A Homogeneous Ensemble of Robust Pre-defined Neural Network Enables Automated Annotation of Human Embryo Morphokinetics.

Background: The purpose of the current study was to reduce the risk of human bias in assessing embryos by automatically annotating embryonic development based on their morphological changes at specified time-points with convolutional neural network (CNN) and artificial intelligence (AI). Methods: Time-lapse videos of embryo development were manually annotated by the embryologist and extracted for use as a supervised dataset, where the data were split into 14 unique classifications based on morphological differences. A compilation of homogeneous pre-trained CNN models obtained via TensorFlow Hub was tested with various hyperparameters on a controlled environment using transfer learning to create a new model. Subsequently, the performances of the AI models in correctly annotating embryo morphologies within the 14 designated classifications were compared with a collection of AI models with different built-in configurations so as to derive a model with the highest accuracy. Results: Eventually, an AI model with a specific configuration and an accuracy score of 67.68% was obtained, capable of predicting the embryo developmental stages (t1, t2, t3, t4, t5, t6, t7, t8, t9+, tCompaction, tM, tSB, tB, tEB). Conclusion: Currently, the technology and research of artificial intelligence and machine learning in the medical field have significantly and continuingly progressed in an effort to develop computer-assisted technology which could potentially increase the efficiency and accuracy of medical personnel's performance. Nonetheless, building AI models with larger data is required to properly increase AI model reliability.

Does elective orthopaedic surgery in pandemic era increase risk of developing COVID-19? A combined analysis of retrospective and prospective study at Cipto Mangunkusumo Hospital, Jakarta, Indonesia.

BACKGROUND: To date, no recommendations have been published on when and how to start again carrying out elective, non-urgent surgery on COVID-19-negative patients after the epidemic peak has been reached in a given country or region and the pressure on healthcare facilities, healthcare workers and resources has been released by so far that elective surgery procedures can be safely and ethically programmed again. This study aims to investigate whether elective orthopaedic surgery will increase the risk of developing COVID-19. MATERIALS AND METHODS: This was a combined retrospective and prospective studies performed at a national tertiary hospital in Jakarta, Indonesia. Subjects were patients who underwent elective orthopaedic surgeries at our institution from April to May 2020. Those who were previously infected with COVID-19 from polymerase chain reaction (PCR) reverse transcriptase (RT) examination obtained via nasopharynx and oropharynx swab, as well as those who were reluctant to participate were excluded from the study. RESULTS: A total of 35 subjects (mean age 32.89 ± 17.42) were recruited. Fifteen (42.9%) subjects were male, and 20 subjects (57.1%) were female. Mean duration of surgery was 240 min with the longest and shortest duration of 690 and 40 min, respectively. General anaesthesia was performed in the majority of cases in 18 surgeries (51.4%) with local anaesthesia as the least in 2 surgeries (5.7%). Length of stay of our study was 6 days of average. None of the patients developed symptoms suggestive of COVID-19 infection. CONCLUSION: We found that elective orthopaedic surgery may not be associated with increased cases of COVID-19 cases. However, our study was limited by short duration of follow-up. Further studies are required in order to investigate the affect of undergoing elective surgery and the number of COVID-19 cases.

Paraganglioma of the filum terminale: review and report of the first case analyzed by CGH.

OBJECTIVE: As a rare tumor paraganglioma of the filum terminale is of diagnostic challenge. A thorough review of all published cases most often reveals a benign course if complete surgically resection is achieved. We report on the first molecular cytogenetic analyses performed on filum termiale paragangliomas. CLINICAL PRESENTATION: A 52-year-old man suffered from low back pain for many years that gradually worsened and was aggravated by sitting and bending. The pain radiated dorsally into both legs. Magnetic resonance imaging (MRI) with and without Gd-DTPA revealed a 12 mm sized, intradural oval mass at the level of L3 with slightly increased T2-signal and a rim of low signal on T2-weighted sequences. The tumor enhanced remarkably after Gd-DTPA. INTERVENTION: The patient underwent a left sided hemilaminectomy of L3. Durotomy revealed a well-delineated, firm and highly vascularized reddish tumor. The proximal terminal filum entered the tumor at the proximal pole and exited its distal pole. Coagulation and dissection of the terminal filum allowed in toto removal of the tumor. DNA was isolated from the formalin-fixed and paraffin-embedded specimen. The tumor was analyzed by comparative genomic hybridization, providing a normal DNA profile without any chromosomal copy number changes. CONCLUSION: The origin of paragangliomas of the CNS and especially of the filum terminale is still unclear. If no complete surgical resection can be achieved, molecular cytogenetic analysis is of additional value to prognostification of paragangliomas of the filum terminale.

The mesopancreas is the primary site for R1 resection in pancreatic head cancer: relevance for clinical trials.

PURPOSE: The prognosis of patients with pancreatic cancer remains poor, even after potentially curative R0 resection. This discrepancy may be due to the histopathological misclassification of R1 cases as curative resections (R0) in the past. MATERIALS AND METHODS: To test this hypothesis, color coding of all resection margins and organ surfaces as part of a standardized histopathological workup was implemented and prospectively tested on 100 pancreatic head specimens. RESULTS: Thirty-five patients were excluded from the analysis owing to the pathohistological diagnosis; only pancreatic ductal adenocarcinoma, distal bile duct adenocarcinoma, and periampullary adenocarcinoma were included. Applying the International Union Against Cancer criteria, 32 cancer resections were classified R0 (49.2%), while 33 cases turned out to be R1 resections (50.8%). The mesopancreas was infiltrated in 22 of the 33 R1 resection specimens (66.6%). It proved to be the only site of tumor infiltration in 17 specimens (51.5%). Applying the Royal College of Pathologists' criteria, 46 resections were classified R1 (70.8%). As expected, the mesopancreas again was the most frequent site of noncurative resection (n = 27; 58.7%). CONCLUSION: Using the intensified histopathological workup for pancreatic head cancer specimens resulted in an increased rate of R1 resections and the mesopancreas represents the primary site for positive resection margins. Such results are of relevance for patients' stratification in clinical trials.

Increased KIT signalling with up-regulation of cyclin D correlates to accelerated proliferation and shorter disease-free survival in gastrointestinal stromal tumours (GISTs) with KIT exon 11 deletions.

Gastrointestinal stromal tumours (GISTs) with deletions in KIT exon 11 are characterized by higher proliferation rates and shorter disease-free survival times, compared to GISTs with KIT exon 11 point mutations. Up-regulation of cyclin D is a crucial event for entry into the G1 phase of the cell cycle, and links mitogenic signalling to cell proliferation. Signalling from activated KIT to cyclin D is directed through the RAS/RAF/ERK, PI3K/AKT/mTOR/EIF4E, and JAK/STATs cascades. ERK and STATs initiate mRNA transcription of cyclin D, whereas EIF4E activation leads to increased translation efficiency and reduced degradation of cyclin D protein. The aim of the current study was to analyse the mRNA and protein expression as well as protein phosphorylation of central hubs of these signalling cascades in primary GISTs, to evaluate whether tumours with KIT exon 11 deletions and point mutations differently utilize these pathways. GISTs with KIT exon 11 deletions had significantly higher mitotic counts, higher proliferation rates, and shorter disease-free survival times. In line with this, they had significantly higher expression of cyclin D on the mRNA and protein level. Furthermore, there was a significantly higher amount of phosphorylated ERK1/2, and a higher protein amount of STAT3, mTOR, and EIF4E. PI3K and phosphorylated AKT were also up-regulated, but this was not significant. Ultimately, GISTs with KIT exon 11 deletions had significantly higher phosphorylation of the central negative cell-cycle regulator RB. Phosphorylation of RB is accomplished by activated cyclin D/CDK4/6 complex, and marks a central event in the release of the cell cycle. Altogether, these observations suggest increased KIT signalling with up-regulation of cyclin D as the basis for the unfavourable clinical course in GISTs with KIT exon 11 deletions.

Loss of 9p leads to p16INK4A down-regulation and enables RB/E2F1-dependent cell cycle promotion in gastrointestinal stromal tumours (GISTs).

Loss of chromosome 9p is a reliable predictor of malignant behaviour in gastrointestinal stromal tumours (GISTs). p16INK4A located at 9p21 inhibits the CDK4/6/cyclin D complex from phosphorylating RB. Phosphorylation of RB through CDK4/6/cyclin D in early G(1) phase frees the transcription factor E2F1 from RB and enables mRNA transcription of genes essential for G(1)/S phase transition. This study aims to determine the impact of 9p loss on mRNA and protein expression of p16INK4A and further key cell cycle regulators in the different phases of the cell cycle. Sixty primary GISTs previously characterized for 9p loss by comparative genomic hybridization were analysed for mRNA expression of p16INK4A, p15INK4B, CDK4, CDK6, cyclin D, p21CIP1p27KIP1, CDK2, cyclin E, cyclin B, RB and E2F1, using quantitative RT-PCR. The protein expression of CDK6, CDK2, p21CIP1, p27KIP1 and phosphorylated RB (S807/S811) was evaluated using protein arrays as a novel and highly sensitive platform for profiling of protein abundance and protein phosphorylation. In parallel, the nuclear percentages of immunohistochemical staining for p16INK4A, cyclin D, E2F1 and RB were quantified on a tissue microarray. GISTs with 9p loss had significantly higher proliferation rates, higher metastatic behaviour and shorter disease-free survival. On the molecular level, GISTs with 9p loss had a significantly reduced mRNA as well as nuclear protein expression of p16INK4A. RB was significantly more phosphorylated in these tumours, together with increased mRNA expression and nuclear staining for E2F1. Furthermore, GISTs with 9p loss had up-regulation of the late G1/S phase promoters CDK2 and cyclin E. We conclude that loss of 9p accompanied by early G1 phase inhibitor p16(INK4A) down-regulation in GISTs facilitates phosphorylation of RB, enabling E2F1-dependent transcription of genes essential for late G1/S phase transition. This study provides a possible basis for the accelerated proliferation and particularly malignant behaviour in GISTs with 9p loss.

Characterization of a newly established uterine carcinosarcoma cell line featuring the sarcomatous phenotype of the tumor in vitro.

We describe the newly established cell line CS-99 derived from a uterine carcinosarcoma retaining features of the sarcomatous phenotype in vitro. CS-99 cells exhibit a mesenchymal morphology with predominantly spindle-shaped cells at nonconfluence turning to pleomorphic appearance at confluence. The mesenchymal phenotype was evidenced immunohistochemically by strong vimentin and moderate SM-actin, which was similar to the sarcomatous component of the primary tumor. P53 was overexpressed in a subset of CS-99 cells. Epithelial membrane antigen was moderately expressed whereas other markers including pan CK, CK 5/6, CK 34, epidermal growth factor receptor, desmin, carcinoembryonic antigen, S100, KIT, ERBB2, and the hormone receptors, estrogen receptor and progesterone receptor revealed either weak or no specific staining in CS-99 cells. High self-renewal capacity corresponded to the population doubling time of 23 h in high passage. CS-99 cells were able to develop three-dimensional tumor spheroids in vitro. Cytogenetic analysis and multicolor fluorescence in situ hybridization of CS-99 demonstrated an almost stable karyotype including numerical changes +8, +18, and +20 and translocations, amongst others der(1)t(1;2), der(1)t(1;7), der(2)t(2;19), der(5)t(5;8), and der(5)t(5;14). Taken together, the cell line CS-99 exhibits strong growths dynamics and a complex but stable karyotype in higher passages, and can be further a useful in vitro model system for studying tumor biology of carcinosarcomas.

Knock-in murine models of familial gastrointestinal stromal tumours.

In recent years, gastrointestinal stromal tumour (GIST) has emerged from a poorly understood group of mesenchymal tumours to a distinct pathological entity that has become a leading model for new therapies targeting kinases. GIST pathogenesis is driven by receptor tyrosine kinase-activating mutations most often in KIT or PDGFRA that may be sporadic or result in familial GIST syndromes. In a recent issue of The Journal of Pathology (J Pathol 2008;214:302-311), Nakai and colleagues report that mutation of the previously un-modelled tyrosine kinase II domain of KIT generates a spectrum of features very similar to that in two human GIST families. Knock-in mouse models of GIST may prove extremely useful in pre-clinical evaluation of kinase-targeted compounds and of the mechanism of drug resistance, but intriguingly it now seems clearer that the distribution of GIST in mouse models and humans is different.

An oncogenetic tree model in gastrointestinal stromal tumours (GISTs) identifies different pathways of cytogenetic evolution with prognostic implications.

To model the cytogenetic evolution in gastrointestinal stromal tumour (GIST), an oncogenetic tree model was reconstructed using comparative genomic hybridization data from 203 primary GISTs (116 gastric and 87 intestinal GISTs, including 151 newly analysed cases), with follow-up available in 173 cases (mean 40 months; maximum 133 months). The oncogenetic tree model identified three major cytogenetic pathways: one initiated by -14q, one by -1p, and another by -22q. The -14q pathway mainly characterized gastric tumours with predominantly stable karyotypes and more favourable clinical course. On the other hand, the -1p pathway was more characteristic of intestinal GISTs, with an increased capacity for cytogenetic complexity and more aggressive clinical course. Loss of 22q, more closely associated with -1p than -14q, appeared to initiate the critical transition to an unfavourable cytogenetic subpathway. This -22q pathway included accumulation of +8q, -9p, and -9q, which could all predict disease-free survival in addition to tumour site. Thus, insights into the cytogenetic evolution obtained from oncogenetic tree models may eventually help to gain a better understanding of the heterogeneous site-dependent biological behaviour of GISTs.

The efficacy and safety of epsilon-aminocaproic acid treatment in patients with cirrhosis and hyperfibrinolysis.

BACKGROUND: Patients with decompensated cirrhosis are at risk for hyperfibrinolysis; this is potentially fatal. epsilon-aminocaproic acid has been used to treat patients with hyperfibrinolysis; however, the data about its benefit in the setting of cirrhosis are minimal. AIM: To analyse the efficacy of epsilon-aminocaproic acid and its safety in cirrhotic patients with hyperfibrinolysis. METHODS: All patients with an abnormal euglobin lysis time who were admitted to Rancho Los Amigos Medical Center from 1 January 2001 to 31 December 2002 were included in the study. Their medical records were reviewed and analysed. RESULTS: There were 60 cirrhotic patients with shortened euglobin lysis time. Fifty-two patients received epsilon-aminocaproic acid. Of the 52 patients, seven had one or more bleeding episodes with the subcutaneous or soft tissue bleeding as the most common indication for epsilon-aminocaproic acid use. Of the 37 patients, 34 (92%) had improvement or resolution of their bleeding. Only two (3%) patients had epsilon-aminocaproic acid treatment discontinued because of minor side effects, rash and lightheadedness. There were no thromboembolic complications of treatment. CONCLUSIONS: epsilon-aminocaproic acid was found to be effective and safe for treatment of hyperfibrinolysis in patients with cirrhosis.

Increased number of mature dendritic cells in Crohn's disease: evidence for a chemokine mediated retention mechanism.

BACKGROUND AND AIMS: Activation of T cells by dendritic cells (DC) is thought to play a pivotal role in induction and maintenance of Crohn's disease. Detailed analyses however concerning the phenotype and maturation of DC as well as the mechanisms underlying their recruitment are still lacking for Crohn's disease. METHODS: Different myeloid and plasmacytoid DC subsets were characterised by immunohistochemistry. Expression of the so-called "lymphoid" chemokines CCL19, CCL20, and CCL21 was determined by real time reverse transcription-polymerase chain reaction in Crohn's disease and normal controls. Furthermore, expression of CCL19, CCL20, and CCL21 as well as their receptors CCR6 (for CCL20) and CCR7 (for CCL19 and CCL21) was characterised by immunohistochemistry and, in addition, their cellular localisation was determined by double immunofluorescence investigations. RESULTS: Colonic tissue affected by Crohn's disease was characterised by an increased number of mature myeloid DC forming clusters with proliferating T cells. In keeping with their advanced maturation, DC possess the chemokine receptor CCR7. Increased expression of the CCR7 ligands CCL19 by DC themselves as well as CCL21 by reticular cells and lymphatic vessels was observed in Crohn's disease, thereby causing the matured DC to be trapped at the site of inflammation. CONCLUSION: Our results demonstrate that autocrine and paracrine actions of lymphoid chemokines in Crohn's disease may lead to increased numbers of mature DC away from their usual migration to lymphoid organs and result in the development of a tertiary lymphatic tissue within the bowel wall maintaining the autoimmune inflammation in Crohn's disease.

Multi-source water pollution in the Upper Citarum watershed, Indonesia, with special reference to its spatiotemporal variation.

The Citarum River in West Java is the largest water supplier to the Saguling Dam, which plays a major role in electric power generation for the entire Java Island and is used for the aquaculture of marketed fish. To elucidate the extent of degradation in water quality and its causes in the Upper Citarum watershed, physical, chemical and biological parameters for water samples collected from various sites were analyzed. The results demonstrate large site-to-site variations in water qualities and pollutant loads derived from various human activities such as agriculture, cattle raising and the textile industry. To halt worsening conditions of the Citarum watershed, integrated mitigation efforts should be made, taking biophysical pollution mechanisms and local socioeconomic conditions into account.

Gastrointestinal stromal tumor (GIST) -- single center experience of prolonged treatment with imatinib.

BACKGROUND: The tyrosine kinase inhibitor imatinib has been introduced into the treatment of gastrointestinal stromal tumors (GIST). Here we report our results of prolonged treatment in comparison to a similar group of GIST patients who had died before imatinib became available. METHODS: Fourteen patients with recurrent or metastatic GIST were treated with imatinib. Clinical data and tumor samples of ten patients from the pre-imatinib era were available for comparison. Comparative genomic hybridisation (CGH) was performed on tumors to identify changes that may predict response to treatment. RESULTS: Fourteen patients were treated, mean treatment time 22.3 months (1 non-response, 2 progression after initial response, 2 stable diseases, 8 partial responses, 1 complete response). Adverse side effects were mild in general. Survival was higher in the treated group (41.1 months vs. 34.8 months in the historical group). Eleven treated patients are alive. CGH analysis showed comparable numbers of chromosomal aberations in both groups. CONCLUSION: Prolonged treatment with imatinib is safe and effective in patients with recurrent or metastatic GIST.

Simultaneous tumour-like, atypical basal cell hyperplasia and acinar adenocarcinoma of the prostate: a comparative morphological and genetic approach.

Basal cell tumours of the prostatic gland are rare, and the classification is difficult. In the present case report, a large, tumour-like proliferation of atypical basaloid cells was found incidentally in a prostatectomy specimen that otherwise contained a conventional acinar adenocarcinoma. The basaloid cells displayed a solid or adenoid-cystic growth pattern and strongly expressed high-molecular-weight cytokeratins and bcl-2. A high Ki-67 index was recorded within the atypical basaloid cells, by far exceeding the one counted in the conventional adenocarcinoma. However, there were no definite criteria for a malignant behaviour of the basal cell tumour. Comparative genomic hybridisation from microdissected tumour cells yielded losses at the short arms of chromosomes 8 and 12 in the conventional adenocarcinoma and a normal karyotype in the basal cell tumour. The pathological findings favoured the diagnosis of an atypical basal cell hyperplasia.

CD4+ CD56+ blastic tumor of the skin: cytogenetic observations and further evidence of an origin from plasmocytoid dendritic cells.

We here report on a case of a blastic tumor, recently described to belong to a new entity sharing phenotypic similarities with blood derived plasmocytoid dendritic cells and formerly regarded as belonging to the group of natural killer cell lymphomas. Besides immunophenotypic characteristics such as the absence of T- cell markers and almost complete absence of markers of the myeloid lineage, these tumors express CD4, CD56 and CD123, the receptor for interleukin-3. Moreover, using the comparative genomic hybridisation technique, CGH, we demonstrate a gain of chromosome 7q, 22 and a loss of chromosome 3p and 13q. Since this type of hematologic disorder often shows its primary manifestation in the skin and often runs a rapidly fatal course, it is important to distinguish this from other types of primary cutaneous lymphomas.

