The Escherichia coli uropathogenic-specific-protein- associated immunity protein 3 (Imu3) has nucleic acid -binding activity

Miha Črnigoj
Zdravko Podlesek
Maruška Budič
Darja Žgur-Bertok
The Escherichia coli uropathogenic-specific-protein- associated immunity protein 3 (Imu3) has nucleic acid -binding activity
R E S E A R C H A R T I C L E Open Access
Background:The Escherichia coli uropathogenic-specific protein (Usp) is a bacteriocin-like genotoxin, active against mammalian cells and associated with E. coli strains that provoke pyelonephritis, prostatitis and bacteraemia. Usp is encoded by a small pathogenicity island with three downstream small open reading frames (Imu1-3) that are believed to provide immunity to the producer. To prevent host suicide, colicins, bacteriocins of E. coli, form tight complexes with their cognate immunity proteins. Colicinimmunity protein complexes are among the strongest protein complexes known. Here, the Usp associated immunity protein 3 (Imu3) was partially characterized to gain insight into its role and mechanism of activity.Results: Isolation and partial characterisation of the Usp-associated immunity protein-3 (Imu3) revealed that, while Usp and Imu3 do not form a high affinity complex, Imu3 exhibits DNA and RNA binding activity. Imu3 was also shown to protect DNA against degradation by colicin E7.Conclusions: Our data infer that nonspecific DNA binding of the Imu3 immunity protein, prevents suicide of E. coli producing the genotoxin Usp.

Background

The Escherichia coli uropathogenic-specific protein (Usp) has been shown to be associated with E. coli strains that provoke pyelonephritis, prostatitis and bacteraemia, and with increased virulence and fitness of pathogenic strains of E. coli [1][2][3][4]. Nucleotide sequence analysis has shown approximately 45% sequence identity of the Usp C-terminal region with that of the E. coli bacteriocin colicin E7, which has nuclease activity, while the Usp N-terminal region is similar to the Type VI protein secretion system component (Hcp like) [5][6][7]. It has been proposed that Usp acts as a bacteriocin against competing E. coli strains and that it also enhances infectivity in the urinary tract. Recently, we demonstrated the genotoxic activity of Usp against mammalian cells [5,8]. To protect the colicin-producing cell from its own toxin, colicin-encoding operons generally harbour one cognate immunity gene [9]. Colicins and their immunity proteins have some of the strongest protein-protein affinities, which result in the formation of stable colicin-immunity protein complexes [10,11]. In contrast, downstream of the usp gene, there are three short open reading frames designated orfU1-3 (288, 294, and 291 bp long, respectively), that are believed to be involved in the protection of the Usp-producing cell from its own nuclease activity [5]. The immunity proteins coded by the usp gene operon have a characteristic two-histidine region which appears to enable the inactivation of the Usp DNase activity [10]. However, Usp-encoding strains that do not have all three orfU immunity protein genes have been described. All three immunity proteins are thus not essential for the protection of the Usp producers, although Usp is lethal when it is expressed alone in E. coli. It has been postulated that none of the three proteins is exclusively required for Usp protein synthesis [6]. As protection of the Usp-producing bacterial cell might be provided by a mechanism that is different from that of the colicins, we have investigated the E. coli Usp-associated immunity protein Imu3, previously designated OrfU3. Our study indicates that Imu3 has protective non specific DNA-binding abilities that could have possible biotechnological potential.

Results and discussion

Isolation of Immunity protein 3 (Imu3) with Ni-NTA affinity chromatography provided protein fractions with appropriate purity; ( Figure 1A). DNA binding ability was not affected by the presence or absence of the his-tag, as both precipitated linear DNA (Additional file 1: Figure  S1). The theoretical and actual mass (11.497 kDa) of the purified Imu3 differed by 1.5 Da (measured by ESI + and Q-Tof; Waters-Micromass, United Kingdom, data not shown), indicating that Imu3 is not post-translationally modified. Parret and DeMot [5] previously described an approximately 45% sequence identity of the C-terminal region of the Usp protein with known nuclease colicins, such as colicins E7 and E9. Although it has been shown that colicin E7 and its immunity protein form a highaffinity complex [11], we were not able to confirm the formation of a high affinity complex between Usp and any of the three smaller proteins encoded downstream of the usp gene (data not shown) which were previously proposed to protect the Usp-producing cell against its endonucleolytic activity [5]. Nevertheless, our results showed that Imu3 protects isolated DNA from digestion by the nuclease colicin E7, indicating a nonspecific protection mechanism that is distinct from that of the colicin immunity proteins ( Figure 2).

Induction of the cloned usp gene (without the immunity protein genes) was either lethal (liquid media) or resulted in severely diminished growth (plates). Of the three potential immunity proteins, when cloned separately downstream of the usp gene, Imu3 showed the greatest degree of protection as the number of transformants obtained was repeatedly higher, with larger colonies than for the other two ( Figure 3, Table 1). We therefore focused our further investigation on Imu3.

Imu3 dimerisation and USP binding

Imu3 has fairly high sequence similarity to the colicin E7 immunity protein Cei, approximately 66% sequence identity as established with the MEGA program package, which was previously reported to form monomers [12]. We investigated potential dimer formation by Imu3, using the cross-linking glutaraldehyde assay, native PAGE electrophoresis and size exclusion chromatography (HPLC). Native PAGE as well as HPLC experiments clearly showed that, Imu3 does not form dimers or multimers since a single peak of size between 11 and 13 kDa was observed regardless of the presence or absence of DNA ( Figure 1B). Cross-linking studies of equimolar mixtures of Imu3 and Usp also showed no complex formation (Additional file 2: Figure S2).

DNA/RNA binding

Our data thus indicate that the Usp-producing cell is protected from the DNase activity of its own Usp by a mechanism that is distinct from that of colicin-producing cells. Surprisingly, EMSA showed that Imu3 binds linear and circular ( Figure 4B) DNA as well as RNA molecules. When Imu3 reached a critical concentration (ca. 1 μg Imu3 per 100 ng double-stranded linear or circular DNA), it repeatedly precipitated the DNA, which resulted in total retardation/precipitation of DNA in the electrophoresis ( Figure 4A). When Imu3 was subjected to treatment with increasing concentrations of ions (NaCl or Mg 2+ ), the effects of DNA retardation were decreased ( Figure 4A and C). Incubations at higher temperatures (70-100°C) also reduced the gel shift effects of Imu3 on DNA ( Figure 4B). The EMSA studies with DNA or E. coli total RNA clearly showed that Imu3 has DNA-binding as well as RNAbinding abilities. No such activity was observed with Imu1 or Imu2 (data not shown).

Furthermore, thermal denaturation curves (A 260 ) showed a stabilising effect of Imu3 on the linear double-stranded DNA molecule. The melting temperature (determined graphically) of DNA alone was 73°C, which increased by 3°C (T m = 76°C) when an aliquot of 0.3 μg Imu3 was added in the EMSA studies. The DNA melting temperature was further raised by an additional 13°C (T m = 89°C) when a 1 μg aliquot of Imu3 was added. This concentration of Imu3 saturated the DNA, and the melting curve revealed a two-phase thermal transition. One transition showed a  stabilisation effect (89°C), whereas the other transition (at 63°C) was shown to be destabilising (in terms of thermal stability), most probably due to partial DNA precipitation ( Figure 5).

Minimal DNA length for Imu3 binding

Binding of short DNA fragments to Imu3 occupied all its free DNA binding sites, and therefore prevented subsequent binding of Imu3 to indicator DNA (EcoRI linearised pUC19). These EMSA tests showed that free Imu3 starts to bind to oligonucleotides longer than 11 base pairs, observed as the reappearance of unbound indicator DNA (absence of precipitation). These results indicate that 11 base pairs is the minimal DNA length required for Imu3 binding ( Figure 6).

EMSA tests with short double stranded DNA fragments (re-annealed oligonucleotides) were also performed however, the results were inconclusive since we repeatedly observed the recurring effect of unbound Imu3 that re-/dis-appeared every 3-5 nucleotides of the oligonucleotide length; however, the underlying basis of this phenomena is unclear.

Separation of Imu3 from DNA and subsequent DNA integrity analysis

Separation of the DNA-Imu3 complex, was examined under different conditions. Exposure of the DNA-Imu3 complex to pH values between pH 3 and pH 13 showed separation at values between pH 11 and pH 12. However, at pH values higher than pH 12.5, DNA degradation was also observed. When the DNA-Imu3 complex was heated to 100°C for 5 min in the presence of  different NaCl concentrations, separation of Imu3 from DNA was observed at 0.5 M NaCl or higher (Additional file 3: Figure S3). Incubation of Imu3-DNA complexes with proteinase K resulted in unbound DNA due to degradation of Imu3.

