The most suitable immunohistochemical criterion to identify basal-like breast carcinomas (BLBC), a molecular subgroup of breast cancer associated with poor prognosis, is the triple negative phenotype along with CK5 and/or EGFR immunoreactivity. However, several putative basal markers have been suggested as alternatives to identify BLBC with more accuracy.

The expression of CK5, EGFR, P-cadherin, CK14, Vimentin and p63 were evaluated in 462 invasive breast carcinomas to determine their sensitivity and specificity for BLBC identification.

P-cadherin and CK5 showed higher sensitivity values, while EGFR, Vimentin and CK14 were the most specific markers. The combination of CK5 with P-cadherin, Vimentin or CK14 proved to be a reliable option for distinguishing the basal phenotype, compared to the "gold standard" pair CK5/EGFR. Furthermore, P-cadherin was still able to recognize a large number of putative BLBC among the "unclassified" group (ER-/PR-/HER2-/CK5-/EGFR-).

P-cadherin, Vimentin and CK14 can recognize BLBC already identified in triple negative/ CK5 and/or EGFR+ tumors, and due to P-cadherin sensitivity for BLBC identification this marker can reliably recruit a large number of breast carcinomas with basal phenotype among immunohistochemistry triple negative/ CK5 and/or EGFR - pool of tumors. Although they need GEP validation, our results can introduce the idea of these markers as additional options in the daily workup of breast pathology laboratories to identify BLBC.

BACKGROUND

Patients with DLBCL exhibit widely divergent outcomes despite harboring histologically identical tumors. Currently, GEP and IHC algorithms assign patients to 1 of 2 main subtypes: germinal center B cell-like (GCB), or activated B cell-like (ABC), the latter of which historically carries a less favorable prognosis. However, it remains controversial as to whether these prognostic groupings remain valid in the era of rituximab therapy.

METHODS

A systematic literature review identified 24 articles from which meta-analyses were conducted, comparing survival outcomes for patients assigned to either GCB or ABC/non-GCB subtype using GEP and/or Hans, Choi, or Muris IHC algorithms.

RESULTS

Patients designated as GCB DLBCL using GEP fared significantly better in terms of overall survival than those with ABC DLBCL (hazard ratio, 1.85; P < .0001). In contrast, the Hans and Choi algorithms failed to identify significant differences in overall survival (P = .07 and P = .76, respectively) between GCB and non-GCB groups.

CONCLUSIONS

Our study illustrates a lack of evidence supporting the use of the Hans and Choi algorithms for stratifying patients into distinct prognostic groups. Rather, GEP remains the preferred method for predicting the course of a patient's disease and informing decisions regarding treatment options.

Multiple myeloma (MM) is malignancy of isotype-switched, BM-localized plasma cells that frequently results in bone destruction, BM failure, and death. Important molecular subgroups are identified by three classes of recurrent immunoglobulin gene translocations and hyperdiploidy, both of which affect disease course. From a clinical standpoint, it is critical to identify MM patients carrying the t(4;14) translocation, which is present in 15% of myelomas and is associated with dysregulation of WHSC1/MMSET and often FGFR3. These patients should all receive bortezomib as part of their initial induction treatment because this has been shown to significantly prolong survival. In contrast, patients with translocations affecting the MAF family of transcription factors, del17p, or gene-expression profiling (GEP)-defined high-risk disease appear to have a worse prognosis that is not dramatically improved by any intervention. These patients should be enrolled in innovative clinical trials. The remaining patients with cyclin D translocations or hyperdiploidy do well with most therapies, and the goal should be to control disease while minimizing toxicity.

Clinical trials indicate efficacy of drugs inhibiting the mammalian target of rapamycin (mTOR) in the treatment of gastroenteropancreatic neuroendocrine tumours (GEP-NET); however, information on detailed expression and activity patterns of mTOR in these tumours is sparse. We investigated the expression of mTOR and expression as well as phosphorylation of its downstream targets 4EBP1, S6K and eIF4E in a cohort of 99 human GEP-NET by immunohistochemistry. We correlated our findings with clinicopathological variables and patient prognosis. We found that 61, 93, 80, 69, 57 and 79% of GEP-NET were positive for mTOR, 4EBP1, cytoplasmic phospho-4EBP1 (p-4EBP1), nuclear p-4EBP1, phospho-S6K (p-S6K) and phospho-eIF4E (p-eIF4E) respectively. mTOR expression and activity were higher in foregut than in midgut tumours. In foregut tumours, expression of mTOR was higher when distant metastases were present (P=0.035). Strong mTOR activity was associated with higher proliferative capacity. In patients with stage IV midgut tumours, strong p-S6K expression was associated with poor disease-specific survival (P=0.048). In conclusion, mTOR shows considerable variations in expression and activity patterns in GEP-NET in dependence of tumour location and metastatic status. We hypothesise that these differences in mTOR expression and activity might possibly influence response to mTOR inhibitors.

