal tubulopapillary neoplasm of pancreas with stromal osseous and cartilaginous metaplasia; a case report.

Recently, a surgically resected case of intraductal tubulopapillary neoplasm (ITPN) with stromal osseous and cartilaginous metaplasia was encountered. A CT scan showed calcification at the tail of the pancreas two years before the operation. In the resected specimen, macroscopically, the main pancreatic duct was dilated and filled with a whitish solid mass without mucinous material. The tumor showed mainly a solid and papillary growth pattern. The tumor cells had no evidence of acinar differentiation. The tumor cells, at the tail of the pancreas, invaded focally to surrounding pancreatic parenchyma with stromal desmoplastic and fibrosclerotic reaction and also formed nodular stromal osseous and cartilaginous metaplasia. The tumor did not invade extrapancreatic tissue and showed no lymph node metastasis. As there were no signs of chronic calcifying pancreatitis, it is hypothesized that the metaplastic stroma was formed by a stromal reaction due to the tumor growth. It is thought, therefore, that the intraducal component of the tumor had existed at least for two years. This case suggests that ITPN is a relatively indolent tumor with a better prognosis than that of other types of invasive ductal adenocarcinoma of the pancreas.

Detection of lymph node metastasis in non-small cell lung cancer using the new system of one-step nucleic acid amplification assay.

INTRODUCTION: The prognosis of non-small cell lung cancer greatly depends on the presence of lymph node metastasis, which limits the need for surgery and adjuvant therapy for advanced cancer. One-step nucleic acid amplification of cytokeratin19 (CK19) mRNA was used to detect lymph node metastasis. Automated Gene Amplification Detector RD-200 and the LYNOAMP CK19 gene amplification reagent as components of the new one-step nucleic acid amplification system, which has increased gene amplification efficiency by improving the reagent composition, have shorter preprocessing and measurement times than conventional systems. We aimed to compare the clinical performance of the new system with that of histopathology and the conventional system. MATERIALS AND METHODS: 199 lymph nodes from 58 non-small cell lung cancer patients who underwent lymph node dissection were examined intraoperatively using the new system, conventional system, and histopathology. RESULTS: Lymph node metastasis was diagnosed in 32, 42, and 44 patients using histopathological analysis, the new system, and the conventional system, respectively. Compared with histopathological analysis, the concordance rate, sensitivity, specificity, positive predictive value, and negative predictive value of the new system were 92.0%, 90.6%, 92.2%, 69.0%, and 98.1%, respectively, and compared with the conventional system, the values were 95.0%, 86.4%, 97.4%, 90.5%, and 96.2%, respectively. CONCLUSION: The clinical performance of the new one-step nucleic acid amplification system in detecting lymph node metastasis of lung cancer is comparable to that of histopathology and the conventional system; its performance was sufficient for determining the appropriate clinical treatment. The new rapid system can be effectively utilized during lung cancer treatment intraoperatively and postoperatively.

Pembrolizumab-Induced Meningoencephalitis: A Brain Autopsy Case.

Encephalitis is very rare, but often fatal immune-related adverse event (irAE) of immune checkpoint inhibitors (ICIs). A 65-year-old Japanese woman was admitted to our hospital because of general fatigue, chillness and high-grade fever for 4 days, 8 months after the initiation of the first-line pembrolizumab monotherapy for metastatic pulmonary adenocarcinoma. On the hospital day 3, she suddenly presented delirium and uncontrollable impaired consciousness. Although the magnetic resonance imaging (MRI) did not suggest a diagnosis of encephalitis and meningitis, the spinal fluid showed abnormally elevated levels of protein (317.6 mg/L) and cell count (197 cells/µL) with increased mononuclear cells (93%). An empirical and intravenous administration of acyclovir in doses of 10 mg/kg body weight every 8 h and steroid pulse therapy in dose of 1 g/body/day from the hospital day 5 until her death failed to improve her conditions. She died on the hospital day 8. The postmortem autopsy showed viable cancer cells in the metastatic tumor in the left occipital lobe and in the spinal fluids. However, many inflammatory cells infiltration in the meninges and perivascular cuffing were prominent especially in the brain stem and medial part of the temporal lobe. Infiltrating lymphocytes in the meninges and parenchyma of the brain stem were mainly composed of cluster of differentiation (CD)8-positive lymphocytes. For irAE encephalitis, early recognition of early signs and symptoms and subsequent early therapeutic intervention are necessary. It is important for oncologists to keep in mind of the possible adverse effects of immunotherapies on the nervous system.

Pigmented spindle cell variant of a thymic atypical carcinoid in an octogenarian.

BACKGROUND: A pigmented carcinoid is an extremely rare variant of carcinoid characterized by melanin pigmentation of the tumor, with only five cases described in the literature. In addition, thymic carcinoids are rare in elderly patients and their prognosis after resection of the carcinoid tumor is unclear. CASE PRESENTATION: An anterior mediastinal tumor was incidentally found in an 82-year-old man who had been diagnosed with acute thoracic empyema. The tumor was considered most likely to be a noninvasive thymoma or thymic carcinoma for which surgery was indicated after the resolution of the empyema. The tumor was completely resected 4 months after the empyema surgery, and the patient had an uneventful postoperative course. A cut surface of the resected specimen was extensively pigmented and appeared dark-brownish, with abundant melanin pigmentation later confirmed in the spindle-shaped tumor cells. Based on the histologic examination and immunohistochemical study, melanoma was eliminated as a differential diagnosis and the tumor was diagnosed as a pigmented atypical carcinoid of the thymus. CONCLUSIONS: This report provides additional knowledge on thymic pigmented carcinoids and thymic atypical carcinoids in elderly patients.

