Exploring p53 protein expression and its link to TP53 mutation in myelodysplasia-related malignancies-Interpretive challenges and potential field of applications.

AIMS: TP53 alterations have a significant prognostic effect in myeloid neoplasms. Our objective was to investigate the TP53 gene mutation status, p53 protein expression and their relationship in dysplasia-related myeloid neoplasms with varying levels of myeloblast counts. METHODS AND RESULTS: A total of 76 bone marrow biopsy samples with different blast counts were analysed. Total and strong (3+) p53 expression was determined. Dual immunohistochemical staining was performed to determine the cell population associated with p53 expression. NGS analysis was performed using the Accel-Amplicon Comprehensive TP53 panel. Both p53 expression and TP53 VAF showed a significant correlation with the myeloblast ratio (P < 0.0001); however, p53 expression was also present in other cell lineages. The VAF value exhibited a significant correlation with p53 expression. A high specificity (0.9800) was observed for TP53 mutation using the ≥ 10% strong (3+) p53 cut-off value, although the sensitivity (0.4231) was low. CONCLUSIONS: Strong (3+) p53 expression using a ≥ 10% cut-off value accurately predicts TP53 mutation but does not reveal the allelic state. The p53 expression is significantly influenced by myeloblast count, and histological interpretation should consider the presence of intermixed non-neoplastic marrow cells with varying physiological p53 expression.

Eculizumab Treatment of Massive Hemolysis Occurring in a Rare Co-Existence of Paroxysmal Nocturnal Hemoglobinuria and Myasthenia Gravis.

The co-occurrence of myasthenia gravis (MG) and paroxysmal nocturnal hemoglobinuria (PNH) is rare; only one case has been published so far. We report a 63-year-old Caucasian female patient who was diagnosed with MG at the age of 43. Thymoma was also detected, and so it was surgically resected, which resulted in reasonable disease control for nearly 20 years. Slight hemolysis began to emerge, and then myasthenia symptoms progressed, so immunosuppressive therapy was started. Due to progressive disease and respiratory failure, the patient underwent plasmapheresis, and ventilatory support was stopped. Marked hemolysis was present, and diagnostic tests confirmed PNH with type III PNH cells. Her myasthenia symptoms aggravated, mechanical ventilation had to be started again, and due to the respiratory acidosis, massive hemolysis occurred. After two plasmapheresis sessions, the patient received eculizumab at 600 mg, resulting in prompt hemolysis control. After the second dose of the treatment, the patient was extubated. Still, due to their inability to cough, she developed another respiratory failure and pneumonia-sepsis, resulting in the patient's death. This case highlights the rare association between these two serious diseases and similar immune-mediated pathophysiology mechanisms involving the complement system.

Proton Logic Gate Based on a Gramicidin-ATP Synthase Integrated Biotransducer.

Ion channels are membrane proteins that allow ionic signals to pass through channel pores for biofunctional modulations. However, biodevices that integrate bidirectional biological signal transmission between a device and biological converter through supported lipid bilayers (SLBs) while simultaneously controlling the process are lacking. Therefore, in this study, we aimed to develop a hybrid biotransducer composed of ATP synthase and proton channel gramicidin A (gA), controlled by a sulfonated polyaniline (SPA) conducting polymer layer deposited on a microelectrode, and to simulate a model circuit for this system. We controlled proton transport across the gA channel using both electrical and chemical input signals by applying voltage to the SPA or introducing calcium ions (inhibitor) and ethylenediaminetetraacetic acid molecules (inhibitor remover). The insertion of gA and ATP synthase into SLBs on microelectrodes resulted in an integrated biotransducer, in which the proton current was controlled by the flux of adenosine diphosphate molecules and calcium ions. Lastly, we created an XOR logic gate as an enzymatic logic system where the output proton current was controlled by Input A (ATP synthase) and Input B (calcium ions), making use of the unidirectional and bidirectional transmission of protons in ATP synthase and gA, respectively. We combined gA, ATP synthase, and SPA as a hybrid bioiontronics system to control bidirectional or unidirectional ion transport across SLBs in biotransducers. Thus, our findings are potentially relevant for a range of advanced biological and medical applications.