To determine whether DNA exposed to Imu3 could subsequently be used for molecular biological manipulations, linear plasmid pBR322 DNA that had been previously complexed with Imu3 was purified with the QIAgen commercial kit. This DNA could be re-ligated, transformed into E. coli, and again subjected to restriction enzyme activity. The integrity of precipitated and religated plasmid DNA was confirmed on the basis of expression of the ampicillin resistance gene among 500 analysed transformants (described in Methods). All procedures were also performed with DNA that had not been previously complexed as a control, and no apparent losses in quantity or quality of DNA were observed (with exception of losses originating from the DNA purification procedure) (Figure 7). Further, we found that Imu3 precipitated DNA from highly (1.5 × 10 -4 fold) diluted solutions, where 1 μL (100 ng) of linear plasmid DNA was diluted in 15 mL. This procedure yielded less DNA as the control but could without doubt be optimised with appropriate protocol modification (Additional file 4). The colicin DNases and their cognate immunity proteins are known to form high affinity complexes with the DNase domain [11,12]. In the present study, despite its two preserved histidines, as nuclease inactivation motifs that are present throughout the DNase immunity protein family, Imu3 showed no coupling with the USP protein, and Imu3 alone was shown to be sufficient for protection of Usp-producing cells. Not unexpectedly due to the sequence similarity of Imu3 with the colicin E7 immunity protein, which was shown by Dennis et al. [12] to be monomeric, we demonstrated, on the basis of different experiments that Imu3 does not undergo dimerisation or multimerisation.   To the best of our knowledge, no known functions have been described yet for the protein products of orfU1, orfU2 and orfU3 (here referred to as Imu1, Imu2 and Imu3). Although all three immunity proteins of the Usp cluster share a high degree of sequence similarity, the present study shows that Imu3 has a distinct RNAand DNA-binding ability. Nevertheless, additional as of yet unresolved mechanisms could be involved in protection of Usp producing cells by its cognate immunity proteins. Interestingly, protein-mediated DNA precipitation has been reported in studies describing eukaryotic histones and the E. coli global regulator, protein HU, a known DNA-binding protein [13,14]. Operons, such as those of colicins, that encode proteins that can be detrimental to the producing cell are regulated precisely to ensure appropriate timing of synthesis and avoid untimely death of the producer [15][16][17]. We can thus speculate that synthesis of Usp and its associated Imu1-3 proteins could also be tightly regulated, limiting their production to avoid overt degradation and masking of the producers' genome. Indeed high expression levels of imu3 (IPTG induced for protein isolation) are toxic for producing cells. DNAbinding (basic) proteins usually have an overall positive charge that facilitates their binding to DNA. The Imu3 protein, has a theoretical isoelectric point of ca. 4.4, which implies that the DNA-binding region must be localised only on part of the tertiary structure of the molecule. Different online DNA-binding motif search tools were used to identify a potential Imu3 DNA binding motif [18,19]. The results imply that the DNA-binding ability of Imu3 probably originates from the helix-turn-helix motif.

Conclusions

In conclusion, our study shows that Imu3 like the colicin E7 immunity protein Cei, does not form dimers and in addition, does not form a tight complex with the Usp protein. However, in contrast to the two other small proteins of the Usp pathogenicity island, Imu1 and 2, Imu3 does bind DNA and RNA. We propose that Usp producing cells are protected from genome fragmentation by Imu3 DNA masking. Further, as Imu3 precipitates but does not damage DNA we believe that could have biotechnological potential.

Methods

Plasmid construction and protein expression

The nucleotide sequences that encode Imu3 (USP-associated immunity protein 3 from E. coli) were amplified from the genomic DNA of the uropathogenic E. coli strain TA211 using standard PCR reactions. The Imu3n-F 5′-T TTCTCGAGCTATAATTTTAAAGATGAAATAG-3′ and Imu3n.R 5′-TTTACGCGTTATTTAGAGTCTTTAAACA AG-3′ primers were used, with XhoI and MluI restriction sites, respectively (in italics). The PCR product was cloned into the blunted pJET1.2 plasmid (Fermentas), and Usp-coding sequences were subsequently excised and re-cloned into the XhoI and MluI sites of the pET8c expression plasmid, with an N-terminal His tag (Novagen). Subsequently, Imu3 was expressed in the E. coli strain BL21(DE3) pLysE as described by the manufacturer (QIAgen). Briefly, an overnight culture of E. coli BL21(DE3) pLysE was diluted in liquid Luria Bertani medium supplemented with 120 mg/L ampicillin (LBAp medium) to an OD 600 of 0.05 at 37°C, and grown to an OD 600 of 0.6. Imu3 production was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to 0.8 mM final concentration. The culture was grown for an additional 4 h, and then the biomass was collected by 10 min centrifugation at 4,000× g.

All of the isolation steps were carried out at 4°C. The collected biomass was treated with DNase, RNase and lysozyme on ice for 1 h, as described by the manufacturer (QIAgen), and complete EDTA-free protease inhibitor cocktail (Roche) was added. The cells were ruptured with 12 consecutive ultrasonication bursts (alternating 30 s pulse, 30 s pause) at the 55 setting (Sonics Vibra Cell). The cell lysates were cleared by three 20 min centrifugations at 20,000× g. All of the other protein isolation steps were carried out. When needed, Imu3 was further purified with size-exclusion FPLC chromatography (Superdex 75 HR 10/30, Amersham Biosciences) equilibrated with 50 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl. Buffer exchanges were carried out using Amicon MWCO 3 kDa microconcentrators (Millipore). The his-tag was removed with the Thrombin Cleavage Capture Kit (Novagen) as described by the manufacturer. Actual mass of Imu3 protein was determined via mass spectrometry ESI + and Q-Tof (Waters-Micromass, United Kingdom).

The degree of Usp-producing cell protection provided by each of the three individual immunity proteins (Imu1-3) was examined in E. coli BL21(DE3) pLysE cells that were transformed with the plasmid pET8c carrying the combination of Usp and either Imu1, Imu2 or Imu3. The transformants were isolated on LB Ap plates with IPTG (0.8 mM final concentration) after being grown overnight at 37°C.

Imu3 and Usp binding

Formation of a Imu3 dimer was checked using the cross-linking glutaraldehyde assay as previously described [20], native PAGE and size exclusion chromatography (HPLC).

Imu3 samples (2 mg/mL) with or without the addition of 2.7 kbp double-stranded linear DNA (pUC19/EcoRI) were initially incubated at 37°C for 30 min, to allow for potential multimerization. Samples were then subjected to either native PAGE resolution or to the glutaraldehyde cross-linking procedure and SDS-PAGE resolution, with the LexA protein as a dimerisation-positive control. Aditionally, Imu3 was checked for dimerisation with size exclusion chromatography (HPLC, Phenomenex Biosep SEC-S2000 column, flow rate: 1 mL/min, 50 mM NaH 2 PO 4 , 300 mM NaCl, pH8), self-cleaved LexA protein was used as a standard (11 kDa, 13 kDa and 26 kDa).

Formation of the Imu3-USP complex was also investigated using the glutaraldehyde assay, after Imu3 and Usp had been mixed in equimolar ratios.

DNA/RNA binding

Various concentrations of either EcoRI linearised pUC19 DNA or total RNA (isolated from E. coli) and the Imu3 protein were used to establish the nucleic-acid-binding ability of Imu3. The Imu3 was incubated with either the DNA or RNA in TE buffer (10 mM Tris, 1 mM EDTA, pH 8) at 37°C for 30 min, prior to the electromobility shift assays (EMSAs) with 0.8% agarose gels. The effects of temperature (10 min incubation at 70°C, 80°C, 90°C and 100°C), pH (6,7,8,9), NaCl (0-500 mM) and Mg 2+ ions concentrations (0-50 mM) on the DNA binding ability of Imu3 were studied.

Thermal denaturation curves of linearised pUC19 DNA and the Imu3 protein were carried out in 5 mM cacodylic buffer (pH 6.5) using a UV-vis spectrophotometer (Cary Varian Cary 100 Bio, Australia) equipped with a thermoelectrically controlled cell holder. UV absorption was measured as a function of temperature (UV melting curves) for different ratios of linear DNA and Imu3 (0, 0.3 and 1.0 μg per 100 ng DNA), at 260 nm. The UV melting temperature ranged from 25°C to 99°C, with a heating rate of 1°C•min -1 and an equilibration time of 1 min. The melting curves of buffer and of the Imu3 protein alone were subtracted from the melting curves of the DNA-Imu3 protein complex, providing curves that show only the changes in the thermal stability of the DNA.

Further, the influences of pH, temperature and ionic strength on the separation of the DNA-Imu3 complex were examined. The effects of pH, were examined in the range from pH 3 to pH 13. Buffers used for these pH values were the following: pH 3-5, citric buffer; pH 6, MES buffer; pH 7-9, TRIS buffer; pH 10-12, glycine/ NaOH buffer; pH 13, NaOH. The impacts of various ions on the separation of the DNA-Imu3 complex were studied as 0-1 M NaCl, 350 mM KCl, 350 mM NaSCN, 70 mM MgCl 2 , 0.7% SDS, 1-3 M (NH 4 ) 2 SO 4 and 2.3 M guanidinium thiocyanate. The effects of temperature were studied 80°C and 95°C, with a 10 min incubation of the complex, and at 100°C, with a 5 min incubation.

To examine whether Imu3 binding to DNA triggers any DNA damage, religation experiments were performed. Initially, the linear plasmid DNA (pUC19) was incubated with the Imu3 protein at 37°C for 30 min, to allow for the DNA-Imu3 complex to form. The samples were subsequently purified using the QIAprep Spin Miniprep kits (QIAgen). To check DNA integrity, the linearised DNA was used for a (self ) ligation reaction (Fermentas); half of the ligation mixture was transformed into E. coli DH5α, while the other half was subjected to a second restriction (EcoRI).

The structural integrity of the Imu3 precipitated plasmid DNA was also investigated on the basis of detection of potential mutations within a non-selected marker, the ampicillin resistance gene. For this purpose, plasmid pBR322 carrying both tetracycline and ampicillin resistance genes was employed. Plasmid DNA was digested with PstI, with a single restriction site within the ampicillin resistance gene to yield one linear DNA fragment. Following gel electrophoresis the linear plasmid DNA was precipitated with Imu3 and centrifuged for 10 minutes at 4°C, followed by washing with 0.5 ml of TE buffer. The pellet was subsequently treated with the PCR Cleaning Kit (Thermo Scientific) and several μl of the isolate were employed for re-ligation. In control experiments, ligase was omitted. Transformants were isolated upon selection for tetracycline resistance. Five hundred transformants were further screened and all found to express ampicillin resistance.

Minimum DNA length for Imu3 binding

The minimum length of single-stranded DNA required for Imu3 binding was studied by initially saturating Imu3 with short DNA fragments of known lengths. The reaction mixtures of Imu3 (10 μg) and an at least 5-fold excess of oligonucleotides, were incubated for 30 min at 37°C (allowing Imu3 to bind to oligonucleotides of sufficient length) prior to the addition of the indicator DNA Table 2 Oligonucleotide sequences used to determine the minimal length of single-stranded and double-stranded DNA for DNA-Imu3 complex formation The lengths of the oligonucleotides spanned from 6 to 32 nucleotides.

