Melanoma is the most fatal skin cancer, but the etiology of this devastating disease is still poorly understood. Recently, the transcription factor Sox10 has been shown to promote both melanoma initiation and progression. Reducing SOX10 expression levels in human melanoma cells and in a genetic melanoma mouse model, efficiently abolishes tumorigenesis by inducing cell cycle exit and apoptosis. Here, we show that this anti-tumorigenic effect functionally involves SOX9, a factor related to SOX10 and upregulated in melanoma cells upon loss of SOX10. Unlike SOX10, SOX9 is not required for normal melanocyte stem cell function, the formation of hyperplastic lesions, and melanoma initiation. To the contrary, SOX9 overexpression results in cell cycle arrest, apoptosis, and a gene expression profile shared by melanoma cells with reduced SOX10 expression. Moreover, SOX9 binds to the SOX10 promoter and induces downregulation of SOX10 expression, revealing a feedback loop reinforcing the SOX10 low/SOX9 high ant,m/ii-tumorigenic program. Finally, SOX9 is required in vitro and in vivo for the anti-tumorigenic effect achieved by reducing SOX10 expression. Thus, SOX10 and SOX9 are functionally antagonistic regulators of melanoma development.
For the development of future cancer therapies it is imperative to understand the molecular processes underlying tumor initiation and expansion. Many key factors involved in these processes have been identified based on cell culture and transplantation experiments, but their relevance for tumor formation and disease progression in the living organism is often unclear. Therefore, genetically modified mice spontaneously developing tumors present indispensable models for cancer research. Here, we address this issue by studying the formation of melanoma, the most fatal skin tumor in industrialized countries. To this end, we use a transgenic mouse model to elucidate cellular and molecular mechanisms regulating congenital nevus and melanoma initiation. We show that a transcription factor called SOX10 promotes melanoma formation by repressing an anti-tumorigenic program involving the activity of a related factor, SOX9. When SOX10 is inactivated, SOX9 becomes upregulated and induces cell cycle arrest and death in melanoma cells. Furthermore, upon experimental elevation of SOX9 levels, SOX10 activity is suppressed, revealing an antagonistic relationship between SOX9 and SOX10 in melanoma initiation. Knowledge of how an anti-tumorigenic program can be stimulated by modulating the activities of these key factors might help to design novel therapeutic strategies.
Cancer genome characterization has revealed driver mutations in genes that govern ubiquitylation; however, the mechanisms by which these alterations promote tumorigenesis remain incompletely characterized. Here, we analyzed changes in the ubiquitin landscape induced by prostate cancer-associated mutations of SPOP, an E3 ubiquitin ligase substrate binding protein. SPOP mutants impaired ubiquitylation of a subset of proteins in a dominant-negative fashion. Of these, DEK and TRIM24 emerged as effector substrates consistently up-regulated by SPOP mutants. We highlight DEK as a SPOP substrate that exhibited decreases in ubiquitylation and proteasomal degradation resulting from heteromeric complexes of wild-type and mutant SPOP protein. DEK stabilization promoted prostate epithelial cell invasion, implicating DEK as an oncogenic effector. More generally, these results provide a framework to decipher tumorigenic mechanisms linked to dysregulated ubiquitylation.
Follicular variant of papillary thyroid carcinoma (FVPTC) shares features of papillary (PTC) and follicular (FTC) thyroid carcinomas on a clinical, morphological, and genetic level. MicroRNA (miRNA) deregulation was extensively studied in PTCs and FTCs. However, very limited information is available for FVPTC. The aim of this study was to assess miRNA expression in FVPTC with the most comprehensive miRNA array panel and to correlate it with the clinicopathological data.
Forty-four papillary thyroid carcinomas (17 FVPTC, 27 classic PTC) and eight normal thyroid tissue samples were analyzed for expression of 748 miRNAs using Human Microarray Assays on the ABI 7900 platform (Life Technologies, Carlsbad, CA). In addition, an independent set of 61 tumor and normal samples was studied for expression of novel miRNA markers detected in this study.
Overall, the miRNA expression profile demonstrated similar trends between FVPTC and classic PTC. Fourteen miRNAs were deregulated in FVPTC with a fold change of more than five (up/down), including miRNAs known to be upregulated in PTC (miR-146b-3p, -146-5p, -221, -222 and miR-222-5p) and novel miRNAs (miR-375, -551b, 181-2-3p, 99b-3p). However, the levels of miRNA expression were different between these tumor types and some miRNAs were uniquely dysregulated in FVPTC allowing separation of these tumors on the unsupervised hierarchical clustering analysis. Upregulation of novel miR-375 was confirmed in a large independent set of follicular cell derived neoplasms and benign nodules and demonstrated specific upregulation for PTC. Two miRNAs (miR-181a-2-3p, miR-99b-3p) were associated with an adverse outcome in FVPTC patients by a Kaplan–Meier (p<0.05) and multivariate Cox regression analysis (p<0.05).
