The Relationship between Tumor Budding and Tumor Deposits in Patients with Stage III Colorectal Carcinoma.

Background/Objectives: Recently, some new morphological features of colorectal cancer have been discovered as important prognostic factors; in this paper, we study the relationship between tumor budding (TB) and tumor deposits (TDs). Methods: The retrospective cohort study included 90 patients with pathohistologically confirmed stage III CRC who were treated with radical surgical resection. All hematoxylin and eosin (H and E)-stained slides from each patient were reviewed, and histological parameters were recorded. The samples were divided into two groups with similar sizes: a group without TDs (N = 51) and a control group with TDs (N = 39). The presence and TB grade were further analyzed in these groups and compared with other clinical and histological features. Results: The prevalence of TB in the investigated cohort was unexpectedly high (94.4%). Overall, there were 23 (25.6%) Bd1, 20 (22.2%) Bd2, and 47 (52.2%) Bd3 cases. The presence of TDs was significantly associated with a higher number of TB (p < 0.001, OR 16.3) and, consequently, with a higher TB grade (p = 0.004, OR 11.04). A higher TB grade (p = 0.001, HR 2.28; 95% CI 1.93-4.76) and a growing number of TDs (p = 0.014, HR 1.52; 95% CI 1.09-2.1) were statistically significantly associated with shorter survival. Conclusions: TDs appear more often in patients with higher TB grades in stage III CRC. A higher TB grade and a growing number of TDs were statistically significantly associated with shorter overall survival. These results could give additional emphasis to the importance of TB as an adverse prognostic factor since a strong relationship with TDs has been demonstrated.

Testicular Germ Cell Tumor Tissue Biomarker Analysis: A Comparison of Human Protein Atlas and Individual Testicular Germ Cell Tumor Component Immunohistochemistry.

The accurate management of testicular germ cell tumors (TGCTs) depends on identifying the individual histological tumor components. Currently available data on protein expression in TGCTs are limited. The human protein atlas (HPA) is a comprehensive resource presenting the expression and localization of proteins across tissue types and diseases. In this study, we have compared the data from the HPA with our in-house immunohistochemistry on core TGCT diagnostic genes to test reliability and potential biomarker genes. We have compared the protein expression of 15 genes in TGCT patients and non-neoplastic testicles with the data from the HPA. Protein expression was converted into diagnostic positivity. Our study discovered discrepancies in three of the six core TGCT diagnostic genes, POU5F1, KIT and SOX17 in HPA. DPPA3, CALCA and TDGF1 were presented as potential novel TGCT biomarkers. MGMT was confirmed while RASSF1 and PRSS21 were identified as biomarkers of healthy testicular tissue. Finally, SALL4, SOX17, RASSF1 and PRSS21 dysregulation in the surrounding testicular tissue with complete preserved spermatogenesis of TGCT patients was detected, a potential early sign of neoplastic transformation. We highlight the importance of a multidisciplinary collaborative approach to fully understand the protein landscape of human testis and its pathologies.

Expression and Prognostic Significance of PD-L1 and NY-ESO1 in Gallbladder Carcinoma.

BACKGROUND/AIM: Gallbladder cancer is a rare malignancy with a very high mortality, usually due to diagnosis in an advanced stage of the disease. Therefore, the aim of this study was to evaluate the clinical significance of cancer/testis antigen 1A (CTAG1A, NY-ESO1) and CD274 molecule (PD-L1, the ligand for programmed cell death protein 1) and their impact on the overall survival of patients with gallbladder cancer. PATIENTS AND METHODS: Using immunohistochemical staining, we determined the expression of NY-ESO1 in tumor cells (positivity: cytoplasmic/nuclear staining of any intensity in ≥50%) and PD-L1 in tumor cells and intratumoral immune cells (positivity: cytoplasmic/membranous staining of any intensity in ≥1%). RESULTS: The median overall survival (OS) of 58 patients with gallbladder cancer in our cohort was 7 months, and depended on the clinical stage of the disease; the 5-year OS rate was 10%. NY-ESO1 was expressed in 69.1% of cases. Immune cells were PD-L1-positive in 36.4% of cases, while tumor cells expressed PD-L1 in only 10.9% of cases. In six cases (10.9%), neither of the studied proteins were expressed. NY-ESO1 expression was negatively correlated with PD-L1 expression in immune cells (p=0.021). NY-ESO1 showed no correlation with any clinicopathological parameters or OS. PD-L1 expression in immune cells was significantly higher in tumors with perineural invasion (rs=0.318; p=0.018) and higher clinical disease stage (rs=0.339; p=0.013) but showed no correlation with OS. CONCLUSION: Patients whose gallbladder cancer expresses NY-ESO1 or PD-L1 might be candidates for immunotherapy.

