Evolution of the neurosurgical management of progestin‐associated meningiomas: a 23-year single‐center experience

Journal of Neuro-Oncology - Approximately 10% of IDH-mutant gliomas harbour non-canonical IDH mutations (non-p.R132H IDH1 and IDH2 mutations). The aim of this study was to analyse the...     

Incremental cost‐effectiveness of double‐reading mammograms

Background. Double reading is a widely used criterion standard in breast cancer screening despite a lack of evidence of the costeffectiveness of the second reading. This study evaluates the incremental costeffectiveness of such a strategy.Design. Costeffectiveness analysis: Nationwide populationbased semiannual screening program for women aged 50–59 in Finland. Participation rate was 91%. All mammograms (95,423) performed during 1990–1995 in three screening centers of the Finnish Cancer Society were read by two radiologists with gradings recorded. The effectiveness of the double reading was the difference in cancers detected in the double compared to that of the single reading. Incremental costs of the double reading for the health care and nonhealth care and the time costs were estimated. The main outcome measure was the incremental cost per additional cancer found as a result of the doublereading strategy.Results. The total number of cancers detected with the double and single reading were 290 and 261, respectively. A significantly higher ratio of carcinoma in situ was the causative pathology in cancers detected only by the second reader. The cost per cancer detected with a single reading was US$ 18,340. The incremental cost of any additional cancer found was US$ 25,523, that is, a 39% higher cost per additional cancer found by double reading.Conclusions. The additional cost per cancer detected by double reading is not drastically higher than with single reading. However, the additional cost per life year saved may be much higher.     

The methodology of quantitation of microvessel density and prognostic value of neovascularization associated with long‐term survival in Japanese patients with breast cancer

The present study updates results on methodology of quantitation of tumor neovascularization and those on the prognostic value of microvessel density (MVD) in breast cancer tissue previously published in the World J. Surg. 21: 49–56, 1997. The followup period of observation of the series was extended to 20 years, and new biological indicators (i.e., proliferating cell nuclear antigen (PCNA), cerbB2, and p53) were included in the analysis. There were 109 patients with primary breast cancer, from 1971 to 1979, followed up for a median of 14 years (range, 1–20). A representative median longitudinal section of each breast tumor was immunohistochemically stained with factor VIIIrelated antigen and analyzed. The three methods of identifying MVD were: (1) average microvessel count (AMC)/mm2, (2) central microvessel count (CMC)/mm2, and (3) highest microvessel count (HMC)/mm2. Thirtyone patients (28.4%) died of breast cancer. There was a relationship between MVD and peritumor blood vessel invasion (AMC: p = 0.0114, CMC: p = 0.0319, and HMC: p = 0.0009). However, there was no relationship between MVD and other factors. Univariate analysis showed that node status (p < 0.0001), histological grade (p < 0.0001), clinical tumor size (T) (p = 0.0002), PCNA (p = 0.0033), p53 (p = 0.0043), mitotic grade (p = 0.0092), AMC (p = 0.0214), and peritumor lymphatic vessel invasion (p = 0.0467) were significantly predictive of overall survival. HMC was borderline significant (p = 0.0702), while CMC and cerbB2 were not significant. Multivariate analysis showed that T (p = 0.0005), node status (p = 0.0053), and AMC (p = 0.0485) were independent factors, but neither CMC nor HMC was independent. AMC, a significant independent prognostic factor, might be a better method than the others for evaluating angiogenesis, but further and larger studies are warranted.     

High level PHGDH expression in breast is predominantly associated with keratin 5‐positive cell lineage independently of malignancy

We have previously reported the 2D PAGE‐based proteomic profiling of a prospective cohort of 78 triple negative breast cancer (TNBC) patients, and the establishment of a cumulative TNBC protein database. Analysis of this database identified a number of proteins as being specifically overexpressed in TNBC samples. One such protein was D‐3‐phosphoglycerate dehydrogenase (Phgdh), a candidate oncogene. We analysed expression of Phgdh in normal and TNBC mammary tissue samples by 2D gel‐based proteomics and immunohistochemistry (IHC), and show here that high‐level expression of Phgdh in mammary epithelial cells is primarily associated with cell lineage, as we found that Phgdh expression was predominant in CK5‐positive cells, normal as well as malignant, thus identifying an association of this protein with the basal phenotype. Quantitative IHC analysis of Phgdh expression in normal breast tissue showed high‐level expression of Phgdh in normal CK5‐positive mammary epithelial cells, indicating that expression of this protein was not associated with malignancy, but rather with cell lineage. However, proteomic profiling of Phgdh showed it to be expressed in two major protein forms, and that the ratio of expression between these variants was associated with malignancy. Overexpression of Phgdh in CK5‐positive cell lineages, and differential protein isoform expression, was additionally found in other tissues and cancer types, suggesting that overexpression of Phgdh is generally associated with CK5 cells, and that oncogenic function may be determined by isoform expression.     

