Ruza Arsenic

Clinical relevance of the de novo production of anti‐HLA antibodies following intestinal and multivisceral transplantation

Despite a negative pretransplant cross‐match, intestinal transplant recipients can mount humoral immune responses soon after transplantation. Moreover, the development of donor‐specific anti‐HLA antibodies (DSAs) is associated with severe graft injury. Between June 2000 and August 2011, 30 patients (median age 37.6 ± 9.8 years) received isolated intestinal transplantations (ITX, n = 18) or multivisceral transplantations (MVTXs, n = 12) at our center. We screened for human leukocyte antigen (HLA) antibodies pre‐ and post‐transplant. If patients produced DSAs, treatment with plasmapheresis and intravenous immunoglobulin (IVIG) was initiated. In the event of DSA persistence and/or treatment‐refractory rejection, rituximab and/or bortezomib were added. Ten patients developed DSAs and simultaneously showed significant signs of rejection. These patients received plasmapheresis and IVIG. Eight patients additionally received rituximab, and two patients were treated with bortezomib. DSA values decreased upon antirejection therapy in 8 of the 10 patients. The development of DSAs following ITX is often associated with acute rejection. We observed that the number of mismatched antigens and epitopes correlates with the probability of developing de novo DSAs. Early diagnosis and therapy, including B‐cell depletion and plasma cell inhibition, are crucial to preventing further graft injury.

Axon Guidance Factor SLIT2 Inhibits Neural Invasion and Metastasis in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) metastasizes by neural, vascular, and local invasion routes, which limit patient survival. In nerves and vessels, SLIT2 and its ROBO receptors constitute repellent guidance cues that also direct epithelial branching. Thus, the SLIT2-ROBO system may represent a key pinch point to regulate PDAC spread. In this study, we examined the hypothesis that escaping from repellent SLIT2-ROBO signaling is essential to enable PDAC cells to appropriate their local stromal infrastructure for dissemination. Through immunohistochemical analysis, we detected SLIT2 receptors ROBO1 and ROBO4 on epithelia, nerves, and vessels in healthy pancreas and PDAC specimens, respectively. SLIT2 mRNA expression was reduced in PDAC compared with nontransformed pancreatic tissues and cell lines, suggesting a reduction in SLIT2-ROBO pathway activity in PDAC. In support of this interpretation, restoring the SLIT2 expression in SLIT2-deficient PDAC cells inhibited their bidirectional chemoattraction with neural cells, and more specifically, impaired unidirectional PDAC cell navigation along outgrowing neurites in models of neural invasion. Restoring autocrine/paracrine SLIT2 signaling was also sufficient to inhibit the directed motility of PDAC cells, but not their random movement. Conversely, RNA interference-mediated silencing of ROBO1 stimulated the motility of SLIT2-competent PDAC cells. Furthermore, culture supernatants from SLIT2-competent PDAC cells impaired migration of endothelial cells (human umbilical vein endothelial cells), whereas an N-terminal SLIT2 cleavage fragment stimulated such migration. In vivo investigations of pancreatic tumors with restored SLIT2 expression demonstrated reduced invasion, metastasis, and vascularization, with opposing effects produced by ROBO1 silencing in tumor cells or sequestration of endogenous SLIT2. Analysis of clinical specimens of PDAC showed that those with low SLIT2 mRNA expression exhibited a higher incidence and a higher fraction of tumor-infiltrated lymph nodes. Taken together, our findings argue that disrupting SLIT2-ROBO signaling in PDAC may enhance metastasis and predispose PDAC cells to neural invasion.

Analysis of PIK3CA mutations in breast cancer subtypes.

Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit &agr; (PIK3CA) is a central element of a signaling pathway involved in cell proliferation, survival, and growth. Certain mutations in this pathway result in enhanced PI3K signaling, which is associated with oncogenic cellular transformation and cancer. The aims of this study were to characterize different types of PIK3CA mutations in exons 9 and 20 in a series of primary breast carcinomas and to correlate the results with clinicopathologic parameters and survival. We used frozen tissue samples and sequenced exons 9 and 20 for a series of 241 patients with a diagnosis of breast carcinoma. We found that 15.8% of the primary breast carcinomas possessed PIK3CA mutations in either exon 9 or exon 20. The rate of PIK3CA mutations was increased in HR+/HER2− tumors (18.6%), but this difference did not reach a statistical significance. The lowest rate of mutations was observed in HR+/HER2+ tumors (5.3%). No statistically significant association was found between the presence of PIK3CA mutations and the prognostic/clinical features of breast cancer, including histologic subtype, Her2 status, axillary lymph node involvement, tumor grade, and tumor stage. However, the presence of the H1047R mutation in 10 samples was associated with a statistically significantly worse overall survival. PIK3CA mutation was found to be a frequent genetic change in all breast cancer subtypes but occurred with the highest rate in HR+/HER2− tumors. Further studies are needed to validate the prognostic impact of different PIK3CA mutations.

Evaluation of radiological prognostic factors of hepatic metastases in patients with non-functional pancreatic neuroendocrine tumors

PURPOSE There are different therapeutic options in non-functional well to moderately differentiated (G1 and G2) pancreatic neuroendocrine tumors (pNET) with unresectable hepatic metastases including systemic chemotherapy and novel molecular targeted therapies. Treatment with somatostatin analogs (SSA) as antiproliferative agents is optional. At initial diagnosis watchful waiting until tumor progression is a well-established approach. Goal of this study was to evaluate imaging features as potential prognostic factors predicting early tumor progression in order to select patients that might benefit from an earlier initiation of medical treatment. PATIENTS AND METHODS In 44 patients we correlated tumor grade, chromogranin A (CgA) levels, treatment with SSA and imaging features of hepatic metastases on contrast-enhanced multiphase CT and MR imaging with time to tumor progression (TTP) according to RECIST 1.0. RESULTS In the total patient cohort none of the tested imaging features was found to be a statistically significant prognostic factor for TTP. Since treatment with SSA was associated with an increased TTP we also analyzed a subgroup of 30 patients not treated with SSA. In this subgroup of patients hypoenhancement of hepatic metastases during early contrast phases was found to be a negative prognostic factor for early tumor progression within 12 months (p=0.039). The other evaluated parameters including hepatic tumor load, number of metastases, and presence of regressive morphological changes did not reveal significant results. CONCLUSION Hypovascularization of liver metastases from G1 and G2 pNET reflected by hypoenhancement during the early contrast phases seems to be associated with early tumor progression. In patients with hypoenhancing metastases repeated biopsy for reassessment of grading of these metastases, and early initiation of therapy should be considered.

Immunohistochemical analysis of PDK1 expression in breast cancer

Background3-phosphoinositide-dependent protein kinase-1 (PDK1) functions downstream of phosphoinositide 3-kinase (PIK3) and activates members of the AGC family of protein kinases that are known to play crucial roles in physiological processes associated with cell metabolism, growth, proliferation and survival. Changes in the expression and activity of PDK1 and several AGC kinases have been linked to human disease, including cancer.MethodsWe used immunohistochemical analysis to determine PDK1 expression in 241 tumors from patients with breast cancer in which we had previously analyzed PIK3CA mutation status.ResultsModerate or high expression of PDK1 was observed in 213 of the 241 cases (88%). There was no correlation between PIK3CA mutation status and PDK1 overexpression.ConclusionOur findings indicate that PDK1 is independently activated in breast cancer and not only as part of the PIK3CA pathway, suggesting that PDK1 plays a specific and distinct role from the canonical PIK3/Akt pathway and promotes oncogenesis independently of AKT. Our data implicate PDK-1 and downstream components of the PDK-1 signaling pathway as promising therapeutic targets for the treatment of breast cancer.

