Multiple Immunofluorescence Imaging Analysis Reveals Differential Expression of Disialogangliosides GD3 and GD2 in Neuroblastomas.

BACKGROUND: Peripheral neuroblastic tumors (pNTs) are the most common childhood extracranial solid tumors. There are several therapeutic strategies targeting disialoganglioside GD2. Disialoganglioside GD3 has become a potential target. However, the mechanism by which pNTs express GD3 and GD2 remains unclear. We investigated the combined expression status of GD3 and GD2 in pNTs and delineated their clinicopathological values. METHODS: GD3 and GD2 expression was examined in pNT tissue samples (n = 35) using immunohistochemistry and multiple immunofluorescence imaging. RESULTS: GD3 and GD2 expression was positive in 32/35 and 25/35 samples, respectively. Combinatorial analysis of GD3 and GD2 expression in neuroblastoma showed that both were heterogeneously expressed from cell to cell. There were higher numbers of GD3-positive and GD2-negative cells in the low-risk group than in the intermediate-risk (P = 0.014) and high-risk (P = 0.009) groups. Cases with high proportions of GD3-positive and GD2-negative cells were associated with the International Neuroblastoma Staging System stage (P = 0.004), Children's Oncology Group risk group (P = 0.001), and outcome (P = 0.019) and tended to have a higher overall survival rate. CONCLUSION: We demonstrated that neuroblastomas from low-risk patients included more GD3-positive and GD2-negative cells than those from high-risk patients. Clarifying the heterogeneity of neuroblastoma aids in better understanding the biological characteristics and clinical behavior.

Combination of a synthetic retinoid and a DNA demethylating agent induced differentiation of neuroblastoma through retinoic acid signal reprogramming.

BACKGROUND: The CpG island methylator phenotype of neuroblastoma (NBL) is strongly associated with poor prognosis and can be targeted by 5-aza-2'-deoxycytidine (5-aza-dC). Differentiation therapy is a standard maintenance therapy for high-risk NBLs. However, the in vivo effect of tamibarotene, a synthetic retinoic acid, and the efficacy of its combination with 5-aza-dC have not been studied. Here, we conducted a preclinical study to assess the in vivo tamibarotene effect and the combination. METHODS: Treatment effects were analysed by in vitro cell growth and differentiation state and by in vivo xenograft suppression. Demethylated genes were analysed by DNA methylation microarrays and geneset enrichment. RESULTS: Tamibarotene monotherapy induced neural extension and upregulation of differentiation markers of NBL cells in vitro, and tumour regression without severe side effects in vivo. 5-Aza-dC monotherapy suppressed tumour growth both in vitro and in vivo, and induced demethylation of genes related to nervous system development and function. Pre-treatment with 5-aza-dC in vitro enhanced upregulation of differentiation markers and genes involved in retinoic acid signaling. Pre-treatment with 5-aza-dC in vivo significantly suppressed tumour growth and reduced the variation in tumour sizes. CONCLUSIONS: Epigenetic drug-based differentiation therapy using 5-aza-dC and TBT is a promising strategy for refractory NBLs.

Mycotic pseudoaneurysm of a pulmonary artery branch caused by Cladosporium.

We report the case of a 53-year-old male with a history of acute myelogenous leukemia, who suffered the rupturing of a right-sided pulmonary artery pseudoaneurysm combined with pneumonia. He underwent a right-sided lower lobectomy. The resected lung tissue demonstrated a mycotic pseudoaneurysm of a pulmonary artery branch together with a filamentous fungal infection. Pseudoaneurysms are caused by the breaching of all layers of a blood vessel wall. The extravasated blood is trapped by the surrounding extravascular tissue or clots. Cladosporium was detected during a polymerase chain reaction-based analysis followed by DNA sequencing of formalin-fixed paraffin-embedded lung tissue samples. Although previous cases of pulmonary artery pseudoaneurysms caused by fungal infections, e.g., Candida or Aspergillus sp., have been reported, to the best of our knowledge this is the first case to involve cladosporiosis.

Persistence of impulsivity in pediatric and adolescent patients with obsessive-compulsive disorder.