Despite the increasing knowledge about the genetic alterations and molecular pathways involved in gliomas, few studies have investigated the association between the gene expression profiles (GEP) and both cytogenetics and histopathology of gliomas. Here, we analyzed the GEP (U133Plus2.0 chip) of 40 gliomas (35 astrocytic tumors, 3 oligodendrogliomas, and 2 mixed tumors) and their association with tumor cytogenetics and histopathology. Unsupervised and supervised analyses showed significantly different GEP in low- vs high-grade gliomas, the most discriminating genes including genes involved in the regulation of cell proliferation, apoptosis, DNA repair, and signal transduction. In turn, among glioblastoma multiforme (GBM), 3 subgroups of tumors were identified according to their GEP, which were closely associated with the cytogenetic profile of their ancestral tumor cell clones: (i) EGFR amplification, (ii) isolated trisomy 7, and (iii) more complex karyotypes. In summary, our results show a clear association between the GEP of gliomas and tumor histopathology; additionally, among grade IV astrocytoma, GEP are significantly associated with the cytogenetic profile of the ancestral tumor cell clone. Further studies in larger series of patients are necessary to confirm our observations.

OBJECTIVE

Increasing evidence has suggested that hepatocellular carcinoma (HCC) might originate from a distinct subpopulation called cancer stem cells (CSCs), which are responsible for the limited efficacy of conventional therapies. We have previously demonstrated that granulin-epithelin precursor (GEP), a pluripotent growth factor, is upregulated in HCC but not in the adjacent non-tumor, and that GEP is a potential therapeutic target for HCC. Here, we characterized its expression pattern and stem cell properties in fetal and cancerous livers.

METHODS

Protein expression of GEP in fetal and adult livers was examined in human and mouse models by immunohistochemical staining and flow cytometry. Liver cancer cell lines, isolated based on their GEP and/or ATP-dependent binding cassette (ABC) drug transporter ABCB5 expression, were evaluated for hepatic CSC properties in terms of colony formation, chemoresistance and tumorigenicity.

RESULTS

We demonstrated that GEP was a hepatic oncofetal protein that expressed in the fetal livers, but not in the normal adult livers. Importantly, GEP+ fetal liver cells co-expressed the embryonic stem (ES) cell-related signaling molecules including β-catenin, Oct4, Nanog, Sox2 and DLK1, and also hepatic CSC-markers CD133, EpCAM and ABCB5. Phenotypic characterization in HCC clinical specimens and cell lines revealed that GEP+ cancer cells co-expressed these stem cell markers similarly as the GEP+ fetal liver cells. Furthermore, GEP was shown to regulate the expression of ES cell-related signaling molecules β-catenin, Oct4, Nanog, and Sox2. Isolated GEP(high) cancer cells showed enhanced colony formation ability and chemoresistance when compared with the GEP(low) counterparts. Co-expression of GEP and ABCB5 better defined the CSC populations with enhanced tumorigenic ability in immunocompromised mice.

CONCLUSIONS

Our findings demonstrate that GEP is a hepatic oncofetal protein regulating ES cell-related signaling molecules. Co-expression of GEP and ABCB5 further enriches a subpopulation with enhanced CSC properties. The current data provide new insight into the therapeutic strategy.

In 1983, a monoclonal antibody, Ki67, was generated, that labeled the nuclei of proliferating non-neoplastic and neoplastic cells. The name Ki67 derived from the city of Kiel (Ki) where the antibody was produced in the university department of pathology and refers to the number of the original clone (67). Systematic assessment of the proliferative activity of tumors using Ki67 started in the 1990s, when Ki67, which only worked on frozen tissue, was complemented by the antibody MIB-1 that also worked in formalin-fixed tissues. Pancreatic neuroendocrine neoplasms (PanNENs) were the first endocrine tumors whose proliferative activity was assessed with Ki67. This approach was so successful that Ki67 was included as prognostic marker in the 2000 and 2004 WHO classifications of gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs). In 2010, the WHO classification of GEP-NENs introduced a three-tiered grading, originally proposed by ENETS in 2006 that was mainly based on the Ki67 index. As it has subsequently been shown that the Ki67 index is the most reliable factor in the prognostic evaluation of GEP-NENs, especially of PanNENs, the 2017 WHO classification of PanNENs requires its use and strongly recommends exact assessment of the proportion Ki67-labeled cells as basis for the calculation of the Ki67 index. Problems in assessing the Ki67 index include intertumoral and intratumoral staining heterogeneity and counting methods. Despite such problems, the Ki67 index has emerged as indispensable for the prognostic and therapeutic stratification of the majority of GEP-NENs and can barely be replaced by counting mitoses. In future, however, it can be anticipated that the Ki67 cut-offs experience refinement in relation to the type of tumor, its location, and its response to therapy. It is also possible that the prognostic risk of an individual tumor is calculated for each Ki67 unit and not for an "a priori" fixed Ki67 class.