Subtype‐dependent prognostic relevance of an interferon‐induced pathway metagene in node‐negative breast cancer

The majority of gene expression signatures developed to predict the likelihood to relapse in breast cancer (BC) patients assigns a high risk score to patients with Estrogen Receptor (ER) negative or highly proliferating tumors. We aimed to identify a signature of differentially expressed (DE) metagenes, rather than single DE genes, associated with distant metastases beyond classical risk factors.We used 105 gene expression profiles from consecutive BCs to identify metagenes whose prognostic role was defined on an independent series of 92 ESR1+/ERBB2− node‐negative BCs (42 cases developing metastases within 5 years from diagnosis and 50 cases metastasis‐free for more than 5 years, comparable for age, tumor size, ER status and surgery). Findings were validated on publicly available datasets of 684 node‐negative BCs including all the subtypes.Only a metagene containing interferon‐induced genes (IFN metagene) proved to be predictive of distant metastasis in our series of patients with ESR1+/ERBB2− tumors (P = 0.029), and such a finding was validated on 457 ESR1+/ERBB2− BCs from public datasets (P = 0.0424). Conversely, the IFN metagene was associated with a low risk of metastasis in 104 ERBB2+ tumors (P = 0.0099) whereas it did not prove to significantly affect prognosis in 123 ESR1−/ERBB2− tumors (P = 0.2235). A complex prognostic interaction was revealed in ESR1+/ERBB2− and ERBB2+ tumors when the association between the IFN metagene and a T‐cell metagene was considered.The study confirms the importance of analyzing prognostic variables separately within BC subtypes, highlights the advantages of using metagenes rather than genes, and finally identifies in node‐negative ESR1+/ERBB2− BCs, the unfavorable role of high IFN metagene expression.     

Inhibition of PARP1‐dependent end‐joining contributes to Olaparib‐mediated radiosensitization in tumor cells

Poly‐ADP‐ribose‐polymerase inhibitors (PARPi) are considered to be optimal tools for specifically enhancing radiosensitivity. This effect has been shown to be replication‐dependent and more profound in HR‐deficient tumors. Here, we present a new mode of PARPi‐mediated radiosensitization which was observed in four out of six HR‐proficient tumor cell lines (responders) investigated, but not in normal cells. This effect is replication‐independent, as the radiosensitization remained unaffected following the inhibition of replication using aphidicolin. We showed that responders are radiosensitized by Olaparib because their DSB‐repair is switched to PARP1‐dependent end‐joining (PARP1‐EJ), as evident by (i) the significant increase in the number of residual γH2AX foci following irradiation with 3Gy and treatment with Olaparib, (ii) the enhanced enrichment of PARP1 at the chromatin after 3Gy and (iii) the inhibition of end‐joining activity measured by a specific reporter substrate upon Olaparib treatment. This is the first study which directly demonstrates the switch to PARP1‐EJ in tumor cells and its contribution to the response to Olaparib as a radiosensitizer, findings which could widen the scope of application of PARPi in tumor therapy.     