(100 ng pUC19/EcoRI). Oligonucleotides too short to form a complex with Imu3, bind to the indicator DNA provoking an electro-mobility shift. Samples were incubated for another 30 min at 37°C, to allow potential binding of Imu3 to the indicator DNA. The samples were later resolved on 0.8% agarose Tris-borate gels. The short DNA fragments were all synthesised as singlestranded oligonucleotides, the sequences of which are given in Table 2.

Figure 1
1Purified Imu3 protein. (A) SDS PAGE gel of Imu3 isolated using Ni-NTA agarose affinity chromatography, M: PageRuler Prestained Protein Ladder (Fermentas). (B) Superimposed chromatograms of Imu3 protein monomers (darker line) (HPLC, size-exlusion) with absorption values at 280 nm normalised. LexA protein self-cleavage products were used as standards (lighter line).

Figure 2
2Imu3 protection against colicin E7 DNase activity. Lane 1 shows linear dsDNA, lane 2 colicin E7 with linear dsDNA, and lane 3 linear DNA with colicin E7 activity and Imu3.

Figure 3
3Protection of E. coli Usp producing cells by Imu proteins. Colonies encoding: A) usp imu1, imu2 and imu3, B) only usp C) usp imu1, D) usp imu2, and E) usp imu3 gene. The concentrations of the plated transformation mixtures were adjusted to obtain a comparable number of transformants for each strain.

Figure 4
4Representative electromobility shift assays on 0.8% agarose gels. Effects of Imu3 binding to DNA ("a" and "c" with pUC19/EcoRI, "b" with pUC19 and "d" RNA).(A) DNA: effect of NaCl (0 to 500 mM), Imu3 concentrations 0.3, 0.6 and 1.2 μg. (B) DNA: effect of temperature (10-min incubation), Imu3 additions 0.0625 to 1.0 μg (two fold increase/step). (C) DNA: effect of Mg 2+ ions, Imu3 concentrations 0.3, 0.6 and 1.2 μg. (D) RNA: Imu3 additions 0.312 to 10 μg (two fold concentration increase/step). (A, B, C, D) M: λ/PstI DNA marker; C: control (pUC19/EcoRI alone).

Figure 5
5Thermal denaturation curves of 100 ng pUC19/EcoRI DNA. DNA alone (solid line); DNA with Imu3 at 0.3 μg (dashed line) and 1.0 μg (dotted line). Signal of Imu3 alone was subtracted where necessary, and all curves were normalised. The arrows indicate the Tm values.

Figure 6
6Electromobility shift assay with short DNA fragments on 0.8% agarose gel. pUC19, plasmid alone; pUC19 + I, plasmid with Imu3 protein. Lane numbers correspond to number of bases in single-stranded DNA oligonucleotides used (i.e.6,7,8,9,10,11,12,13,14 and 15 bases long). Lanes 6-15, after incubation of Imu3 and corresponding oligonucleotide, 100 ng linear pUC19/EcoRI DNA (target) was added. M: λ/PstI marker.

Figure 7
7Representative electromobility shift assays of religated DNA previously complexed with Imu3 (0.8% agarose gels).Lane 1, 100 ng pUC19/EcoRI DNA; lane 2: 100 ng pUC19/EcoRI DNA purified with the QIAprep kit; lane 3: 100 ng pUC19/EcoRI DNA-Imu3 complex purified with the QIAprep kit; lane 4: ligation reaction of purified DNA; lane 5: ligation reaction of purified DNA-Imu3 complex; lane 6: restriction (EcoRI) of ligation reaction of purified DNA (from lane 4); lane 7: restriction (EcoRI) of ligation reaction of purified DNA-Imu3 complex (from lane 5). M: λ/PstI marker.

Table 1
1Protection of Usp producing E. coli by the individual Imu proteinsRelative numbers of transformants obtained with plasmids carrying the usp gene without and with the individual imu genes.Strain
% of transformants relative
to control (usp + imu1-3)

usp +
1.7 ± 1.2

usp + imu1
2.4 ± 1.2

usp + imu2
4.1 ± 2.0

usp + imu3
10.6 ± 4.0

http://www.biomedcentral.com/1471-2180/14/16
AcknowledgementsThis work was financed by the Slovene Research Agency (ARRS). We would like to thank Dušan Žigon for help with mass spectroscopy, Nataša Poklar Ulrih with DNA melting experiments and Luka Ausec for assistance with bioinformatics issues.Additional filesAdditional file 1:Figure S1. Effect of His-tag presence on Imu3 DNA-binding ability. M: PageRuler Prestained Protein Ladder (Fermentas); 1. Imu3 with His-tag (SDS-PAGE); 2. Imu3 with His-tag removed (SDS-PAGE); 3. 100 ng of pUC19/EcoRI (agarose electrophoresis, AE); 4. 100 ng of pUC19/ EcoRI complexed with Imu3 with His-tag removed (AE).Additional file 2:Figure S2. Dimerisation of Imu3 and USP proteins, 10% SDS PAGE gel. M: PageRuler Prestained Protein Ladder (Fermentas); 1. Imu3 protein (11.5 kDa); 2. USP protein (67 kDa). Samples following cross-linking with glutaraldehyde (3-7); 3. Imu3 protein, 4. USP protein, 5. Imu3 and USP, 6. Imu3 and USP with addition of DNA, 7. LexA protein mono/dimer (24/48 kDa).Additional file 3:Figure S3. Representative electromobility shift assays on 0.8% agarose gels. Effects of pH, NaCl and temperature on DNA-Imu3 complex relaxation. Lane 1: pUC19/EcoRI DNA (100 ng); lane 2: Imu3-pUC19/ EcoRI untreated complex; lanes 3-7: Imu3-pUC19/EcoRI complex treated with pH values 10, 11, 12, 12.5, 13; lanes 8-11: Imu3-pUC19/EcoRI complex incubated 5 min at 100°C in the presence 0, 0.2, 0.5 and 1 M NaCl; lanes 12-14: complex treated with 0, 0.5 and 1 M NaCl at pH 12.Additional file 4:Figure S4. DNA precipitation from diluted systems. On agarose gel: 1. pUC19/EcoRI (100 ng); 2. pUC19/EcoRI purified with GeneJet PCR purification Kit (Fermentas); 3. 100 ng of pUC19/EcoRI diluted 1.5 × 10 -4 in 15 mL buffer and salvaged with Imu3 precipitation and subsequent GeneJet PCR purification Kit Imu3 removal.Competing interestsThe authors declare that they have no competing interests.Authors' contributions
Pathogenicity island markers, virulence determinants malx and usp, and the capacity of Escherichia coli to persist in infants' commensal microbiotas. A Ostblom, I Adlerberth, A E Wold, F L Nowrouzian, Appl Environ Microbiol. 777Ostblom A, Adlerberth I, Wold AE, Nowrouzian FL: Pathogenicity island markers, virulence determinants malx and usp, and the capacity of Escherichia coli to persist in infants' commensal microbiotas. Appl Environ Microbiol 2011, 77(7):2303-2308.

Molecular epidemiology of 3 putative virulence genes for Escherichia coli urinary tract infection -usp, iha, and iroN(E-coli). R J Bauer, L X Zhang, B Foxman, A Siitonen, M E Jantunen, H Saxen, C F Marrs, J Infect Dis. 18510Bauer RJ, Zhang LX, Foxman B, Siitonen A, Jantunen ME, Saxen H, Marrs CF: Molecular epidemiology of 3 putative virulence genes for Escherichia coli urinary tract infection -usp, iha, and iroN(E-coli). J Infect Dis 2002, 185(10):1521-1524.

Distribution and genetic association of putative uropathogenic virulence factors iroN, iha, kpsMT, ompT and usp in Escherichia coli isolated from urinary tract infections in Japan. S Kanamaru, H Kurazono, S Ishitoya, A Terai, T Habuchi, M Nakano, O Ogawa, S Yamamoto, J Urol. 1706Kanamaru S, Kurazono H, Ishitoya S, Terai A, Habuchi T, Nakano M, Ogawa O, Yamamoto S: Distribution and genetic association of putative uropathogenic virulence factors iroN, iha, kpsMT, ompT and usp in Escherichia coli isolated from urinary tract infections in Japan. J Urol 2003, 170(6):2490-2493.

Characterization of a putative virulence island in the chromosome of uropathogenic Escherichia coli possessing a gene encoding a uropathogenic-specific protein. H Kurazono, S Yamamoto, M Nakano, G B Nair, A Terai, W Chaicumpa, H Hayashi, Microb Pathog. 283Kurazono H, Yamamoto S, Nakano M, Nair GB, Terai A, Chaicumpa W, Hayashi H: Characterization of a putative virulence island in the chromosome of uropathogenic Escherichia coli possessing a gene encoding a uropathogenic-specific protein. Microb Pathog 2000, 28(3):183-189.

Escherichia coli's uropathogenic-specific protein: a bacteriocin promoting infectivity? Microbiol-Sgm. Aha Parret, De Mot, R , 148Parret AHA, De Mot R: Escherichia coli's uropathogenic-specific protein: a bacteriocin promoting infectivity? Microbiol-Sgm 2002, 148:1604-1606.

Structural and sequence diversity of the pathogenicity island of uropathogenic Escherichia coli which encodes the USP protein. M Nakano, S Yamamoto, A Terai, O Ogawa, S Makino, H Hayashi, G B Nair, H Kurazono, FEMS Microbiol Lett. 2051Nakano M, Yamamoto S, Terai A, Ogawa O, Makino S, Hayashi H, Nair GB, Kurazono H: Structural and sequence diversity of the pathogenicity island of uropathogenic Escherichia coli which encodes the USP protein. FEMS Microbiol Lett 2001, 205(1):71-76.

Nuclease colicins and their immunity proteins. G Papadakos, J A Wojdyla, C Kleanthous, Q Rev Biophys. 20121Papadakos G, Wojdyla JA, Kleanthous C: Nuclease colicins and their immunity proteins. Q Rev Biophys 2012, 45(1):57-103.