Despite high similarity in miRNA expression between FVPTC and classic PTC, several miRNAs were uniquely expressed in each tumor type, supporting their histopathologic differences. Highly upregulated miRNA identified in this study (miR-375) can serve as a novel marker of papillary thyroid carcinoma, and miR-181a-2-3p and miR-99b-3p can predict relapse-free survival in patients with FVPTC thus potentially providing important diagnostic and predictive value.
HER2 status assessment became a mandatory test assay in breast cancer, giving prognostic and predictive information including eligibility for adjuvant anti-HER2 therapy. Precise and reliable assessment of HER2 status is therefore of utmost importance. In this study we analyzed breast cancer samples by a novel technology for concomitant detection of the HER2 protein and gene copy number.
Tissue microarrays containing 589 invasive breast cancer samples were analyzed with a double immunohistochemistry (IHC) and silver labeled in situ hybridization (SISH) assay simultaneously detecting HER2 protein and gene copy number in the same tumor cells. This bright-field assay was analyzed using scores according to the modified ASCO guidelines and the results were correlated with patient prognosis.
Overall concordance rate between protein expression and the presence of gene amplification was 98%. Fifty-seven of 60 tumors (95%) with IHC score 3+, 6 of 10 tumors with IHC score 2+ (60%) and only 3 of 519 tumors (0.6%) with IHC score 0/1+ were amplified by SISH. Patients with gene amplification despite IHC score 0/1+ had a tendency for worse overall survival (p = 0.088, reaching nearly statistical significance) compared to IHC score 0/1+ without amplification. In contrast, there was no difference in overall survival in IHC score 3+/2+ tumors with and without gene amplification.
The novel double IHC and SISH assay for HER2 is efficient in the identification of breast cancer with discordant HER2 protein and HER2 gene status, especially for the prognostically relevant groups of HER2 protein negative tumors with HER2 amplification and HER2 protein positive tumors without HER2 amplification. Breast cancer without HER2 amplification among IHC score 2+/3+ tumors (10% in our cohort) suggests that other mechanisms than gene amplification contribute to protein overexpression in these cells.
The diagnosis of conventional and oncocytic poorly differentiated thyroid carcinomas is difficult. The aim of this study was to characterize their largely unknown miRNA expression profile and to compare it to well differentiated thyroid tumors as well as to identify miRNAs which could potentially serve as diagnostic and prognostic markers.
A total of 14 poorly differentiated, 13 oncocytic poorly differentiated, 72 well differentiated thyroid carcinomas and 8 normal thyroid specimens were studied for expression of 768 miRNAs using PCR-Microarrays.
MiRNA expression was different between poorly differentiated and oncocytic poorly differentiated thyroid carcinomas demonstrating individual clusters on the clustering analysis. Both tumor types showed upregulation of miR-125a-5p, -15a-3p, -182, -183-3p, -222, -222-5p and downregulation of miR-130b, -139-5p, -150, -193a-5p, -219-5p, -23b, -451, -455-3p and of miR-886-3p as compared to normal thyroid tissue. In addition, the oncocytic poorly differentiated thyroid carcinomas demonstrated upregulation of miR-221 and miR-885-5p. The difference in expression was also observed between miRNA expression in poorly differentiated and well differentiated tumors. The CHAID algorithm allowed to separate poorly differentiated from well differentiated thyroid carcinomas with a 73–79% accuracy using miR-23b and miR-150 as a separator. Kaplan-Meier and multivariate analysis showed a significant association with tumor relapses (for miR-23b) and with tumor specific death (for miR-150) in poorly differentiated and oncocytic poorly differentiated thyroid carcinomas.
MiRNA expression is different in conventional and oncocytic poorly differentiated thyroid carcinomas in comparison to well differentiated thyroid cancers and can be used for discrimination between these tumor types. The newly identified deregulated miRNAs (miR-150, miR-23b) bear the potential to be used in a clinical setting delivering prognostic and diagnostic information.
thyroid cancer; poorly differentiated carcinoma carcinoma; follicular thyroid carcinoma; papillary carcinoma; miRNA profiling; diagnosis; prognosis
Systemic mastocytosis (SM) is a heterogenous, clonal mast cell (MC) proliferation, rarely associated with clonal hematologic non-mast cell lineage disease (SM-AHNMD). KITD816V is regarded as driver-mutation in SM-AHNMD.
DNA isolated from peripheral blood (PB) of an SM-CMML patient was investigated with targeted next generation sequencing. Variants were verified by Sanger sequencing and further characterized in the SM part of the bone marrow trephine (BMT), normal tissue, and FACS sorted PB cell subpopulations.
