István Takács

Abstract S1-04: Avelumab (MSB0010718C), an anti-PD-L1 antibody, in patients with locally advanced or metastatic breast cancer: A phase Ib JAVELIN solid tumor trial

Background: The programmed death-1 receptor (PD-1) and its ligand (PD-L1) are key therapeutic targets in the reactivation of the immune response against cancer. Avelumab* (MSB0010718C) is a fully human anti-PD-L1 IgG1 antibody being investigated in clinical trials. We report clinical activity of avelumab in a cohort of patients (pts) with locally advanced (LA) or metastatic breast cancer (MBC) refractory to or progressing after standard-of-care therapy (NCT01772004). Methods: Pts received avelumab at 10 mg/kg Q2W until confirmed progression, unacceptable toxicity, or any criterion for withdrawal occurred. Tumors were assessed every 6 wks (RECIST 1.1). Unconfirmed best overall response was evaluated. Adverse events (AEs) were graded by NCI-CTCAE v4.0. Biopsy or surgical specimens were collected within 90 days prior to 1st dose of avelumab for biomarker analyses. Tumor PD-L1 expression was assessed by immunohistochemistry using various cutoff criteria. Results: As of 27 Feb 2015, 168 pts (167 female, 1 male) with MBC, including ductal (56.5%), carcinoma NOS (9.5%), lobular (3.6%), or other (30.4%), were treated with avelumab and followed for a median of 10 mo (range 6-15). Median age was 55y (range 31-81), ECOG performance status was 0 (49.4%) or 1 (50.6%), and pts had received a median of 3 prior therapies for LA/M disease (range 0-10; pts must have received prior treatment with taxane and anthracycline, unless contraindicated). Pts were HER2–/ER+ or PR+ (69 [41.1%]), triple negative (TNBC = HER2–/ER–/PR–; 57 [33.9%]), HER2+ (26 [15.5%]), or had unknown biomarker status (16 [9.5%]). Median duration of treatment was 8 wks (range 2-50), and 9 pts (5.4%) remained on avelumab. Any grade treatment-related treatment-emergent AEs (TEAEs) occurred in 120 pts (71.4%); the most common (>10%) were fatigue (33 [19.6%]), nausea (24 [14.3%]), and infusion-related reactions (20 [11.9%]). Treatment-related grade ≥3 TEAEs occurred in 24 pts (14.3%) and included (≥1%) fatigue, anemia, increased GGT, and autoimmune hepatitis (each 3 [1.8%]), and arthralgia (2 [1.2%]). There were 2 treatment-related deaths (acute liver failure, respiratory distress). Unconfirmed objective response rate (ORR) in the entire cohort was 5.4% (9 pts; 95% CI: 2.5, 9.9), with 1 CR and 8 PRs. Five of 9 responses were ongoing at time of cutoff. Stable disease was observed in additional 40 pts (23.8%), for an overall disease control rate of 29.2%. Evidence of tumor reduction by ≥30% was seen in 15 pts (8.9%). There were responders in all biomarker subgroups, including 5 PRs in TNBC (n=57 [8.8%; 95% CI: 2.9, 19.3]). PD-L1 expression was evaluable in 136 pts. Among all pts with PD-L1 expressing immune cells within the tumor, 33.3% (4 of 12) had PRs. In pts with TNBC who had PD-L1+ immune cells within the tumor, 44.4% (4 of 9) had PRs, compared with 2.6% (1 of 39) for TNBC and PD-L1– immune cells. Conclusions: Avelumab showed an acceptable safety profile and had clinical activity in a subset of pts with MBC. In pts with TNBC, presence of PD-L1 expressing immune cells within the tumor may be associated with clinical responses to avelumab. Further analyses of PD-L1 expression and clinical activity of avelumab in MBC are ongoing. *Proposed INN. Citation Format: Dirix LY, Takacs I, Nikolinakos P, Jerusalem G, Arkenau H-T, Hamilton EP, von Heydebreck A, Grote H-J, Chin K, Lippman ME. Avelumab (MSB0010718C), an anti-PD-L1 antibody, in patients with locally advanced or metastatic breast cancer: A phase Ib JAVELIN solid tumor trial. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S1-04.