Minimally differentiated acute myeloid leukemia (AML-M0) is defined by immature morphology and expression of early hematologic markers. By gene expression profiling (GEP) and subsequent unsupervised analysis of 35 AML-M0 samples and 253 previously reported AML cases, we demonstrate that AML-M0 cases express a unique signature that is largely separated from other molecular subtypes. Hematologic transcription regulators such as CEBPA, CEBPD, and ETV6, and the differentiation associated gene MPO appeared strongly down-regulated, in line with the primitive state of this leukemia. AML-M0 frequently carries loss-of-function RUNX1 mutation. Unsupervised analyses revealed a subdivision between AML-M0 cases with and without RUNX1 mutations. RUNX1 mutant AML-M0 samples showed a distinct up-regulation of B cell-related genes such as members of the B-cell receptor complex, transcription regulators RUNX3, ETS2, IRF8, or PRDM1, and major histocompatibility complex class II genes. Importantly, prediction with high accuracy of the AML-M0 subtype and prediction of patients carrying RUNX1 mutation within this subtype were possible based on the expression level of only a few transcripts. We propose that RUNX1 mutations in this AML subgroup cause lineage infidelity, leading to aberrant coexpression of myeloid and B-lymphoid genes. Furthermore, our results imply that AML-M0, although originally determined by morphology, constitutes a leukemia subgroup.

BACKGROUND

Rab3A, a member of the Rab3 small G protein family, regulates Ca2+-dependent exocytosis of neurotransmitter. The cyclical activation and inactivation of Rab3A are essential for the Rab3A action in exocytosis. GDP-Rab3A is activated to GTP-Rab3A by Rab3 GDP/GTP exchange protein (Rab3 GEP) and GTP-Rab3A is inactivated to GDP-Rab3A by Rab3 GTPase-activating protein (Rab3 GAP). We have recently found a novel protein, named rabconnectin-3, which is co-immunoprecipitated with Rab3 GEP or GAP from the extract of the crude synaptic vesicle (CSV) fraction of rat brain. Rabconnectin-3 is abundantly expressed in the brain where it is associated with synaptic vesicles. We have found that two more proteins are co-immunoprecipitated with Rab3 GEP from the CSV fraction of rat brain. We attempted here to isolate and characterize one of them.

RESULTS

We determined its partial amino acid sequence, cloned its cDNA from a human cDNA library, and determined its primary structure. The protein consisted of 1490 amino acids (aa) and showed a calculated molecular weight of 163808. The protein had 7 WD domains. The protein was abundantly expressed in the brain where it co-localized with rabconnectin-3 on synaptic vesicles. The protein formed a stable complex with rabconnectin-3. We named this protein rabconnectin-3beta and renamed rabconnectin-3 rabconnectin-3alpha. Rabconnectin-3beta, but not rabconnectin-3alpha, directly bound Rab3 GEP. Neither rabconnectin-3alpha nor -3beta directly bound Rab3 GAP.

CONCLUSIONS

These results indicate that rabconnectin-3 consists of the alpha and beta subunits and binds directly Rab3 GEP through the beta subunit and indirectly Rab3 GAP through an unidentified molecule(s).

In patients with gastroenteropancreatic (GEP) neuroendocrine tumors, we investigated the usefulness of somatostatin receptor scintigraphy (SRS) in the detection of liver metastasis, which represents the most important prognostic factor in these tumors, and in the management of affected patients.

METHODS

We enrolled 149 patients with GEP tumors, 69 during initial staging and 80 in follow-up. All patients underwent whole-body scanning at 4 and 24 h, followed by abdominal planar and SPECT imaging after intravenous injection of 250 MBq (111)In-pentetreotide. The patients had previously been submitted to 2 of 3 conventional imaging procedures (CIP), such as CT, MRI, and ultrasound of the abdomen within 1 mo before SRS; on the basis of liver CIP data, the patients had been classified into 3 groups as follows: no evidence of liver metastases, the presence of resectable metastases, or the presence of nonresectable metastases.