Low coverage deep-sequencing (mean 10x) on a GS 454 Junior revealed two as yet unreported SNVs (CBFA2T3 and CLTCL1), both germ-line mutations. High coverage (mean 1674x) targeted re-sequencing on an Ion Proton revealed 177 variants in coding regions. Excluding SNPs, the final list comprised 11 variants. Among these, TET2 (p.Thr1027fs, p.Cys1263Ser) and RUNX1 (p.Asn109Ser) were identified in in the peripheral blood and the SM part of BMT, but not in normal tissue. Furthermore, Sanger sequencing of PB cells revealed similar signal intensities for both TET2 mutations in FACS sorted CD34+ precursor cells and CD16+ granulocytes comparable to signals in the SM part of BMT. In contrast, RUNX1 exhibited a double intensity in CD34+ cells compared to the SM part of BMT and a homozygous variant signal in granulocytes. Both TET2 and RUNX1 mutations were not detectable in B- and T-cells.
We present a heterozygous triple-mutation pattern (KIT, TET2, RUNX1) in mast cells (SM disease part) with additional LOH of RUNX1 in granulocytes (CMML disease part). These identified mutations allow a more detailed insight into a multistep pathogenesis which suggests a common tumor progenitor in SM-CMML.
Chronic myelomonocytic leukemia; Systemic mastocytosis; SM-CMML; Next Generation Sequencing; c-KIT mutation; TET-2 mutation; RUNX1 mutation
Aim of the study
To evaluate the frequency of MRE11/RAD50/NBS1 (MRN)-complex loss of protein expression in endometrial cancers (EC) and to determine whether loss of MRE11 renders the cancer cells sensitive to Poly(ADP-ribose) polymerase (PARP)-inhibitory treatment.
MRN expression was examined in 521 samples of endometrial carcinomas and in 10 cancer cell lines. A putative mutation hotspot in the form of an intronic poly(T) allele in MRE11 was sequenced in selected cases (n = 26). Sensitivity to the PARP-inhibitor, BMN673 was tested in colony formation assays before and after MRE11 silencing using siRNA. Homologous recombination (HR) DNA repair was evaluated by RAD51-foci formation assay upon irradiation and drug treatment.
Loss of MRE11 protein was found in 30.7% of EC tumours and significantly associated with loss of RAD50, NBS1 and mismatch repair protein expression. One endometrial cell line showed a markedly reduced MRE11 expression due to a homozygous poly(T) mutation of MRE11, thereby exhibiting an increased sensitivity to BMN673. MRE11 depletion sensitizes MRE11 expressing EC cell lines to the treatment with BMN673. The increased sensitivity to PARP-inhibition correlates with reduced RAD51 foci formation upon ionizing radiation in MRE11-depleted cells.
Loss of the MRE11 protein predicts sensitivity to PARP-inhibitor sensitivity in vitro, defining it as an additional synthetic lethal gene with PARP. The high incidence of MRE11 loss in ECs can be potentially exploited for PARP-inhibitor therapy. Furthermore, MRE11 protein expression using immunohistochemistry could be investigated as a predictive biomarker for PARP-inhibitor treatment.
In this study we aimed to establish an unbiased automatic quantification pipeline to assess islet specific features such as β-cell area and density per islet based on immunofluorescence stainings. To determine these parameters, the in vivo protein expression levels of TMEM27 and BACE2 in pancreatic islets of 32 patients with type 2 diabetes (T2D) and in 28 non-diabetic individuals (ND) were used as input for the automated pipeline. The output of the automated pipeline was first compared to a previously developed manual area scoring system which takes into account the intensity of the staining as well as the percentage of cells which are stained within an islet. The median TMEM27 and BACE2 area scores of all islets investigated per patient correlated significantly with the manual scoring and with the median area score of insulin. Furthermore, the median area scores of TMEM27, BACE2 and insulin calculated from all T2D were significantly lower compared to the one of all ND. TMEM27, BACE2, and insulin area scores correlated as well in each individual tissue specimen. Moreover, islet size determined by costaining of glucagon and either TMEM27 or BACE2 and β-cell density based either on TMEM27 or BACE2 positive cells correlated significantly. Finally, the TMEM27 area score showed a positive correlation with BMI in ND and an inverse pattern in T2D. In summary, automated quantification outperforms manual scoring by reducing time and individual bias. The simultaneous changes of TMEM27, BACE2, and insulin in the majority of the β–cells suggest that these proteins reflect the total number of functional insulin producing β–cells. Additionally, β–cell subpopulations may be identified which are positive for TMEM27, BACE2 or insulin only. Thus, the cumulative assessment of all three markers may provide further information about the real β–cell number per islet.