Different Gene Expression Patterns in the Bone Tissue of Aging Postmenopausal Osteoporotic and Non-osteoporotic Women

PurposeTo identify genes that are differently expressed in osteoporotic and non-osteoporotic human bone and to describe the relationships between these genes using multivariate data analysis.MethodsSeven bone tissue samples from postmenopausal osteoporotic patients and 10 bone tissue samples from postmenopausal non-osteoporotic women were examined in our study. Messenger RNA was prepared from each sample and reverse transcribed to cDNA. The expression differences of 87 selected genes were analyzed in a Taqman probe-based quantitative real-time RT-PCR system.ResultsA Mann-Whitney U-test indicated significant differences in the expression of nine genes (pxa0≤xa00.05). Seven of these nine genes–ALPL, COL1A1, MMP2, MMP13, MMP9, PDGFA, NFKB1—were significantly downregulated in the bone tissue of osteoporotic women, while CD36 and TWIST2 were significantly upregulated in osteoporotic patients. Principal components analysis was used to evaluate data structure and the relationship between osteoporotic and non-osteoporotic phenotypes based on the multiple mRNA expression profiles of 78 genes. Canonical variates analysis demonstrated further that osteoporotic and non-osteoporotic tissues can be distinguished by expression analysis of genes coding growth factors/non-collagen matrix molecules, and genes belonging to the canonical TGFB pathway.ConclusionSignificant differences observed in gene expression profiles of osteoporotic and non-osteoporotic human bone tissues provide further insight into the pathogenesis of this disease. Characterization of the differences between osteoporotic and non-osteoporotic bones by expression profiling will contribute to the development of diagnostic tools in the future.

Lack of association between calcium-sensing receptor gene “A986S” polymorphism and bone mineral density in Hungarian postmenopausal women

Calcium-sensing receptor (CaSR) is an attractive candidate gene for osteoporosis susceptibility. The CaSR A986S genotype has been shown to have an effect on serum calcium. Recently, an association has been reported between the CaSR gene A986S polymorphism and bone mineral density in healthy white girls. In this study, we examined whether CaSR gene A986S polymorphism is associated with decreased bone mass in 230 Hungarian postmenopausal women. From this cohort, 108 osteoporotic patients were compared with 122 healthy control women. Bone mineral density (BMD) was measured at the lumbar spine (L2-4) and femoral neck using dual-energy X-ray absorptiometry. Allele-specific polymerase chain reaction was used to amplify A986S polymorphisms of the CaSR gene. We found no difference in the distribution of different alleles or genotypes between groups (p = 0.762). No significant effect of CaSR genotype on BMD was observed either in the whole population or in the subgroups. Our data do not support the idea that CaSR gene A986S polymorphism has an impact on bone mass.

Oestrogen and vitamin D receptor (VDR) genotypes and the expression of ErbB-2 and EGF receptor in human rectal cancers

Oestrogen/oestrogen receptor (ER) and vitamin D/vitamin D receptor (VDR) systems have been implicated in the pathogenesis of colorectal cancers. The expression of erbB-2 and epidermal growth factor receptor (EGFR) in colorectal cancers has been suggested to have diagnostic and prognostic significance. In our study, XbaI and PvuII polymorphisms of the ER gene and the BsmI polymorphism of the VDR gene were studied in 56 Caucasian patients with rectal cancer. The relationship between the ER and VDR genotypes and the expression of oncogenes was also investigated. The presence of the x allele of ER gene significantly correlated with the overexpression of the erbB-2 and EGFR oncogenes. Significantly increased erbB-2 expression was observed in patients with the VDR B allele. The XXbb allelic combination of the ER/VDR genes was associated with a significantly lower erbB-2 expression, whereas in the other genotypes significantly higher oncogene expression was seen. Our data raise the possibility that ER/VDR gene polymorphisms accompanied by variable oncogene expression might influence the pathogenetic processes of colorectal cancers.

Lack of association between interleukin-1 receptor antagonist protein gene polymorphism and bone mineral density in Hungarian postmenopausal women.