RESULTS

Liver metastases were histologically proven in 65 cases. SPECT identified malignant lesions in 60 of 65 patients with metastases (sensitivity, 92.3%), planar imaging identified malignant lesions in 38 of 65 patients (sensitivity, 58.5%), and CIP identified malignant lesions in 52 of 65 patients (sensitivity, 80%). Only SPECT demonstrated liver involvement in 13 patients, whereas CIP showed liver involvement in 5 other cases. Moreover, SPECT was significantly more sensitive than planar imaging and CIP in identifying patients with single lesions. Neither SPECT nor planar imaging showed false-positive results in patients with no evidence of liver metastases, including 21 patients with hemangiomas (specificity, 100%), 12 of which were false-positive on CIP (specificity, 85.7%). SPECT per-lesion sensitivity (92.4%) was significantly higher than that of planar imaging (52.4%) and CIP (79.4%). Moreover, SPECT correctly changed patient classification and, thus, management in 28 of 149 patients (18.8%), whereas planar imaging changed classification in 13 patients (8.7%), identifying new or additional metastases not evident on CIP or excluding metastases on CIP of patients with false-positive findings, thus avoiding unnecessary surgery; however, SPECT classification was incorrect in 3.3% of patients, and planar imaging was incorrect in 17.4%.

CONCLUSIONS

(111)In-Pentetreotide SRS is a useful diagnostic tool in the detection of liver metastases in GEP tumor patients. In particular, SPECT proved to be significantly more sensitive and accurate than both planar imaging and CIP. Moreover, SPECT was also the most reliable procedure to obtain correct patient classification, thus guiding the most appropriate therapeutic strategy.

Prognostic models for multiple myeloma have been fraught with tremendous heterogeneity in outcome among subgroups. In the context of Total Therapy 2, a tandem transplant trial for newly diagnosed myeloma, comprehensive information was available in 220 patients on standard prognostic factors (SPF), magnetic resonance imaging (MRI)-defined focal lesions, cytogenetic abnormalities (CA), fluorescence-in-situ-hybridisation (FISH)-derived amplification of chromosome 1q21 (amp1q21) and deletion of 13q14, as well as gene expression profiling (GEP). Five multivariate analysis-based survival models were derived, utilising SPF only (model 1), with progressive addition of CA (model 2), MRI (model 3), FISH (model 4) and GEP (model 5). The R(2) value, a measure of accounting for clinical outcome variability, increased progressively from 18% in model 1 to 38% in model 5. The hazard ratio for overall survival was highest for GEP (3.07, P < 0.001) followed by amp1q21 (1.71, P = 0.05). According to the presence of none (49%), one (35%) or both of these two risk features (16%), 3-year survival decreased progressively from 92% to 78% to 43% (P < 0.0001). Thus, the dominance over other prognostic parameters of molecular genetics justifies the generation of quantitative reverse transcription polymerase chain reaction methodology ('MM genetic kit') for the optimal risk stratification of patients participating in therapeutic trials.

Carcinoid tumors are the most frequent gut neuroendocrine tumors accounting for more than 50% of all tumors of the gastroenteropancreatic (GEP) axis. These tumors appear to derive from a stem cell line capable of differentiating into a variety of malignant cells that secrete many different peptides and amines. The symptoms of carcinoid tumors are often non-specific, vague abdominal pain that may precede the diagnosis by a median of 9 years. Carcinoid syndrome occurs in less than 10% of patients. We evaluated the effects of SMS 201-995 in 14 such patients, 12 with diarrhea, 8 with flushing, 3 with wheezing, one with tricuspid valve incompetence, 6 with facial telangiectasia, 3 with a pellagra type dermatosis and one with myopathy. Diarrhea was abolished or significantly reduced in 83%, flushing in 100%, wheezing in 100%, and myopathy improved in the one patient. Blood serotonin was resistant to change, urine 5HIAA fell in 75%, and most gut neuropeptide hormones apart from somatostatin were suppressed. Tumor growth appeared to be slowed in 2/3 of cases treated for up to 4 years. The analog of somatostatin appears to be a useful addition to the therapeutic armamentarium for carcinoid tumors and the symptom complex.

OBJECTIVE

Somatostatin (sst) are present in the majority of gastro-entero-pancreatic (GEP) tumours. Effects of somatostatin receptor (sst) analogues are partial and of limited duration. Cell lines derived from GEP express dopaminergic receptors D(2). New chimeric analogues simultaneously recognising sst(2) and sst(5) or sst(2) and D(2) have additive effects in inhibition of GH and prolactin secretion in pituitary adenomas. Our aim was to quantify the expression of sst and D(2) mRNA in human GEP tumours.