Immunohistochemical validation of PSMA-expression measured by 68Ga-PSMA PET/CT in primary prostate cancer

68Ga-labeled prostate-specific membrane antigen (68Ga-PSMA) PET/CT has a proven role in staging and restaging of prostate cancer (PCA). The aims of this study were to evaluate the association of intraprostatic 68Ga-PSMA PET/CT findings and PSMA expression in immunohistochemical staining and generate a cutoff value for differentiation between normal prostate (PN) and PCA. Methods: The data of 31 patients (mean age, 67.2 y) who underwent prostatectomy and preoperative PET were retrospectively analyzed. On PET, focally increased uptake in the prostate was suggestive of tumor. A region of interest was placed on the suggestive area to generate an SUVmax; a similar region of interest was placed on adjacent visually PN. Both PCA and PN were stained with monoclonal anti-PSMA antibody (clone 3E6, 1:100, M3620). Results: All intraprostatic PCA lesions on PET could be confirmed histopathologically. In PN sections (n = 31), median staining intensity was mild, median percentage of stained cells was 20% ± 14.24%, and median immunoreactive score (IRS) was 1. In PCA sections (n = 31), median IRS was 3, median staining intensity was strong, and median percentage of stained cells was 80% ± 16.46%. The mean SUVmax (±SD) of PCA (14.06 ± 15.56) was significantly higher than that of PN (2.43 ± 0.63; P < 0.001). Receiver-operating-characteristic curve analyses of the SUVmax of PCA, validated by immunohistochemical staining in 62 tissue samples, showed the best cutoff to be 3.15 (sensitivity, 97%; specificity, 90%; area under curve, 0.987). Applied to multifocal PCA, it resulted in sensitivity and specificity of 87% and 97% respectively. The mean SUVmax of PCA and PN for an IRS of less than 2 (n = 26; 2.52 ± 0.64) was significantly lower than the mean SUVmax for an IRS of 2 or more (n = 36; 12.38 ± 15.02; P < 0.001). The mean SUVmax was significantly lower in PCA samples with fewer than 50% stained cells (n = 30; 2.81 ± 2.35) than in samples with 50% or more (n = 32; 13.34 ± 15.55; P < 0.001). There was no correlation between the SUVmax of PCA and Gleason score (P = 0.54). Conclusion: This study showed that SUVmax on 68Ga-PSMA PET/CT correlates significantly with PSMA expression in primary PCA, enabling the detection of PCA with a high sensitivity and specificity.

Evaluation of somatostatin, CXCR4 chemokine and endothelin A receptor expression in a large set of paragangliomas

Paragangliomas are predominantly benign tumors, but in some cases invasive growth and also metastasis are observed. Given the limited number of nonsurgical treatment options, novel target structures for diagnostics and therapy of this tumor entity are urgently needed. In the present study, expression of all five somatostatin receptor (SST) subtypes, chemokine receptor CXCR4 and endothelin receptor type A (ETA) was assessed by means of immunohistochemistry in a total of 66 paraffin-embedded paraganglioma samples from 55 patients. The stainings were rated by means of the Immunoreactive Score and correlated to clinical data and to succinate dehydrogenase subunit B (SDHB) expression. SST2A was by far the most prominent receptor in the paragangliomas investigated. It was present in 89% of the tumors at a high intensity, followed by SST5, SST3, SST1 and SST4, which were detected in 47%, 35%, 35% and 13% of the samples, respectively. SDHB positive tumors exhibited significantly higher SST2A and SST3 expression as compared to SDHB negative cases. There was no correlation between SST and Ki-67 expression or grading of the tumors and no difference in SST expression between primary tumors and metastases. Cell surface expression of CXCR4 and ETA was detected only in few samples. On tumor capillaries, however, exceptionally strong staining for these two receptors was noticed in the vast majority of the tumors. In conclusion, paragangliomas are well suited for SST2A-based diagnostics and treatment modalities. An indirect targeting of these highly vascularized tumors via CXCR4 or ETA may also represent a promising future strategy.

Proteasome inhibitor bortezomib enhances the effect of standard chemotherapy in small cell lung cancer

Small cell lung cancer (SCLC) is an aggressive cancer showing a very poor prognosis because of metastasis formation at an early stage and acquisition of chemoresistance. One key driver of chemoresistance is the transcription factor Forkhead box protein M1 (FOXM1) that regulates cell cycle proliferation, maintenance of genomic stability, DNA damage response, and cell differentiation in numerous tumor entities. In this study we investigated the role of FOXM1 in SCLC progression and analyzed the effect of FOXM1 inhibition using two proteasome inhibitors, bortezomib and siomycin A. FOXM1 was strongly expressed in patient-derived SCLC samples (n=123) and its nuclear localization was associated with the proliferation marker Ki-67. Both proteasome inhibitors successfully inhibited FOXM1 expression leading to a significantly reduced proliferation and a decreased mitotic rate along with cell cycle arrest and apoptosis induction. These effects were further enhanced by addition of bortezomib to standard chemotherapy. Treatment of mice bearing chemoresistant SCLC xenografts with bortezomib reduced the mean bioluminescence signal of tumors by 54%. Similarly, treatment with cisplatin as a standard chemotherapy reduced the mean bioluminescence signal of tumors by 58%. However, in combination with standard chemotherapy bortezomib further reduced the mean bioluminescence signal by 93% (p=0.0258). In conclusion, we demonstrate the effect of bortezomib in inhibiting FOXM1 expression and thus in sensitizing resistant SCLC cells to standard chemotherapy. Thus, addition of bortezomib to standard chemotherapy might potently improve SCLC therapy, particularly in an extensive cancer stage.

Increased activity of the immunoregulatory enzyme indoleamine-2,3-dioxygenase (IDO) with consecutive tryptophan depletion predicts death in patients with neuroendocrine neoplasia

Background/Aims: Data from a considerable number of malignancies demonstrate that depletion of the essential amino acid tryptophan via induction of the immunoregulatory enzyme indoleamine-2,3-dioxygenase (IDO) serves as an important tumour escape strategy and is of prognostic importance. Here we investigate the predictive value of the activity of IDO as well as levels of tryptophan and respective downstream catabolites in a large cohort of patients with neuroendocrine neoplasms (NEN). Methods: 142 consecutive Caucasian patients (62 male, aged 60.3 ± 11.9 years) with histologically confirmed NEN were systematically analysed in a retrospective blinded end point analysis. Patients were followed up for a mean period of about 3.9 ± 1.9 years. Clinical outcome, levels of established biomarkers, and tryptophan degradation markers (assessed using tandem mass spectrometry) including estimated IDO activity were recorded. Cox proportional hazards regression models were performed for the assessment of prognostic power. Results: We found that baseline tryptophan levels were significantly lower and IDO activity was significantly increased in non-survivors. The risk for death inclined stepwise and was highest in patients in the upper tertile of IDO activity. Cox proportional regression models identified IDO activity as an independent predictor of death. Conclusions: In this retrospective analysis, we observed that baseline activity of the immunoregulatory enzyme IDO was significantly increased in non-survivors. IDO activity was identified as an independent predictor of death in this cohort of NEN patients. Whether IDO activity or tryptophan depletion serves to guide future therapeutic interventions in NEN remains to be established.

Fascin-1 expression as stratification marker in borderline epithelial tumours of the ovary

Aims To evaluate the actin-bundling protein fascin-1 (FSCN1) as marker for borderline ovarian tumours (BOTs). Methods We analysed a retrospective cohort of 140 BOTs with validated diagnosis by an independent pathologist. Immunohistochemical detection of FSCN1 was quantified as combined immunoreactive score (CIS) blinded to clinical patient data. Analyses were first performed for FSCN1 positive versus negative, and then verified using three categories derived from the observed distribution (negative, weak, strong; CIS 0, 1–2, 3–9). Results We detected FSCN1 positivity in 51.4%, and strong expression (CIS 3–9) in 14.3% of the samples. FSCN1 positivity was associated with serous subtype (p<0.001) and micropapillary pattern (p<0.001). Correlation with micropapillary pattern remained significant within the serous BOT (SBOT) subgroup (p=0.022). Strong FSCN1 expression (CIS 3–9) was associated both with the presence of implants (p=0.022), and a higher International Federation of Gynecology and Obstetrics (FIGO) stage (p=0.020). Conclusions Our analysis links FSCN1 with SBOT with micropapillary pattern. Strong expression is associated with higher FIGO stage and the presence of implants, both related to elevated risk of recurrence. Hence, FSCN1 is an interesting marker worth further analyses of its prognostic value in BOTs.