AIM: Increasing clinical evidence points to impulsivity as a symptom of obsessive-compulsive disorder (OCD). However, little is known about its persistence over time. METHODS: In this study, we evaluated the performance of 12 pediatric patients with OCD on the Stroop color-word task, which assesses impulsivity, and compared this with age- and sex-matched controls. In parallel, we measured changes in hemodynamic responses during the task, using near-infrared spectroscopy. As patients in the OCD group were naïve to treatment, we compared results before and after 3-year medication with serotonin reuptake inhibitors. RESULTS: We report that, compared with controls, the OCD group had significantly poorer performance and less activation in the prefrontal cortex during the Stroop color-word task. Surprisingly, while serotonin-reuptake-inhibitors treatment reduced OCD symptomology, it did not improve the diminished hemodynamic responses or task performance of these patients. CONCLUSION: Our findings suggest that a persistent deficit exists in the inhibitory control of pediatric patients with OCD; they also provide insight into the pathophysiology of OCD.

Event-Related Potentials Correlate with the Severity of Child and Adolescent Patients with Attention Deficit/Hyperactivity Disorder.

BACKGROUND: Attention deficit/hyperactivity disorder (ADHD) symptoms can continue through adolescence and adulthood, including difficulty in staying focused, paying attention, and controlling behavior, as well as hyperactivity. While children and adolescents with ADHD have functional impairments at multiple dimensions, there are no objective biological indicators to assess the severity of ADHD. Event-related potentials (ERPs) are widely used as a noninvasive method for evaluating sensory and cognitive processes involved in attention tasks. Previous studies have shown that P300 amplitude or latency, a main component in ERPs, is altered in patients with ADHD. However, little is known about the relationship between P300 and the severity of ADHD symptoms. METHOD: We sought to measure both P300 amplitude and latency in ERPs during auditory oddball tasks in 44 patients with ADHD (mean age ± SD 10.28 ± 3.43 years) and 15 age- and gender-matched normally developing children (11.40 ± 3.02 years). In ADHD patients, we also assessed symptom severity using the ADHD rating scale-IV-Japanese version. RESULT: In ADHD groups, P300 amplitude and latency were attenuated and prolonged compared to controls at the frontocentral, centroparietal, and parietal positions. Furthermore, levels of P300 latency at these positions are positively correlated with the inattention subscale scores measured by the ADHD rating scale-IV-Japanese version. CONCLUSIONS: The present study revealed that the degree of P300 latency might reflect the severity of ADHD symptoms with children and adolescents, suggesting that ERPs are a useful technique to evaluate the severity of ADHD symptoms.

Early onset of cardiomyopathy and intellectual disability in a girl with Danon disease associated with a de novo novel mutation of the LAMP2 gene.

Danon disease, primary lysosome-associated membrane protein-2 (LAMP-2) deficiency, is characterized clinically by cardiomyopathy, myopathy and intellectual disability in boys. Because Danon disease is inherited in an X-linked dominant fashion, males are more severely affected than females, who usually have only cardiomyopathy without myopathy or intellectual disability; moreover, the onset of symptoms in females is usually in adulthood. We describe a girl with Danon disease who presented with hypertrophic cardiomyopathy and Wolff-Parkinson-White (WPW) syndrome at 12 years of age. Subsequently, she showed signs of mild learning disability and intellectual disability on psychological examinations. She had a de novo novel mutation in the LAMP-2 gene and harbored an identical c.749C > A (p.Ser250X) variant, resulting in a stop codon in exon 6. She showed decreased, but not completely absent LAMP-2 expression on immunohistochemical and Western blot analyses of a skeletal muscle biopsy specimen, which has been suggested to be caused by a 50% reduction in LAMP-2 expression (LAMP-2 haploinsufficiency) in female patients with Danon disease caused by a heterozygous null mutation. To our knowledge, our patient is one of the youngest female patients to have been given a diagnosis of Danon disease. In addition, this is the first documented case in a girl that was clearly associated with intellectual disability, which is very rare in females with Danon disease. Our findings suggest that studies of female patients with Danon disease can extend our understanding of the clinical features of this rare disease.

Immunohistochemical co-expression status of cytokeratin 5/6, androgen receptor, and p53 as prognostic factors of adjuvant chemotherapy for triple negative breast cancer.

