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Uterine leiomyosarcomas (uLMS) are rare, aggressive malignancies for which limited treatment options are available. To gain novel molecular insights into uLMS and identify potential novel therapeutic targets, we characterized 84 uLMS samples for genome-wide somatic copy number alterations, mutations, gene fusions and gene expression and performed a data integration analysis. We found that alterations affecting TP53, RB1, PTEN, MED12, YWHAE and VIPR2 were present in the majority of uLMS. Pathway analyses additionally revealed that the PI3K/AKT/mTOR, estrogen-mediated S-phase entry and DNA damage response signaling pathways, for which inhibitors have already been developed and approved, frequently harbored genetic changes. Furthermore, a significant proportion of uLMS was characterized by amplifications and overexpression of known oncogenes (CCNE1, TDO2), as well as deletions and reduced expression of tumor suppressor genes (PTEN, PRDM16). Overall, it emerged that the most frequently affected gene in our uLMS samples was VIPR2 (96%). Interestingly, VIPR2 deletion also correlated with unfavorable survival in uLMS patients (multivariate analysis; HR = 4.5, CI = 1.4-14.3, p = 1.2E-02), while VIPR2 protein expression was reduced in uLMS vs. normal myometrium. Moreover, stimulation of VIPR2 with its natural agonist VIP decreased SK-UT-1 uLMS cell proliferation in a dose-dependent manner. These data suggest that VIPR2, which is a negative regulator of smooth muscle cell proliferation, might be a novel tumor suppressor gene in uLMS. Our work further highlights the importance of integrative molecular analyses, through which we were able to uncover the genes and pathways most frequently affected by somatic alterations in uLMS.

Anti-EGFR antibodies are used for the treatment of RAS wild type metastatic colorectal cancer. We previously showed that EGFR gene copy number (GCN) predicts response to anti-EGFR therapy in KRAS exon 2 wild type metastatic colorectal cancer. The aim of our study was to analyse the predictive role of EGFR GCN in RAS/BRAF/PIK3CA wild type metastatic colorectal cancer. The material included 102 patients with KRAS exon 2 wild type metastatic colorectal cancer treated with anti-EGFR ± cytotoxic therapy. Next generation sequencing was used for KRAS, NRAS, BRAF and PIK3CA gene mutation analyses. EGFR GCN was analysed by EGFR immunohistochemistry guided automated silver in situ hybridisation. Increased EGFR GCN (≥4.0) predicted a better response and prolonged progression free survival in anti-EGFR treated RAS/BRAF/PIK3CA wild type patients (Log-rank test, p = 0.0004). In contrast, survival of RAS/BRAF/PIK3CA wild type, EGFR GCN below 4.0 patients did not differ from patients with mutant RAS, BRAF or PIK3CA. Our study indicates that EGFR GCN predicts anti-EGFR treatment efficacy in patients with RAS/BRAF/PIK3CA wt metastatic CRC. Tumours with EGFR GCN below 4.0 appear to be as refractory to anti-EGFR treatment as tumours with mutation in any of the RAS/RAF/PIK3CA pathway genes.

Uterine leiomyosarcomas (uLMS) are rare, aggressive malignancies for which limited treatment options are available. To gain novel molecular insights into uLMS and identify potential novel therapeutic targets, we characterized 84 uLMS samples for genome-wide somatic copy number alterations, mutations, gene fusions and gene expression and performed a data integration analysis. We found that alterations affecting TP53, RB1, PTEN, MED12, YWHAE and VIPR2 were present in the majority of uLMS. Pathway analyses additionally revealed that the PI3K/AKT/mTOR, estrogen-mediated S-phase entry and DNA damage response signaling pathways, for which inhibitors have already been developed and approved, frequently harbored genetic changes. Furthermore, a significant proportion of uLMS was characterized by amplifications and overexpression of known oncogenes (CCNE1, TDO2), as well as deletions and reduced expression of tumor suppressor genes (PTEN, PRDM16). Overall, it emerged that the most frequently affected gene in our uLMS samples was VIPR2 (96%). Interestingly, VIPR2 deletion also correlated with unfavorable survival in uLMS patients (multivariate analysis; HR = 4.5, CI = 1.4-14.3, p = 1.2E-02), while VIPR2 protein expression was reduced in uLMS vs. normal myometrium. Moreover, stimulation of VIPR2 with its natural agonist VIP decreased SK-UT-1 uLMS cell proliferation in a dose-dependent manner. These data suggest that VIPR2, which is a negative regulator of smooth muscle cell proliferation, might be a novel tumor suppressor gene in uLMS. Our work further highlights the importance of integrative molecular analyses, through which we were able to uncover the genes and pathways most frequently affected by somatic alterations in uLMS.