Significance of TP53 mutations as predictive markers of adjuvant cisplatin‐based chemotherapy in completely resected non‐small‐cell lung cancer

Adjuvant cisplatin‐based chemotherapy only marginally improves survival in patients with completely resected non‐small‐cell lung cancer (NSCLC). We have evaluated the predictive value of mutations in TP53, encoding the tumour suppressor p53, in the International Adjuvant Lung Cancer Trial (IALT), a randomized trial of adjuvant cisplatin‐based chemotherapy against observation. TP53 (exons 4–8) was sequenced in 524 archived specimens of IALT patients with a median follow‐up of 7.5 years. Predictive analyses were based on Cox models adjusted for clinical and pathological variables. P‐values ≤0.01 were considered as significant. Mutations were detected in 221 patients (42%) and had no predictive value for the effect of chemotherapy (interaction between TP53 and treatment: p = 0.17 for Overall Survival (OS); p = 0.06 for Disease‐Free Interval, (DFS)). However, among patients with mutations, outcome appeared worse in treatment compared to observation arms (HR for OS = 1.36 (95% CI [0.97–1.31), p = 0.08; DFS = 1.40 (95% CI [1.01–1.95]), p = 0.04). When grouping mutations into classes according to predicted effects on protein structure, the tendency towards worse outcomes was restricted to “structure” mutations affecting residues of the hydrophobic core that are not located at the p53 protein‐DNA interface (HR for death in this class vs wild‐type T53 = 1.66; 95% CI [1.10–2.52], p = 0.02). Overall, TP53 mutations are not significant predictors of outcome in this trial of cisplatin‐based chemotherapy, although a specific class of structural mutations may be associated with a tendency towards worse outcomes upon treatment.     

Selective activity over a constitutively active RET‐variant of the oral multikinase inhibitor dovitinib: Results of the CNIO‐BR002 phase I‐trial

Background

Given our preclinical data showing synergy between dovitinib and paclitaxel in preclinical models we conducted this phase I trial aiming to define the recommended phase II‐dose (RP2D) on the basis of toxicity and pharmacodynamic criteria while searching for genetic variants that could sensitize patients to the regimen under study.

Patients and methods

A 3+3 escalation schedule was adopted. Seriated FGF23 and dovitinib and paclitaxel pharmacokinetic profiles were determined along a single‐agent dovitinib “priming‐phase” followed by a dovitinib + paclitaxel combination phase. RECIST 1.1 criteria and NCI CTCAE V.4.0 were used. In fresh pre‐treatment tumor biopsy samples, FGFR1, 2 and 3 amplifications were revealed by FISH probes; 32 missense variants were genotyped in tumors and peripheral blood mononuclear cells with Taqman genotyping assays (FGFR1‐3 and RET). Constructs encoding for wild‐type and variant genes associated with clinical benefit were transfected into HEK‐293 cells for preclinical experiments checking constitutive activation and dovitinib sensitivity of the variants.

Results

twelve patients were recruited in three dose‐levels. At level 1B (200 mg dovitinib 5‐days‐on/2‐days‐off plus 60 mg/m 2‐week of paclitaxel) more than 50% FGF23 upregulation was observed and no dose‐limiting‐toxicities (DLTs) occurred. The most frequent toxicities were asthenia, neutropenia, nausea/vomiting and transaminitis. Two patients with progressive disease prior to trial inclusion achieved prolonged disease stabilization. Both had the germline variant G2071A in the RET gene, which led to constitutive activation of the protein product and Y‐905 phosphorylation, both in transfectants and in patients with the alteration. This variant was sensitive to dovitinib; in addition both patients experienced progression upon medication withdrawal.

Conclusions

Level 1B was the RP2D as it provided adequate pharmacodynamic exposure to dovitinib. The G2071A germline variant act as a genetic modifier that renders different tumors sensitive to dovitinib.     

Targeting of VEGF‐dependent transendothelial migration of cancer cells by bevacizumab

Cancer progression is often associated with the formation of malignant effusions. Vascular endothelial growth factor (VEGF) is a major regulator of vascular permeability and has been implicated as mediator of tumor progression.We examined the production and secretion of VEGF(165) in various primary cancer cells derived from malignant effusions, and the role of exogenous VEGF165 as a mediator of effusion formation. VEGF165 was constantly secreted by all cultured tumor cells in an mTOR‐dependent manner, as it was inhibited by the mTOR inhibitor rapamycin. Secreted VEGF165 showed functional activity by inducing endothelial leakiness and tumor cell‐transendothelial migration in vitro, effects which could be reverted by the anti‐VEGF antibody bevacizumab.Thus, mTOR inhibitors as well as bevacizumab should be considered as potential agents in cancer patients suffering from malignant effusions.     