Triple negative breast cancer (TNBC) is immunohistochemically characterised by the lack of expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor type 2 (HER2). TNBC is known for its poor prognosis and high recurrence probability. There is no effective targeted treatment for TNBC, but only adjuvant chemotherapies. There are two TNBC subtypes, basal-like and non-basal-like, which are defined based on positive cytokeratin (CK) 5/6 and/or epidermal growth factor receptor (EGFR) expression. In particular, CK5/6 expression is reported to correlate with TNBC recurrence. TNBC lacks ER-α expression, but some TNBCs are known to express the androgen receptor (AR). Moreover, although p53 accumulation is detected in various malignant tumors, its influence on adjuvant chemotherapy for patients with TNBC remains unclear. The aim of this study was to assess the combined immunohistochemical expression of CK 5/6, AR, and p53 as a potential prognostic marker of adjuvant chemotherapy for patients with TNBC. The expression of CK5/6, AR, and p53 in formalin-fixed and paraffin-embedded (FFPE) surgical sections from 52 patients with TNBC was analysed by immunohistochemistry (IHC) and the co-expression patterns in individual cells were investigated by immunofluorescent (IF) staining. Low AR expression was correlated with high clinical stage (P < 0.05) and low nuclear grade (P < 0.05). The expression of CK5/6 and p53 did not correlate with clinicopathological features. Patients who needed adjuvant chemotherapy presented the worst prognosis. In particular, when the IHC expression pattern was CK5/6 (-), AR (-), and p53 (+), the disease free survival (DFS) and overall survival (OS) were the worst. On the other hand, patients with AR (+) and p53 (-) TNBC presented a good prognosis. The analysis of the co-expression status of these three markers showed that no cells presented both AR and CK5/6 expression. Furthermore, TP53 mRNA expression was higher in patients with AR-negative TNBC (P < 0.05) and in patients with the worst prognosis (P < 0.05) than in the other patients. These results suggested that, in patients with CK5/6-negative TNBC, AR expression correlated with good prognosis, but p53 accumulation correlated with poor prognosis. The present IHC markers allowed us to predict the post-surgery prognosis of patients with TNBC. In conclusion, TNBCs are heterogeneous. Patients with the CK5/6 (-), AR (-), and p53 (+) TNBC subtype, evaluated by IHC, presented the worst prognosis. These IHC markers will be helpful to follow patients with TNBC.

Increased phosphorylation of ERK1/2 is associated with worse chemotherapeutic outcome and a poor prognosis in advanced lung adenocarcinoma.

Constitutive activation of extracellular signal-regulated kinase (ERK)1/2 pathway, that is activated by various stimuli including growth factors and oncogenic driver mutations, is observed in various cancers. However, the difference of the activated levels of the pathway is still unclear in clinical significances. The aim of this study was to investigate the effect of different ERK1/2 pathway activation, assessed by the expression levels of phosphorylated (p) ERK1/2, on the prognosis of advanced lung adenocarcinoma patients. Paraffin-embedded lung biopsy samples were obtained from 85 lung adenocarcinoma patients. Correlation between pERK1/2 expression levels that were assessed by immunohistochemistry (IHC) analysis and oncogenic driver mutation status, clinicopathological factors, outcome from standard anticancer therapies, and prognosis was investigated. Varying levels of pERK1/2 expression were observed in 68 (80.0 %) patients. The overall survival was significantly reduced in patients with higher pERK1/2 expression in comparison to those with lower expression levels (P = 0.03). In particular, higher pERK1/2 expression levels correlated with worse performance status and worse clinical outcome. Thus, the IHC analysis of pERK1/2 expression levels may predict patient prognosis in advanced lung adenocarcinoma. Inhibition of ERK1/2 pathway activated by various signals may improve the effects of standard chemotherapies and the clinical condition of patients with advanced cancer.

Altered intracellular region of MUC1 and disrupted correlation of polarity-related molecules in breast cancer subtypes.