Molecular cytogenetic analysis of chromosomal breakpoints in the IGH, MYC, BCL6, and MALT1 gene loci in primary cutaneous B-cell lymphomas.

Chromosomal translocations affecting the IGH locus and various oncogene loci are recurrent in many types of systemic B-cell lymphomas. Hardly any data exist, however, on such translocations in primary cutaneous B-cell lymphomas (PCBCL). Here, a series of 29 PCBCL was investigated by interphase fluorescence in situ hybridization with probes for the IGH, MYC, BCL6, and MLT1 loci. None of the six follicle center cell lymphomas and nine marginal zone lymphomas showed evidence for any translocation affecting these loci. In contrast, 11 of 14 large B-cell lymphomas of the leg harbored breakpoints in at least one of the loci. Translocations involving the MYC locus were detected in six cases, five of them derived from a MYC/IGH juxtaposition and one from a translocation involving a non-IG gene partner. Rearrangements of the BCL6 locus were detected in five B-cell lymphomas of the leg, and involved IGH (two cases), IGL (one case), and non-IG genes (two cases). This study shows that large B-cell lymphomas of the leg display a pattern of chromosomal translocations similar to their systemic counterparts whereas primary cutaneous follicle center cell lymphomas and marginal zone lymphomas lack these typical chromosomal translocations.

Prognostic factors influencing surgical management and outcome of gastrointestinal stromal tumours.

BACKGROUND: The purpose of this study was to review surgical experience with gastrointestinal stromal tumours (GISTs) at a single tertiary university hospital, and to identify morphological and genetic prognostic markers of tumour progression. METHODS: Forty-eight GISTs from 39 patients were reviewed retrospectively. The prognostic significance of DNA copy number changes, measured by comparative genomic hybridization (CGH), and morphological markers in low-risk and high-risk tumours were investigated. RESULTS: Significantly more patients died from disease after incomplete tumour resection than after complete primary resection (P = 0.020). Tumour size of 5 cm or greater, mitotic count of 2 or more, and proliferative activity greater than 10 per cent were significantly associated with a shorter recurrence-free survival (P = 0.020, P = 0.001 and P = 0.002 respectively). Patients with low-risk tumours had a significantly better outcome than those with high-risk GISTs, both in terms of overall and recurrence-free survival (P < or = 0.001). CGH performed on 16 tumours revealed fewer DNA sequence copy number changes in low-risk than in high-risk GISTs. Non-progressive GISTs contained significantly fewer genetic alterations than recurrent or metastatic tumours (P < 0.001). Only tumours with more than five changes showed disease progression. CONCLUSION: Complete surgical resection is the most important means of cure for GISTs. DNA copy number changes are related to the behaviour of these tumours and may serve as additional prognostic markers.

Mathematical tree models for cytogenetic development in solid tumors.

We present a new approach for modeling the occurrence of genetic changes in human tumors over time. In solid tumors, data on genetic alterations are usually only available at a single point in time, allowing no direct insight into the sequential order of genetic events. In our approach, genetic tumor development and progression is assumed to follow a probabilistic tree model. We use maximum likelihood estimation to reconstruct a tree model for the genetic evolution of a given tumor type. The use of the proposed method is illustrated by an application to cytogenetic data from 173 cases of clear cell renal cell carcinoma, which results in a model for the karyotypic evolution of this tumor.

Ultrastructural changes of cardiac valves in bacterial endocarditis.

AIM: The principal objective of this study was to document morphological changes in valves with acute endocarditis in order to gain further knowledge of the pathogenesis of these diseases. METHODS: Scanning and transmission electron microscopic investigations were carried out on explanted human heart valves to reveal ultrastructural changes due to bacterial endocarditis. RESULTS: Bacterial inflammation endocarditis initially induced metaplasia of the endothelial cells which then lose contact with each other. In the 2nd phase of the disease, the collagen fibres are systematically removed whereby large cavities appear. In the 3rd phase, localised hyperplasia of collagen fibres was observed often resulting in the development of vegetation. The ultrastructural changes are uniform and independent of the bacterial species. CONCLUSION: Bacterial endocarditis is therefore a set of complex interactions between endothelial cells and bacteria which should be taken into consideration for the development of new therapeutic approaches.