Lung cancer remains one of the leading causes for cancer-related death in developed countries. In lung adenocarcinomas, EGFR mutations and EML4-ALK fusions are associated with response to EGFR and ALK inhibition. By contrast, therapeutically exploitable genetic alterations have been lacking in squamous-cell lung cancer. We conducted a systematic search for alterations that are therapeutically amenable and performed high-resolution gene-copy number analyses in a set of 232 lung cancer specimens. We identified frequent and focal FGFR1 amplification in squamous-cell lung cancer (n=155), but not in other lung cancer subtypes, and confirmed its presence in an independent cohort of squamous-cell lung cancer samples employing FISH (22% of cases). Using cell-based screening with the FGFR inhibitor (PD173074) in a large (n=83) panel of lung cancer cell lines, we demonstrated that this compound inhibited growth (p=0.0002) and induced apoptosis (p=0.008) specifically in those lung cancer cells carrying amplified FGFR1. We validated the dependency on FGFR1 of FGFR1-amplified cell lines by knockdown of FGFR1 and by ectopic expression of a resistance allele of FGFR1 (FGFR1V561M), which rescued FGFR1-amplified cells from PD173074-mediated cytotoxicity. Finally we showed that inhibition of FGFR1 with a small molecule led to significant tumor shrinkage in vivo. Focal FGFR1 amplification is common in squamous-cell lung cancer and associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in this cohort of patients.
Histone deacetylases (HDACs) are known to be associated with an overexpression in different types of cancer such as colon and prostate cancer. In this study we aimed to evaluate the protein expression of class I HDACs in urothelial carcinoma of the bladder.
A tissue microarray containing 348 tissuesamples from 174 patients with a primary urothelial carcinoma of the bladder was immunohistochemically stained for HDAC 1, 2 and 3. Intensity of staining was evaluated and the association with clinico-pathological features and prognosis was assessed.
High HDAC expression levels were found in 40 to 60% of all investigated urothelial carcinomas (HDAC-1: 40%, HDAC-2: 42%, HDAC-3: 59%).
HDAC-1 and HDAC-2 were significantly associated with higher tumour grades.
Although all three markers could not predict progression in univariate analyses, high HDAC-1 expression was associated with a trend toward poorer prognosis. Patients with high-grade tumours and high expression levels of HDAC-1 were more likely to progress compared to all other patients (p < 0.05).
High-grade noninvasive papillary bladder tumours are associated with high expression levels of HDAC-1 and HDAC-2. High grade tumours in combination with high expression of HDAC-1 showed a worse prognosis than the other tumours. The high expression levels of HDACs observed particularly in high grade urothelial bladder cancer clearly warrant subsequent studies on the potential use of HDAC inhibitors as a novel therapeutic approach.
Class I HDACs; Urothelial cancer; Molecular markers
Despite existing vaccination strategies targeting TRP-2, its function is not yet fully understood. TRP-2 is an enzyme involved in melanin biosynthesis and therefore discussed as a differentiation antigen. However, in mice Trp-2 was shown to be expressed in melanocyte stem cells of the hair follicle and therefore also considered as an indicator of stemness. A proper understanding of the TRP-2 function is crucial, considering a vaccination targeting cells with stemness properties would be highly effective in contrast to a therapy targeting differentiated melanoma cells.
Analysing over 200 melanomas including primaries, partly matched metastases and patients’ cell cultures we show that TRP-2 is correlated with Melan A expression and decreases with tumor progression. In mice it is expressed in differentiated melanocytes as well as in stem cells. Furthermore, we identify a TRP-2 negative, proliferative, hypoxia related cell subpopulation which is significantly associated with tumor thickness and diseases progression. Patients with a higher percentage of those cells have a less favourable tumor specific survival.
Our findings underline that TRP-2 is a differentiation antigen, highlighting the importance to combine TRP-2 vaccination with other strategies targeting the aggressive undifferentiated hypoxia related subpopulation.
DCT, Dopachrome tautomerase; TMA, Tissue microarray; TRP-2, Tyrosinase related protein-2
Recurrent mutations in the Speckle-Type POZ Protein (SPOP) gene occur in up to 15% of prostate cancers. However, the frequency and features of cancers with these mutations across different populations is unknown.
To investigate SPOP mutations across diverse cohorts and validate a series of assays employing high-resolution melting (HRM) analysis and Sanger sequencing for mutational analysis of formalin-fixed paraffin-embedded material.
Design, Setting, and Participants
720 prostate cancer samples from six international cohorts spanning Caucasian, African American, and Asian patients, including both prostate-specific antigen-screened and unscreened populations, were screened for their SPOP mutation status. Status of SPOP was correlated to molecular features (ERG rearrangement, PTEN deletion, and CHD1 deletion) as well as clinical and pathologic features.