Circulating Tumor Cells in Colorectal Cancer: Detection Systems and Clinical Utility.

Colorectal cancer (CRC) is the third most common cancer worldwide. The high mortality from CRC is mainly related to metastasis affecting distant organs and their function. Dissemination of tumor cells from the primary tumor and hematogeneous spread are considered crucial in the formation of tumor metastases. The analysis of circulating tumor cells (CTCs) and CTC clusters in the blood can be used for the early detection of invasive cancer. Moreover, CTCs have a prognostic significance in the monitoring of a malignant disease or the response to chemotherapy. This work presents an overview of the research conducted on CTCs with the aim of finding suitable detection systems and assessing the possibility of clinical applications in patients with CRC.

Profiling Colorectal Cancer in the Landscape Personalized Testing-Advantages of Liquid Biopsy.

Drug-specific therapeutic approaches for colorectal cancer (CRC) have contributed to significant improvements in patient health. Nevertheless, there is still a great need to improve the personalization of treatments based on genetic and epigenetic tumor profiles to maximize the quality and efficacy while limiting cytotoxicity. Currently, CEA and CA 19-9 are the only validated blood biomarkers in clinical practice. For this reason, laboratories are trying to identify new specific prognostics and, more importantly, predictive biomarkers for CRC patient profiling. Thus, the unique landscape of personalized biomarker data should have a clinical impact on CRC treatment strategies and molecular genetic screening tests should become the standard method for diagnosing CRC. This review concentrates on recent molecular testing in CRC and discusses the potential modifications in CRC assay methodology with the upcoming clinical application of novel genomic approaches. While mechanisms for analyzing circulating tumor DNA have been proven too inaccurate, detecting and analyzing circulating tumor cells and protein analysis of exosomes represent more promising options. Blood liquid biopsy offers good prospects for the future if the results align with pathologists' tissue analyses. Overall, early detection, accurate diagnosis and treatment monitoring for CRC with specific markers and targeted molecular testing may benefit many patients.

Somatostatin receptor SSTR2A and SSTR5 expression in neuroendocrine breast cancer.

Neuroendocrine breast cancer (NEBC) is a group of rare tumors, which could benefit from therapy targeting the somatostatin receptors (SSTRs). In particular, SSTR2A and SSTR5 are potential targets given their consistent expression in gastrointestinal and pancreatic primary and metastatic neuroendocrine cancers. Currently, there are no studies describing the expression of SSTRs in NEBC. The purpose of our study was to characterize the immunohistochemical expression of SSTR2A and SSTR5 in a cohort of NEBC. Thirty-one primary NEBC cases were analyzed, and SSTR2A and SSTR5 immunohistochemistry performed and scored using the modified immunoreactive score proposed by Remmele and Stanger. All patients were females with a mean age of 66.6 years (SD = 14). 77% of cases were histological grade 2. SSTR2A showed a weak positivity in 11 cases (35.5%), moderate positivity in 6 cases (19.4%) and strong positivity in 5 cases (16.1%). Nine cases were negative for SSTR2A (29%). SSTR5 showed a weak positivity in 16 cases (51.6%), moderate positivity in 6 cases (19.4%), while no cases showed strong positivity. Nine cases were negative for SSTR5 (29%). Five cases were negative for both SSTR2A and SSTR5. A weak to moderate SSTR2A and SSTR5 expression was observed in 50-70% of the cases. A subset of NEBCs with strong SSR2A expression may benefit from SSTRs targeted therapy. These results need further validation in a larger series including metastatic NEBC, to provide potential therapeutic targets for patients with advanced disease.

