MAPK pathway activation and the origins of pediatric low‐grade astrocytomas

Low‐grade astrocytomas (LGAs) are the most common type of brain tumor in children. Until recently, very little was known about the underlying biology and molecular genetics of these tumors. However, within the past year a number of studies have shown that the MAPK pathway is constitutively activated in a high proportion of LGAs. Several genetic aberrations which generate this deregulation of the MAPK pathway have been identified, most notably gene fusions between KIAA1549 and BRAF. In this review we summarize these findings, discuss how these gene fusions may arise and consider possible implications for diagnosis and treatment. J. Cell. Physiol. 222: 509–514, 2010. © 2009 Wiley‐Liss, Inc.     

Pathophysiological roles of chronic low‐grade inflammation mediators in polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is the most common hormonal imbalance disease in reproductive‐aged women. Its basic characteristics are ovulatory dysfunction and ovarian overproduction of androgens that lead to severe symptoms such as insulin resistance, hirsutism, infertility, and acne. Notwithstanding the disease burden, its underlying mechanisms remain unknown, and no causal therapeutic exists. In recent years, further studies showed that inflammation processes are involved in ovulation and play a key role in ovarian follicular dynamics. Visceral adipose tissue can cause inflammatory response and maintenance of the inflammation state in adipocytes by augmented production of inflammatory cytokines, monocyte chemoattractant proteins, and recruitment of the immune cell. Therefore, the PCOS can be related to a low‐grade inflammation state and inflammatory markers. Investigating the inflammatory processes and mediators that contribute to the commencement and development of PCOS can be a critical step for better understanding the pathophysiology of the disease and its treatment through inhibition or control of related pathways. In the present review, we discuss the pathophysiological roles of chronic low‐grade inflammation mediators including inflammasome‐related cytokines, interleukin‐1β (IL‐1β), and IL‐18 in PCOS development.     

Atypical squamous cells and low‐grade squamous intraepithelial lesion in cervical cytology: cytohistological correlation and implication for management in a low‐resource setting

N. Gupta, R. Srinivasan, R. Nijhawan, A. Rajwanshi, P. Dey, V. Suri and L. Dhaliwal Atypical squamous cells and low‐grade squamous intraepithelial lesion in cervical cytology: cytohistological correlation and implication for management in a low‐resource setting Objectives: To perform an audit of all cervical smears reported as atypical squamous cells (ASC) and low‐grade squamous intraepithelial lesion (LSIL) as in the Bethesda system (TBS) 2001, and determine their histological follow‐up and outcome when available, in order to define the threshold for colposcopic referral. Material and methods: A total of 25 203 cervical smears were screened over a period of 3 years (January 2006 – December 2008) and all ASC and LSIL smears were reviewed with the corresponding histological follow‐up. All cervical intraepithelial neoplasia (CIN) grade 2 lesions and above (CIN2+) were considered as clinically significant lesions for analysis. Results: Out of 25 203 cervical smears, 424 (1.7%) were reported as ASC and 113 (0.4%) as LSIL. Additionally, three were reported as atypical cells, not otherwise specified. The ASC : SIL ratio was 2.18 : 1. Follow‐up histology was available in 153 (36.8%) of the ASC cases and revealed CIN2+ lesions in 22 (14.4%). Follow‐up histology was available in 50 (44.2%) of LSIL cases and revealed clinically significant abnormalities in five (10%), all of which were CIN2. CIN3 and invasive squamous carcinomas were seen in 5.9% and 1.4%, respectively, of cases of ASC, and not seen in LSIL. Reclassification of ASC smears into ASC‐US (ASC‐undetermined significance) and ASC‐H (ASC‐ high grade SIL not excluded) revealed ASC‐H in 2.6% of all ASC smears, with a clinically significant outcome in 45.4%. Conclusion: In a low‐resource setting where human papillomavirus testing is unaffordable, the threshold for colposcopic referral and follow‐up histology should be ASC rather than SIL.     