Results and Limitations
Overall frequency of SPOP mutations was 8.1% (4.6% to 14.4%), SPOP mutation was inversely associated with ERG rearrangement (P < .01), and SPOP mutant (SPOPmut) cancers had higher rates of CHD1 deletions (P < .01). There were no significant differences in biochemical recurrence in SPOPmut cancers. Limitations of this study include missing mutational data due to sample quality and lack of power to identify a difference in clinical outcomes.
SPOP is mutated in 4.6% to 14.4% of patients with prostate cancer across different ethnic and demographic backgrounds. There was no significant association between SPOP mutations with ethnicity, clinical, or pathologic parameters. Mutual exclusivity of SPOP mutation with ERG rearrangement as well as a high association with CHD1 deletion reinforces SPOP mutation as defining a distinct molecular subclass of prostate cancer.
The gold standard of HER2 status assessment in breast cancer is still debated. Immunohistochemistry (IHC) and in-situ technology as fluorescent-labeled methodology (FISH) can be influenced by pre-analytical factors, assay-conditions and interpretation of test results. We retrospectively conducted this quality control study and analyzed HER2 test results in breast cancer within the routine diagnostic service in a single institution over a period of 12 years. We addressed the question how stable and concordant IHC and FISH methods are and whether HER2 positivity rate has changed over this period.
Data of 7714 consecutive HER2-FISH-assays in a period of 12 years (2001–2012) on breast cancer biopsies and excision specimens were retrospectively analyzed. From 2001 to 2004, FISH tests were performed from all cases with IHC score 3+ and 2+ (and in some tumors with IHC score 1+ and 0). From 2005–2010, HER2 status was only determined by FISH. From 2011–2012, all breast carcinomas were analyzed by both IHC and FISH. Scoring and cut-off-definition were done according to time-current ASCO-CAP and FDA-guidelines.
Between 2001–2004, IHC score 3+ was diagnosed in 22% of cases, 69% of these 3+ cases were amplified by FISH. 6% of IHC score 0/1+ cases were amplified by FISH. There was a mean amplification rate of 15.8% (range 13 -19%) using FISH only HER2-assays (2005–2010). Starting 2008, a slight drop in the amplification rate from 17% to 14% was noticed due to the modified ASCO-criteria in 2007. From 2011–2012, 12% of cases were 3+ by IHC, 84% of them were amplified by FISH. Less than 1% of IHC score 0/1+ cases were amplified by FISH. Concordance between FISH and IHC increased from 83% to 97%.
Our quality control study demonstrates that HER2 positivity rate remained stable by FISH-technology but showed a significant variation by IHC over the analyzed 12 years. Improvement in concordance rate was due to standardization of pre-analytical factors, scoring and interpretation.
HER2; Fluorescence in situ hybridization (FISH); Immunohistochemistry; Breast cancer
TFE3 translocation renal cell carcinoma (tRCC) is defined by chromosomal translocations involving the TFE3 transcription factor at chromosome Xp11.2. Genetically proven TFE3 tRCCs have a broad histologic spectrum with overlapping features to other renal tumor subtypes. In this study, we aimed for characterizing RCC with TFE3 protein expression. Using next-generation whole transcriptome sequencing (RNA-Seq) as a discovery tool, we analyzed fusion transcripts, gene expression profile, and somatic mutations in frozen tissue of one TFE3 tRCC. By applying a computational analysis developed to call chimeric RNA molecules from paired-end RNA-Seq data, we confirmed the known TFE3 translocation. Its fusion partner SFPQ has already been described as fusion partner in tRCCs. In addition, an RNA read-through chimera between TMED6 and COG8 as well as MET and KDR (VEGFR2) point mutations were identified. An EGFR mutation, but no chromosomal rearrangements, was identified in a control group of five clear cell RCCs (ccRCCs). The TFE3 tRCC could be clearly distinguished from the ccRCCs by RNA-Seq gene expression measurements using a previously reported tRCC gene signature. In validation experiments using reverse transcription-PCR, TMED6-COG8 chimera expression was significantly higher in nine TFE3 translocated and six TFE3-expressing/non-translocated RCCs than in 24 ccRCCs (P < .001) and 22 papillary RCCs (P < .05–.07). Immunohistochemical analysis of selected genes from the tRCC gene signature showed significantly higher eukaryotic translation elongation factor 1 alpha 2 (EEF1A2) and Contactin 3 (CNTN3) expression in 16 TFE3 translocated and six TFE3-expressing/non-translocated RCCs than in over 200 ccRCCs (P < .0001, both).
During cancer progression, malignant cells may evade immunosurveillance. However, evidence for immunological escape in humans is scarce. We report here the clinical course of a melanoma patient whose initial tumor was positive for the antigens NY-ESO-1, MAGE-C1, and Melan-A. Upon immunization with a recombinant vaccinia/fowlpox NY-ESO-1 construct, the patient experienced a mixed clinical response and spreading of the NY-ESO-1 epitopes in the CD4+ T cell compartment. After NY-ESO-1 protein + CpG immunization, the patient’s anti-NY-ESO-1 IgG response increased. Over the following years, progressing lesions were resected and found to be NY-ESO-1-negative while being positive for MAGE-C1, Melan-A, and MHC-I. The fatal, inoperable brain metastasis was analyzed after his death and also proved to be NY-ESO-1-negative, while being positive for MAGE-C1 and Melan-A, as well as MHC-I. We propose that cancer control and cancer escape in this patient were governed by NY-ESO-1-specific immunological pressure. Our findings provide evidence for the existence of immunoediting and immunoescape in this cancer patient.
NY-ESO-1; Cancer/Testis antigen; melanoma; immunosurveillance; escape
Prostate cancer is the second most common cancer in men worldwide and causes over 250,000 deaths each year1. Overtreatment of indolent disease also results in significant morbidity2. Common genetic alterations in prostate cancer include losses of NKX3.1 (8p21)3,4 and PTEN (10q23)5,6, gains of the androgen receptor gene (AR)7,8 and fusion of ETS-family transcription factor genes with androgen-responsive promoters9–11. Recurrent somatic base-pair substitutions are believed to be less contributory in prostate tumorigenesis12,13 but have not been systematically analyzed in large cohorts. Here we sequenced the exomes of 112 prostate tumor/normal pairs. Novel recurrent mutations were identified in multiple genes, including MED12 and FOXA1. SPOP was the most frequently mutated gene, with mutations involving the SPOP substrate binding cleft in 6–15% of tumors across multiple independent cohorts. SPOP-mutant prostate cancers lacked ETS rearrangements and exhibited a distinct pattern of genomic alterations. Thus, SPOP mutations may define a new molecular subtype of prostate cancer.
The combinations of genetic alterations that cooperate with von Hippel–Lindau (VHL) mutation to cause clear cell renal cell carcinoma (ccRCC) remain poorly understood. We show that the TP53 tumour suppressor gene is mutated in approximately 9% of human ccRCCs. Combined deletion of Vhl and Trp53 in primary mouse embryo fibroblasts causes proliferative dysregulation and high rates of aneuploidy. Deletion of these genes in the epithelium of the kidney induces the formation of simple cysts, atypical cysts and neoplasms, and deletion in the epithelia of the genital urinary tract leads to dysplasia and tumour formation. Kidney cysts display a reduced frequency of primary cilia and atypical cysts and neoplasms exhibit a pro-proliferative signature including activation of mTORC1 and high expression of Myc, mimicking several cellular and molecular alterations seen in human ccRCC and its precursor lesions. As the majority of ccRCC is associated with functional inactivation of VHL, our findings suggest that for a subset of ccRCC, loss of p53 function represents a critical event in tumour development.
ccRCC; cyst; p53; VHL
Several multigene expression-based tests offering prognostic and predictive information in hormone-receptor positive early breast cancer were established during the last years. These tests provide prognostic information on distant recurrences and can serve as an aid in therapy decisions. We analyzed the recently validated reverse-transcription-quantitative-real-time PCR-based multigene-expression Endopredict (EP)-test on 34 hormone-receptor positive breast-cancer cases and compared the EP scores with the Oncotype DX Recurrence-scores (RS) obtained from the same cancer samples.
Formalin-fixed, paraffin-embedded invasive breast-cancer tissues from 34 patients were analyzed by the EP-test. Representative tumor blocks were analyzed with Oncotype DX prior to this study. Tumor tissue was removed from unstained slides, total-RNA was isolated and EP-analysis was performed blinded to Oncotype DX results.
Extraction of sufficient amounts of RNA and generation of valid EP-scores were possible for all 34 samples. EP classified 11 patients as low-risk and 23 patients as high-risk. RS Score defined 15 patients as low-risk, 10 patients as intermediate-risk in and 9 patients as high-risk. Major-discrepancy occurred in 6 of 34 cases (18%): Low-risk RS was classified as high-risk by EP in 6 cases. Combining the RS intermediate-risk and high-risk groups to a common group, the concordance between both tests was 76%. Correlation between continuous EP and RS-scores was moderate (Pearson-coefficient: 0.65 (p<0.01).
We observed a significant but moderate concordance (76%) and moderate correlation (0.65) between RS and EP Score. Differences in results can be explained by different weighting of biological motives covered by the two tests. Further studies are needed to explore the clinical relevance of discrepant test results with respect of outcome.
Although malignant cells can be recognized and controlled by the immune system, in patients with clinically apparent cancer immunosurveillance has failed. To better understand local immunoregulatory processes that impact on cancer progression, we correlated intratumoral immunological profiles with the survival of patients affected by primary clear cell renal cell carcinoma (ccRCC). A retrospective analysis of 54 primary ccRCC samples for 31 different immune response-related transcripts, revealed a negative correlation of CD68 (a marker of tumor-associated macrophages, TAMs) and FOXP3 (a marker of regulatory T cells, Tregs) with survival. The subsequent analysis of 12 TAM-related transcripts revealed an association between the genes coding for CD163, interferon regulatory factor 4 (IRF4) and fibronectin 1 (FN1), all of which have been linked to the M2 TAM phenotype, with reduced survival and increased tumor stage, whereas the opposite was the case for the M1-associated gene coding for inducible nitric oxide synthetase (iNOS). The M2 signature of (CD68+) TAMs was found to correlate with CD163 expression, as determined in prospectively collected fresh ccRCC tissue samples. Upon co-culture with autologous tumor cells, CD11b+ cells isolated from paired blood samples expressed CD163 and other M2-associated proteins, suggesting that the malignant cells promote the accumulation of M2 TAMs. Furthermore, the tumor-associated milieu as well as isolated TAMs induced the skewing of autologous, blood-derived CD4+ T cells toward a more immunosuppressive phenotype, as shown by decreased production of effector cytokines, increased production of interleukin-10 (IL-10) and enhanced expression of the co-inhibitory molecules programmed death 1 (PD-1) and T-cell immunoglobulin mucin 3 (TIM-3). Taken together, our data suggest that ccRCC progressively attracts macrophages and induces their skewing into M2 TAMs, in turn subverting tumor-infiltrating T cells such that immunoregulatory functions are increased at the expense of effector functions.
T-cell response; clear cell renal cell carcinoma; immunoregulation; tumor immunology; tumor-associated macrophages
Malignant transformation describes the phenomenon in which a somatic component of a germ cell teratoma undergoes malignant differentiation. A variety of different types of sarcoma and carcinoma, all non-germ cell, have been described as a result of malignant transformation.
A 33-year-old man presented with a left testicular mass and elevated tumour markers. Staging investigations revealed retroperitoneal lymphadenopathy with obstruction of the left ureter and distant metastases. Histopathology from the left radical orchiectomy showed a mixed germ cell tumour (Stage III, poor prognosis). The ureter was stented and four cycles of cisplatin, etoposide and bleomycin chemotherapy administered. After initial remission, the patient recurred four years later with a large retroperitoneal mass involving the renal vessels and the left ureter. Left retroperitoneal lymph node dissection with en-bloc resection of the left kidney was performed.
Histopathology revealed a germ cell tumour metastasis consisting mainly of mature teratoma. Additionally, within the teratoma a papillary renal cell carcinoma was found. The diagnosis was supported by immunohistochemistry showing positivity for AMACR, CD10 and focal expression of RCC and CK7. There was no radiological or histo-pathological evidence of a primary renal cell cancer.
To the best of our knowledge, malignant transformation into a papillary renal cell carcinoma has not been reported in a testicular germ cell tumour metastasis following platinum-based chemotherapy. This histological diagnosis might have implications for potential future therapies. In the case of disease recurrence, renal cell cancer as origin of the recurrent tumour has to be excluded because renal cell carcinoma metastases would not respond well to the classical germ cell tumour chemotherapy regimens.
Retroperitoneal teratoma; Malignant transformation; Germ cell tumour metastasis; Renal cell cancer
Several classification systems are available to assess pathological response to neoadjuvant chemotherapy in breast cancer, but reliable biomarkers to predict the efficiency of primary systemic therapy (PST) are still missing. Deregulation of gap junction channel forming connexins (Cx) has been implicated in carcinogenesis and tumour progression through loss of cell cycle control. In this study we correlated Cx expression and cell proliferation with disease survival and pathological response to neoadjuvant chemotherapy in breast cancers using existing classification systems.
The expression of Cx26, Cx32, Cx43, Cx46 and Ki67 was evaluated in 96 breast cancer patients prior to and after neoadjuvant chemotherapy using duplicate cores in tissue microarrays (TMA). Cx plaques of <1μm were detected with multilayer, multichannel fluorescence digital microscopy. Current classifications to assess residual tumour burden after primary systemic therapy included the EWGBSP, CPS-EG, Miller-Payne, Sataloff and NSABP systems.
In our cohort dominated by hormone receptor (ER/PR) positive and HER2 negative cases, only the CPS-EG classification showed prognostic relevance: cases with scores 1–2 had significantly better overall survival (p=0.015) than cases with scores 3–5. Pre-chemotherapy Cx43 expression correlated positively with hormone receptor status both before and after chemotherapy and had a negative correlation with HER2 expression pre-chemotherapy. There was a positive correlation between Cx32 and HER2 expression pre-chemotherapy and between Cx32 and Ki67 expression post-chemotherapy. A negative correlation was found between post-chemotherapy Cx46 and Ki67 expression. Decreased post-chemotherapy Cx26 expression (<5%) statistically correlated with better overall survival (p=0.011). Moderate or higher Cx46 expression (>20%) pre- and post-chemotherapy correlated with significantly better survival in the intermediate prognostic subgroups of EWGBSP TR2b (ppre-chemo=0.006; Sataloff TB (ppre-chemo=0.005; ppost-chemo=0.029) and in Miller-Payne G3 (ppre-chemo=0.002; ppost-chemo=0.012) classifications. Pre-chemotherapy, Cx46 expression was the only marker that correlated with overall survival within these subgroups.
Our results suggest that Cx46 and Cx26 expression in breast cancer may improve the assessment of pathological response and refine intermediate prognostic subgroups of residual tumour classifications used after neoadjuvant chemotherapy.
Breast cancer; Connexin; Gap junction; Preoperative chemotherapy; Prognosis
Chronic kidney disease (CKD) represents a major health burden1. Its central feature of renal fibrosis is not well understood. By whole exome resequencing in a model disorder for renal fibrosis, nephronophthisis (NPHP), we identified mutations of Fanconi anemia-associated nuclease 1 (FAN1) as causing karyomegalic interstitial nephritis (KIN). Renal histology of KIN is indistinguishable from NPHP except for the presence of karyomegaly2. FAN1 has nuclease activity, acting in DNA interstrand crosslinking (ICL) repair within the Fanconi anemia pathway of DNA damage response (DDR)3–6. We demonstrate that cells from individuals with FAN1 mutations exhibit sensitivity to the ICL agent mitomycin C. However, they do not exhibit chromosome breakage or cell cycle arrest after diepoxybutane treatment, unlike cells from patients with Fanconi anemia. We complement ICL sensitivity with wild type FAN1 but not mutant cDNA from individuals with KIN. Depletion of fan1 in zebrafish revealed increased DDR, apoptosis, and kidney cysts akin to NPHP. Our findings implicate susceptibility to environmental genotoxins and inadequate DNA repair as novel mechanisms of renal fibrosis and CKD.
Pathologists are highly trained medical professionals who play an essential part in the diagnosis and therapy planning of malignancies and inflammatory diseases. Their work is associated with potential health hazards including injuries involving infectious human tissue, chemicals which are assumed to be carcinogenic or long periods of microscope and computer work. This study aimed to provide the first comprehensive assessment of the health situation of pathologists in Switzerland.
Pathologists in Switzerland were contacted via the Swiss Society of Pathologists and asked to answer an ethically approved, online anonymous questionnaire comprising 48 questions on occupational health problems, workplace characteristics and health behaviour.
163 pathologists participated in the study. Forty percent of pathologists reported musculoskeletal problems in the previous month. The overall prevalence was 76%. Almost 90% of pathologists had visual refraction errors, mainly myopia. 83% of pathologists had experienced occupational injuries, mostly cutting injuries, in their professional career; more than one fifth of participants reported cutting injuries in the last year. However, long lasting injuries and infectious diseases were rare. Depression and burnout affected every eighth pathologist. The prevalence of smoking was substantially below that of the general Swiss population.
The results of this study suggest that more care should be taken in technical and personal protective measures, ergonomic workplace optimisation and reduction of work overload and work inefficiencies. Despite the described health risks, Swiss pathologists were optimistic about their future and their working situation. The high rate of ametropia and psychological problems warrants further study.
Occupational; Health risk; Pathologist; Musculoskeletal; Injury; Questionnaire
Type II endometrial carcinomas are a highly aggressive group of tumour subtypes that are frequently associated with inactivation of the TP53 tumour suppressor gene. We show that mice with endometrium-specific deletion of Trp53 initially exhibited histological changes that are identical to known precursor lesions of type II endometrial carcinomas in humans and later developed carcinomas representing all type II subtypes. The mTORC1 signalling pathway was frequently activated in these precursor lesions and tumours, suggesting a genetic cooperation between this pathway and Trp53 deficiency in tumour initiation. Consistent with this idea, analyses of 521 human endometrial carcinomas identified frequent mTORC1 pathway activation in type I as well as type II endometrial carcinoma subtypes. mTORC1 pathway activation and p53 expression or mutation status each independently predicted poor patient survival. We suggest that molecular alterations in p53 and the mTORC1 pathway play different roles in the initiation of the different endometrial cancer subtypes, but that combined p53 inactivation and mTORC1 pathway activation are unifying pathogenic features among histologically diverse subtypes of late stage aggressive endometrial tumours.
clear cell; endometrial carcinoma; mouse model; p53; serous