MUC1 glycoprotein is overexpressed and its intracellular localization altered during breast carcinoma tumorigenesis. The present study aimed to clarify the relationship of cytoplasmic localization of MUC1 with the breast cancer subtype and the correlation of 10 molecules associated with cell polarity in breast cancer subtypes. We immunostained 131 formalin-fixed and paraffin-embedded breast cancer specimens with an anti-MUC1 antibody (MUC1/CORE). For 48 of the 131 tumor specimens, laser-assisted microdissection and real-time quantitative RT-PCR were performed to analyze mRNA levels of MUC1 and 10 molecules, ß-catenin, E-cadherin, claudin 3, claudin 4, claudin 7, RhoA, cdc42, Rac1, Par3 and Par6. Localization of MUC1 protein varied among breast cancer subtypes, that is, both the apical domain and cytoplasm in luminal A-like tumors (P < 0.01) and both the cytoplasm and cell membrane in luminal B-like (growth factor receptor 2 [HER2]+) tumors (P < 0.05), and no expression was found in triple negative tumors (P < 0.001). Estrogen receptor (ER)+ breast cancers showed higher MUC1 mRNA levels than ER- breast cancers (P < 0.01). The incidence of mutual correlations of expression levels between two of the 10 molecules (55 combinations) was 54.5% in normal breast tissue and 38.2% in luminal A-like specimens, 16.4% in luminal B-like (HER2+), 3.6% in HER2 and 18.2% in triple negative specimens. In conclusion, each breast cancer subtype has characteristic cytoplasmic localization patterns of MUC1 and different degrees of disrupted correlation of the expression levels between the 10 examined molecules in comparison with normal breast tissue.

Involvement of Chemokines and a CD4-Positive T Cell Subset in the Development of Conjunctival Secondary Lymphoid Follicles in an Atopic Keratoconjunctivitis Mouse Model.

BACKGROUND: Massive B cell lymphoid hyperplasia and its associated factors may play a role in exacerbating inflammation in allergic disorders. We here investigated the chemokines and CD4-positive T cell subset involved in the development of secondary lymphoid follicles (iCALT) in conjunctival tissues in an atopic keratoconjunctivitis mouse model (AKC mouse). METHODS: NC/Nga mice were divided into three groups: AKC (percutaneous sensitization and instillation of crude house dust mite antigen), AD (percutaneous sensitization only) and C (untreated control). Pathological changes in the conjunctival tissues of each group were investigated using histological and immunohistochemical detection of CD4 and CD20. Furthermore, tissue sections of iCALT (AKC-iCALT subgroup) and conjunctiva without iCALT (AKC-conjunctiva subgroup) were obtained from AKC mice using laser-assisted microdissection. mRNA expression of chemokine and T cell subset-related transcription factors were compared between the AKC-iCALT and AKC-conjunctiva subgroups using polymerase chain reaction (PCR) array and real-time reverse transcription-PCR (RT-PCR) methods. RESULTS: iCALT with central aggregation of CD20-positive B cells and CD4-positive T cell infiltration surrounding B cells was observed in the palpebral conjunctival tissue of the AKC group, but not in that of the AD and C groups. Chemokine and T cell subset-related transcription factor expression was confirmed using real-time RT-PCR, with significant increases in Ccl5, Ccl17, Cxl20, Cxcl3, Ccr7, Foxp3 and T-bet mRNA expression in the AKC-iCALT subgroup compared with those in the AKC-conjunctiva subgroup. CONCLUSIONS: We concluded that CCL5, CCL17 and CCL20, as well as T-bet- and Foxp3-positive lymphocytes may be iCALT-related factors and that iCALT-related chemokines are worth evaluating as biomarkers.

Prefrontal dysfunction in pediatric Tourette's disorder as measured by near-infrared spectroscopy.

BACKGROUND: Tourette's disorder (TD) is a chronic childhood-onset disorder characterized by the presence of multiple motor and vocal tics. Despite strong evidence that the pathophysiology of TD involves structural and functional disturbances of the basal ganglia and cortical frontal areas, in vivo imaging studies have produced conflicting results. Recent developments in near-infrared spectroscopy (NIRS) technology have enabled noninvasive assessment of brain function in people with psychiatric disorders. METHODS: We asked 10 individuals with pediatric TD and 10 healthy controls who were age- and sex- matched to perform the Stroop color-word task during NIRS. We used prefrontal probes and a 24-channel NIRS machine to measure the relative concentrations of oxyhemoglobin (oxy-Hb) every 0.1 s during the task. RESULTS: We found that oxy-Hb changes in the prefrontal cortex were significantly smaller in the TD group compared with the control group, especially in the left dorsolateral prefrontal cortex. CONCLUSIONS: Our data suggest that individuals with pediatric TD have a reduced prefrontal hemodynamic response as measured by NIRS.