The major determinant for risk of osteoporosis in later life is bone mineral density (BMD) attained during early adulthood. Bone mineral density is a complex trait that is presumably influenced by multiple genes. Interleukin-1 receptor antagonist protein (IL-1RN) is an attractive candidate gene for osteoporosis susceptibility, because IL-1RN completely inhibits the stimulatory effects of interleukin-1 (IL-1) on bone resorption in organ cultures and has been implicated in the pathogenesis of osteoporosis. In addition, the IL-1RN gene contains a variable-number tandem repeat polymorphism (VNTR) in intron 2 with three potential protein-binding sites. Recently, an association has been found between this polymorphism and postmenopausal bone loss in the spine. In this study, we use the previously described IL-1RN polymorphism to test for an association between this polymorphism and bone mineral density in our population of postmenopausal women. There was no correlation between alleles or genotypes and BMD in the 286 subjects. Dividing subjects into osteoporotic and healthy groups (osteoporotics and controls), we found no difference in the distribution of alleles or genotypes between groups. We found no association between IL-1RN alleles or genotypes and BMD either at the lumbar spine or the femoral neck within groups. Our data do not support the hypothesis that this IL-1RN gene VNTR polymorphism has an impact on bone mass in postmenopausal women.

Avelumab, an anti-PD-L1 antibody, in patients with locally advanced or metastatic breast cancer: a phase 1b JAVELIN Solid Tumor study.

PurposeAgents targeting programmed death receptor 1 (PD-1) or its ligand (PD-L1) have shown antitumor activity in the treatment of metastatic breast cancer (MBC). The aim of this study was to assess the activity of avelumab, a PD-L1 inhibitor, in patients with MBC.MethodsIn a phase 1 trial (JAVELIN Solid Tumor; NCT01772004), patients with MBC refractory to or progressing after standard-of-care therapy received avelumab intravenously 10xa0mg/kg every 2xa0weeks. Tumors were assessed every 6xa0weeks by RECIST v1.1. Adverse events (AEs) were graded by NCI-CTCAE v4.0. Membrane PD-L1 expression was assessed by immunohistochemistry (Dako PD-L1 IHC 73-10 pharmDx).ResultsA total of 168 patients with MBC, including 58 patients with triple-negative breast cancer (TNBC), were treated with avelumab for 2–50xa0weeks and followed for 6–15xa0months. Patients were heavily pretreated with a median of three prior therapies for metastatic or locally advanced disease. Gradexa0≥xa03 treatment-related AEs occurred in 13.7% of patients, including two treatment-related deaths. The confirmed objective response rate (ORR) was 3.0% overall (one complete response and four partial responses) and 5.2% in patients with TNBC. A trend toward a higher ORR was seen in patients with PD-L1+ versus PD-L1−xa0tumor-associated immune cells in the overall population (16.7% vs. 1.6%) and in the TNBC subgroup (22.2% vs. 2.6%).ConclusionAvelumab showed an acceptable safety profile and clinical activity in a subset of patients with MBC. PD-L1 expression in tumor-associated immune cells may be associated with a higher probability of clinical response to avelumab in MBC.

Calcium-sensing receptor A986S polymorphism in human rectal cancer

Abstract.Background and aims: In vivo and in vitro experiments show the protective role of calcium ions (Ca2+) against colorectal cancer. The calcium-sensing receptor (CaSR) detects extracellular Ca2+ concentration. An association between the CaSR A986S polymorphism and serum calcium in healthy adults has been reported. Subjects with AA genotype had lower serum concentrations of Ca2+ than other genotypes. The expression of erbB-2, epidermal growth factor receptor (EGFR), p53, and ras in colorectal cancer has been suggested to have diagnostic and prognostic significance. Patients and methods: We investigated the relationship between the CaSR A986S polymorphism and the expression of erbB-2, EGFR, p53, and ras as well as the UICC stage in 56 patients with rectal cancer. Results: The occurrence of the genotype AA was not different in cancer patients and in 112 controls. In the presence of the coexpression of major oncogenes, patients with genotype AA were in significantly higher UICC stages than in the case of AS genotype. During the follow-up period AA genotype showed a tendency for poor prognosis. Conclusions: Our observation raises the possibility that genetic alterations of CaSR influence the pathogenesis of rectal cancer.