A score derived from routine biochemical parameters increases the diagnostic accuracy of chromogranin A in detecting patients with neuroendocrine neoplasms.

BACKGROUND: Chromogranin A (CgA) is a valuable biomarker for detection and follow-up of patients with neuroendocrine neoplasms (NENs). However, various comorbidities may influence serum CgA, which decreases its diagnostic accuracy. We aimed to investigate which laboratory parameters are independently associated with increased CgA in real-life setting and to develop a scoring system, which could improve the diagnostic accuracy of CgA in detecting patients with NENs. METHODS: This retrospective study included 55 treatment naïve patients with NENs and160 patients with various comorbidities but without NEN (nonNENs). Scoring system (CgA-score) was developed based on z-scores obtained from receiver operating curve analysis for each parameter that was associated with elevated serum CgA in nonNENs. RESULTS: CgA correlated positively with serum BUN, creatinine, α2-globulin, red-cell distribution width, erythrocyte sedimentation rate, plasma glucose and correlated inversely with hemoglobin, thrombocytes and serum albumin. Serum CgA was also associated with the presence of chronic renal failure, arterial hypertension and diabetes and the use of PPI. In the entire study population, CgA showed an area under the curve of 0.656. Aforementioned parameters were used to develop a CgA-score. In a cohort of patients with CgA-score <12.0 (N = 87), serum CgA >156.5 ng/ml had 77.8% sensitivity and 91.5% specificity for detecting NENs (AUC 0.841, 95% CI 0.713-0.969, P < 0.001). Serum CgA had no diagnostic value in detecting NENs in patients with CgA-score >12.0 (AUC 0.554, 95% CI 0.405-0.702, P = 0.430). CONCLUSIONS: CgA-score encompasses a wide range of comorbidities and represents a promising tool that could improve diagnostic performance of CgA in everyday clinical practice.

PATHOLOGICAL CHANGES OF RENAL ARTERIES IN PATIENTS WITH RENAL CELL CARCINOMA.

Renal cell carcinoma is the ninth most common cancer in the world. It may have a varied microscopic appear- ance, and the most common histopathological type is clear cell carcinoma. The most common pathological changes of renal arteries are atherosclerosis and fibromuscular dysplasia (FMD). During histopathological evaluation of a kidney specimen containing carcinoma, the renal vein is routinely analyzed, while the renal artery is usually given little attention. Our stud- ies have shown that pathological changes of renal arteries are significantly more frequent in the group of patients with renal cell carcinoma compared with the control group and the group of patients with non-tumor kidney diseases. These relations led us to the conclusion that the onset of renal artery changes is not prior to the carcinoma or non-tumor diseases and that they are formed simultaneously or as a consequence. Further studies should be aimed at determining the incidence of these changes in a larger number of samples and the detection of their possible correlation with renal cell carcinoma.

Epithelioid hemangioma of the orbit: case report.

Epithelioid hemangioma (EH) and Kimura's disease (KD) were once considered different stages of the same disease, as they share many clinical and histopathologic similarities. Nowadays, they are considered as two different entities, but some authors still confuse these terms. Our objective is to present a case of EH occurring in a very uncommon location and to emphasize the microscopic and clinical differences between EH and KD. We present a case of EH of the orbit in an 83-year-old man diagnosed after histopathologic evaluation of a mass that was surgically removed from the orbit. The tumor showed typical microscopic appearance with pathognomonic epithelioid endothelial cells. The diagnosis was also confirmed by immunohistochemical analysis. Our case clearly illustrates typical appearance of EH and the main differences between EH and KD are thoroughly discussed.