METHODS

mRNA expression of sst(1), sst(2), sst(3) and sst(5) as well as D(2), was analysed using real-time PCR (TaqMan probe) in a series of 35 patients with GEP tumours (pancreas (n = 19) and intestinal (n = 16)). Levels of expression were compared with a group of 13 somatotroph adenomas.

RESULTS

All GEP tumours express sst(1), sst(2) and D(2). Expression of sst(3) and sst(5) was observed in 89 and 76% of tumours respectively with highly variable levels. sst(2) mRNA expression was higher in nonfunctional tumours (P < 0.009) and sst5 was higher in pancreatic than in intestinal tumours (P < 0.02). Whereas sst(2) levels were similar between GEP and somatotroph tumours, levels of sst(5) and D(2) were higher in the former (394.9 +/- 156.1 x 10(-2) vs 69.7 +/- 19.5 x 10(-2) copy/copy beta-Gus (P < 0.0036) and 519.6 +/- 121.2 x 10(-2) vs 50.0 +/- 21.6 x 10(-2) copy/copy beta-Gus (P < 0.0001) respectively). In small tumours ( < 30 mm), sst(2) density appeared as a crucial parameter in somatostatin receptor scintigraphy results, whereas in big tumours, a consistent bias in SRS results was introduced by the size. In pancreatic GEP, high-level sst(3) expression was found in tumours with more active angiogenesis (higher microvessel density and vascular endothelial growth factor expression (P < 0.03)).

CONCLUSIONS

GEP tumours co-express sst(2) and D(2) in 100% of cases and sst(5) in 89% thus supporting the testing of bi-specific agonists (sst(2)/sst(5) or sst(2)/D(2)) in these tumours.

An association between exposure to ambient particulate matter (PM) and increased incidence of mortality and morbidity due to lung cancer and cardiovascular diseases has been demonstrated by recent epidemiological studies. Reactive oxygen species (ROS), especially hydroxyl radicals, generated by PM, have been suggested by many studies as an important factor in the oxidative damage of DNA by PM. The purpose of this study was to characterize quantitatively hydroxyl radical generation by various transition metals in the presence of H2O2 in aqueous buffer solution (pH 7.4) and hydroxylation of 2'-deoxyguanosine (dG) to 8-hydroxy-2'-deoxyguanosine (8-OHdG) under similar conditions. The order of metals' redox reactivity and hydroxyl radical production was Fe(II), V(IV), Cu(I), Cr(III), Ni(II), Co(II), Pb(II), Cd(II). Then, we investigated the generation of hydroxyl radicals in the presence of H2O2 by various airborne PM samples, such as total suspended particulate (TSP), PM10, PM2.5 (PM with aerodynamic diameter 10 and 2.5 microm), diesel exhaust particles (DEP), gasoline exhaust particles (GEP) and woodsmoke soot under the same conditions. When suspensions of PMs were incubated with H2O2 and dG at pH 7.4, all particles induced hydroxylation of dG and formation of 8-OHdG in a dose-dependent increase. Our findings demonstrated that PM's hydroxyl radical (HO radical) generating ability and subsequent dG hydroxylation is associated with the concentration of water-soluble metals, especially Fe and V and other redox or ionizable transition metals and not their total metal content, or insoluble metal oxides, via a Fenton-driven reaction of H2O2 with metals. Additionally, we observed, by Electron paramagnetic resonance (EPR), that PM suspensions in the presence of H2O2 generated radical species with dG, which were spin-trapped by 2-methyl-2-nitroso-propane (MNP).

The dbl oncogene product (Dbl) showed the GDP/GTP exchange protein (GEP) activity on all the rho family small GTP-binding protein (G protein) members including RhoA and Rac1 as well as mCdc42. Dbl was active on both the lipid-modified and -unmodified forms of these small G proteins, but was much more active on the former form than on the latter form. In the presence of Rho GDI, an inhibitory GEP for the rho family members, the GEP activity of Dbl was markedly reduced. These properties of Dbl were partly different from those of Smg GDS, another GEP which is active not only on the rho family members but also on Ki-Ras and Rap1 and is active only on their lipid-modified form.