The Escherichia coli uropathogenic specific protein Usp, is a bacteriocin-like genotoxin. D Nipič, Z Podlesek, M Črnigoj, M Budič, D Žgur-Bertok, J Infect Dis. In pressNipič D, Podlesek Z, Črnigoj M, BudiČ M, Žgur-Bertok D: The Escherichia coli uropathogenic specific protein Usp, is a bacteriocin-like genotoxin. J Infect Dis 2013. In press.

. E Cascales, S K Buchanan, D Duche, C Kleanthous, R Lloubes, K Postle, M Riley, S Slatin, D Cavard, Colicin biology. Microbiol Mol Biol Rev. 711Cascales E, Buchanan SK, Duche D, Kleanthous C, Lloubes R, Postle K, Riley M, Slatin S, Cavard D: Colicin biology. Microbiol Mol Biol Rev 2007, 71(1):158-229.

Protein-protein interactions in colicin E9 DNase-immunity protein complexes .2. Cognate and noncognate interactions that span the millimolar to femtomolar affinity range. R Wallis, K Y Leung, A J Pommer, H Videler, G R Moore, R James, C Kleanthous, Biochemistry. 3442Wallis R, Leung KY, Pommer AJ, Videler H, Moore GR, James R, Kleanthous C: Protein-protein interactions in colicin E9 DNase-immunity protein complexes .2. Cognate and noncognate interactions that span the millimolar to femtomolar affinity range. Biochemistry 1995, 34(42):13751-13759.

The crystal structure of the DNase domain of colicin E7 in complex with its inhibitor Im7 protein. T P Ko, C C Liao, W Y Ku, K F Chak, H S Yuan, Struct Fold Des. 7Ko TP, Liao CC, Ku WY, Chak KF, Yuan HS: The crystal structure of the DNase domain of colicin E7 in complex with its inhibitor Im7 protein. Struct Fold Des 1999, 7(1):91-102.

A structural comparison of the colicin immunity proteins Im7 and Im9 gives new insights into the molecular determinants of immunity-protein specificity. C A Dennis, H Videler, R A Pauptit, R Wallis, R James, G R Moore, C Kleanthous, Biochem J. 333Dennis CA, Videler H, Pauptit RA, Wallis R, James R, Moore GR, Kleanthous C: A structural comparison of the colicin immunity proteins Im7 and Im9 gives new insights into the molecular determinants of immunity-protein specificity. Biochem J 1998, 333:183-191.

Spiral structure of Escherichia coli HU alpha beta provides foundation for DNA supercoiling. F S Guo, S Adhya, Proc Natl Acad Sci U S A. 10411Guo FS, Adhya S: Spiral structure of Escherichia coli HU alpha beta provides foundation for DNA supercoiling. Proc Natl Acad Sci U S A 2007, 104(11):4309-4314.

The effect of Superhelicity on the interaction of Histone f1 with closed circular duplex DNA. T Vogel, M F Singer, J Biol Chem. 8Vogel T, Singer MF: The effect of Superhelicity on the interaction of Histone f1 with closed circular duplex DNA. J Biol Chem 1976, 251(8):2334-2338.

Codon-usage based regulation of colicin K synthesis by the stress alarmone ppGpp. I Kuhar, Jpm Van Putten, D Zgur-Bertok, W Gaastra, B Jordi, Mol Microbiol. 411Kuhar I, van Putten JPM, Zgur-Bertok D, Gaastra W, Jordi B: Codon-usage based regulation of colicin K synthesis by the stress alarmone ppGpp. Mol Microbiol 2001, 41(1):207-216.

Zgur-Bertok D: A cka-gfp transcriptional fusion reveals that the colicin K activity gene is induced in only 3 percent of the population. J Mulec, Z Podlesek, P Mrak, A Kopitar, A Ihan, J Bacteriol. 1852Mulec J, Podlesek Z, Mrak P, Kopitar A, Ihan A, Zgur-Bertok D: A cka-gfp transcriptional fusion reveals that the colicin K activity gene is induced in only 3 percent of the population. J Bacteriol 2003, 185(2):654-659.

Double locking of an Escherichia coli promoter by two repressors prevents premature colicin expression and cell lysis. M Butala, S Sonjak, S Kamensek, M Hodoscek, D F Browning, D Zgur-Bertok, Sjw Busby, Mol Microbiol. 861Butala M, Sonjak S, Kamensek S, Hodoscek M, Browning DF, Zgur-Bertok D, Busby SJW: Double locking of an Escherichia coli promoter by two repressors prevents premature colicin expression and cell lysis. Mol Microbiol 2012, 86(1):129-139.

NPS@: network protein sequence analysis. C Combet, C Blanchet, C Geourjon, G Deleage, Trends Biochem Sci. 253Combet C, Blanchet C, Geourjon C, Deleage G: NPS@: network protein sequence analysis. Trends Biochem Sci 2000, 25(3):147-150.

BindN: a web-based tool for efficient prediction of DNA and RNA binding sites in amino acid sequences. L J Wang, S J Brown, Nucleic Acids Res. 34Wang LJ, Brown SJ: BindN: a web-based tool for efficient prediction of DNA and RNA binding sites in amino acid sequences. Nucleic Acids Res 2006, 34:W243-W248.

Determination of quaternary structure of an active enzyme using chemical cross-linking with glutaraldehyde. W S Craig, 10.1186/1471-2180-14-16Methods Enzymol. 156Craig WS: Determination of quaternary structure of an active enzyme using chemical cross-linking with glutaraldehyde. Methods Enzymol 1988, 156:333-345. doi:10.1186/1471-2180-14-16

The Escherichia coli uropathogenic-specificprotein-associated immunity protein 3 (Imu3) has nucleic acid -binding activity. Črnigoj, BMC Microbiology. 1416Cite this article asCite this article as: Črnigoj et al.: The Escherichia coli uropathogenic-specific- protein-associated immunity protein 3 (Imu3) has nucleic acid -binding activity. BMC Microbiology 2014 14:16.


Avelumab in patients with previously treated metastatic adrenocortical carcinoma: phase 1b results from the JAVELIN solid tumor trial

Christophe Le Tourneau
Christopher Hoimes
†
Corrine Zarwan
Deborah J Wong
Sebastian Bauer
Rainer Claus
Martin Wermke
Subramanian Hariharan
Anja Von Heydebreck
Vijay Kasturi
Vikram Chand
James L Gulley
Avelumab in patients with previously treated metastatic adrenocortical carcinoma: phase 1b results from the JAVELIN solid tumor trial
10.1186/s40425-018-0424-9R E S E A R C H A R T I C L E Open Access
Background:We assessed the efficacy and safety of avelumab, an anti-programmed death ligand 1 (PD-L1) antibody, in patients with previously treated metastatic adrenocortical carcinoma (mACC).Methods: In this phase 1b expansion cohort, patients with mACC and prior platinum-based therapy received avelumab at 10 mg/kg intravenously every 2 weeks. Continuation of mitotane was permitted; however, mitotane levels during the study were not recorded. Tumor response was assessed by Response Evaluation Criteria In Solid Tumors v1.1.Results: Fifty patients received avelumab and were followed for a median of 16.5 months. Prior treatment included ≥2 lines in 74.0%; mitotane was continued in 50.0%. The objective response rate (ORR) was 6.0% (95% CI, 1.3% to 16.5%; partial response in 3 patients). Twenty-one patients (42.0%) had stable disease as best response (disease control rate, 48.0%). Median progression-free survival was 2.6 months (95% CI, 1.4 to 4.0), median overall survival (OS) was 10.6 months (95% CI, 7.4 to 15.0), and the 1-year OS rate was 43.4% (95% CI, 27.9% to 57.9%). In evaluable patients with PD-L1+ (n = 12) or PD-L1− (n = 30) tumors (≥5% tumor cell cutoff), ORR was 16.7% vs 3.3% (P = .192). Treatment-related adverse events (TRAEs) occurred in 82.0%; the most common were nausea (20.0%), fatigue (18.0%), hypothyroidism (14.0%), and pyrexia (14.0%). Grade 3 TRAEs occurred in 16.0%; no grade 4 to 5 TRAEs occurred. Twelve patients (24.0%) had an immune-related TRAE of any grade, which were grade 3 in 2 patients (4.0%): adrenal insufficiency (n = 1), and pneumonitis (n = 1). Conclusions: Avelumab showed clinical activity and a manageable safety profile in patients with platinumtreated mACC.

Background

Adrenocortical carcinoma (ACC) is a rare endocrine tumor with an incidence of approximately 0.7 cases/1 million population per year [1]. Genomic studies indicate that mutations in defined driver genes, including zinc and ring finger 3 (ZNRF3), telomerase reverse transcriptase (TERT), protein kinase cAMP-dependent type I regulatory subunit alpha (PRKAR1A), ribosomal protein L22 (RPL22), telomeric repeat binding factor 2 (TERF2), cyclin E1 (CCNE1), and neurofibromin 1 (NF1), may have a major role in the etiology of ACC [2], and that ACC has a moderate overall mutation burden compared with other tumor types [3]. Patients with ACC have a poor prognosis, with an estimated 5-year survival rate for metastatic ACC (mACC) of < 20% [4][5][6]. Therapeutic options for mACC are limited and include surgery; platinum agents, mitotane, streptozocin, and other systemic therapies; and locoregional radiotherapy [7]. Although cytotoxic treatments are widely administered, they are associated with limited efficacy and high toxicity, and targeted agents have not shown clinically meaningful activity in this disease [8]. Thus, there is an urgent need for new treatment options.

Cancer cells can exploit the programmed death 1 (PD-1)/ programmed death ligand 1 (PD-L1) immune checkpoint pathway to promote an immunosuppressive environment and allow tumor growth. Specifically, PD-L1 expressed on tumor cells binds to PD-1 expressed on activated T cells, resulting in downregulation of antitumor immune responses [9,10]. Monoclonal antibodies targeting the PD-1/ PD-L1 axis have shown clinical activity in multiple tumor types [11]. PD-L1 is expressed in ACC tissues throughout various stages of disease, providing a rationale for assessing anti-PD-1/PD-L1 antibodies in mACC [12].