Somatic Genomic and Transcriptomic Characterization of Primary Ovarian Serous Borderline Tumors and Low-Grade Serous Carcinomas.

Low-grade serous carcinoma (LGSC) may develop from serous borderline tumor (SBT) tissue, where the micropapillary type (mSBT) presents the highest risk for progression. The sensitivity of LGSC to standard chemotherapy is limited, so alternative therapeutic approaches, including targeted treatment, are needed. However, knowledge about the molecular landscape of LGSC and mSBT is limited. A sample set of 137 pathologically well-defined cases (LGSC, 97; mSBT, 40) was analyzed using capture DNA next-generation sequencing (727 genes) and RNA next-generation sequencing (147 genes) to show the landscape of somatic mutations, gene fusions, expression pattern, and prognostic and predictive relevance. Class 4/5 mutations in the main driver genes (KRAS, BRAF, NRAS, ERBB2, USP9X) were detected in 48% (14/29) of mSBT cases and 63% (47/75) of LGSC cases. The USP9X mutation was detected in only 17% of LGSC cases. RNA next-generation sequencing revealed gene fusions in 6 of 64 LGSC cases (9%) and 2 of 33 mSBT cases (9%), and a heterogeneous expression profile across LGSC and mSBT. No molecular characteristics were associated with greater survival. The somatic genomic and transcriptomic profiles of 35 mSBT and 85 LGSC cases are compared for the first time. Candidate oncogenic gene fusions involving BRAF, FGFR2, or NF1 as a fusion partner were identified. Molecular testing of LGSC may be used in clinical practice to reveal therapeutically significant targets.

Neuroendocrine Marker Expression in Primary Non-neuroendocrine Epithelial Tumors of the Ovary: A Study of 551 Cases.

Expression of neuroendocrine (NE) markers in primary ovarian non-NE epithelial tumors has rarely been evaluated. The aim of our study was to evaluate the expression of the most widely used NE markers in these neoplasms and to determine any prognostic significance of NE marker expression. The cohort consisted of 551 primary ovarian tumors, including serous borderline tumors, low-grade serous carcinomas, high-grade serous carcinomas (HGSC), clear cell carcinomas, endometroid carcinomas, mucinous borderline tumors, and mucinous carcinomas. Immunohistochemical analysis was performed using antibodies against INSM1, synaptophysin, chromogranin, and CD56 on tissue microarray. Positivity for INSM1, synaptophysin, chromogranin, and CD56 was most frequently observed in mucinous tumors (48.7%, 26.0%, 41.5%, and 100%, respectively). The positivity for these NE markers was mostly restricted to nonmucinous elements distributed throughout the tumor. The mucinous borderline tumor and mucinous carcinomas groups had similar proportions of positivity (mucinous borderline tumor: 53%, mucinous carcinomas: 39%). In the other tumor types, except for HGSC, there was only focal expression (5%-10%) or negativity for NE markers. HGSC showed high CD56 expression (in 26% of cases). Survival analysis was only performed for CD56 in HGSC as this was the only group with sufficient positive cases, and it showed no prognostic significance. Except for mucinous tumors, expression of NE markers in non-NE ovarian epithelial tumors is low. CD56 expression in HGSC occurs frequently but is without diagnostic or prognostic value.

Loss of Uncoupling Protein 1 Expression in the Subcutaneous Adipose Tissue Predicts Childhood Obesity.

Stimulation of thermogenesis by inducing uncoupling protein 1 (UCP1) expression in adipocytes is thought to promote weight loss by increasing energy expenditure, and it is postulated that the human newborn has thermogenic subcutaneous fat depots. However, it remains unclear whether a relevant number of UCP1-expressing (UCP1+) adipocytes exist in the early postnatal life. Here we studied the distribution of UCP1 and the expression of thermogenic genes in the subcutaneous adipose tissues of the human fetus, infant and child. We show that the deep layer of human fetal and neonatal subcutaneous fat, particularly the abdominal wall, is rich in UCP1+ adipocytes. These adipocytes develop in the late third trimester and persist throughout childhood, expressing a panel of genes linked to mitochondrial biogenesis and thermogenesis. During the early childhood adiposity rebound-a critical phase that determines obesity risk later in life-the absence of adipose tissue UCP1 expression in children with normal body mass index (BMI) correlates with an obesity-associated gene expression signature. Finally, UCP1 expression is negatively correlated with BMI z-score and adipocyte size in infants and children. Overall, our results show that the absence of UCP1 expression in adipose tissue is an early indicator of adipose tissue expansion in children.