Potent immunogenicity in BRCA1‐mutated patients with high‐grade serous ovarian carcinoma

High‐grade serous ovarian carcinomas (HGSOCs) were among the tumours with an unsatisfactory outcome of immune checkpoint inhibitors (ICIs). It is imperative to develop feasible biomarker for identifying responsive candidates and guiding precise immunotherapy for HGSOC patients. Here, we analysed genomic data of patients with HGSOCs to depict their immunological phenotype of tumour microenvironment (TME) and figure out the major determinants of immunogenicity. In comparison with other solid tumours, we observed the lowest levels of PD‐L1, total mutation burden (TMB) and cytolytic molecules in HGSOCs. Surprisingly, TMB is not certainly positively related to tumour immune response as it failed to predict the response to ICIs in a considerable portion of patients in previous clinical trials. By a machine learning approach in search of biomarkers for immunotherapy implications for HGSOCs, we identified the ten most dominant factors determining the immunogenicity of HGSOCs. Interestingly, we found that BRCA1 mutated tumours presented a potent immunogenic phenotype, independent of TMB, meeting the criteria of both our dominant factors and the determinants of immunogenicity established before. Our findings provide evidence that BRCA1‐mutation may be served as a predictive biomarker in guiding ICI therapies for the patients with HGSOCs.     

Characterization of low‐abundance species in the active pharmaceutical ingredient of CIGB‐300: A clinical‐grade anticancer synthetic peptide

CIGB‐300 is a first‐in‐class synthetic peptide‐based drug of 25 amino acids currently undergoing clinical trials in cancer patients. It contains an amidated disulfide cyclic undecapeptide fused to the TAT cell‐penetrating peptide through a beta‐alanine spacer. CIGB‐300 inhibits the CK2‐mediated phosphorylation leading to apoptosis of tumor cells in vitro, and in vivo in cancer patients. Despite the clinical development of CIGB‐300, the characterization of peptide‐related impurities present in the active pharmaceutical ingredient has not been reported earlier. In the decision tree of ICHQ3A(R2) guidelines, the daily doses intake, the abundance, and the identity of the peptide‐related species are pivotal nodes that define actions to be taken (reporting, identification, and qualification). For this, purity was first assessed by reverse‐phase chromatography (>97%) and low‐abundance impurities (≤0.27%) were collected and identified by mass spectrometry. Most of the impurities were generated during peptide synthesis, the spontaneous air oxidation of the reduced peptide, and the lyophilization step. The most abundant impurity, with no biological activity, was the full‐length peptide containing Met¹⁷ transformed into a sulfoxide residue. Interestingly, parallel and antiparallel dimers of CIGB‐300 linked by 2 intermolecular disulfide bonds exhibited a higher antiproliferative activity than the CIGB‐300 monomer. Likewise, very low abundance trimers and tetramers of CIGB‐300 linked by disulfide bonds (≤0.01%) were also detected. Here we describe for the first time the presence of active dimeric species whose feasibility as novel CIGB‐300 derived entities merits further investigation.     

Identification of novel biomarker candidates for immunohistochemical diagnosis to distinguish low‐grade chondrosarcoma from enchondroma

Chondrosarcoma is the third most common primary bone cancer, requiring surgical resection. However, differentiation of low‐grade chondrosarcoma (grade 1) from enchondroma that is benign and only requires regular follow‐up is one of the most frequent diagnostic dilemmas facing orthopedic oncologists in clinical management. Although multiple techniques are applied to make the distinction, immunohistochemistry is an important ancillary technique, especially when a histopathological stain of specimen must be obtained in order to guarantee an accurate confirmation. Currently, no adequate immunohistochemical diagnostic protein biomarkers are available to distinguish low‐grade chondrosarcoma from enchondroma. To discover novel protein biomarker candidates, an LC‐MS/MS approach was applied to directly compare formalin‐fixed, paraffin‐embedded low‐grade chondrosarcoma with enchondroma tissue samples. The proteomics analysis revealed 17 protein biomarker candidates. A principle was developed to prioritize the candidates using category and ranking. An algorithm, prioritization index of biomarker candidates for immunohistochemistry on tissue specimens, was developed to rank the candidates inside each category. Using the proteomics data and bioinformatics results, the prioritization index of biomarker candidates for immunohistochemistry on tissue revealed periostin as a top candidate. Immunohistochemical staining of periostin in 23 low‐grade chondrosarcoma and 31 enchondroma tissue specimens disclosed 87% specificity and 70% sensitivity.     

Value of computer‐assisted quantitative nuclear morphometry for differentiation of reactive renal tubular cells from low‐grade urothelial carcinoma

H. Ohsaki, E. Hirakawa, K. Kagawa, M. Nakamura, H. Kiyomoto and R. Haba Value of computer‐assisted quantitative nuclear morphometry for differentiation of reactive renal tubular cells from low‐grade urothelial carcinoma Objective: To assess whether computer‐assisted quantitative morphological parameters can be an effective tool for objectively distinguishing reactive renal tubular cells from low‐grade urothelial carcinoma cells (LG‐UCs) in voided urine. Methods: Nuclear morphometry was performed by a computer‐assisted image analyser system on Papanicolaou‐stained cytological specimens. The circumference of reactive renal tubular cells (n = 40) or LG‐UC (n = 20) nuclei were manually traced, and the following nuclear morphometric parameters were analysed: (i) area, (ii) perimeter, (iii) roundness factor, (iv) maximum length, and (v) linear factor. For each nuclear measurement, we calculated the maximum, minimum, mean and standard deviation. Results: The mean nuclear area and nuclear perimeter were higher in reactive renal tubular cells compared to the LG‐UCs. The mean of roundness and linear factors (reflecting a tendency for the nuclear outline to be regular and oval, respectively) were higher in LG‐UCs compared with reactive renal tubular cells. Among nuclear areas, the nuclear perimeter, roundness factors and maximum length did not show any significant differences between reactive renal tubular cells and LG‐UCs. On the other hand, the linear factor showed a mean higher value among LG‐UCs than reactive renal tubular cells (P = 0.023). Conclusions: Of five quantitative nuclear morphological parameters, only linear factor was statistically significant in differentiating reactive renal tubular cells in renal disease from LG‐UCs.     

Recurrent adaptation in a low‐dispersal trait

The study of natural populations from contrasting environments has greatly enhanced our understanding of ecological‐dependent selection, adaptation and speciation. Cases of parallel evolution in particular have facilitated the study of the molecular and genetic basis of adaptive variation. This includes the type and number of genes underlying adaptive traits, as well as the extent to which these genes are exchanged among populations and contribute repeatedly to parallel evolution. Yet, surprisingly few studies provide a comprehensive view on the evolutionary history of adaptive traits from mutation to widespread adaptation. When did key mutations arise, how did they increase in frequency, and how did they spread? In this issue of Molecular Ecology, Van Belleghem et al. ( 2015 ) reconstruct the evolutionary history of a gene associated with wing size in the salt marsh beetle Pogonus chalceus. Screening the entire distribution range of this species, they found a single origin for the allele associated with the short‐winged ecotype. This allele seemingly evolved in an isolated population and rapidly introgressed into other populations. These findings suggest that the adaptive genetic variation found in sympatric short‐ and long‐winged populations has an allopatric origin, confirming that allopatric phases may be important at early stages of speciation.     

Can cytological features differentiate reactive renal tubular cells from low‐grade urothelial carcinoma cells?

H. Ohsaki, E. Hirakawa, Y. Kushida, S. Yokoshita, M. Nakamura, H. Kiyomoto and R. Haba Can cytological features differentiate reactive renal tubular cells from low‐grade urothelial carcinoma cells? Objective: To compare the cytomorphological and immunocytochemical features of reactive renal tubular cells and low‐grade urothelial carcinoma cells (LG‐UCs). Methods: We examined 15 cytological parameters in 38 cases with reactive renal tubular cells in renal disease and 20 cases of LG‐UCs from bladder cancer that had been diagnosed by histological examination. Voided urine cytological parameters evaluated were as follows: (i) maximum cell numbers of clusters, (ii) cannibalism, (iii) rosette‐like arrangement, (iv) hobnail‐shaped cells, (v) vacuolated cytoplasm, (vi) intracytoplasmic haemosiderin, (vii) irregular nuclear contours, (viii) chromatin pattern, (ix) prominent nucleoli, (x) cast encasement, (xi) casts, (xii) dysmorphic erythrocytes, (xiii) isomorphic erythrocytes, (xiv) necrosis, and (xv) vimentin reactivity. The above parameters were determined using Mann–Whitney U‐test and chi‐square test, with differences considered significant at P < 0.05. Results: In reactive renal tubular cells, low to moderate cell numbers of clusters (fewer than 50 cells), rosette‐like arrangement, hobnail‐shaped cells, vacuolated cytoplasm, intracytoplasmic haemosiderin, euchromatin pattern, prominent nucleoli, dysmorphic erythrocytes and vimentin reactivity were present in significantly higher proportions compared with those in LG‐UCs. In LG‐UCs, high cell numbers of clusters (50 cells or more), cannibalism, heterochromatin pattern, isomorphic erythrocytes and necrosis were seen in significantly higher proportions. No significant differences were observed in irregular nuclear contours, cast encasement or casts. Conclusions: Based on results of the present study, maximum cell numbers of clusters, cannibalism, rosette‐like arrangement, hobnail‐shaped cells, vacuolated cytoplasm, intracytoplasmic haemosiderin, chromatin pattern, prominent nucleoli, dysmorphic erythrocytes, isomorphic erythrocytes, necrosis, and vimentin reactivity were capable of distinguishing reactive renal tubular cells from LG‐UCs.     