Avelumab is a human IgG1 monoclonal antibody with a wild-type Fc region that was designed to specifically bind and block PD-L1 [13]. Unlike other anti-PD-L1/PD-1 antibodies in clinical practice, avelumab is unique in that preclinical models show that it can induce antibody-dependent cellular cytotoxicity of tumor cells [14,15]. Thus, avelumab may engage both the adaptive and innate immune systems. Avelumab treatment has been associated with durable responses in several tumor types [16][17][18], and avelumab has been approved in various countries for the treatment of patients with metastatic Merkel cell carcinoma and in the United States and Canada for treatment of locally advanced or metastatic urothelial carcinoma that progressed during or after platinum-containing chemotherapy [19]. Here we report results from a phase 1b cohort of patients with previously treated mACC who received avelumab.

Methods

Study design and treatment

JAVELIN Solid Tumor (ClinicalTrials.gov ID NCT01772004) in an international, multicenter, phase 1, open-label trial designed to investigate the clinical activity, safety, and pharmacokinetics of avelumab in patients with metastatic solid tumors, with expansion in selected tumor types. Findings from the dose-escalation portion of this study (phase 1a) were reported previously [13]. In this phase 1b dose-expansion cohort, eligible patients were aged ≥18 years and had an Eastern Cooperative Oncology Group performance status (ECOG PS) of ≤1; histologically or cytologically confirmed mACC with ≥1 measurable lesion; ≥1 line of prior systemic therapy for metastatic disease (including ≥1 platinum-based therapy); and relapsed, refractory, or progressive disease following last line of treatment. Patients receiving mitotane were permitted to continue treatment during the trial; however, details relating to ongoing mitotane treatment was not systematically collected and mitotane levels during the study were not recorded. Patients were also required to have adequate hematologic, hepatic, and renal function and availability of a fresh or archival tumor specimen. Exclusion criteria included prior treatment with a T cell-targeting immune checkpoint inhibitor, other cancer diagnosis within 5 years, rapidly progressive disease, CNS metastases, known autoimmune disease, or ongoing steroid treatment (except for patients with adrenal insufficiency, who could continue treatment at a physiological replacement dose).

The trial was conducted in accordance with the ethics principles of the Declaration of Helsinki and the International Council for Harmonisation Guidelines on Good Clinical Practice. The protocol was approved by the institutional review board or independent ethics committee of each center. All patients provided written informed consent before enrollment.

Procedures

Avelumab (EMD Serono, Rockland, MA) 10 mg/kg was administered intravenously over 60 mins every 2 weeks until progression, unacceptable toxicity, or other protocol-based criteria for withdrawal occurred. Dose modifications were not permitted. Treatment was discontinued following: 1) any grade 4 adverse event (AE) except for single laboratory values out of the normal range that were unrelated to study treatment, without clinical correlate, and resolved within 7 days with medical management; 2) any grade 3 AE except for: (i) transient (≤6 h) influenza-like symptoms or pyrexia controlled with medical management; (ii) fatigue, local infusion-related reaction, headache, nausea, or emesis that resolved to grade ≤ 1 within 24 h; (iii) single laboratory values out of the normal range that were unrelated to study treatment and without clinical correlate (excluding grade ≥ 3 increase in liver enzyme concentrations) that resolved to grade ≤ 1 within 7 days; (iv) tumor flare phenomena (local pain, irritation, or localized rash at a tumor site); (v) grade ≥ 3 amylase or lipase abnormality not associated with symptoms or clinical manifestations of pancreatitis; or 3) increase in Eastern Cooperative Oncology Group performance status to ≥3 not resolved to ≤2 by Day 14 of the following cycle. Grade 2 AEs were managed by dose delays; events that did not resolve to grade ≤ 1 by the end of the next treatment cycle or recurred led to permanent discontinuation of avelumab (except for hormone insufficiencies that could be managed by replacement therapy). A premedication regimen of diphenhydramine and acetaminophen was administered 30 to 60 min before all avelumab infusions.

Safety was assessed at each biweekly trial visit and included assessment of AEs, physical examination, and clinical laboratory tests. AEs and laboratory abnormalities were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events v4.0. A serious AE (SAE) was defined as any untoward event that was life-threatening, required hospitalization, resulted in disability, was a congenital anomaly, or resulted in death. Immune-related AEs were identified using a prespecified list of Medical Dictionary for Regulatory Activities terms.

Clinical activity was assessed by investigators using Response Evaluation Criteria In Solid Tumors (RECIST) v1.1 [20] and modified immune-related response criteria [21]. Radiographic tumor assessments were completed at baseline and then every 6 weeks. For patients who had a partial response (PR) or complete response (CR), a confirmatory computed tomography or magnetic resonance imaging scan was completed ≥28 days later, preferably at the scheduled 6-week interval. PD-L1 expression was assessed using a proprietary immunohistochemistry assay (Dako PD-L1 IHC 73-10; Dako, Carpinteria, CA) [18].

Outcomes

Primary endpoints for the whole JAVELIN Solid Tumor trial are the occurrence of dose-limiting toxicities during the first 3 weeks of treatment in the phase 1a dose-escalation part (reported previously) [13] and confirmed best overall response adjudicated by an independent review committee in specified expansion cohorts. Prespecified endpoints assessed in this cohort included best overall response per investigator assessment (defined as best response per RECIST v1.1 obtained among all tumor assessments after the start of treatment with avelumab until documented disease progression), immune-related best overall response, duration of response (defined as the time from first documented PR or CR until progressive disease or death, whichever occurred first), progression-free survival (PFS), overall survival (OS), evaluation of PD-L1 expression, and safety (including incidence and severity of AEs). Change in the sum of target lesion diameters from baseline over time was evaluated in patients with baseline tumor assessments and ≥ 1 postbaseline assessment.

Statistical analysis

Enrollment of 50 patients was planned for this cohort; this sample size was selected to provide point estimates and 95% Clopper-Pearson CIs for an objective response rate (ORR; proportion of patients with a PR or CR) of 10% (95% CI, 3.3% to 21.8%) in the case of 5 responders, and 20% (95% CI, 10.0% to 33.7%) in the case of 10 responders. Safety and clinical activity were analyzed in all patients who received ≥1 dose of avelumab. Time-to-event endpoints were estimated with the Kaplan-Meier method, and CIs for the median were calculated using the Brookmeyer-Crowley method. P values for association between categorical variables were determined using the Fisher exact test.

Results

Between September 9, 2014, and the data cutoff date of December 31, 2016, 50 patients were enrolled in 6 countries. Median age was 50 years (range, 21 to 71 years) ( Table 1). Patients were heavily pretreated; the median number of prior lines of systemic therapy was 2 (range, 1 to 6), and 37 patients (74.0%) had received ≥2 prior lines. Date of disease progression before enrollment was documented in 49 patients, and median time since last disease progression was 0.92 months (range, 0.33 to 8.61 months). Twenty-five patients (50%) continued to receive concurrent mitotane. Median duration of avelumab treatment was 3.4 months (range, 0.5 to 24.8 months), and median follow-up was 16.5 months (range, 11.7 to 27.6 months). Patients received a median 7 doses of avelumab (range, 1 to 52). At data cutoff, 5 patients Of 50 patients enrolled, 3 had confirmed PRs, and the ORR was 6.0% (95% CI, 1.3% to 16.5%) ( Table 2). Of the 3 responding patients, 2 were receiving concomitant mitotane (both had prior progression while receiving mitotane), and 2 had PD-L1+ tumors (based on 1% or 5% expression cutoffs). In patients who had received 1 (n = 13), 2 (n = 18), or ≥ 3 (n = 19) prior lines of systemic therapy since diagnosis, ORR was 15.4%, 5.6%, and 0%, respectively. Response was ongoing in 1 patient, who had a duration of response of 19.4 months at data cutoff (Fig. 1); this was a man aged 63 years with a PD-L1− tumor who had received prior treatment with cisplatin, etoposide, and mitotane and had target lesions in the liver and lymph node (Additional file 1: Figure S1). The patient received avelumab with concurrent mitotane, had a PR documented at the second assessment (week 13), and remained on treatment without progression throughout 22.0 months of follow-up. Twenty-one patients had a best response of stable disease, resulting in a disease control rate of 48.0%, which was the same in patients with or without concomitant mitotane. Using immune-related response criteria, ORR was 6.0% (95% CI, 1.3% to 16.5%), and the disease control rate was 58.0% (Additional file 1: Table S1). Of 48 patients evaluable for change in tumor size, 16 (33.3%) had a reduction of any level vs baseline, including a reduction of ≥30% in 4 patients (8.0%) (Fig. 2a). One patient had an initial assessment showing progressive disease (~100% increase vs baseline and new lesion at week 7) but later assessments showed a decrease vs baseline (nadir − 74%; Fig. 2b).

Median PFS was 2.6 months (95% CI, 1.4 to 4.0), and PFS rates at 6 months and 1 year were 20.9% (95% CI, 10.6% to 33.5%) and 8.7% (95% CI, 2.6% to 19.6%), respectively (Fig. 3a). Using immune-related criteria, median PFS was 3.8 months (95% CI, 2.4 to 5.5). Median OS was 10.6 months (95% CI, 7.4 to 15.0), and the 1-year OS rate was 43.4% (95% CI, 27.9% to 57.9%) (Fig. 3b).

PD-L1 expression was evaluable in tumor samples from 42 patients (84%). In patients with PD-L1+ (n = 12) or PD-L1-(n = 30) tumors based on a 5% PD-L1 expression cutoff, ORR (95% CI) was 16 . 3c and Fig. 3d). Findings were similar based on analyses of PD-L1 expression using a 1% cutoff (Additional file 1: Table S2 and Figure S2). Analyses of higher tumor cell PD-L1 cutoffs and PD-L1 expression on tumor-infiltrating immune cells were not informative because few patients had PD-L1+ tumors (0 to 5 patients).