With advent of several treatment options in multiple myeloma (MM), a selection of effective regimen has become an important issue. Use of gene expression profile (GEP) is considered an important tool in predicting outcome; however, it is unclear whether such genomic analysis alone can adequately predict therapeutic response. We evaluated the ability of GEP to predict complete response (CR) in MM. GEP from pretreatment MM cells from 136 uniformly treated MM patients with response data on an IFM, France led study were analyzed. To evaluate variability in predictive power due to microarray platform or treatment types, additional data sets from three different studies (n=511) were analyzed using same methods. We used several machine learning methods to derive a prediction model using training and test subsets of the original four data sets. Among all methods employed for GEP-based CR predictive capability, we got accuracy range of 56-78% in test data sets and no significant difference with regard to GEP platforms, treatment regimens or in newly diagnosed or relapsed patients. Importantly, permuted P-value showed no statistically significant CR predictive information in GEP data. This analysis suggests that GEP-based signature has limited power to predict CR in MM, highlighting the need to develop comprehensive predictive model using integrated genomics approach.

Metastatic tumors with an uncertain primary site can be a difficult clinical problem. In tens of thousands of patients every year, no confident diagnosis is ever issued, making standard-of-care treatment impossible. Gene expression profiling (GEP) tests currently available to analyze these difficult-to-diagnose tumors have never been directly compared with the diagnostic standard of care, immunochemistry (IHC). This prospectively conducted, blinded, multicenter study compares the diagnostic accuracy of GEP with IHC in identifying the primary site of 157 formalin-fixed paraffin-embedded specimens from metastatic tumors with known primaries, representing the 15 tissues on the GEP test panel. Four pathologists rendered diagnoses by selecting from 84 stains in 2 rounds. GEP was performed using the Pathwork Tissue of Origin Test. Overall, GEP accurately identified 89% of specimens, compared with 83% accuracy using IHC (P=0.013). In the subset of 33 poorly differentiated and undifferentiated carcinomas, GEP accuracy exceeded that of IHC (91% to 71%, P=0.023). In specimens for which pathologists rendered their final diagnosis with a single round of stains, both IHC and GEP exceeded 90% accuracy. However, when the diagnosis required a second round, IHC significantly underperformed GEP (67% to 83%, P<0.001). GEP has been validated as accurate in diagnosing the primary site in metastatic tumors. The Pathwork Tissue of Origin Test used in this study was significantly more accurate than IHC when used to identify the primary site, with the most pronounced superiority observed in specimens that required a second round of stains and in poorly differentiated and undifferentiated metastatic carcinomas.

Loss of ATRX leads to epigenetic alterations, including abnormal levels of DNA methylation at repetitive elements such as telomeres in murine cells. We conducted an extensive DNA methylation and mRNA expression profile study on a cohort of 82 patients with astrocytic tumors to study whether ATRX expression was associated with DNA methylation level in astrocytic tumors and in which cellular functions it participated. We observed that astrocytic tumors with lower ATRX expression harbored higher DNA methylation level at chromatin end and astrocytic tumors with ATRX-low had distinct gene expression profile and DNA methylation profile compared with ATRX-high tumors. Then, we uncovered that several ATRX associated biological functions in the DNA methylation and mRNA expression profile (GEP), including apoptotic process, DNA-dependent positive regulation of transcription, chromatin modification, and observed that ATRX expression was companied by MGMT methylation and expression. We also found that loss of ATRX caused by siRNA induced apoptotic cells increasing, reduced tumor cell proliferation and repressed the cell migration in glioma cells. Our results showed ATRX-related regulatory functions of the combined profiles from DNA methylation and mRNA expression in astrocytic tumors, and delineated that loss of ATRX impacted biological behaviors of astrocytic tumor cells, providing important resources for future dissection of ATRX role in glioma.

Somatostatin receptors are expressed by a large variety of human tumors. In vitro receptor autoradiographic studies have shown that these tumors can express more than one somatostatin receptor subtype. Whereas the majority of tumors bind octreotide with high affinity, some, i.e., prostate tumors, bind octreotide with low affinity only. The discovery of five somatostatin receptor subtypes, sst1-5, by gene cloning has increased our understanding of somatostatin receptor structure and function. Using in situ hybridization techniques, we found that various human tumors, identified as somatostatin receptor-positive in binding studies, expressed sst2 mRNA in the majority of cases, whereas sst1 and sst3 were less frequent. Often, all three sst were expressed simultaneously. In another recent in situ hybridization study, primary prostate cancers were shown to preferentially express sst1, rather than sst2 or sst3. Moreover, a high incidence of sst5 was found in growth hormone (GH)-producing pituitary adenomas and, to a lesser extent, in active pituitary adenomas; gastroenteropancreatic (GEP) tumors showed all possible combinations, but with a predominance of sst2. Overall, the presence of sst2 mRNA and/or sst5 generally correlated with the presence of octreotide-binding sites, but with exceptions. These results indicate the highly variable abundance of sst mRNAs in individual somatostatin receptor-containing tumors. Somatostatin receptors were not only found in tumoural tissue, but also in the peritumoral vascular system. This was particularly well studied in colorectal carcinomas, where the peritumoral veins were shown to express in all cases a high density of somatostatin receptors, probably of the sst2 type, binding octreotide with high affinity. Therefore, the host peritumoral vascular system may be a possible target of somatostatin action in tumor development. Somatostatin may act locally on tumor growth through two different mechanisms dependent on local somatostatin receptor expression: through direct action on tumor cells or through action on peritumoral vessels, which may alter the hemodynamics of the tumoral blood circulation.