Instability of a dinucleotide repeat in the 3′‐untranslated region (UTR) of the microsomal prostaglandin E synthase‐1 (mPGES‐1) gene in microsatellite instability‐high (MSI‐H) colorectal carcinoma

DNA mismatch‐repair gene mutations, with consequent loss of functional protein expression, result in microsatellite instability (MSI). Microsatellite sequences are found in coding regions and in regulatory regions of genes (i.e., 5′‐UTRs and 3′‐UTRs). In addition to being a surrogate marker of defective mismatch repair, deletion or insertion microsatellite sequences can dysregulate gene expression in MSI‐H (microsatellite instability‐high) tumors. The microsomal prostaglandin E synthase‐1 (mPGES‐1) gene product, mPGES‐1, participates in prostaglandin E2 (PGE2) production. Moreover, mPGES‐1 is often overexpressed in human colorectal tumors, and is thought to contribute to progression of these tumors. Here we identified a dinucleotide repeat, (GT)24, in the mPGES‐1 gene 3′ untranslated region (3′‐UTR), and analyzed its mutation frequencies in MSI‐H and microsatellite stable (MSS) tumors. The (GT)24 repeat exhibited instability in all MSI‐H tumors examined (14), but not in any of the MSS tumors (13). In most cases, (GT)24 repeat instability resulted in insertion of additional GT units. We also determined mPGES‐1 mRNA levels in MSI‐H and MSS colorectal cancer cell lines. Three of four previously designated “MSI‐H” cell lines showed higher mPGES‐1 mRNA levels compared to MSS cell lines; correlations between elevated mPGES‐1 mRNA levels and microsatellite (GT)24 repeat characteristics are present for all six cell lines. Our results demonstrate that mPGES‐1 is a target gene of defective mismatch repair in human colorectal cancer, with functional consequence.     

Reversal of estrogen‐resistance in murine mammary adenocarcinomas

From mouse mammary progestindependent (PD) adenocarcinomas induced with medroxyprogesterone acetate (MPA) we developed several in vivo lines that are maintained by subcutaneous syngeneic passages in MPAtreated mice and express estrogen (ER) and progesterone receptors (PR). Although most lines remained PD, with time some progestinindependent (PI) variants arose. Both PD and PI tumors regress with estrogen treatment although estrogenresistant variants may also arise. The object of this paper was to investigate the reversibility of estrogenresistance and its possible relation with hormone receptor downregulation. Tumor regression was induced in a progestinindependent tumor line (BET) by treatment with a 5mg 17estradiol (E2) silastic pellet. One of the tumors started to grow disclosing an estrogenresistant pattern of growth. This tumor line (BETR) was transplanted into E2treated and untreated animals (n= 4–6), selecting for the next passage tumors growing in treated animals. Seven new sublines were obtained at different passages by selecting for the next passage the tumors that had grown in untreated mice (BETRaBETRg), until no tumors grew in E2treated mice. ER and PR were measured by a ligandbinding, dextrancoated charcoal method using a single saturating point. From the seven sublines initiated, the first four proved to be reversible after 3–6 generation transplantation and the last three did not revert. A difference in PR expression between BET and BETR (p < 0.05) was registered, but it did not correlate with the specific hormone behavior since two reverted lines had a pattern similar to that of BET and the other two were similar to BETR. The expression of PR was higher in E2treated mice (p < 0.05) and highly variable in the parental line. This led us to study the expression of PR at different stages of the estrous cycle. Higher levels of PR were observed in proestrous, estrous, and metestrous (p < 0.05) than in diestrous, and undetectable levels were found in ovariectomized mice. No differences in ER expression were detected during the estrous cycle. It can be concluded that under certain experimental conditions, estrogenresistance is a reversible phenomenon. The experimental manipulation of hormone resistance may help develop strategies to modify the response to antihormones in humans.     

Multi‐marker analysis of circulating cell‐free DNA toward personalized medicine for colorectal cancer

Development of a Q‐PCR‐based assay for the high‐performance analysis of circulating cell‐free DNA (ccfDNA) requires good knowledge of its structure and size.In this work, we present the first visual determination of ccfDNA by Atomic Force Microscopy (AFM) on plasma samples from colorectal cancer (CRC) patients and healthy donors. In addition to the examination of fragment size distribution profile as performed by Q‐PCR, this analysis confirms that ccfDNA is highly fragmented and that more than 80% of ccfDNA fragments in CRC plasma are below 145 bp. We adapted an Allele‐Specific Blocker (ASB) Q‐PCR to small ccfDNA fragments to determine simultaneously the total ccfDNA concentration, the presence of point mutation, the proportion of mutated allele, and a ccfDNA integrity index. The data validated analytically these four parameters in 124 CRC clinical samples and 71 healthy individuals. The multi‐marker method, termed Intplex, enables sensitive and specific non‐invasive analysis of tumor ccfDNA, which has great potential in terms of cost, quality control, and easy implementation in every clinical center laboratory.     