Correlation Analyses between Histological Staging and Molecular Alterations in Tumor-Derived and Cell-Free DNA of Early-Stage Primary Cutaneous Melanoma.

Here, we investigate the correlation and statistical analyses between histological staging and molecular alterations in tumor-derived (tdDNA) and cell-free DNA (cfDNA) obtained from early-stage primary cutaneous melanoma (PCM) patients using digital PCR (dPCR) for the detection of the BRAF p.V600E somatic pathogenic variant. In the prospective study, a total of 68 plasma and paired tdDNA samples, and in the retrospective cohort, a total of 100 tdDNA samples were analyzed using dPCR and reverse hybridization StripAssay. The Breslow depth (BD) and Clark level were applied to categorize the study population. Our results demonstrate that dPCR is a highly sensitive and specific method for the detection of BRAF p.V600E somatic variants in cfDNA samples from PCM patients. A strong correlation was detected between BD and cfDNA concentration in all mutant and negative cases, between the tdDNA concentration and the tumor-derived variant allele frequency (VAF) of BRAF p.V600E, between the tdVAF and the cfVAF in all cases, and between the cfDNA and cfVAF in mutant cases. The tdVAF and cfVAF of BRAF p.V600E and cfDNA concentration were the highest in Clark's V category. The cfDNA concentration was statistically significantly higher in Clark's III, IV, and V groups compared to cases with a better prognosis. It can also be explained by the fact that cases with a more advanced stage classification release more cfDNA into the peripheral circulation.

HER2 status as a potential predictive biomarker for ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) is a subtype of ovarian carcinoma characterized by unique biological features and highly malignant characteristics including low chemosensitivity. Therefore, new therapeutic targets are needed. These could include the downstream pathways of receptor tyrosine kinases, especially the human epidermal growth factor receptor 2 (HER2). Our main objective was to characterize the HER2 status using immunohistochemistry (IHC) and FISH on 118 OCCCs, also considering the novel paradigm of HER2-zero and HER2-low status. Other aims included determination of the association between HER2 status and survival, HER2 gene DNA and RNA NGS analysis, HER2 gene expression analysis, and correlation between IHC and gene expression in HER2-zero and HER2-low cases. Cases with HER2 overexpression/amplification accounted for 5.1% (6/118), with additional 3% harbouring HER2 gene mutation. The remaining 112 (94.9%) cases were HER2-negative. Of these, 75% were classified as HER2-zero and 25% as HER2-low. This percentage of HER2 aberrations is significant concerning their possible therapeutic influence. Cases from the HER2-zero group showed significantly better survival. Although this relationship lost statistical significance in multivariate analysis, the results have potential therapeutic significance. HER2 gene expression analysis showed a significant correlation with HER2 IHC status in the entire cohort (HER2-positive vs. HER2-negative), while in the cohort of only HER2-negative cases, the results did not reach statistical significance, suggesting that gene expression analysis would not be suitable to confirm the subdivision into HER2-low and HER2-zero. Our results also emphasize the need for standardized HER2 testing in OCCC to determine the best predictor of clinical response.

Immunohistochemical expression of PRAME in 485 cases of epithelial tubo-ovarian tumors.

Preferentially expressed antigen of melanoma (PRAME) is a cancer/testis antigen selectively expressed in somatic tissues and various solid malignant tumors and is associated with poor prognostic outcome. Our research aimed to comprehensively compare its expression in a large cohort of tubo-ovarian epithelial tumors and examine its correlation with our clinico-pathologic data, as well as to assess its potential use in diagnostics and therapy.We examined 485 cases of epithelial tubo-ovarian tumors including 107 clear cell carcinomas (CCC), 52 endometroid carcinomas (EC), 103 high grade serous carcinomas (HGSC), 119 low grade serous carcinomas (LGSC)/micropapillary variant of serous borderline tumors (mSBT), and 104 cases of mucinous carcinomas (MC)/mucinous borderline tumors (MBT). The immunohistochemical analysis was performed using TMAs.The highest levels of expression were seen in EC (60%), HGSC (62%), and CCC (56%), while expression in LGSC/mSBT (4%) and MC/MBT (2%) was rare. The clinico-pathologic correlations and survival analysis showed no prognostic significance.The results of our study showed that PRAME is neither prognostic nor a suitable ancillary marker in the differential diagnosis of tubo-ovarian epithelial tumors. Nevertheless, knowledge about the PRAME expression may be important concerning its potential predictive significance, because targeting PRAME as a potential therapeutic option is currently under investigation.

Refined criteria for p53 expression in ovarian mucinous tumours are highly concordant with TP53 mutation status, but p53 expression/TP53 status lack prognostic significance.

In gynecological neoplasms, immunohistochemical (IHC) expression of p53 is generally an accurate predictor of TP53 mutation status if correctly interpreted by the pathologist. However, the literature concerning cut-offs, frequency and prognostic significance of p53 staining in ovarian mucinous tumours is limited and heterogeneous. We performed an analysis of 123 primary ovarian mucinous tumours including mucinous borderline tumours (MBT), mucinous carcinomas (MC), and tumours with equivocal features between MBT and MC. We assessed p53 expression for the three recognised patterns of aberrant staining in ovarian carcinoma [overexpression ('all'), null and cytoplasmic] but using a recently suggested cut-off for aberrant overexpression in ovarian mucinous tumours (strong nuclear p53 staining in ≥12 consecutive tumour cells) and correlated the results with next generation sequencing (NGS) in all qualitatively sufficient cases (92/123). Aberrant p53 expression was present in 25/75 (33.3%) MBT, 23/33 (69.7%) MC (75% of MC with expansile invasion and 61.5% with infiltrative invasion), and 10/15 (66.7%) tumours equivocal between MBT and MC. Regarding the 92 tumours with paired IHC and mutation results, 86 showed concordant results and six cases were discordant. Three discordant MBT cases showed aberrant expression but were TP53 wild-type on sequencing. Three cases had normal p53 expression but contained a TP53 mutation. Overall, IHC predicted the TP53 mutation status with high sensitivity (94.1%) and specificity (92.7%). The accuracy of IHC was 93.5% with a positive predictive value of 94.1% and a negative predictive value of 92.7%. When comparing MC cases with wild-type TP53 versus those with TP53 mutation, there were no significant differences concerning disease-free survival, local recurrence-free survival, or metastases-free survival (p>0.05). In the MBT subgroup, there were no events for survival analyses. In conclusion, using an independent large sample set of ovarian mucinous tumours, the results of our study confirm that the suggested refined cut-off of strong nuclear p53 staining in ≥12 consecutive tumour cells reflect high accuracy, sensitivity and specificity for an underlying TP53 mutation but the TP53 mutation status has no prognostic significance in either MC or MBT.

Grade Group accuracy is improved by extensive prostate biopsy sampling, but unrelated to prostatectomy specimen sampling or use of immunohistochemistry.

Assessing the accurate Grade Group of a prostate needle biopsy specimen is essential for choosing the adequate therapeutic modality for prostate cancer patients. However, it is well-known that biopsy Grade Group tends to up- or downgrade significantly at radical prostatectomy. We aimed to investigate the correlation between accuracy and biopsy core number, performed immunohistochemical staining (IHC) or prostatectomy specimen sampling, with the latest also being correlated with higher detection rates of adverse pathological features, e.g., positive surgical margins, higher pathological stage or presence of perineural invasion (PnI status). The study cohort consisted of 315 consecutive patients diagnosed with prostate adenocarcinoma via transrectal ultrasound-guided needle biopsy who later underwent radical prostatectomy. We grouped and compared patients based on Grade Group accuracy, presence of IHC on biopsy, margin status, pathological stage, and PnI status. Inter-observer reproducibility was also calculated. Statistical analyzes included ANOVA, Tukey's multiple comparisons post hoc test, Chi-squared test, and Fleiss kappa statistics. Undergraded cases harboured a significantly lower number of biopsy cores (p < 0.05), than accurately graded cases. Using IHC did not affect grading accuracy significantly, nor did the number of slides from prostatectomy specimens. The mean number of slides was virtually identical when margin status, pathological stage and PnI status of prostatectomy specimens were compared. Inter-observer reproducibility at our institute was calculated as fair (overall kappa = 0.29). Grade Group accuracy is significantly improved by obtaining more cores at biopsy but is unrelated to performed IHC. The extent of sampling prostatectomy specimens, however, did not affect accuracy and failed to significantly improve detection of adverse pathological features.

Compound heterozygous variants in MAPK8IP3 were detected in severe congenital hypotonia mimicking lethal spinal muscular atrophy.

Mitogen-activated protein kinase 8-interacting protein 3 gene (MAPK8IP3) encodes the c-Jun-amino-terminal kinase-interacting protein 3 (JIP3) and is involved in retrograde axonal transport. Heterozygous de novo pathogenic variants in MAPK8IP3 result in a neurodevelopmental disorder with or without brain abnormalities and possible axonal peripheral neuropathy. Whole-exome sequencing was performed on an individual presenting with severe congenital muscle hypotonia of neuronal origin mimicking lethal spinal muscular atrophy. Compound heterozygous rare variants (a splice and a missense) were detected in MAPK8IP3, inherited from the healthy parents. Western blot analysis in a muscle biopsy sample showed a more than 60% decrease in JIP3 expression. Here, we suggest a novel autosomal recessive phenotype of a lower motor neuron disease caused by JIP3 deficiency.

Parallel testing of liquid biopsy (ctDNA) and tissue biopsy samples reveals a higher frequency of EZH2 mutations in follicular lymphoma.

BACKGROUND: Recent genomic studies revealed enhancer of zeste homolog 2 (EZH2) gain-of-function mutations, representing novel therapeutic targets in follicular lymphoma (FL) in around one quarter of patients. However, these analyses relied on single-site tissue biopsies and did not investigate the spatial heterogeneity and temporal dynamics of these alterations. OBJECTIVES: We aimed to perform a systematic analysis of EZH2 mutations using paired tissue (tumor biopsies [TB]) and liquid biopsies (LB) collected prior to treatment within the framework of a nationwide multicentric study. METHODS: Pretreatment LB and TB samples were collected from 123 patients. Among these, 114 had paired TB and LB, with 39 patients characterized with paired diagnostic and relapse samples available. The EZH2 mutation status and allele burden were assessed using an in-house-designed, highly sensitive multiplex droplet digital PCR assay. RESULTS: EZH2 mutation frequency was found to be 41.5% in the entire cohort. In patients with paired TB and LB samples, EZH2 mutations were identified in 37.8% of the patients with mutations exclusively found in 5.3% and 7.9% of TB and LB samples, respectively. EZH2 mutation status switch was documented in 35.9% of the patients with paired diagnostic and relapse samples. We also found that EZH2 wild-type clones may infiltrate the bone marrow more frequently compared to the EZH2 mutant ones. CONCLUSION: The in-depth spatio-temporal analysis identified EZH2 mutations in a considerably higher proportion of patients than previously reported. This expands the subset of FL patients who most likely would benefit from EZH2 inhibitor therapy.

A comprehensive molecular analysis of 113 primary ovarian clear cell carcinomas reveals common therapeutically significant aberrations.

BACKGROUND: Molecular aberrations occurring in primary ovarian clear cell carcinoma (OCCC) can be of diagnostic, predictive, and prognostic significance. However, a complex molecular study including genomic and transcriptomic analysis of large number of OCCC has been lacking. METHODS: 113 pathologically confirmed primary OCCCs were analyzed using capture DNA NGS (100 cases; 727 solid cancer related genes) and RNA-Seq (105 cases; 147 genes) in order to describe spectra and frequency of genomic and transcriptomic alterations, as well as their prognostic and predictive significance. RESULTS: The most frequent mutations were detected in genes ARID1A, PIK3CA, TERTp, KRAS, TP53, ATM, PPP2R1A, NF1, PTEN, and POLE (51,47,27,18,13,10,7,6,6, and 4%, respectively). TMB-High cases were detected in 9% of cases. Cases with POLEmut and/or MSI-High had better relapse-free survival. RNA-Seq revealed gene fusions in 14/105 (13%) cases, and heterogeneous expression pattern. The majority of gene fusions affected tyrosine kinase receptors (6/14; four of those were MET fusions) or DNA repair genes (2/14). Based on the mRNA expression pattern, a cluster of 12 OCCCs characterized by overexpression of tyrosine kinase receptors (TKRs) AKT3, CTNNB1, DDR2, JAK2, KIT, or PDGFRA (p < 0.00001) was identified. CONCLUSIONS: The current work has elucidated the complex genomic and transcriptomic molecular hallmarks of primary OCCCs. Our results confirmed the favorable outcomes of POLEmut and MSI-High OCCC. Moreover, the molecular landscape of OCCC revealed several potential therapeutical targets. Molecular testing can provide the potential for targeted therapy in patients with recurrent or metastatic tumors.

Comparative Analysis of Bone Ingrowth in 3D-Printed Titanium Lattice Structures with Different Patterns.

In this study, metal 3D printing technology was used to create lattice-shaped test specimens of orthopedic implants to determine the effect of different lattice shapes on bone ingrowth. Six different lattice shapes were used: gyroid, cube, cylinder, tetrahedron, double pyramid, and Voronoi. The lattice-structured implants were produced from Ti6Al4V alloy using direct metal laser sintering 3D printing technology with an EOS M290 printer. The implants were implanted into the femoral condyles of sheep, and the animals were euthanized 8 and 12 weeks after surgery. To determine the degree of bone ingrowth for different lattice-shaped implants, mechanical, histological, and image processing tests on ground samples and optical microscopic images were performed. In the mechanical test, the force required to compress the different lattice-shaped implants and the force required for a solid implant were compared, and significant differences were found in several instances. Statistically evaluating the results of our image processing algorithm, it was found that the digitally segmented areas clearly consisted of ingrown bone tissue; this finding is also supported by the results of classical histological processing. Our main goal was realized, so the bone ingrowth efficiencies of the six lattice shapes were ranked. It was found that the gyroid, double pyramid, and cube-shaped lattice implants had the highest degree of bone tissue growth per unit time. This ranking of the three lattice shapes remained the same at both 8 and 12 weeks after euthanasia. In accordance with the study, as a side project, a new image processing algorithm was developed that proved suitable for determining the degree of bone ingrowth in lattice implants from optical microscopic images. Along with the cube lattice shape, whose high bone ingrowth values have been previously reported in many studies, it was found that the gyroid and double pyramid lattice shapes produced similarly good results.

Microsatellite instability in non-endometrioid ovarian epithelial tumors: a study of 400 cases comparing immunohistochemistry, PCR, and NGS based testing with mutation status of MMR genes.

Testing of microsatellite instability is not only used as a triage for possible Lynch syndrome, but also to predict immunotherapy treatment response. The aim of this study was to assess the frequency of mismatch repair deficiency (MMR-D)/microsatellite instability (MSI) in 400 cases of non-endometrioid ovarian tumors (high-grade serous, low-grade serous, mucinous and clear cell), to compare different methodological approaches of testing, and to assess the optimal approach for next generation sequencing (NGS) MSI testing. For all tumors, we evaluated immunohistochemical (IHC) expression of MMR proteins and assessed microsatellite markers by PCR-based method. Except for high-grade serous carcinoma, we correlated the findings of IHC and PCR with NGS-based MSI testing. We compared the results with somatic and germline mutation in MMR genes. Among the whole cohort, seven MMR-D cases, all clear cell carcinomas (CCC), were found. On PCR analysis, 6 cases were MSI-high and one was MSS. In all cases, mutation of an MMR gene was found; in 2 cases, the mutation was germline (Lynch syndrome). An additional 5 cases with a mutation in MMR gene(s) with MSS status and without MMR-D were identified. We further utilized sequence capture NGS for MSI testing. Employing 53 microsatellite loci provided high sensitivity and specificity. Our study shows that MSI occurs in 7% of CCC while it is rare or absent in other nonendometrioid ovarian neoplasms. Lynch syndrome was present in 2% of patients with CCC. However, some cases with MSH6 mutation can evade all testing methods, including IHC, PCR, and NGS-MSI.

A comprehensive immunohistochemical analysis of 26 markers in 250 cases of serous ovarian tumors.

BACKGROUND: We examined a large cohort of serous tubo-ovarian tumors with 26 immunohistochemical markers, with the aim to assess their value for differential diagnosis and prognosis. METHODS: Immunohistochemical analyses with 26 immunomarkers were performed on 250 primary tubo-ovarian tumors including 114 high grade serous carcinomas (HGSC), 97 low grade serous carcinomas (LGSC), and 39 serous borderline tumors (micropapillary variant, mSBT). The associations of overall positivity with clinicopathological characteristics were evaluated using the chi-squared test or Fisher's Exact test. RESULTS: We found significantly different expression of p53, p16, ER, PR, PTEN, PAX2, Mammaglobin, RB1, Cyclin E1, stathmin, LMP2, L1CAM, CD44, and Ki67 in HGSCs compared to LGSCs. No significant differences were found between LGSC and mSBT. None of the other included markers (PAX8, ARID1A, HNF1B, Napsin A, CDX2, SATB2, MUC4, BRG1, AMACR, TTF1, BCOR, NTRK) showed any differences between the investigated serous tumors. Regarding the prognosis, only PR and stathmin showed a statistically significant prognostic meaning in LGSCs, with better overall survival (OS) and recurrence-free survival (RFS) in cases positive for PR, and worse outcome (RFS) for stathmin. None of the study markers showed prognostic significance in HGSCs. CONCLUSION: We provided an extensive immunohistochemical analysis of serous ovarian/tubo-ovarian tumors. Although we found some differences in the expression of some markers in HGSCs compared to LGSCs, only p53, p16, and Ki67 seem to be useful in real diagnostic practice. We also suggested the best discriminative cut-off for Ki67 (10% of positive tumor cells) for distinguishing HGSC from LGSC. We found prognostic significance of PR and stathmin in LGSCs. Moreover, the high expression of stathmin could also be of predictive value in ovarian carcinomas as target-specific anti-stathmin effectors are potential therapeutic targets.

Pan-tumor T-lymphocyte detection using deep neural networks: Recommendations for transfer learning in immunohistochemistry.

The success of immuno-oncology treatments promises long-term cancer remission for an increasing number of patients. The response to checkpoint inhibitor drugs has shown a correlation with the presence of immune cells in the tumor and tumor microenvironment. An in-depth understanding of the spatial localization of immune cells is therefore critical for understanding the tumor's immune landscape and predicting drug response. Computer-aided systems are well suited for efficiently quantifying immune cells in their spatial context. Conventional image analysis approaches are often based on color features and therefore require a high level of manual interaction. More robust image analysis methods based on deep learning are expected to decrease this reliance on human interaction and improve the reproducibility of immune cell scoring. However, these methods require sufficient training data and previous work has reported low robustness of these algorithms when they are tested on out-of-distribution data from different pathology labs or samples from different organs. In this work, we used a new image analysis pipeline to explicitly evaluate the robustness of marker-labeled lymphocyte quantification algorithms depending on the number of training samples before and after being transferred to a new tumor indication. For these experiments, we adapted the RetinaNet architecture for the task of T-lymphocyte detection and employed transfer learning to bridge the domain gap between tumor indications and reduce the annotation costs for unseen domains. On our test set, we achieved human-level performance for almost all tumor indications with an average precision of 0.74 in-domain and 0.72-0.74 cross-domain. From our results, we derive recommendations for model development regarding annotation extent, training sample selection, and label extraction for the development of robust algorithms for immune cell scoring. By extending the task of marker-labeled lymphocyte quantification to a multi-class detection task, the pre-requisite for subsequent analyses, e.g., distinguishing lymphocytes in the tumor stroma from tumor-infiltrating lymphocytes, is met.

A comprehensive immunohistochemical analysis of IMP2 and IMP3 in 542 cases of ovarian tumors.

BACKGROUND: IMP2 and IMP3 are mRNA binding proteins involved in carcinogenesis. We examined a large cohort of ovarian tumors with the aim to assess the value of IMP2 and IMP3 for differential diagnosis, and to assess their prognostic significance. METHODS: Immunohistochemical analyses with antibodies against IMP2 and IMP3 were performed on 554 primary ovarian tumors including 114 high grade serous carcinomas, 100 low grade serous carcinomas, 124 clear cell carcinomas, 54 endometrioid carcinomas, 34 mucinous carcinomas, 75 mucinous borderline tumors, and 41 serous borderline tumors (micropapillary variant). The associations of overall positivity with clinicopathological characteristics were evaluated using the chi-squared test or Fisher's Exact test. RESULTS: We found IMP2 expression (in more than 5% of tumor cells) in nearly all cases of all tumor types, so the prognostic meaning could not be analyzed. The positive IMP3 expression (in more than 5% of tumor cells) was most common in mucinous carcinomas (82%) and mucinous borderline tumors (81%), followed by high grade serous (67%) and clear cell carcinomas (67%). The expression was less frequent in endometrioid carcinomas (39%), low grade serous carcinomas (23%), and micropapillary variant of serous borderline tumors (20%). Prognostic significance of IMP3 could be evaluated only in low grade serous carcinomas in the case of relapse-free survival, where negative cases showed better RFS (p = 0.033). CONCLUSION: Concerning differential diagnosis our results imply that despite the differences in expression in the different ovarian tumor types, the practical value for diagnostic purposes is limited. Contrary to other solid tumors, we did not find prognostic significance of IMP3 in ovarian cancer, with the exception of RFS in low grade serous carcinomas. However, the high expression of IMP2 and IMP3 could be of predictive value in ovarian carcinomas since IMP proteins are potential therapeutical targets.

Molecular Identification and In Silico Protein Analysis of a Novel BCOR-CLGN Gene Fusion in Intrathoracic BCOR-Rearranged Sarcoma.

BCOR (BCL6 corepressor)-rearranged sarcomas (BRSs) are a heterogeneous group of sarcomas previously classified as part of the group of "atypical Ewing" or "Ewing-like" sarcomas, without the prototypical ESWR1 gene translocation. Due to their similar morphology and histopathological features, diagnosis is challenging. The most common genetic aberrations are BCOR-CCNB3 fusion and BCOR internal tandem duplication (ITD). Recently, various new fusion partners of BCOR have been documented, such as MAML3, ZC3H7B, RGAG1, and KMT2D, further increasing the complexity of such tumor entities, although the molecular pathogenetic mechanism remains to be elucidated. Here, we present an index case of intrathoracic BRS that carried a novel BCOR-CLGN (calmegin) gene fusion, exhibited by a 52-year-old female diagnosed initially by immunohistochemistry due to the positivity of a BCOR stain; the fusion was identified by next-generation sequencing and was confirmed by Sanger sequencing. In silico protein analysis was performed to demonstrate the 3D structure of the chimera protein. The physicochemical properties of the fusion protein sequence were calculated using the ProtParam web-server tool. Our finding further broadens the fusion partner gene spectrum of BRS. Due to the heterogeneity, molecular ancillary tests serve as powerful tools to discover these unusual variants, and an in silico analysis of the fusion protein offers an appropriate approach toward understanding the exact pathogenesis of such a rare variant.