Overexpression of PGC1α and accumulation of p62 in apocrine carcinoma of the breast.

Apocrine carcinoma is categorized as a special type of breast carcinoma because of its specific morphological features. To clarify the characteristics of apocrine carcinoma from the point of view of the mitochondrial profile, we conducted a comparative study between apocrine and non-apocrine carcinomas. The expressions of mitochondrial related factors (PGC1α, Nrf1, Nrf2, mtTFA and COX4) were examined in a testing set of breast cancer tissue. Apocrine carcinomas showed a clear tendency towards higher mRNA expression levels of PGC1α than non-apocrine carcinomas. The expression of the selected factor, PGC1α, as well as that of p62 was further examined. The results revealed that apocrine carcinomas showed a higher immunohistochemical positivity rate for PGC1α (21.3% vs. 3.2%; P = 0.008), and that the mRNA expression level of PGC1α was significantly higher in apocrine carcinoma than in non-apocrine carcinoma (P = 0.007). The immunohistochemical positivity rate for p62 protein was also higher in apocrine carcinomas (44.7% vs. 21.0%; P = 0.015), although no significant difference in the p62 mRNA expression level was detected between the two types of carcinoma (P = 0.633). In conclusion, this study revealed that apocrine carcinoma overexpressed PGC1α contributing to mitochondrial biogenesis, and also p62 protein accumulation.

Increased prefrontal hemodynamic change after atomoxetine administration in pediatric attention-deficit/hyperactivity disorder as measured by near-infrared spectroscopy.

AIM: Atomoxetine, approved in Japan for the treatment of pediatric attention-deficit/hyperactivity disorder (ADHD) in April 2009, is a nonstimulant that is thought to act presynaptically via the inhibition of norepinephrine reuptake. Near-infrared spectroscopy is a non-invasive optical tool that can be used to study oxygenation and hemodynamic changes in the cerebral cortex. The present study examined the effects of a clinical dose of atomoxetine on changes in prefrontal hemodynamic activity in children with ADHD, as measured by near-infrared spectroscopy using the Stroop Color-Word Task. METHODS: Ten children with ADHD participated in the present study. We used 24-channel near-infrared spectroscopy to measure the relative concentrations of oxyhemoglobin in the frontal lobes of participants in the drug-naïve condition and those who had received atomoxetine for 8 weeks. Measurements were conducted every 0.1 s during the Stroop Color-Word Task. We used the ADHD Rating Scale-IV-Japanese version (Home Version) to evaluate ADHD symptoms. RESULTS: We found a significant decrease in ADHD Rating Scale-IV-Japanese version scores, from 30.7 to 22.6 (P=0.003). During the Stroop Color-Word Task, we found significantly higher levels of oxyhemoglobin changes in the prefrontal cortex of participants in the atomoxetine condition compared with those in the drug-naïve condition. CONCLUSIONS: This increase in oxyhemoglobin changes might indicate an intensified prefrontal hemodynamic response induced by atomoxetine. Near-infrared spectroscopy is a sensitive tool for measuring the pharmacological effects of atomoxetine in children with ADHD.

Proximal tubule morphology in rats with renal congestion: a study involving the in vivo cryotechnique.

The present study aimed to examine the changes induced in proximal tubules by renal congestion using the in vivo cryotechnique (IVCT). Twelve male Wistar rats were divided into four equal groups: group 1 (the control); groups 2 and 3, which were subjected to 2 and 5 min of congestion, respectively; and group 4, which was subjected to 5 min of congestion followed by 10 min of recirculation. Under anesthesia, renal congestion was induced in the bilateral kidneys by ligating the inferior vena cava just above the branching renal veins. The left kidneys, which were subjected to the IVCT, were then compared with the right kidneys, which underwent a conventional fixation method. Among the left kidneys, the proximal tubules in group 1 consisted of cuboidal cells and had open lumina. In the congestive groups, the diameters of the proximal tubules were increased, and their lumina were obstructed by swollen cells and ischemia-associated cell debris. In group 4, the proximal tubules were still dilated, as seen in the congestive groups; however, the swollen cells had recovered their cuboidal form, and the cell debris had disappeared from the tubules' lumina. The present study demonstrated the in vivo morphology of proximal tubules in living rats subjected to congestion, which was unclear using conventional fixation methods.