Identification of stably expressed reference small non‐coding RNAs for microRNA quantification in high‐grade serous ovarian carcinoma tissues

MicroRNAs (miRNAs) belong to a family of small non‐coding RNAs (sncRNAs) playing important roles in human carcinogenesis. Multiple investigations reported miRNAs aberrantly expressed in several cancers, including high‐grade serous ovarian carcinoma (HGS‐OvCa). Quantitative PCR is widely used in studies investigating miRNA expression and the identification of reliable endogenous controls is crucial for proper data normalization. In this study, we aimed to experimentally identify the most stable reference sncRNAs for normalization of miRNA qPCR expression data in HGS‐OvCa. Eleven putative reference sncRNAs for normalization (U6, SNORD48, miR‐92a‐3p, let‐7a‐5p, SNORD61, SNORD72, SNORD68, miR‐103a‐3p, miR‐423‐3p, miR‐191‐5p, miR‐16‐5p) were analysed on a total of 75 HGS‐OvCa and 30 normal tissues, using a highly specific qPCR. Both the normal tissues considered to initiate HGS‐OvCa malignant transformation, namely ovary and fallopian tube epithelia, were included in our study. Stability of candidate endogenous controls was evaluated using an equivalence test and validated by geNorm and NormFinder algorithms. Combining results from the three different statistical approaches, SNORD48 emerged as stably and equivalently expressed between malignant and normal tissues. Among malignant samples, considering groups based on residual tumour, miR‐191‐5p was identified as the most equivalent sncRNA. On the basis of our results, we support the use of SNORD48 as best reference sncRNA for relative quantification in miRNA expression studies between HGS‐OvCa and normal controls, including the first time both the normal tissues supposed to be HGS‐OvCa progenitors. In addition, we recommend miR‐191‐5p as best reference sncRNA in miRNA expression studies with prognostic intent on HGS‐OvCa tissues.     

Improving the cytological diagnosis of high‐grade serous carcinoma in ascites with a panel of complementary biomarkers in cell blocks

Introduction

Precise cytological diagnosis of pelvic high‐grade serous carcinoma (HGSC) in ascites is important for tumour staging, therapeutic decision‐making and prognostic evaluation. However, it can often be difficult to distinguish metastatic HGSC cells from reactive mesothelial cells based on morphology alone. Immunocytochemical analysis of ascites cell blocks has been used to obtain accurate diagnosis and provide a reliable basis for treatment decisions in the clinic. This study was performed to determine whether a panel of antibodies is necessary to achieve high specificity and sensitivity for the identification of HGSC cells.

Methods

Ascites samples from 70 cases (70/253, 27.7%) of histologically confirmed HGSC were postoperatively collected from 2012 to 2015 and were immunocytochemically analysed.

Results

The sensitivity and specificity of Ber‐EP4 (a marker of HGSC) for detecting HGSC was 85.7% and 82.1%, respectively, whereas the sensitivity and specificity of HBME‐1 for identifying mesothelial cells was 100% and 68.3%, respectively. To improve the rate of detection further of HGSC, 29 cases of ascites were also stained for E‐cadherin (a marker of HGSC) and calretinin (a marker of mesothelial cells). The combination of Ber‐EP4 and E‐cadherin as markers of adenocarcinoma cells increased the sensitivity and specificity for HGSC detection to 100% and 88.9%, respectively. Meanwhile, the sensitivity and specificity for mesothelial cell identification increased to 100% and 90%, respectively, when HBME‐1 and calretinin were combined.

Conclusion

This panel of complementary biomarkers is valuable and ideal for the differential diagnosis of HGSC based on ascites cytology.