Correlation of vascular endothelial growth factor and hypoxia-inducible factor-1α expression with pathological renal artery changes in patients with renal cell carcinoma.

OBJECTIVE: The aim of this study was to correlate the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) with pathological renal artery changes in patients with renal cell carcinoma (RCC). A further aim was to correlate intratumoral microvessel density (MVD) with VEGF and HIF-1α expression and prognostic factors for RCC, including tumour necrosis. MATERIAL AND METHODS: Formalin-fixed and paraffin-embedded tissue blocks from 150 patients with RCC and 50 patients with non-tumorous kidney diseases were analysed. The control group consisted of specimens from both renal arteries obtained from 25 decedents at routine autopsy (50 cases in total). Immunohistochemistry was performed using primary antibodies to VEGF, HIF-1α and CD31. RESULTS: Pathological renal artery changes were more common in patients with RCC and non-tumorous kidney diseases than in the control group. MVD was higher in the RCCs of patients with pathological renal artery changes. Tumours with higher HIF-1α expression had higher MVD; however, VEGF expression was not associated with MVD. A significant association was also found between MVD and the extent of tumour necrosis, in that less necrotic tumours had higher MVD. No association between renal artery changes and VEGF and HIF-1α expression was established. CONCLUSION: Considering the results of this study, the evaluation of renal artery changes in forthcoming research on RCC would be helpful for several reasons: to estimate their incidence in a larger number of patients, to clarify their connection with RCC and to reveal their relationship with MVD in RCC.

Extensive retraction artefact predicts biochemical recurrence-free survival in prostatic carcinoma.

AIMS: To determine whether the presence and extent of peritumoral retraction artefact could be used to predict biochemical recurrence-free survival in prostatic carcinoma. METHODS AND RESULTS: The study included 162 consecutive patients treated by radical retropubic prostatectomy and bilateral lymphadenectomy for clinically localized prostatic carcinoma. A variable degree of retraction artefact was present in all 162 analysed tumours. The extent of retraction artefact in prostatic carcinomas ranged from 5% to 55% with a median value of 15% (interquartile range 10-25%). We found no correlation between the extent of retraction artefact in the tumours and patient's age (P=0.608), preoperative (P=0.362) and postoperative (P=0.279) Gleason score or lymph node metastases (P=0.084). In contrast, the extent of retraction artefact correlated with high preoperative prostate-specific antigen (P<0.001), short follow-up time (P<0.001), seminal vesicle invasion and/or extracapsular extension of the tumour (T3 stage tumours) (P<0.001) and positive surgical margins (P<0.001). Furthermore, extensive retraction artefact was associated with poor biochemical recurrence-free survival in both univariate (P<0.001) and multivariate analyses (P=0.013). CONCLUSION: The presence of extensive retraction artefact in prostatic carcinoma correlates with tumour characteristics signifying aggressive behaviour and indicates poor biochemical recurrence-free survival.

Synchronous rectal adenocarcinoma and bilateral clear cell renal carcinoma.

A 69-year-old man was admitted for resection of rectal adenocarcinoma diagnosed by colonoscopy. Preoperative computed tomography scan and abdominal ultrasonography revealed bilateral renal tumors measuring up to 2 and 2.8 cm in largest diameter, respectively. The patient underwent partial colectomy and bilateral partial nephrectomy. Microscopically, rectal adenocarcinoma penetrated the submucosa, without invasion of the muscularis propria. Both renal tumors were clear cell renal carcinomas of Fuhrmann nuclear grade 2. To our knowledge, this is the first case of synchronous adenocarcinoma of the rectum and bilateral clear cell renal cell carcinoma described in the literature to date.

Immunohistochemical expression of tumor antigens MAGE-A3/4 and NY-ESO-1 in renal oncocytoma and chromophobe renal cell carcinoma.