Peptide receptor radionuclide therapy (PRRT) is an established treatment of metastatic neuroendocrine tumors grade 1-2 (G1-G2). However, its possible benefit in high-grade gastroenteropancreatic (GEP) neuroendocrine neoplasms (NEN G3) is largely unknown. We therefore aimed to assess the benefits and side effects of PRRT in patients with GEP NEN G3. We performed a retrospective cohort study at 12 centers to assess the efficacy and toxicity of PRRT in patients with GEP NEN G3. Outcomes were response rate, disease control rate, progression-free survival (PFS), overall survival (OS) and toxicity. We included 149 patients (primary tumor: pancreatic n = 89, gastrointestinal n = 34, unknown n = 26). PRRT was first-line (n = 30), second-line (n = 62) or later-line treatment (n = 57). Of 114 patients evaluated, 1% had complete response, 41% partial response, 38% stable disease and 20% progressive disease. Of 104 patients with documented progressive disease before PRRT, disease control rate was 69%. The total cohort had median PFS of 14 months and OS of 29 months. Ki-67 21-54% (n = 125) vs Ki-67 ≥55% (n = 23): PFS 16 vs 6 months (P < 0.001) and OS 31 vs 9 months (P < 0.001). Well (n = 60) vs poorly differentiated NEN (n = 62): PFS 19 vs 8 months (P < 0.001) and OS 44 vs 19 months (P < 0.001). Grade 3-4 hematological or renal toxicity occurred in 17% of patients. This large multicenter cohort of patients with GEP NEN G3 treated with PRRT demonstrates promising response rates, disease control rates, PFS and OS as well as toxicity in patients with mainly progressive disease. Based on these results, PRRT may be considered for patients with GEP NEN G3.

Programmed death-ligand 1 (PD-L1), which is expressed on many cancer cells, interacts with PD1 expressed on the surface of T cells, inhibiting the T cells and blocking the antitumor immune response. Expression of PD-L1 in gastroenteropancreatic neuroendocrine tumors (GEP-NETs) has not been studied. We investigated the impact of PD-L1 expression in 32 patients with metastatic GEP-NET. The expression of PD-L1 was evaluated using an anti-PD-L1 immunohistochemistry (IHC) antibody optimized for staining of formalin-fixed paraffin-embedded (FFPE) tissue samples. The correlation between PD-L1 and clinicopathological data including survival and response to systemic treatments was analyzed. Primary sites were 24 foregut-derived GEP-NETs, including stomach (n=1), duodenum (n=2), biliary tract (n=7), and pancreas (n=14), and 8 hindgut-derived GEP-NETs of the distal colon and rectum. Among the 32 patients with metastatic GEP-NET analyzed in this study, 7 (21.9%) had expression of PD-L1 in tumor tissues. Expression of PD-L1 was significantly associated with high-grade WHO classification (grade 3) (p=0.008) but not with gender, primary site, and number of metastatic sites (p>0.05). The status of PD-L1 expression was statistically associated with progression-free survival (PFS) for first-line systemic treatment (p=0.047). Moreover, the status of PD-L1 expression could significantly predict overall survival (p=0.037). The expression of PD-L1 was associated with higher WHO tumor grade (grade 3) in metastatic GEP-NETs. PD-L1 expression had both predictive and prognostic value for survival of patients with metastatic GEP-NETs.