[Progressive multifocal leukoencephalopathy in a patient with rheumatoid arthritis under salazosulfapyridine treatment].

Progressive multifocal leukoencephalopathy (PML) is a rare opportunistic infection caused by JC virus (JCV) activation. We report an 85-years old man who had been diagnosed to have rheumatoid arthritis (RA) 1.5 years prior to diagnosis of PML, and had been treated with salazosulfapyridine (SASP). He developed weakness of the left upper limb, which progressed gradually for two months. A neurological examination on admission revealed severe palsy of the left upper limb without sensory disturbance, cognitive decline or gait disturbance. Brain MRI revealed white matter lesions in the right frontal lobe around the precentral gyrus. Cerebrospinal fluid (CSF) examination and peripheral lymphocyte counts were normal. HIV was ruled out serologically. There were no findings suggestive of malignancy. We suspected PML and stopped SASP. JCV-DNA was detected in CSF. There were enlarged nuclei positive with VP-1 immunostaining in the brain biopsy materials. Thus, the diagnosis of PML was definitive. Paralysis of the left upper limb began to improve one week after discontinuing SASP. Treatment with mefloquine and mirtazapine was initiated, but he developed severe interstitial pneumonia, which might be caused by mefloquine. Therefore, he underwent rehabilitation without medication. JCV-DNA became undetectable and white matter lesions decreased 6 months later. Paralysis improved and he had no problem with activities of daily living a year later. The risk factor for PML has changed over the last decade, and drugs such as biologics became significant risk factors for patients with autoimmune diseases. There are reports suggesting that systemic lupus erythematosus (SLE) and RA themselves might be independent risk factors for PML. Although there is no previous report of SASP inducing PML, SASP might be the culprit in our case. However, there is another possibility that SAPS and RA worked synergistically for the onset of PML.

The Ebbinghaus illusion deceives adults but not young children.

The sensitivity of size perception to context has been used to distinguish between 'vision for action' and 'vision for perception', and to study cultural, psychopathological, and developmental differences in perception. The status of that evidence is much debated, however. Here we use a rigorous double dissociation paradigm based on the Ebbinghaus illusion, and find that for children below 7 years of age size discrimination is much less affected by surround size. Young children are less accurate than adults when context is helpful, but more accurate when context is misleading. Even by the age of 10 years context-sensitivity is still not at adult levels. Therefore, size contrast as shown by the Ebbinghaus illusion is not a built-in property of the ventral pathway subserving vision for perception but a late development of it, and low sensitivity to the Ebbinghaus illusion in autism is not primary to the pathology. Our findings also show that, although adults in Western cultures have low context-sensitivity relative to East Asians, they have high context-sensitivity relative to children. Overall, these findings reveal a gradual developmental trend toward ever broader contextual syntheses. Such developments are advantageous, but the price paid for them is that, when context is misleading, adults literally see the world less accurately than they did as children.

Durvalumab-Induced Diffuse Alveolar Hemorrhage: An Autopsy Case Report.

Durvalumab, a programmed cell death ligand 1 inhibitor, induces various immune-related adverse events (irAEs), including lung injury. However, diffuse alveolar hemorrhage (DAH) is a rare type of lung injury due to immune checkpoint inhibitors. A 76-year-old man with c-stage IIIA squamous cell carcinoma of the lung received maintenance durvalumab therapy after chemoradiotherapy. He developed dyspnea and malaise after 11 cycles of durvalumab. Chest computed tomography showed rapidly spreading bilateral ground-glass opacity in the lungs. We diagnosed DAH by hemosiderin-laden macrophages in bloody bronchoalveolar lavage fluid. Despite mechanical ventilation, steroids, and cyclophosphamide, he died of respiratory failure. The autopsy revealed that fresh and old bleeding areas coexisted, and neither pulmonary vasculitis nor diffuse alveolar damage was detected microscopically. Furthermore, CD3+ and CD8+ lymphocytes were observed in the lung interstitium, whereas CD20+ and CD4+ lymphocytes were scarcely detected. We report the first case of durvalumab-induced DAH. We should be alert to irAEs with DAH as a potential differential diagnosis of lung injury during durvalumab treatment.

Pembrolizumab-Induced Delayed-Onset Hepatitis.

Hepatitis is one of the serious immune-related adverse events (irAEs). However, delayed-onset hepatitis induced by immune-checkpoint inhibitors (ICIs) is rare, and the histopathological features remain to be clarified. A 65-year-old woman with advanced lung adenocarcinoma in the right upper lobe (cT4N3M1c, c-stage IVB) received four courses of pembrolizumab. Her hepatic and biliary tract enzyme levels started increasing 2 months after the final administration of pembrolizumab, and the elevated levels of these enzymes prolonged. Liver biopsy revealed panlobular infiltration of inflammatory cells, and most of the infiltrating inflammatory cells were lymphocytes; however, there were a small number of neutrophils, eosinophils, and plasma cells. There was no confluent necrosis. Furthermore, immunohistochemical analyses proved that infiltrating lymphocytes were predominantly CD3-positive (CD3+) and CD8+, and few CD20+ and CD4+ lymphocytes were observed. Based on these findings, she was diagnosed with a case of hepatitis as an irAE. Administration of prednisolone (0.5 mg/kg/day) as well as the addition of azathioprine failed to suppress the deterioration. However, an increase in the dose of prednisolone (up to 1 mg/kg/day) enabled us to control hepatitis. This case showed that hepatitis can occur even after discontinuation of ICIs, and that liver biopsy may be useful in the diagnosis. Clinicians should not hesitate to perform liver biopsy for confirmation of the diagnosis.