The distinction between renal oncocytoma (RO) and chromophobe renal cell carcinoma (ChRCC), especially the eosinophilic variant, can often be difficult. Our study has documented for the first time the expression of MAGE-A3/4 and NY-ESO-1 cancer testis antigens (CTAs) in these tumors. A total of 35 patients (17 ROs and 18 ChRCCs) were included in the study. Two antibodies were used for immunohistochemical staining: 57B recognizing multiple MAGE-A and D8.38 recognizing NY-ESO-1 CTAs. Fifteen (88.2%) samples of RO stained positively for both MAGE-A3/4 and NY-ESO-1 antigens. Regarding ChRCC, seven (38.9%) stained positively for MAGE-A3/4 and six (33.3%) for NY-ESO-1 antigens. Median MAGE-A3/4 expression was moderately positive in RO and negative in ChRCC. The difference in MAGE-A3/4 expression between two tumor groups was significant (P=0.0013). Median NY-ESO-1 expression was strongly positive in RO and negative in ChRCC. The difference in NY-ESO-1 expression between two tumor groups was also significant (P=0.0008). Our study has shown that RO had a significantly higher expression of both CTAs. However, additional research is needed to clarify their potential diagnostic implications.

CD138-positive plasmacytoid urothelial carcinoma of urinary bladder with focal micropapillary features.

Both the plasmacytoid and micropapillary types of urothelial carcinoma of the urinary bladder are uncommon, distinct clinical and pathological findings. To date, several reports in the English medical literature have been published on either of these variants. CD138 is commonly used as a marker for tumors of plasma cell origin. However, few authors have described positive immunoreactivity of plasmacytoid cells in urothelial carcinoma. Mixed histological differentiation is thought to be a phenotype of locally aggressive and advanced urothelial carcinoma. Therefore, a precise histopathological diagnosis should be made and awareness of all the entities is crucial. We report a case of CD138-positive plasmacytoid urothelial carcinoma of the bladder with focal micropapillary features. To our knowledge this is the first case of these two rare subtypes of urothelial carcinoma combined in a single cystectomy specimen.

Intensity of stromal changes predicts biochemical recurrence-free survival in prostatic carcinoma.

OBJECTIVE: The reactive stroma of prostate cancer contains a mixture of myofibroblasts and fibroblasts, while fully differentiated smooth-muscle cells are very rare or absent. In experimental prostate cancer models, prostatic stromal cells promote angiogenesis and stimulate prostate tumorigenesis. The aim of this study is to analyse whether the intensity of stromal changes can predict survival in patients with prostatic carcinoma. MATERIAL AND METHODS: Stromal reaction was quantified histochemically and imunohistochemically in 50 patients treated with radical prostatectomy for clinically localized prostate carcinoma and its relationship with established prognostic factors was assessed. RESULTS: Kaplan-Meier analysis showed a significant association between the pattern of vimentin and desmin expression and the length of disease-free period; patients with a higher vimentin or lower desmin expression had a shorter disease-free period. On multivariate analysis only vimentin expression (odds ratio 4.06, 95% confidence interval 1.01-16.26, p = 0.049) was a significant predictor of biochemical recurrence. In patients with identical Gleason pattern and Gleason score the level of vimentin expression could identify patients with a higher risk of disease recurrence. CONCLUSIONS: Intensity of stromal changes could serve as an independent prognostic factor in the assessment of biochemical recurrence-free survival. Among prostate cancer patients with an identical Gleason score, it could identify patients with a higher risk of biochemical recurrence. Thus, stromal changes and their intensity could serve as a novel marker for the recognition of patients with an increased risk of disease recurrence.

Can renal oncocytoma be distinguished from chromophobe renal cell carcinoma by the presence of fibrous capsule?