Interleukin‐6 drives melanoma cell motility through p38α‐MAPK‐dependent up‐regulation of WNT5A expression

Extensive research has demonstrated a tumor‐promoting role of increased WNT5A expression in malignant melanoma. However, very little light has been shed upon how WNT5A expression is up‐regulated in melanoma. A potential regulator of WNT5A expression is the pro‐inflammatory cytokine Interleukin (IL)‐6, which shares the ability of WNT5A to increase melanoma cell invasion. Here, we investigate whether IL‐6 can promote melanoma cell motility through an increased expression of WNT5A. We clearly demonstrate that the WNT5A‐antagonistic peptide Box5 could inhibit IL‐6‐induced melanoma cell migration and invasion. Furthermore, IL‐6 stimulation of the human melanoma cell lines HTB63 and A375 increased the expression of WNT5A in a dose‐dependent manner. To identify the signaling mechanism responsible for this up‐regulation, we explored the involvement of the three main signals induced by IL‐6; STAT3, Akt and ERK 1/2. Of these, only STAT3 was activated by IL‐6 in the melanoma cell lines tested. However, the STAT3 inhibitor S3I‐201 failed to inhibit IL‐6‐induced WNT5A up‐regulation in HTB63 and A375 cells. Nor did STAT3 siRNA silencing affect the expression of WNT5A. In search of an alternative signaling mechanism, we detected IL‐6‐induced activation of p38‐MAPK in HTB63 and A375 cells. The p38‐MAPK inhibitor SB203580 abolished the IL‐6‐induced WNT5A up‐regulation and blocked IL‐6‐induced melanoma cell invasion. The latter effect could be rescued by the addition of recombinant WNT5A. Notably, immunoprecipitation analysis revealed that only the p38α‐MAPK isoform was activated by IL‐6, and subsequent siRNA silencing of p38α‐MAPK abolished the IL‐6‐induced up‐regulation of WNT5A. Taken together, we demonstrate a novel link between the two melanoma pro‐metastatic agents IL‐6 and WNT5A explaining how IL‐6 can increase melanoma cell invasion and thus promote the metastatic process. This finding provides a basis for future therapeutic intervention of melanoma progression.     

Antibody‐independent targeted quantification of TMPRSS2‐ERG fusion protein products in prostate cancer

Fusions between the transmembrane protease serine 2 (TMPRSS2) and ETS related gene (ERG) represent one of the most specific biomarkers that define a distinct molecular subtype of prostate cancer. Studies of TMPRSS2‐ERG gene fusions have seldom been performed at the protein level, primarily due to the lack of high‐quality antibodies suitable for quantitative studies. Herein, we applied a recently developed PRISM (high‐pressure high‐resolution separations with intelligent selection and multiplexing)‐SRM (selected reaction monitoring) strategy for quantifying ERG protein in prostate cancer cell lines and tumors. The highly sensitive PRISM‐SRM assays provided confident detection of 6 unique ERG peptides in both TMPRSS2‐ERG positive cell lines and tissues, but not in cell lines or tissues lacking the TMPRSS2‐ERG rearrangement, clearly indicating that ERG protein expression is significantly increased in the presence of the TMPRSS2‐ERG gene fusion. Significantly, our results provide evidence that two distinct ERG protein isoforms are simultaneously expressed in TMPRSS2‐ERG positive samples as evidenced by the concomitant detection of two mutually exclusive peptides in two patient tumors and in the VCaP prostate cancer cell line. Three peptides, shared across almost all fusion protein products, were determined to be the most abundant peptides, providing “signature” peptides for detection of ERG over‐expression resulting from TMPRSS2‐ERG gene fusion. The PRISM‐SRM assays provide valuable tools for studying TMPRSS2‐ERG gene fusion protein products in prostate cancer.