Tumor-associated trypsin inhibitor (TATI) is a 6-kDa peptide, which is identical to the pancreatic-secretory-trypsin inhibitor (PSTI). TATI is produced by several tumors and cancer cell lines, and is used as a serum marker for mucinous ovarian cancer. Elevated serum levels of TATI have also been observed in renal-cell carcinoma (RCC). However, it is unclear whether the increase of serum TATI in this disease is caused by production of TATI by the tumor tissue, by the acute-phase reaction frequently associated with cancer, or by impaired renal function. We examined the expression of TATI in malignant and histologically normal renal tissue by immunohistochemistry, in situ hybridization and reverse-transcriptase-polymerase-chain reaction (RT-PCR). Furthermore, we measured pre-operative serum TATI levels in 21 patients with RCC. Immunohistochemically, TATI was detected in 13 of 20 histologically normal renal-tissue samples, but not in 32 tissue samples from RCC. By RT-PCR, TATI mRNA was detected in all of 10 histologically normal kidneys and in 6 of 11 RCCs, while in situ hybridization analysis gave negative results. Pre-operative serum TATI was elevated in 57% of RCC patients. We also studied expression of TATI mRNA and protein in 7 renal-cancer cell lines, by RT-PCR and immunofluorometric assay respectively: 6 cancer cell lines were positive for TATI mRNA, while 4 of them also produced TATI protein at low levels. These results indicate that TATI is synthesized by the histologically normal renal tissue and by some renal cancers, and suggest that the elevation of serum TATI associated with renal-cell carcinoma may be caused by the release of TATI produced by the tumor.

We have investigated, using flow cytometry, the expression of 19 adhesion molecules on fresh and IL-2-activated NK cells. The study included beta 1, beta 2 and beta 3 integrins, CD2, CD54 and CD58 (belonging to the immunoglobulin superfamily), and CD44 and L-selectin (homing receptors). alpha 1 and alpha 2 of the beta 1 integrins were non-existent and alpha 3 was weak on freshly isolated NK cells, but their expression increased after 4 weeks in culture with IL-2. On the other hand, some down-regulation of alpha 4 and alpha 5 and disappearance of alpha 6 was detected. CD 11a/CD18 was upregulated by IL-2, whereas CD11b-c/CD18 were down-regulated. As a novel finding we detected beta 3 on IL-2-activated T and NK cells. CD2, CD44, CD54 and CD58 were increased by IL-2 but L-selectin was strongly down-regulated on the long-term-activated NK cells. Although IL-2-activated lymphocytes are potent tumor-lysing killer cells in vitro and therefore a potential modality in cancer treatment, the IL-2 induced changes in lymphocyte adhesion molecule expression may also lead to undesired effects, such as altered untargeted distribution and compromised migratory capacity.

We have studied the presence of plasminogen activators in large granular lymphocytes and other peripheral blood cells. After immunofluorescence staining with polyclonal antiserum against urokinase- type plasminogen activator, the majority of LGLs showed granular staining which was located in Golgi- derived vesicles. LGLs were negative for tissue-type activator. The presence of only urokinase-type PA in LGLs was also confirmed by determining the molecular weight of the intracellular activator and by immunoblotting the antigen from solubilized cell preparations. Monocytes and granulocytes were more intensely stained with anti-u-PA than LGLs. In LGL/ K562 cell conjugates the fluorescence was often located close to the contact area and the vesicular fluorescence polarized during conjugate formation. Inhibitors of PA and other serine proteinases are known to abolish NK-cell activity. We now show that they affect a later stage than programming for lysis in the cytotoxic action, suggesting a role for u-PA or other serine proteases in the lethal-hit stage of NK activity.