The granulin-epithelin precursor (GEP/PCDGF), a 68-88 kDa secreted glycoprotein, has been shown to be an important growth and survival factor for ovarian cancer cells. Furthermore, GEP expression is a predictor of patient survival in metastatic ovarian cancer cells. Up to this point, however, the molecular mechanisms and clinical relevance of a GEP-mediated prosurvival phenotype remain poorly characterized. We hypothesize that the prosurvival function of GEP is important in ovarian cancer tumor progression and chemoresponse. To explore this hypothesis, we examined the effects of GEP overexpression on migration, invasion and cisplatin (CDDP) chemosensitivity in the ovarian cancer cell line A2780. Full length GEP transfectants demonstrated an increased capacity to migrate and invade their substratum when compared to empty vector controls. In addition, GEP overexpression was associated with CDDP chemoresistance. Finally, GEP overexpression increased tumor formation and protected cells from tumor regression in response to CDDP treatment in vivo. Taken together, these data support a role for GEP in tumor progression and development of drug resistance.

OBJECTIVE

Renal radiation during peptide receptor radionuclide therapy (PRRT) may result in glomerular damage, a potential reduction of glomerular filtration rate (GFR) and ultimately lead to renal failure. While reported PRRT nephrotoxicity is limited to data derived from serum creatinine-allowing only approximate estimates of GFR-the aim of this study is to accurately determine PRRT-induced long-term changes of renal function and associated risk factors according to state-of-the-art GFR measurement.

METHODS

Nephrotoxicity was analysed using (99m)Tc-diethylenetriaminepentaacetic acid (DTPA) clearance data of 74 consecutive patients with gastroenteropancreatic neuroendocrine tumours (GEP NET) undergoing PRRT with (177)Lu-octreotate. The mean follow-up period was 21 months (range 12-50) with a median of five GFR measurements per patient. The change of GFR was analysed by linear curve fit. Potential risk factors including diabetes mellitus, arterial hypertension, previous chemotherapy, renal impairment at baseline and cumulative administered activity were analysed regarding potential impact on renal function loss. In addition, Common Terminology Criteria for Adverse Events (CTCAE) v3.0 were used to compare nephrotoxicity determined by (99m)Tc-DTPA clearance versus serum creatinine.

RESULTS

The alteration in GFR differed widely among the patients (mean -2.1 ± 13.1 ml/min/m(2) per year, relative yearly reduction -1.8 ± 18.9%). Fifteen patients (21%) experienced a mild (2-10 ml/min/m(2) per year) and 16 patients (22%) a significant (>10 ml/min/m(2) per year) decline of GFR following PRRT. However, 11 patients (15%) showed an increase of >10 ml/min/m(2) per year. Relevant nephrotoxicity according to CTCAE (grade ≥3) was observed in one patient (1.3%) with arterial hypertension and history of chemotherapy. Nephrotoxicity according to serum creatinine was discordant to that defined by GFR in 15% of the assessments and led to underestimation in 12% of patients. None of the investigated factors including cumulative administered activity contributed to the decline of renal function.

CONCLUSIONS

Serious nephrotoxicity after PRRT with (177)Lu-octreotate is rare (1.3%). However, slight renal impairment (GFR loss >2 ml/min/m(2) per year) can frequently (43%) be detected by (99m)Tc-DTPA clearance assessments. Cumulative administered activity of (177)Lu-octreotate is not a major determinant of renal impairment in our study.

Primary plasma cell leukemia (pPCL) is a rare, yet aggressive form of de novo plasma cell tumor, distinct from secondary PCL (sPCL) which represents a leukemic transformation of pre-existing multiple myeloma (MM). Herein, we performed a comprehensive molecular analysis of a prospective series of pPCLs by means of FISH, single nucleotide polymorphism (SNP) array and gene expression profiling (GEP). IGH@ translocations were identified in 87% of pPCL cases, with prevalence of t(11;14) (40%) and t(14;16) (30.5%), whereas the most frequent numerical alterations involved 1p (38%), 1q (48%), 6q (29%), 8p (42%), 13q (74%), 14q (71%), 16q (53%), and 17p (35%). We identified a minimal biallelic deletion (1.5 Mb) in 8p21.2 encompassing the PPP2R2A gene, belonging to a family of putative tumor suppressors and found to be significantly down-regulated in deleted cases. Mutations of TP53 were identified in four cases, all but one associated with a monoallelic deletion of the gene, whereas activating mutations of the BRAF oncogene occurred in one case and were absent in N- and K-RAS. To evaluate the influence of allelic imbalances in transcriptional expression we performed an integrated genomic analysis with GEP data, showing a significant dosage effect of genes involved in transcription, translation, methyltransferase activity, apoptosis as well as Wnt and NF-kB signaling pathways. Overall, we provide a compendium of genomic alterations in a prospective series of pPCLs which may contribute to improve our understanding of the pathogenesis of this aggressive form of plasma cell dyscrasia and the mechanisms of tumor progression in MM.