All 50 patients (100.0%) had an AE of any grade; 41 patients (82.0%) had a treatment-related AE (TRAE) ( Table 3). The most common TRAEs of any grade were nausea (n = 10; 20.0%), fatigue (n = 9; 18.0%), hypothyroidism (n = 7; 14.0%), and pyrexia (n = 7; 14.0%). Five patients (10%) had an infusion-related reaction; all were grade 1 or 2. Eight patients (16.0%) had a grade 3 TRAE and no patient had a grade 4 or 5 TRAE. Of patients with or without concomitant  mitotane treatment during avelumab treatment (n = 25 in each group), 6 (24.0%) vs 2 (8.0%) patients had a grade 3 TRAE, respectively. Increase in alanine aminotransferase level (n = 2) was the only grade 3 TRAE that occurred in more than 1 patient; both patients received concomitant mitotane. Treatment-related grade 3 elevations in liver enzyme levels occurred only in patients who received concomitant mitotane. Twelve patients (24.0%) had an immune-related TRAE of any grade; those occurring in ≥1 patient were hypothyroidism (n = 7), adrenal insufficiency (n = 3), and pneumonitis (n = 2). Two patients (4.0%) had a grade 3 immune-related TRAE: adrenal insufficiency (n = 1) and pneumonitis (n = 1). Thirty-two patients (64.0%) had an SAE; in 4 patients (8.0%), the SAE was treatment related: pneumonitis (n = 1), adrenal insufficiency and transaminitis (n = 1), thyroiditis (n = 1), and pyrexia (n = 1). Six patients (12.0%) had AEs that led to death, but none were treatment related: disease progression (n = 4), failure to thrive (n = 1), and respiratory failure (n = 1).

Discussion

In this single-arm phase 1b study, which to our knowledge is the largest prospective trial of a checkpoint inhibitor in mACC, avelumab showed antitumor activity with an acceptable safety profile in a platinum-treated population. Three patients (6.0%) had an objective response, including patients with PD-L1+ and PD-L1− tumors, and all of whom had received only 1 (n = 2) or 2 (n = 1) prior lines of treatment. This suggests that the activity of avelumab might be greatest in patients with limited pretreatment, although the small patient numbers in this study prevent any definitive conclusion. Reasons for improved response in patients with less pretreatment may include a smaller tumor burden, decreased proportion of treatment-resistant cells within the tumor, and reduced immunosuppression associated with multiple prior lines of chemotherapy. Although the ORR and median PFS were modest in this heavily pretreated population, the disease control rate was 48.0%, median OS was 10.6 months, and the 1-year OS rate was 43.0%. No association was seen between concomitant mitotane treatment and clinical activity of avelumab, although the absence of detailed patient data relating to ongoing mitotane treatment, including drug levels, is a limitation of the study. Apart from the current study, other data reported in ACC with anti-PD-L1/PD-1 agents are preliminary findings from phase 2 studies of nivolumab and pembrolizumab in patients with previously treated advanced ACC. Of 7 patients who received nivolumab, 5 had a best response of disease progression and 2 were awaiting evaluation [22]. Of 11 patients who received pembrolizumab, 2 had a PR, 1 achieved stable disease, and the 6-month PFS rate was 27% [23]. In addition, in the phase 1a study of avelumab in patients with various advanced malignancies, a PR occurred in a patient with ACC [13]. The manageable safety profile of avelumab seen in patients with mACC was consistent with experience in other tumor types [16][17][18]. Patients receiving concomitant mitotane had a higher rate of grade 3 TRAEs than those not receiving mitotane (24.0% vs 8.0%), particularly liver enzyme elevations. This reflects the known toxicity profile of mitotane, which includes hepatic, gastrointestinal, neurological, and hematologic AEs [24]. However, our study showed that the tolerability of avelumab and mitotane in combination is acceptable.

Current treatment options for patients with mACC are highly limited. In the first-line setting, response rates with mitotane monotherapy are estimated to be approximately 10% to 30%, although data from prospective trials are lacking [7]. In a randomized phase 3 study of mitotane combined with either etoposide, doxorubicin, and cisplatin or streptozocin in patients with unresectable ACC without prior treatment (except mitotane), the ORR was 23.2% vs 9.2% (P < .001), the disease-control rate was 58.3% vs 31.4% (P < .001), median PFS was 5.0 vs 2.1 months (P < .001), median OS was 14.8 vs 12.0 months (P = .07), and serious AEs occurred in 58.1% vs 41.6% of patients [25]. In a phase 2 trial of gemcitabine plus metronomic fluoropyrimidine as second-/third-line treatment in patients with advanced ACC who were receiving ongoing mitotane treatment (n = 28), the ORR was 7.1%, disease control rate was 46.4%, median time to progression was 5.3 months, and median OS was 9.8 months; grade 3/4 AEs were leukopenia (21.4%), thrombocytopenia (3.5%), and mucositis (3.5%) [26]. Thus, the results from our study indicate that avelumab has comparable clinical activity and may be better tolerated than existing treatment options for this hard-to-treat cancer. A randomized phase 2 study in non-small-cell lung cancer has shown that combining an anti-PD-1 antibody with platinum-based chemotherapy increased the ORR and prolonged PFS vs chemotherapy alone [27]. This suggests that studies in ACC of avelumab in combination with chemotherapy or as maintenance therapy after first-line induction chemotherapy are warranted.

Targeted molecular therapies have been explored in patients with previously treated advanced ACC, but efficacy has been poor or nonexistent. In a phase 3 trial of linsitinib (insulin-like growth factor receptor 1 and insulin receptor inhibitor) vs placebo in 139 patients with previously treated ACC, of whom 55% had received prior cisplatin, ORR was 3.3% vs 0%, disease control rate was 32.2% vs 34.7%, median PFS was 44 vs 46 days (P = .30), and median OS was 323 vs 356 days (P = .77) [28].  Treatment with erlotinib (tyrosine kinase inhibitor [TKI] of epidermal growth factor receptor) plus gemcitabine in 10 patients resulted in tumor-size reduction in 1 patient, whereas 8 patients had progressive disease at first evaluation [29]. In other studies, no patient treated with combinations of bevacizumab (anti-VEGF antibody) plus capecitabine [30] or sorafenib (multi-TKI) plus paclitaxel [31] had an objective response or stable disease. In a study of axitinib (VEGF receptor inhibitor), none of 13 patients had an objective response, 4 patients showed decreased tumor growth, and median PFS was 5.5 months [32]. Among 35 evaluable patients who received sunitinib (multi-TKI), 14.2% had stable disease as best response and median PFS was 2.8 months [33]. In a study assessing cixutumumab (anti-insulin growth factor 1 receptor antibody) plus temsirolimus (mTOR inhibitor), 11 of 26 patients (42.3%) had a best response of stable disease [34]. Thus, the activity of immunotherapy may be more promising than seen to date in studies of targeted therapies.

Conclusions

In patients with mACC previously treated with chemotherapy, avelumab had moderate clinical activity either as monotherapy or with concurrent mitotane (50% of patients), particularly in those with limited pretreatment or PD-L1+ tumors, and with an acceptable safety profile. These findings provide a rationale for studies of avelumab-based combination treatment in patients with advanced ACC.

Additional file

Additional file 1: Table S1. Confirmed best objective response based on modified immune-related response criteria. Table S2. Antitumor activity based on PD-L1 expression on tumor cells (1% and 5% cutoffs) in evaluable patients (n = 42). Figure S1. Computed tomography scans of a patient with adrenocortical carcinoma who experienced a long-term tumor response with avelumab treatment. The patient had a partial response documented at the second assessment (week 13) and remained on treatment without progression until last follow-up. Figure S2.

(10.0%) remained on treatment. Reasons for treatment discontinuation were disease progression (n = 32 [64.0%]), AE (n = 5 [10.0%]), death (n = 3 [6.0%]), withdrawal of consent (n = 2 [4.0%]), protocol noncompliance (n = 1 [2.0%]), and other (n = 2 [4.0%]).

Fig. 1 Fig. 2 a
12Time to and duration of response or stable disease (n = 24). Bars represent individual patients with partial response or stable disease. At time of data cutoff, partial response was ongoing in 1 patient Best change in target lesions from baseline (%) Best change and b change over time from baseline in sum of target lesion diameters in evaluable patients (n = 48). In a bars for individual patients are color-coded according to tumor PD-L1 status based on a 5% PD-L1 expression cutoff in tumor cells. In b lines for individual patients are color-coded based on confirmed best overall response

Fig. 3 a
3PFS and b OS in the overall population (N = 50), and c PFS and d OS based on PD-L1 expression on tumor cells (5% cutoff) in evaluable patients (n = 42). NE,

3
3TRAEs by Concomitant Mitotane Treatment (any grade in ≥10% of patients in either group or grade 3 Abbreviations: ALT Alanine aminotransferase, AST Aspartate aminotransferase, GGT γ-glutamyltransferase

(A) Progression-free survival (PFS) and (B) overall survival (OS) based on programmed death ligand 1 (PD-L1) expression on tumor cells (1% cutoff) in evaluable patients (n = 42). ACC, adrenocortical carcinoma; NE, nonevaluable. (DOCX 498 kb)

.7% (2.1% to 48.4%) vs 3.3% (0.1% to 17.2%) (P = .192), median PFS was 5.5 (1.3 to 8.2) vs 1.7 (1.4 to 4.0) months (HR, 0.66 [95% CI, 0.32 to 1.39]), and median OS was 14.4 (7.4 to 14.4) vs 10.6
(7.3 to not evaluable) months (HR, 0.91 [95% CI, 0.33 to
2.49]) (Fig

Table

Table

Abbreviations ACC: Adrenocortical carcinoma; AE: Adverse event; ALT: Alanine aminotransferase; AST: Asparate aminotransferase; CCNE1: Cyclin E1; CI: Confidence interval; CNS: Central nervous system; CR: Complete response; ECOG PS: Eastern Cooperative Oncology Group performance status; GGT: γ-glutamyltransferase; HR: Hazard ratios; mACC: Metastatic adrenocortical carcinoma; mTOR: Mammalian Target of Rapamycin; NE: Not evaluable; NF1: Neurofibromin 1; ORR: Objective response rate; OS: Overall survival; PD-1: Programmed death 1; PD-L1: Programmed death ligand 1; PFS: Progression-free survival; PRKAR1A: Protein kinase cAMP-dependent type 1 regulatory subunit alpha; RECIST: Response Evaluation Criteria In Solid Tumors; RPL22: Ribosomal protein L22; SAE: Serious adverse event; TERF2: Telomeric repeat binding factor 2; TERT: Telomerase reverse transcriptase; TKI: Tyrosine-kinase inhibitor; TRAE: Treatment-related adverse event; VEGF: Vascular endothelial growth factor; ZNRF3: Zinc and ring finger 3
Le Tourneau et al. Journal for ImmunoTherapy of Cancer (2018) 6:111
AcknowledgmentsWe thank the patients and their families, the investigators, co-investigators, and study teams at each of the participating centers and at Merck KGaA, Darmstadt, Germany, EMD Serono Research & Development Institute (Billerica, MA; a business of Merck KGaA, Darmstadt, Germany), and Quintiles (Durham, NC).FundingThis trial was sponsored by Merck KGaA, Darmstadt, Germany, and is part of an alliance between Merck KGaA, Darmstadt, Germany, and Pfizer, Inc., New York, NY, USA. Medical writing support was provided by ClinicalThinking, and funded by Merck KGaA and Pfizer.Availability of data and materialsThe datasets used and/or analyzed during this study are available from the corresponding author on reasonable request. More information can be found at https://www.merckgroup.com/en/research/our-approach-toresearch-and-development/healthcare/clinical-trials/commitment-responsibledata-sharing.html.Authors' contributionsConception and design: CLT, AvH, VC, JLG. Provision of study materials or patients: CLT, CH, CZ, DJW, SB, RC, MW, JLG. Collection and assembly of data: All authors. Data analysis and interpretation: All authors. Manuscript writing: All authors. Final approval of manuscript: All authors.Ethics approval and consent to participateThe trial was conducted in accordance with the ethics principles of the Declaration of Helsinki and the International Council for Harmonisation Guidelines on Good Clinical Practice. The protocol was approved by the institutional review board or independent ethics committee of each center. All patients provided written informed consent before enrollment.Consent for publication Not applicable.Competing interests C. Le Tourneau has served in consulting or advisory roles for Merck & Co., Bristol-Myers Squibb, Novartis, and AstraZeneca and has received honoraria from EMD Serono and AstraZeneca. C. Hoimes has served in consulting or advisory roles for Eisai and Seattle Genetics and has served on speakers bureaus for Bristol-Myers Squibb and Roche-Genentech. C. Zarwan has served in Author details
Bauer has served in consulting or advisory roles for Bayer, Blueprint Medicines, Deciphera, and Eli Lilly; has received honoraria from Bayer, Novartis, GSK, Pfizer, and PharmaMar; has received research funding from Blueprint Medicines, Incyte, and Novartis; and has received reimbursement for travel and accommodation expenses from PharmaMar. R. Claus has served in consulting or advisory roles for AbbVie, Gilead, Janssen Oncology, and Roche-Genentech and has received honoraria from Janssen Oncology and Roche. M. Wermke has served in consulting or advisory roles for AstraZeneca, Bristol-Myers Squibb, Novartis, and Roche-Genentech and has received research funding from Boehringer, Celgene, Novartis, and Pfizer. S. Hariharan reports employment with and stock ownership in Pfizer. A. von Heydebreck reports employment with and stock ownership in Merck KGaA. Bristol-Myers Squibb, Serono, Kura Oncology, & Merck, Co, S Roche-Genentech, or advisory roles for Perceptive Informatics and Revere Pharmaceutics. D.J. Wong has served in consulting or advisory roles for and has received honoraria from Bristol-Myers Squibb and has received research funding from Armo Biosciences, AstraZeneca/MedImmune, BioMed Valley Discoveries. Bristol-Myers Squibb. J.L. GulleyBavarian NordicCelgene, EMD Serono, and Pfizeror advisory roles for Perceptive Informatics and Revere Pharmaceutics. D.J. Wong has served in consulting or advisory roles for and has received honoraria from Bristol-Myers Squibb and has received research funding from Armo Biosciences, AstraZeneca/MedImmune, BioMed Valley Discoveries, Bristol-Myers Squibb, EMD Serono, KURA Oncology, Merck & Co., and Roche-Genentech. S. Bauer has served in consulting or advisory roles for Bayer, Blueprint Medicines, Deciphera, and Eli Lilly; has received honoraria from Bayer, Novartis, GSK, Pfizer, and PharmaMar; has received research funding from Blueprint Medicines, Incyte, and Novartis; and has received reimbursement for travel and accommodation expenses from PharmaMar. R. Claus has served in consulting or advisory roles for AbbVie, Gilead, Janssen Oncology, and Roche-Genentech and has received honoraria from Janssen Oncology and Roche. M. Wermke has served in consulting or advisory roles for AstraZeneca, Bristol-Myers Squibb, Novartis, and Roche-Genentech and has received research funding from Boehringer, Celgene, Novartis, and Pfizer. S. Hariharan reports employment with and stock ownership in Pfizer. A. von Heydebreck reports employment with and stock ownership in Merck KGaA. V. Kasturi reports employment with EMD Serono. V. Chand reports employment with EMD Serono and stock ownership in Bristol-Myers Squibb. J.L. Gulley has received research funding from Astellas Medivation, Bavarian Nordic, Celgene, EMD Serono, and Pfizer.

Extent of disease at presentation and outcome for adrenocortical carcinoma: have we made progress?. E Kebebew, E Reiff, Q Y Duh, O H Clark, A Mcmillan, World J Surg. 30Kebebew E, Reiff E, Duh QY, Clark OH, McMillan A. Extent of disease at presentation and outcome for adrenocortical carcinoma: have we made progress? World J Surg. 2006;30:872-8.

Comprehensive pan-genomic characterization of adrenocortical carcinoma. S Zheng, A D Cherniack, N Dewal, R A Moffitt, L Danilova, B A Murray, Cancer Cell. 29Zheng S, Cherniack AD, Dewal N, Moffitt RA, Danilova L, Murray BA, et al. Comprehensive pan-genomic characterization of adrenocortical carcinoma. Cancer Cell. 2016;29:723-36.

Burden of nonsynonymous mutations among TCGA cancers and candidate immune checkpoint inhibitor responses. L M Colli, M J Machiela, T A Myers, L Jessop, K Yu, S J Chanock, Cancer Res. 76Colli LM, Machiela MJ, Myers TA, Jessop L, Yu K, Chanock SJ. Burden of nonsynonymous mutations among TCGA cancers and candidate immune checkpoint inhibitor responses. Cancer Res. 2016;76:3767-72.

Prognostic parameters of metastatic adrenocortical carcinoma. G Assié, Antoni G Tissier, F Caillou, B Abiven, G Gicquel, C , J Clin Endocrinol Metab. 92Assié G, Antoni G, Tissier F, Caillou B, Abiven G, Gicquel C, et al. Prognostic parameters of metastatic adrenocortical carcinoma. J Clin Endocrinol Metab. 2007;92:148-54.

Limited prognostic value of the 2004 International Union Against Cancer staging classification for adrenocortical carcinoma: proposal for a revised TNM classification. M Fassnacht, S Johanssen, M Quinkler, P Bucsky, H S Willenberg, F Beuschlein, Cancer. 115Fassnacht M, Johanssen S, Quinkler M, Bucsky P, Willenberg HS, Beuschlein F, et al. Limited prognostic value of the 2004 International Union Against Cancer staging classification for adrenocortical carcinoma: proposal for a revised TNM classification. Cancer. 2009;115:243-50.

The European Network for the Study of Adrenal Tumors staging system is prognostically superior to the International Union Against Cancer-staging system: a North American validation. G Lughezzani, M Sun, P Perrotte, C Jeldres, A Alasker, H Isbarn, Eur J Cancer. 46Lughezzani G, Sun M, Perrotte P, Jeldres C, Alasker A, Isbarn H, et al. The European Network for the Study of Adrenal Tumors staging system is prognostically superior to the International Union Against Cancer-staging system: a North American validation. Eur J Cancer. 2010;46:713-9.

NCCN Clinical Practice Guidelines in Oncology. Neuroendocrine Tumors. V3. 2017. NCCN Clinical Practice Guidelines in Oncology. Neuroendocrine Tumors. V3. 2017. https://www.nccn.org/professionals/physician_gls/pdf/ neuroendocrine.pdf. Accessed 15 Mar 2018.

Adrenocortical carcinoma (ACC): diagnosis, prognosis, and treatment. R Libé, Front Cell Dev Biol. 345Libé R. Adrenocortical carcinoma (ACC): diagnosis, prognosis, and treatment. Front Cell Dev Biol. 2015;3:45.

Tumorassociated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. H Dong, S E Strome, D R Salomao, H Tamura, F Hirano, D B Flies, Nat Med. 8Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, et al. Tumor- associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med. 2002;8:793-800.

Targeting the PD-1/B7-H1(PD-L1) pathway to activate anti-tumor immunity. S L Topalian, C G Drake, D M Pardoll, Curr Opin Immunol. 24Topalian SL, Drake CG, Pardoll DM. Targeting the PD-1/B7-H1(PD-L1) pathway to activate anti-tumor immunity. Curr Opin Immunol. 2012;24:207-12.

PD-1/PD-L1 inhibitors. J Sunshine, J M Taube, Curr Opin Pharmacol. 23Sunshine J, Taube JM. PD-1/PD-L1 inhibitors. Curr Opin Pharmacol. 2015;23: 32-8.

Programmed death ligand-1 expression in adrenocortical carcinoma: an exploratory biomarker study. A P Fay, S Signoretti, M Callea, G H Telό, R R Mckay, J Song, J Immunother Cancer. 33Fay AP, Signoretti S, Callea M, Telό GH, McKay RR, Song J, et al. Programmed death ligand-1 expression in adrenocortical carcinoma: an exploratory biomarker study. J Immunother Cancer. 2015;3:3.

Avelumab for metastatic or locally advanced previously treated solid tumours (JAVELIN Solid Tumor): a phase 1a, multicohort, dose-escalation trial. C R Heery, G O'sullivan-Coyne, R A Madan, L Cordes, A Rajan, M Rauckhorst, Lancet Oncol. 18Heery CR, O'Sullivan-Coyne G, Madan RA, Cordes L, Rajan A, Rauckhorst M, et al. Avelumab for metastatic or locally advanced previously treated solid tumours (JAVELIN Solid Tumor): a phase 1a, multicohort, dose-escalation trial. Lancet Oncol. 2017;18:587-98.

Antibody-dependent cellular cytotoxicity activity of a novel anti-PD-L1 antibody avelumab (MSB0010718C) on human tumor cells. B Boyerinas, C Jochems, M Fantini, C R Heery, J L Gulley, K Y Tsang, Cancer Immunol Res. 3Boyerinas B, Jochems C, Fantini M, Heery CR, Gulley JL, Tsang KY, et al. Antibody-dependent cellular cytotoxicity activity of a novel anti-PD-L1 antibody avelumab (MSB0010718C) on human tumor cells. Cancer Immunol Res. 2015;3:1148-57.

Malignant mesothelioma effusions are infiltrated by CD3 + T cells highly expressing PD-L1 and the PD-L1 + tumor cells within these effusions are susceptible to ADCC by the anti-PD-L1 antibody avelumab. S Khanna, A Thomas, D Abate-Daga, J Zhang, B Morrow, S M Steinberg, J Thorac Oncol. 11Khanna S, Thomas A, Abate-Daga D, Zhang J, Morrow B, Steinberg SM, et al. Malignant mesothelioma effusions are infiltrated by CD3 + T cells highly expressing PD-L1 and the PD-L1 + tumor cells within these effusions are susceptible to ADCC by the anti-PD-L1 antibody avelumab. J Thorac Oncol. 2016;11:1993-2005.

Avelumab in patients with chemotherapy-refractory metastatic Merkel cell carcinoma: a multicentre, single-group, open-label, phase 2 trial. H L Kaufman, J Russell, O Hamid, S Bhatia, P Terheyden, D' Angelo, S P , Lancet Oncol. 17Kaufman HL, Russell J, Hamid O, Bhatia S, Terheyden P, D'Angelo SP, et al. Avelumab in patients with chemotherapy-refractory metastatic Merkel cell carcinoma: a multicentre, single-group, open-label, phase 2 trial. Lancet Oncol. 2016;17:1374-85.

Avelumab for patients with previously treated metastatic or recurrent nonsmall-cell lung cancer (JAVELIN Solid Tumor): dose-expansion cohort of a multicentre, open-label, phase 1b trial. J L Gulley, A Rajan, D R Spigel, N Iannotti, J Chandler, Djl Wong, Lancet Oncol. 18Gulley JL, Rajan A, Spigel DR, Iannotti N, Chandler J, Wong DJL, et al. Avelumab for patients with previously treated metastatic or recurrent non- small-cell lung cancer (JAVELIN Solid Tumor): dose-expansion cohort of a multicentre, open-label, phase 1b trial. Lancet Oncol. 2017;18:599-610.

Avelumab, an anti-programmed death-ligand 1 antibody, in patients with refractory metastatic urothelial carcinoma: results from a multicenter, phase Ib study. A B Apolo, J R Infante, A Balmanoukian, M R Patel, D Wang, K Kelly, J Clin Oncol. 35Apolo AB, Infante JR, Balmanoukian A, Patel MR, Wang D, Kelly K, et al. Avelumab, an anti-programmed death-ligand 1 antibody, in patients with refractory metastatic urothelial carcinoma: results from a multicenter, phase Ib study. J Clin Oncol. 2017;35:2117-24.

Bavencio (avelumab). package insertBavencio (avelumab) [package insert].

. M D Rockland, EMD Serono, IncRockland, MD: EMD Serono, Inc; 2017.

New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). E A Eisenhauer, P Therasse, J Bogaerts, L H Schwartz, D Sargent, R Ford, Eur J Cancer. 45Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer. 2009;45:228-47.

Guidelines for the evaluation of immune therapy activity in solid tumors: immunerelated response criteria. J D Wolchok, A Hoos, S O'day, J S Weber, O Hamid, C Lebbé, Clin Cancer Res. 15Wolchok JD, Hoos A, O'Day S, Weber JS, Hamid O, Lebbé C, et al. Guidelines for the evaluation of immune therapy activity in solid tumors: immune- related response criteria. Clin Cancer Res. 2009;15:7412-20.

Preliminary results from a phase II study of nivolumab for patients with metastatic adrenocortical carcinoma (ACC). L Cavalcante, B A Carneiro, Rlb Costa, Y K Chae, A Rademaker, F J Giles, J Clin Oncol. 3596Suppl 7S:abstractCavalcante L, Carneiro BA, Costa RLB, Chae YK, Rademaker A, Giles FJ. Preliminary results from a phase II study of nivolumab for patients with metastatic adrenocortical carcinoma (ACC). J Clin Oncol. 2017;35(Suppl 7S:abstract):96.

Efficacy of pembrolizumab (MK-3475) in patients with adrenocortical carcinoma. M A Habra, M Campbell, C Jimenez, D Karp, D Hong, V Subbiah, J Immunother Cancer. 5424Suppl 2:abstractHabra MA, Campbell M, Jimenez C, Karp D, Hong D, Subbiah V, et al. Efficacy of pembrolizumab (MK-3475) in patients with adrenocortical carcinoma. J Immunother Cancer. 2017;5(Suppl 2:abstract):P424.

Current and emerging therapies for advanced adrenocortical carcinoma. L J Tacon, R S Prichard, P S Soon, B G Robinson, R J Clifton-Bligh, S B Sidhu, Oncologist. 16Tacon LJ, Prichard RS, Soon PS, Robinson BG, Clifton-Bligh RJ, Sidhu SB. Current and emerging therapies for advanced adrenocortical carcinoma. Oncologist. 2011;16:36-48.

Combination chemotherapy in advanced adrenocortical carcinoma. M Fassnacht, M Terzolo, B Allolio, E Baudin, H Haak, A Berruti, N Engl J Med. 366Fassnacht M, Terzolo M, Allolio B, Baudin E, Haak H, Berruti A, et al. Combination chemotherapy in advanced adrenocortical carcinoma. N Engl J Med. 2012;366:2189-97.

Gemcitabine plus metronomic 5-fluorouracil or capecitabine as a second −/third-line chemotherapy in advanced adrenocortical carcinoma: a multicenter phase II study. P Sperone, A Ferrero, F Daffara, A Priola, B Zaggia, M Volante, Endocr Relat Cancer. 17Sperone P, Ferrero A, Daffara F, Priola A, Zaggia B, Volante M, et al. Gemcitabine plus metronomic 5-fluorouracil or capecitabine as a second −/third-line chemotherapy in advanced adrenocortical carcinoma: a multicenter phase II study. Endocr Relat Cancer. 2010;17:445-53.

KEYNOTE-021 investigators. Carboplatin and pemetrexed with or without pembrolizumab for advanced, non-squamous non-smallcell lung cancer: a randomised, phase 2 cohort of the open-label KEYNOTE-021 study. C J Langer, S M Gadgeel, H Borghaei, V A Papadimitrakopoulou, A Patnaik, S F Powell, Lancet Oncol. 17Langer CJ, Gadgeel SM, Borghaei H, Papadimitrakopoulou VA, Patnaik A, Powell SF, et al. KEYNOTE-021 investigators. Carboplatin and pemetrexed with or without pembrolizumab for advanced, non-squamous non-small- cell lung cancer: a randomised, phase 2 cohort of the open-label KEYNOTE- 021 study. Lancet Oncol. 2016;17:1497-508.

OSI-906) versus placebo for patients with locally advanced or metastatic adrenocortical carcinoma: a double-blind, randomised, phase 3 study. M Fassnacht, A Berruti, E Baudin, M J Demeure, J Gilbert, H Haak, Lancet Oncol. 16Fassnacht M, Berruti A, Baudin E, Demeure MJ, Gilbert J, Haak H, et al. Linsitinib (OSI-906) versus placebo for patients with locally advanced or metastatic adrenocortical carcinoma: a double-blind, randomised, phase 3 study. Lancet Oncol. 2015;16:426-35.

Treatment of advanced adrenocortical carcinoma with erlotinib plus gemcitabine. M Quinkler, S Hahner, S Wortmann, S Johanssen, P Adam, C Ritter, J Clin Endocrinol Metab. 93Quinkler M, Hahner S, Wortmann S, Johanssen S, Adam P, Ritter C, et al. Treatment of advanced adrenocortical carcinoma with erlotinib plus gemcitabine. J Clin Endocrinol Metab. 2008;93:2057-62.

Bevacizumab plus capecitabine as a salvage therapy in advanced adrenocortical carcinoma. S Wortmann, M Quinkler, C Ritter, M Kroiss, S Johanssen, S Hahner, Eur J Endocrinol. 162Wortmann S, Quinkler M, Ritter C, Kroiss M, Johanssen S, Hahner S, et al. Bevacizumab plus capecitabine as a salvage therapy in advanced adrenocortical carcinoma. Eur J Endocrinol. 2010;162:349-56.

Phase II study of weekly paclitaxel and sorafenib as second/third-line therapy in patients with adrenocortical carcinoma. A Berruti, P Sperone, A Ferrero, A Germano, A Ardito, A M Priola, Eur J Endocrinol. 166Berruti A, Sperone P, Ferrero A, Germano A, Ardito A, