Treatment outcome of atypical EGFR mutations in the German National Network Genomic Medicine Lung Cancer (nNGM).

BACKGROUND: Atypical EGFR mutations occur in 10%-30% of non-small-cell lung cancer (NSCLC) patients with EGFR mutations and their sensitivity to classical epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKI) is highly heterogeneous. Patients harboring one group of uncommon, recurrent EGFR mutations (G719X, S768I, L861Q) respond to EGFR-TKI. Exon 20 insertions are mostly insensitive to EGFR-TKI but display sensitivity to exon 20 inhibitors. Clinical outcome data of patients with very rare point and compound mutations upon systemic treatments are still sparse to date. PATIENTS AND METHODS: In this retrospective, multicenter study of the national Network Genomic Medicine (nNGM) in Germany, 856 NSCLC cases with atypical EGFR mutations including co-occurring mutations were reported from 12 centers. Clinical follow-up data after treatment with different EGFR-TKIs, chemotherapy and immune checkpoint inhibitors were available from 260 patients. Response to treatment was analyzed in three major groups: (i) uncommon mutations (G719X, S7681, L861Q and combinations), (ii) exon 20 insertions and (iii) very rare EGFR mutations (very rare single point mutations, compound mutations, exon 18 deletions, exon 19 insertions). RESULTS: Our study comprises the largest thus far reported real-world cohort of very rare EGFR single point and compound mutations treated with different systemic treatments. We validated higher efficacy of EGFR-TKI in comparison to chemotherapy in group 1 (uncommon), while most exon 20 insertions (group 2) were not EGFR-TKI responsive. In addition, we found TKI sensitivity of very rare point mutations (group 3) and of complex EGFR mutations containing exon 19 deletions or L858R mutations independent of the combination partner. Notably, treatment responses in group 3 (very rare) were highly heterogeneous. Co-occurring TP53 mutations exerted a non-significant trend for a detrimental effect on outcome in EGFR-TKI-treated patients in groups 2 and 3 but not in group 1. CONCLUSIONS: Based on our findings, we propose a novel nNGM classification of atypical EGFR mutations.

Distribution of tumor-infiltrating-T-lymphocytes and possible tumor-escape mechanisms avoiding immune cell attack in locally advanced adenocarcinomas of the esophagus

Introduction The inflammatory microenvironment has emerged as one of the focuses of cancer research. Little is known about the immune environment in esophageal adenocarcinoma (EAC) and possible tumor-escape mechanisms to avoid immune cell attack. Patients and methods We measured T cell inflammation (CD3, CD8) in the microenvironment using a standardized software-based evaluation algorithm considering different predefined tumor areas as well as expression of MHC class 1 and PD-L1 on 75 analyzable primarily resected and locally advanced (≥ pT2) EACs. We correlated these findings statistically with clinical data. Results Patients with high amounts of T cell infiltration in their tumor center showed a significant survival benefit of 41.4 months compared to 16.3 months in T cell poor tumors (p = 0.025), although CD3 fails to serve as an independent prognostic marker in multivariate analysis. For the invasion zone, a correlation between number of T-cells and overall survival was not detectable. Loss of MHC1 protein expression on tumor cells was seen in 32% and PD-L1 expression using the combined positive score (CPS) in 21.2%. Most likely due to small numbers of cases, both markers are not prognostically relevant, even though PD-L1 expression correlates with advanced tumor stages. Discussion Our analyses reveal an outstanding, though not statistically independent, prognostic relevance of T-cell-rich inflammation in our group of EACs, in particular driven by the tumor center. For the first time, we describe that the inner part of the invasion zone in EACs shows significantly fewer T-cells than other tumor segments and is prognostically irrelevant. We also demonstrate that the loss of antigen presenting ability via MHC1 downregulation by the carcinoma cells is a common escape mechanism in EACs. Future work will need to show whether tumors with MHC class 1 loss respond less well to immunotherapy (AU)

Distribution of tumor-infiltrating-T-lymphocytes and possible tumor-escape mechanisms avoiding immune cell attack in locally advanced adenocarcinomas of the esophagus.

INTRODUCTION: The inflammatory microenvironment has emerged as one of the focuses of cancer research. Little is known about the immune environment in esophageal adenocarcinoma (EAC) and possible tumor-escape mechanisms to avoid immune cell attack. PATIENTS AND METHODS: We measured T cell inflammation (CD3, CD8) in the microenvironment using a standardized software-based evaluation algorithm considering different predefined tumor areas as well as expression of MHC class 1 and PD-L1 on 75 analyzable primarily resected and locally advanced (≥ pT2) EACs. We correlated these findings statistically with clinical data. RESULTS: Patients with high amounts of T cell infiltration in their tumor center showed a significant survival benefit of 41.4 months compared to 16.3 months in T cell poor tumors (p = 0.025), although CD3 fails to serve as an independent prognostic marker in multivariate analysis. For the invasion zone, a correlation between number of T-cells and overall survival was not detectable. Loss of MHC1 protein expression on tumor cells was seen in 32% and PD-L1 expression using the combined positive score (CPS) in 21.2%. Most likely due to small numbers of cases, both markers are not prognostically relevant, even though PD-L1 expression correlates with advanced tumor stages. DISCUSSION: Our analyses reveal an outstanding, though not statistically independent, prognostic relevance of T-cell-rich inflammation in our group of EACs, in particular driven by the tumor center. For the first time, we describe that the inner part of the invasion zone in EACs shows significantly fewer T-cells than other tumor segments and is prognostically irrelevant. We also demonstrate that the loss of antigen presenting ability via MHC1 downregulation by the carcinoma cells is a common escape mechanism in EACs. Future work will need to show whether tumors with MHC class 1 loss respond less well to immunotherapy.

Improved Tissue Processing in Esophageal Adenocarcinoma After Ivor Lewis Esophagectomy Allows Histological Analysis of All Surgically Removed Lymph Nodes with Significant Effects on Nodal UICC Stages.

BACKGROUND: In esophageal carcinoma, the numbers of metastatic and total removed lymph nodes (LN) are well-established variables of long-term prognosis. The overall rate of retrieved LN depends on neoadjuvant treatment, the extent of surgical lymphadenectomy, and the modality of the pathological workup. The question in this study is whether technically extended histopathological preparation can increase the number of detected (metastatic) LN with an impact on nodal UICC staging. PATIENTS AND METHODS: A cohort of 77 patients with esophageal adenocarcinoma was treated with Ivor Lewis esophagectomy including standardized two-field lymphadenectomy. The specimens were grossed, and all manually detectable LN were retrieved. The remaining tissue was completely embedded by the advanced "acetone compression" retrieval technique. The primary outcome parameter was the total number of detected lymph nodes before and after acetone workup. RESULTS: A mean number of 23,1 LN was diagnosed after standard manual LN preparation. With complete embedding of the fatty tissue using acetone compression, the number increased to 40.5 lymph nodes (p < 0.0001). The mean number of metastatic LN increased from 3.2 to 4.2 nodal metastases following acetone compression (p < 0.0001). Additional LN metastases which caused a change in the primary (y)pN stage were found in ten patients (13.0%). CONCLUSIONS: Advanced lymph node retrieval by acetone compression allows a reliable statement on the real number of removed LN. Results demonstrate an impact on the nodal UICC stage. A future multicenter study will examine the prognostic impact of improved lymph node retrieval on long-term oncologic outcome.

ALK immunohistochemistry positive, FISH negative NSCLC is infrequent, but associated with impaired survival following treatment with crizotinib.

OBJECTIVE: Metastasized non-small cell lung cancer (NSCLC) with an anaplastic lymphoma kinase (ALK) rearrangement is usually sensitive to a range of ALK-tyrosine kinase inhibitors. ALK-positive NSCLC have been identified in pivotal phase III trials with fluorescence in situ hybridization (ALK FISH+). These tumors are also expressing the fusion product (ALK immunohistochemistry (IHC)+). However, discrepant cases occur, including ALK IHC + FISH-. The aim of this study was to collect ALK IHC + cases and compare within this group response to crizotinib treatment of ALK FISH + cases with ALK FISH- cases. MATERIALS AND METHODS: In this European prospective multicenter research study patients with Stage IV ALK IHC + NSCLC treated with crizotinib were enrolled. Tumor slides were validated centrally for ALK IHC and ALK FISH. RESULTS: Registration of 3523 ALK IHC tests revealed a prevalence of 2.7% (n = 94) ALK IHC + cases. Local ALK FISH analysis resulted in 48 concordant (ALK IHC+/FISH+) and 16 discordant (ALK IHC+/FISH-) cases. Central validation revealed 37 concordant and 7 discordant cases, 5 of which had follow-up. Validation was hampered by limited amount of tissue in biopsy samples. The PFS at 1 year for ALK concordant and discordant was 58% and 20%, respectively (HR = 2.4; 95% CI: 0.78-7.3; p = 0.11). Overall survival was significantly better for concordant cases than discordant cases after central validation (HR=4.5; 95% CI= 1.2-15.9; p=0.010. CONCLUSION: ALK IHC + FISH- NSCLC is infrequent and associated with a worse outcome on personalized treatment. A suitable predictive testing strategy may be to screen first with IHC and then confirm with FISH instead of considering ALK IHC equivalent to ALK FISH according to the current guidelines.

Therapy susceptible germline-related BRCA 1-mutation in a case of metastasized mixed adeno-neuroendocrine carcinoma (MANEC) of the small bowel.

BACKGROUND: Adenocarcinomas or combined adeno-neuroendocrine carcinomas (MANEC) of small bowel usually have a dismal prognosis with limited systemic therapy options. This is the first description of a patient showing a germline-related BRCA1 mutated MANEC of his ileum. The tumor presented a susceptibility to a combined chemotherapy and the PARP1-inhibitor olaparib. CASE PRESENTATION: A 74-year old male patient presented with a metastasized MANEC of his ileum. Due to clinical symptoms his ileum-tumor and the single brain metastasis were removed. We verified the same pathogenic (class 5) BRCA1 mutation in different tumor locations. There was no known personal history of a previous malignant tumor. Nevertheless we identified his BRCA1 mutation as germline-related. A systemic treatment was started including Gemcitabine followed by selective internal radiotherapy (SIRT) to treat liver metastases and in the further course Capecitabine but this treatment finally failed after 9 months and all liver metastases showed progression. The treatment failure was the reason to induce an individualized therapeutic approach using combined chemotherapy of carboplatin, paclitaxel and the Poly (ADP-ribose) polymerase- (PARP)-inhibitor olaparib analogous to the treatment protocol of Oza et al. All liver metastases demonstrated with significant tumor regression after 3 months and could be removed. In his most current follow up from December 2017 (25 months after his primary diagnosis) the patient is in a very good general condition without evidence for further metastases. CONCLUSION: We present first evidence of a therapy susceptible germline-related BRCA1 mutation in small bowel adeno-neuroendocrine carcinoma (MANEC). Our findings offer a personalized treatment option. The germline background was unexpected in a 74-year old man with no previously known tumor burden. We should be aware of the familiar background in tumors of older patients as well.

Prediction of outcome by lymph node ratio in patients with parotid gland cancer.

OBJECTIVE: Lymph node ratio (LNR) has been shown to be an independent predictor of recurrence risk and survival in different entities of carcinoma. METHODS: In this retrospective chart review, 128 patients with parotid gland cancer (PGC) subsequently treated by primary surgery were included. About 64% (n = 82) of these patients were additionally treated with adjuvant radiotherapy. Five-year overall survival rates were determined by subgroups based on LNR value. RESULTS: Lymph node ratio was found to be significantly associated with overall survival rate (P < 0.001). Using univariate analyses, pathological tumour-node-metastasis (TNM)-stage, UICC-stage grouping and extracapsular spread were found to be significant predictors of overall survival (P < 0.001). However, with a multivariate analyses, LNR remained the only independent predictor of overall survival (P = 0.043). CONCLUSIONS: After surgery for PGC, evaluation of the neck using LNR was found to reliably stratify the overall survival rate.

The role of salvage extended lymph node dissection in patients with rising PSA and PET/CT scan detected nodal recurrence of prostate cancer.

BACKGROUND: Non-prostatic bed recurrence of prostate cancer (PCa) is usually treated with androgen deprivation therapy (ADT). We analyzed the impact of salvage extended lymph node dissection (sLND) on cancer control in patients with rising PSA and lymph node (LN) metastases. METHODS: Between 2009 and 2016 we performed sLND in 87 patients with biochemical recurrence (BCR) and positive LNs on 18FEC and 68Ga-PSMA positron emission tomography/X-ray computer tomography (PET/CT) after primary treatment (PT) of PCa. Intra- and postoperative complications according to Clavien-Dindo were assessed and the rates of biochemical response (BR), BCR-free and clinical recurrence (CR)-free survival, as well as time to initiation of systemic treatment were evaluated. RESULTS: Mean age of patients and mean PSA at sLND was 66.7 years (46-80 years) and 2.63 ng ml-1 (1.27-3.75 ng ml-1), respectively. With 87.4% radical prostatectomy (RP) was the most common PT. In all, 57.9% of patients additionally underwent adjuvant/salvage radiation therapy (RT) and 18.4% received ADT before sLND. Complete BR (cBR) was diagnosed in 27.5% of patients and incomplete BR in 40.6%. In total, 62.2% of patients remained without ADT at follow-up. With a median follow-up of 21 months (1-75 months), the cancer-specific mortality rate was 3.7%. The 3-year BCR-free, systemic therapy-free and CR-free survival rates for patients with cBR were 69.3%, 77.0% and 75%, respectively. CONCLUSIONS: sLND can be performed without significant complications and achieves an immediate BR, thus allowing a significant postponement of systemic therapy in selected patients with BCR and nodal recurrence of PCa. Therefore, sLND following 68Ga-PSMA PET/CT should be considered as part of a multimodal diagnostic and treatment concept for selective patients.

[Biomarker analysis from a pathologist's view. Founding the rationale for personalised treatment of lung cancer]. / Biomarkeranalytik aus Sicht des Pathologen. Grundlage für eine rationale, personalisierte Tumortherapie am Beispiel von Lungenkrebs.

The advent of genomic medicine and sequencing analysis of entire cancer genomes has rapidly improved our understanding of cancer genomics and has defined pathogenetic lesions initiating and driving cancer phenotypes in a causative manner. Moreover rapid development of small molecule inhibitors and highly selective biologicals provided effective tools to intervene with oncogenic signalling resulting from such lesions in the cancer genome. Thereby, the pathologist is now in the position to diagnose causative lesions in the cancer genome as molecular biomarkers directing the selection of patients for effective and highly selective therapies. If oncogenic driver lesions are vigorously validated preclinically and a useful diagnostic test is available, it is possible to provide a proof-of-concept at a very early stage of clinical drug development with the possibility of immediate personal benefit for participants in such phase I/II studies. This approach has significantly changed clinical testing and avoids testing proof for efficacy in large stage III clinical trials at a high failure rate. Therefore, our review summarises recent and paradigmatic progress in lung cancer biomarker diagnostics and defines academic and regulatory requirements for biomarker analysis and selective personalised therapies.

Olive oil attenuates the cholesterol-induced development of nonalcoholic steatohepatitis despite increased insulin resistance in a rodent model.

It is indefinite whether nonalcoholic steatohepatitis (NASH) results as by-product from general metabolic perturbations and adipokine dysregulations or whether defined dietary factors also play a pathogenetic role. Here, we examine the effects of a modification of dietary lipids in a NASH inducing diet on metabolic changes as well as hepatic steatosis, inflammation, and fibrosis in rats. Male Wistar rats were fed with variations of the atherogenic diet (AD), which induces pathophysiological changes resembling human NASH. Dietary variants (AD without cholesterol, cholate, or choline; change of neutral fat to olive oil or coconut oil) were fed for 8 weeks. Insulin resistance, adipokine profile, liver histology, and lipid content as well as expression of proinflammatory and profibrogenic genes were examined. AD led to clear signs of hepatic steatosis and inflammation together with an increase in TNF and collagen type 1 expression. AD without cholesterol showed markedly less liver damage without changes of insulin action and adipokine profile. AD with olive oil and AD without cholate clearly attenuated hepatic inflammation, whereas fat deposition and features of the metabolic syndrome were increased in these animals. Insulin resistance and hepatic fat deposition per se do not cause significant hepatic inflammation in this rodent model. However, dietary cholesterol is an important causal agent for the development of NASH. Olive oil plays a protective role in this respect, which might be due to the high content of monounsaturated fatty acids.

SIRT1 activation enhances HDAC inhibition-mediated upregulation of GADD45G by repressing the binding of NF-κB/STAT3 complex to its promoter in malignant lymphoid cells.

We explored the activity of SIRT1 activators (SRT501 and SRT2183) alone and in combination with panobinostat in a panel of malignant lymphoid cell lines in terms of biological and gene expression responses. SRT501 and SRT2183 induced growth arrest and apoptosis, concomitant with deacetylation of STAT3 and NF-κB, and reduction of c-Myc protein levels. PCR arrays revealed that SRT2183 leads to increased mRNA levels of pro-apoptosis and DNA-damage-response genes, accompanied by accumulation of phospho-H2A.X levels. Next, ChIP assays revealed that SRT2183 reduces the DNA-binding activity of both NF-κB and STAT3 to the promoter of GADD45G, which is one of the most upregulated genes following SRT2183 treatment. Combination of SRT2183 with panobinostat enhanced the anti-growth and anti-survival effects mediated by either compound alone. Quantitative-PCR confirmed that the panobinostat in combination with SRT2183, SRT501 or resveratrol leads to greater upregulation of GADD45G than any of the single agents. Panobinostat plus SRT2183 in combination showed greater inhibition of c-Myc protein levels and phosphorylation of H2A.X, and increased acetylation of p53. Furthermore, EMSA revealed that NF-κB binds directly to the GADD45G promoter, while STAT3 binds indirectly in complexes with NF-κB. In addition, the binding of NF-κB/STAT3 complexes to the GADD45G promoter is inhibited following panobinostat, SRT501 or resveratrol treatment. Moreover, the combination of panobinostat with SRT2183, SRT501 or resveratrol induces a greater binding repression than either agent alone. These data suggest that STAT3 is a corepressor with NF-κB of the GADD45G gene and provides in vitro proof-of-concept for the combination of HDACi with SIRT1 activators as a potential new therapeutic strategy in lymphoid malignancies.

Lysine-specific demethylase 1 restricts hematopoietic progenitor proliferation and is essential for terminal differentiation.

Lysine (K)-specific demethylase 1A (LSD1/KDM1A) has been identified as a potential therapeutic target in solid cancers and more recently in acute myeloid leukemia. However, the potential side effects of a LSD1-inhibitory therapy remain elusive. Here, we show, with a newly established conditional in vivo knockdown model, that LSD1 represents a central regulator of hematopoietic stem and progenitor cells. LSD1 knockdown (LSD1-kd) expanded progenitor numbers by enhancing their proliferative behavior. LSD1-kd led to an extensive expansion of granulomonocytic, erythroid and megakaryocytic progenitors. In contrast, terminal granulopoiesis, erythropoiesis and platelet production were severely inhibited. The only exception was monopoiesis, which was promoted by LSD1 deficiency. Importantly, we showed that peripheral blood granulocytopenia, monocytosis, anemia and thrombocytopenia were reversible after LSD1-kd termination. Extramedullary splenic hematopoiesis contributed to the phenotypic reversion, and progenitor populations remained expanded. LSD1-kd was associated with the upregulation of key hematopoietic genes, including Gfi1b, Hoxa9 and Meis1, which are known regulators of the HSC/progenitor compartment. We also demonstrated that LSD1-kd abrogated Gfi1b-negative autoregulation by crossing LSD1-kd with Gfi1b:GFP mice. Taken together, our findings distinguish LSD1 as a critical regulator of hematopoiesis and point to severe, but reversible, side effects of a LSD1-targeted therapy.

Giant cell hepatitis: an unusual cause of fulminant liver failure.

Giant cell hepatitis is a very rare disease of unknown origin. It has been hypothesized that drugs, viral infections, or autoimmune reactions may play a pathogenetic role. Here, we describe a 33 year old patient with bacterial bronchitis who was treated with doxycycline (100 mg/d) for one week. Furthermore the patient complained of malaise and a distinct jaundice. Liver parameters increased dramatically (AST 4670 U/l, ALT 5350 U/l, bilirubin 226 µmol/l) and liver function was impaired (INR = 1,45). The ultrasound scan showed a hepatomegaly with no signs of cirrhosis, normal spleen size and normal bile ducts; liver perfusion was normal. No evidence of Wilson's disease, hemochromatosis, autoimmune hepatitis, primary sclerosing cholangitis, primary biliary cirrhosis, hepatitis A, B, C and E, HIV, CMV, VZV, adenoviral infections, or paracetamol intoxication was found. Subsequently, the patient developed acute liver failure (AST 2134 U/l, ALT 2820 U/l, bilirubin 380 µmol/l, INR 3.0) and a beginning renal failure. Therefore, he was transferred to our transplant center. Due to increasing confusion and somnolence due to cerebral edema mechanical ventilation was needed. Because of an acute renal failure and severe hepatic encephalopathia MARS-hemodialysis was performed. Three weeks after the appearance of the jaundice he underwent liver transplantation (MELD 40). Surprisingly, in the explanted liver the diagnosis of giant cell hepatitis was made. Today--2 years after successful liver transplantation--the patient is in very good condition with normal liver function. In conclusion, giant cell hepatitis is a rare cause of acute liver failure that is often recognized only histologically.

Dietary folic acid activates AMPK and improves insulin resistance and hepatic inflammation in dietary rodent models of the metabolic syndrome.

The AMP activated kinase plays an important role in metabolic control, and pharmacologic enhancement of AMPK activity is used to improve insulin resistance. We hypothesized that high dose of folic acid supplementation might improve insulin sensitivity and hepatic inflammation and examined this by a dietary intervention in (a) the high fat fed rat model of the metabolic syndrome, which shows sole hepatic steatosis as well as (b) in rats fed with a high cholesterol, high cholate diet inducing nonalcoholic steatohepatitis (NASH). Male Wistar rats were fed with folic acid supplemented (40 mg/kg) high fat diet [based on lard, fat content 25% (wt/wt)] or NASH inducing diet (containing 15% fat, 1.25% cholesterol, 0.5% sodium cholate). Metabolic profiling was performed by measuring the animals' visceral fat pads, fasting plasma glucose, insulin, and adipokines as well as in vivo insulin tolerance tests. Hepatic steatosis and inflammation were analyzed semiquantitatively by histological analysis. Folic acid supplementation reduced visceral obesity and improved plasma adiponectin levels. In vivo insulin sensitivity was improved, and in HF-FA rats folic acid increased activation of hepatic AMPK. Further, folic acid supplementation improved hepatic inflammation in animals fed with NASH-inducing diet. Dietary folic acid improved parameters of insulin resistance and hepatic inflammation in rodent models. This might be due to an increased AMK activation.

[Comparing immunohistochemical diagnosis of cancer of unknown primary with gene expression-based tumor classification]. / Vergleich immunhistologischer mit genexpressionsbasierter Diagnostik: von Karzinomen mit unbekanntem Primarius.

Five percent of cancer cases present as metastases. If the primary tumor cannot be identified after diagnostic workup, the disease is referred to as cancer of unknown primary (CUP) and is classified as C80.9 according to ICD-10. In Germany, CUP is among the ten most common causes of tumor-related death, with mortality similar to mortality in gastric or pancreatic cancer. Biopsies of the tumor manifestation are generally examined histopathologically and by immunohistochemistry (IHC). Gene expression profiling (GEP) is a new diagnostic technique that might further contribute to tumor specification.In a retrospective study, 43 CUP cases underwent central immunohistochemical review and centrally performed GEP using a classifier based on 495 genes. There was concordance between IHC, GEP and clinical picture in 54% of cases. In four cases, the combination of methods led to an unequivocal identification of the primary tumor.In conclusion, we regard detailed IHC workup and complementary GEP advisable for the purposes of targeted therapy, as well as to identify or exclude specific tumors in a CUP situation.

Insulin-like growth factor-1 receptor acts as a growth regulator in synovial sarcoma.

Synovial sarcomas account for 5-10% of all soft tissue sarcomas and the majority of synovial sarcomas display characteristic t(X;18) translocations that result in enhanced transcription of the insulin-like growth factor-2 (IGF-2) gene. IGF-2 is an essential fetal mitogen involved in the pathogenesis of different tumours, leading to cellular proliferation and inhibition of apoptosis. Here we asked whether activation of IGF signalling is of functional importance in synovial sarcomas. We screened human synovial sarcomas for expression of IGF-2 and the phosphorylated IGF-1 receptor (IGF-1R), which mainly mediates the proliferative and anti-apoptotic effects of IGF-2. Since both the phosphatidylinositol 3'-kinase (PI3K)-AKT pathway and the MAPK signalling cascade are known to be involved in the transmission of IGF-1R signals, expression of phosphorylated (p)-AKT and p-p44/42 MAPK was additionally assessed. All tumours expressed IGF-2 and 78% showed an activated IGF-1R. All tumours were found to express p-AKT and 92% showed expression of activated p44/42 MAPK. To analyse the functional and potential therapeutic relevance of IGF-1R signalling, synovial sarcoma cell lines were treated with the IGF-1R inhibitor NVP-AEW541. Growth was impaired by the IGF-1R antagonist, which was consistently accompanied by a dose-dependent reduction of phosphorylation of AKT and p44/42 MAPK. Functionally, inhibition of the receptor led to increased apoptosis and diminished mitotic activity. Concurrent exposure of selected cells to NVP-AEW541 and conventional chemotherapeutic agents resulted in positive interactions. Finally, synovial sarcoma cell migration was found to be dependent on signals transmitted by the IGF-1R. In summary, our data show that the IGF-1R might represent a promising therapeutic target in synovial sarcomas.

Coexisting somatic promoter hypermethylation and pathogenic MLH1 germline mutation in Lynch syndrome.

Somatic epimutations in the MLH1 promoter mimic the phenotype of Lynch syndrome. To date, no somatic hypermethylation of the MLH1 promoter in the carrier of a pathogenic MLH1 germline mutation has been identified, prompting the recommendation that a germline mutation in MLH1 should only be sought in the absence of tumour tissue methylation. We aimed to determine whether methylation of the MLH1 promoter may coexist in carriers of a pathogenic germline mutation in MLH1. We examined the methylation status of the MLH1 promoter in 123 tumour tissue samples, demonstrating high microsatellite instability and loss of expression of a mismatch repair protein (60 cases with MLH1 germline mutation, 25 cases without mutation, 38 cases with MSH2 mutations), using combined bisulphite restriction analysis (COBRA) and SNaPshot analysis. Methylation of the MLH1 promoter was found in two patients with pathogenic germline mutations, one a carrier of a MLH1 mutation and the other a carrier of a MSH2 mutation. Our results demonstrate that methylation of the MLH1 promoter region does not exclude the presence of a germline mutation in a mismatch repair (MMR) gene. Hypermethylation of the MLH1 promoter may be present in most cases of sporadic colorectal cancers, but this does not exclude a diagnosis of Lynch syndrome.

Immunohistochemical expression patterns of AP2alpha and AP2gamma in the developing fetal human breast.

AIMS: AP2alpha (TFAP2A) and AP2gamma (TFAP2G) transcription factors have been implicated in the control of proliferation, differentiation and apoptosis of normal breast epithelium and in breast cancer. The aim of this study was to provide a comprehensive analysis of the expression patterns of TFAP2A and TFAP2G in the developing fetal breast anlage with other relevant markers. METHODS AND RESULTS: Sixty fetal and one infant human breast specimens from 14 weeks of gestational age to 5 months old were examined. The primary breast outgrowth/nipple showed TFAP2A expression by the basal cells (week 14), followed later by cytokeratin (CK) 5 co-expression (week 17). Sprouting of the secondary outgrowths was characterized by HER-2+ invading cells. Preliminary ductal buds were lined by TFAP2G/HER-1-expressing myoepithelial precursors (week 19). Maturation of TFAP2A/CK18+ epithelia and TFAP2G/smooth muscle actin-positive myoepithelia proceeded in a distal-to-proximal manner beginning in the terminal end buds (week 22). CK5+ progenitor cells and CK5/TFAP2A or CK5/TFAP2G co-expressing intermediary glandular or myoepithelial cells were found in the terminal end buds of neonatal fetal breast tissue. CONCLUSIONS: AP2 transcription factors may play decisive pacemaker roles in initiating and coordinating budding and branching processes during formation of the fetal breast anlage, possibly via modulation of an epidermal growth factor receptor.