The protective role of bone morphogenetic protein-8 in the glucocorticoid-induced apoptosis on bone cells

One of the side effects associated with glucocorticoid therapy is glucocorticoid-induced bone loss. Glucocorticoids partly detain bone formation via the inhibition of osteoblastic function, however, the exact mechanism of this inhibition remains elusive. In this study, we examined the effect of dexamethasone, an active glucocorticoid analogue, on cell viability and expression of bone remodelling-related genes in primary mouse calvarial and cloned MC3T3-E1 osteoblasts. Using sensitive biochemical assays, we demonstrated the apoptotic effect of dexamethasone on osteoblastic cells. Then, utilizing Taqman probe-based quantitative RT-PCR technology, gene expression profiles of 111 bone metabolism-related genes were determined. As a result of dexamethasone treatment we have detected significant apoptotic cell death, and six genes, including Smad3, type-2 collagen α-1, type-9 collagen α-1, matrix metalloproteinase-2, bone morphogenetic protein-4 and bone morphogenetic protein-8 showed (BMP-8) significant changes in their expression on a time- and concentration-dependent manner. BMP-8, (a novel player in bone-metabolism) exhibited a two orders of magnitude elevation in its mRNA level and highly elevated protein concentration by Western blot in response to dexamethasone treatment. The knockdown of BMP-8 by RNA interference significantly increased dexamethasone-induced cell death, confirming a protective role for BMP-8 in the glucocorticoid-induced apoptosis of osteoblasts. Our results suggest that BMP-8 might be an essential player in bone metabolism, especially in response to glucocorticoids.

CYP24A1 inhibition facilitates the anti-tumor effect of vitamin D3 on colorectal cancer cells

AIMnThe effects of vitamin D3 have been investigated on various tumors, including colorectal cancer (CRC). 25-hydroxyvitamin-D3-24-hydroxylase (CYP24A1), the enzyme that inactivates the active vitamin D3 metabolite 1,25-dihydroxyvitamin D3 (1,25-D3), is considered to be the main enzyme determining the biological half-life of 1,25-D3. During colorectal carcinogenesis, the expression and concentration of CYP24A1 increases significantly, suggesting that this phenomenon could be responsible for the proposed efficacy of 1,25-D3 in the treatment of CRC. The aim of this study was to investigate the anti-tumor effects of vitamin D3 on the human CRC cell line Caco-2 after inhibition of the cytochrome P450 component of CYP24A1 activity.nnnMETHODSnWe examined the expression of CYP24A1 mRNA and the effects of 1,25-D3 on the cell line Caco-2 after inhibition of CYP24A1. Cell viability and proliferation were determined by means of sulforhodamine-B staining and bromodeoxyuridine incorporation, respectively, while cytotoxicity was estimated via the lactate dehydrogenase content of the cell culture supernatant. CYP24A1 expression was measured by real-time reverse transcription polymerase chain reaction. A number of tetralone compounds were synthesized to investigate their CP24A1 inhibitory activity.nnnRESULTSnIn response to 1,25-D3, CYP24A1 mRNA expression was enhanced significantly, in a time- and dose-dependent manner. Caco-2 cell viability and proliferation were not influenced by the administration of 1,25-D3 alone, but were markedly reduced by co-administration of 1,25-D3 and KD-35, a CYP24A1-inhibiting tetralone. Our data suggest that the mechanism of action of co-administered KD-35 and 1,25-D3 does not involve a direct cytotoxic effect, but rather the inhibition of cell proliferation.nnnCONCLUSIONnThese findings demonstrate that the selective inhibition of CYP24A1 by compounds such as KD-35 may be a new approach for enhancement of the anti-tumor effect of 1,25-D3 on CRC.

Marked Increase in CYP24A1 Gene Expression in Human Papillary Thyroid Cancer

The active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25-D3) inhibits cell growth and induces cell differentiation and apoptosis in numerous tumors, such as colon, breast, and prostate cancers (1–3). However, the anticancer effect of 1,25-D3 cannot always be seen in a clinical setting. In case of colon cancers, a marked increase in the expression of the 1,25D3 inactivating enzyme, the 24-hydroxylase (CYP24A1), has been observed (4). The role of 1,25-D3 in the development of thyroid carcinomas has not yet been established. However, Sharma et al. (5) observed the correlation of high baseline CYP24A1 and relatively low stimulation after calcitriol treatment (compared with sensitive cells) with lack of growth inhibition in numerous thyroid cancer cell lines. We have also examined the gene expression of the inactivating CYP24A1 and the activating 1-alpha-hydroxylase (CYP27B1) enzyme in human thyroid cancers. To date, gene expression analysis of thyroid samples (both normal and papillary tumor tissue of the same patient) was carried out in six unrelated, consecutive, Hungarian, Caucasian patients at our Thyroid Clinic. All surgically removed thyroid tissue samples underwent histological examination, and papillary tumor was identified in all cases. The study was approved by the Regional Committee of Science and Research Ethics, Semmelweis University (SOTE-TUKEB 1160-0/20101018EKU), and all patients gave written informed consent. Total RNA was isolated from each sample with Roche High Pure Total RNA Isolation kit. Five hundred nanograms of total RNA was reverse-transcribed to cDNA. The expression differences of selected genes were analyzed by Taqman probe-based quantitative real-time reverse transcriptase– polymerase chain reaction. Relative quantification was carried out from collected data (threshold cycle numbers) by Applied Biosystems 7500 System SDS software 1.3. CYP24A1 mRNA expression was markedly increased in all but one papillary cancer compared with that of normal thyroid tissue, sometimes reaching 300-fold elevation (Supplementary Fig. S1; Supplementary Data are available online at www.liebertonline.com/thy). No significant alteration was seen in CYP27B1 gene activity between neoplastic and normal tissues. There was no significant difference in serum 25-OHvitamin D3 concentrations among patients (mean: 28.03 ng/ mL, range: 22.7–31.9 ng/mL). In this letter, we report marked increases in the 1,25-D3neutralizing CYP24A1 gene expression in human papillary thyroid cancer. The tumor cell growth-inhibiting role of vitamin D3 has been extensively studied in different malignancies (1–3). The interest in the association of vitamin-D3 serum levels with various cancer incidences has dramatically increased. However, only very limited data are available on the relationship between serum vitamin D3 concentrations and malignant thyroid conditions (6,7). The pilot study of Laney et al. showed levels of vitamin D and rates of vitamin D deficiency to be similar between patients with thyroid nodules, thyroid cancer in remission, and active thyroid cancer (8). However, the results of Stepien et al. revealed significantly lower calcitriol concentrations in patients with papillary, follicular, and anaplastic thyroid cancers (7). This finding could be attributable to higher CYP24A1 levels cleaving calcitriol, as there were no differences in serum 25-OH-vitamin D3. Earlier, calcitriol had also been shown to attenuate thyrotropinstimulated growth and iodide uptake by rat thyroid cells (FRTL-5) (9,10). Papillary cancer is the most common thyroid malignancy. The role of vitamin D3 in the prevention or development of this disorder has to be yet determined. The increase in CYP24A1 expression in this cancer type could be a ‘‘protective’’ mechanism against the well-known anticancer effect of calcitriol. The observation of Sharma et al. (5) regarding increased expression of CYP24A1 in thyroid cancer cell lines corroborates this notion. One of the cell lines they used was human papillary thyroid carcinoma cell line, whereas the other ones showed the features of human anaplastic thyroid carcinoma. Also, the most resistant cell lines to calcitriol had the highest baseline levels of CYP24A1. In our study at this stage, the number of examined tumor tissues does not allow drawing of statistically significant conclusion about the correlation of tumor histopathological stage or aggressiveness with CYP24A1 levels. Khadzkou et al. demonstrated enhanced 1-alphahydroxylase expression but concluded that this elevation did not show large differences in CYP27B1 expression in papillary thyroid cancer and its metastasis compared with normal