AKR1C3-negative high-risk metastatic castration-sensitive prostate cancer has long-term response to first-line treatment with abiraterone: Four case reports.

We experienced four cases of high-risk metastatic castration-sensitive prostate cancer (mCSPC) in which first-line treatment with abiraterone showed a sustained long-term response of over 5 years. We conducted immunohistochemical staining of aldo-keto reductase family 1 member C3 (AKR1C3) expression, which associate with poor prognosis of metastatic castration-resistant prostate cancer (mCRPC), and all prostate cancer tissue from four cases showed negative. These results suggested that AKR1C3-negative high-risk mCSPC cases may respond well to first-line treatment with abiraterone. This is the first report describing association of high-risk mCSPC and negative AKR1C3.

Next-generation sequencing of primary testicular lymphoma and relapse in the glans penis after prophylactic radiation therapy: a rare case report.

BACKGROUND: Primary testicular lymphoma (PTL) is relatively rare. The contralateral testis is a common site of PTL relapse; therefore, once complete remission is achieved, radiation therapy (RT) is administered to the contralateral testis to prevent relapse. CASE PRESENTATION: A 76-year-old man was diagnosed with PTL and received RT as described above. However, despite achieving and maintaining complete remission, a mass diagnosed as diffuse large B-cell lymphoma by tissue biopsy developed in the glans penis 6.5 years after prophylactic RT. We investigated whether the glans penile lymphoma was PTL relapse or a new malignancy by genomic analysis using next-generation sequencing of DNA extracted from two histopathological specimens. CONCLUSIONS: We found the same variant allele fraction in four somatic genes (MYD88, IL7R, BLNK, and FLT3) at similar frequencies, indicating that the glans penile lymphoma had the same origin as the PTL. To the best of our knowledge, this is the first case report of PTL relapse in the glans penis.

Factors Associated with Early and Late Seizure Related to Aneurysmal Subarachnoid Hemorrhage.

Post-stroke epilepsy may occur after aneurysmal subarachnoid hemorrhage (aSAH). Both early and late seizures could cause severe neurocognitive deficits if administration of appropriate antiseizure medication is delayed. Therefore, it is important to elucidate the risk factors for early and late seizures, which could be shared with medical teams to promptly manage seizures. There are aspects of both hemorrhage and ischemia in aSAH, and thus, numerous risk factors are considered for early and late seizures. We examined factors associated with aSAH-related early and late seizures. Among 297 patients who had aSAH and underwent direct or endovascular surgery, 25 had early seizures and 20 had late seizures. Patients who did not experience any seizures in at least 2-years of follow-up (n = 81) were used as the control group. Early seizures were associated with older age and acute severe nonneurological infection, whereas late seizures were associated with intraparenchymal lesion volume >10 mL and shunt placement. In patients with late seizures, consistency was frequently observed between electroencephalogram and the presence of intraparenchymal lesions. The frontopolar electrode on electroencephalogram was highly sensitive to abnormality in early seizures. Early seizures were induced by the patient's systemic factors, which may lower the threshold for cortical excitability. Patients with intraparenchymal lesions who undergo shunt placement should be carefully followed up for late seizures.

Immunohistochemical and molecular profiles of heterogeneous components of metaplastic breast cancer: a squamous cell carcinomatous component was distinct from a spindle cell carcinomatous component.