The most important differential diagnosis of chromophobe renal cell carcinoma (CRCC) is renal oncocytoma. Due to overlapping morphological characteristics of renal oncocytoma and CRCC, particularly its eosinophilic variant, making a correct diagnosis can be challenging. To date, no data are available on the presence of the tumor fibrous capsule as a diagnostic feature in differentiating these tumors. The main purpose of this study was to establish the presence and compare the thickness of the tumor fibrous capsule between two tumor groups. A total of 37 tumors--18 cases of CRCC (three eosinophilic and 15 classic) and 19 cases of renal oncocytoma--were analyzed. Four slides of each tumor stained with hematoxylin and eosin were first scanned at low-power magnification (x40) to assess the presence of the capsule. If present, the capsule was measured in three different thickest areas at higher magnification (x200). The mean value of capsule thickness was calculated and taken into consideration. The capsule was present in 12 (66.7%) cases of CRCCs and in only two (10.5%) cases of renal oncocytomas. Statistical analysis showed significant difference between the presence of fibrous capsule in these two observed tumor groups (P = 0.001). Average thickness of capsule in CRCCs was 337.7 microm, and 115.4 microm in renal oncocytomas, but the median was not statistically significant (P = 0.198). Studies with a larger number of cases are needed to conclude if this characteristic could be a low-cost, reliable microscopic feature in differentiating between CRCC and renal oncocytoma.

Comparison of occurrence of upper urinary tract carcinomas in the region with endemic villages and non-endemic nephropathy region in Croatia.

Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial renal disease of a still unknown etiology, associated with an increased frequency of urothelial carcinoma, particularly of the upper urinary tract (UUT). The aim of the study was to compare the occurrence of UUT carcinomas between Brodsko-Posavska Region (BPR) which is the region with endemic villages and the non-endemic region of Zagreb (ZG) in two six-year periods with a 20 year period separating the two, pointing out a possible difference in occurrence regarding war in Croatia (1991-1995). Comparing BPR and ZG regions we found a more then 5 times higher frequency of UUT carcinomas in BPR in the first period and more than 4.5 times higher frequency in the second period. Women in BPR were more frequently affected with UUT carcinomas.

Pleomorphic ductal carcinoma of the breast with predominant micropapillary features.

An 83-year-old woman with long-standing chronic ischemic cardiac and obstructive pulmonary disease, presented with a painless tumor in her right breast. Microscopically the tumor consisted of micropapillary formations and loosely cohesive nests and strands of large, highly pleomorphic cells. Micropapillary formations were surrounded by peritumoral retraction clefting, and the papillae lacked a true fibrovascular core. Multinucleated giant and bizarre tumor cells were also present and numerous. Within the tumor a high-grade intraductal component with the same cell morphology and necrosis and mucin production was found. Micropapillary pattern occupied approximately 60% of the tumor mass, loosely cohesive nests and strands approximately 20% and an intraductal component was noted in approximately 20% of the tumor mass. On immunohistochemistry the tumor cells were positive for pan-cytokeratin, epithelial membrane antigen (EMA), S100 protein and E-cadherin while estrogen and progesterone receptors, HER2-neu and Bcl2 were negative. EMA staining was diffuse and observed in the outer and inner margins of neoplastic nests. The diagnosis of pleomorphic breast carcinoma with predominant micropapillary features was established. In summary, micropapillary carcinoma can be distinguished from other types of breast carcinoma with micropapillary growth pattern on the basis of reverse cell polarity, which is easily confirmed on immunohistochemistry.

Targeting human cancer cells with VEGF receptor-2-directed liposomes.

Antibodies are among the most versatile tools used today to characterize and target molecules in cells and in biological tissues. The development of phage display libraries encoding a large repertoire of single chain antibodies, scFv, allows the rapid and efficient isolation of antibodies specific for almost any type of molecule. A great advantage of such recombinant antibodies is the possibility to functionalize them by introducing new amino acid sequences. This leads to new features that would be difficult to introduce into naturally occurring antibody molecules. This approach has been successfully applied to create molecules with new biological activities, e.g. by generating chimeric scFv antibodies carrying sequences derived from other biomolecules such as blood clotting factors or enzymes. Here, we describe a new antibody isolated from an M13 phage library that recognizes vascular endothelial growth factor receptor 2, VEGFR-2. This antibody, scFvVR-2H9 was coupled to liposomes and used to specifically target VEGFR-2-expressing human cancer cells in culture.