Metaplastic breast carcinoma (MBC), a category of breast cancer, includes different histological types, which are occasionally mixed and heterogeneous. Considering the heterogeneity of cancer cells in a tumour mass has become highly significant, not only from a biological aspect but also for clinical management of recurrence. This study aimed to analyse the immunohistochemical and molecular profiles of each MBC component of a tumour mass. Twenty-five MBC tumours were histologically evaluated, and the most frequent MBC component (c) was squamous cell carcinoma (SCC), followed by spindle cell carcinoma (SpCC). A total of 69 components of MBC and non-MBC in formalin-fixed paraffin-embedded sections were examined for 7 markers by immunohistochemistry. SCC(c) were significantly PTEN negative and CK14 positive, and SpCC(c) were significantly E-cadherin negative and vimentin positive. Multivariate analyses revealed that immunohistochemical profiles of normal/intraductal (IC)(c), no special type (NST)(c), and MBC(c) differed; moreover, SCC(c) and SpCC(c) were distinctly grouped. PTEN gene mutation was detected only in SCC(c) (2/7), but not in SpCC(c). Next-generation sequence analyses for 2 cases with tumours containing SCC(c) demonstrated that PTEN gene mutation increased progressively from IC(c) to NST(c) to SCC(c). In conclusion, the immunohistochemical and molecular profiles of the SCC(c) of MBC are distinct from those of the SpCC(c).

Complex phenotypic heterogeneity of combined hepatocellular-cholangiocarcinoma with a homogenous TERT promoter mutation.

To clarify the mechanism underlying the development and poor prognosis of combined hepatocellular-cholangiocarcinoma (cHCC-CCA), we characterized liver cancer driver mutations and poor prognostic markers in both the HCC and intrahepatic CCA (iCCA) components of a cHCC-CCA tumor. The telomerase reverse transcriptase (TERT) promoter mutation C228T was quantified by digital polymerase chain reaction using DNA from multiple microdissected cancer components of a single cHCC-CCA nodule. The protein expression of cancer-related markers, including TERT, was examined by serial thin-section immunohistochemistry and double-staining immunofluorescence. TERT promoter mutation and TERT protein expression were detected in all cancer components but not in noncancer regions. TERT promoter mutation frequencies were similar among components; those of TERT protein-positive cancer cells were higher in iCCA and mixed components than in HCC. The frequencies of Ki67- and p53-positive cells were similarly higher in iCCA and mixed components than in HCC. However, double-positive cells for the three proteins were unexpectedly rare; single-positive cells dominated, indicating phenotypic microheterogeneity in cancer cells within a component. Interestingly, HCC and CCA marker protein immunohistochemistry suggested dedifferentiation of HCC and transdifferentiation from HCC to iCCA in HCC and iCCA components, respectively. Such phenotypic intercomponent heterogeneity and intracomponent microheterogeneity were detected in a tumor nodule of cHCC-CCA uniformly carrying the early HCC driver mutation. Moreover, poor prognostic markers were randomly expressed without a regular pattern, consistent with the poor prognosis.

Correlation between pathology and quantitative ultrasonographic evaluation of pancreatic fat with ultrasonographic classification.

PURPOSE: No studies of the relationship between grayscale sonographic findings and pancreatic fat content have been reported to date. This study aimed to investigate the correlation between echogenicity and fat content of resected specimens using quantitative analysis. METHODS: Forty-two consecutive patients who underwent pancreatoduodenectomy or distal pancreatectomy for pancreatic tumors were enrolled in this study. Ultrasonographic images were compared with quantitative pathological analysis. Subjective evaluation of echogenicity was classified as hypoechoic, isoechoic, hyperechoic, and super hyperechoic. The total and intralobular fat areas were measured. RESULTS: The mean, median, modal, minimum, and maximum ultrasound gray values correlated with the proportion of total fat area (r = 0.349; 0.357, 0.486, 0.466, and 0.347; p = 0.024, 0.020, 0.014, 0.019, and 0.089, respectively), but did not correlate with the proportion of intralobular fat area. Subjective classification was correlated with median gray value (p < 0.001), intralobular fat area (p = 0.118), and total fat area (p = 0.011). Cases were classified as hypoechoic (n = 3), isoechoic (n = 7), hyperechoic (n = 30), and super hyperechoic (n = 2). The subjective classification was correlated with the median gray value (p < 0.001) and total fat area (p = 0.005), and not correlated with the intralobular fat area (p = 0.118). Hyperechoic or super hyperechoic pancreatic parenchyma contains over 19.7% fat. Computed tomography values correlated with the proportion of intralobular fat area (r = - 0.479, p = 0.004) and total fat area (r = - 0.541, p < 0.001). CONCLUSION: Echogenicity classified based on subjective evaluation and image analysis were correlated with the proportion of fat in the pancreas.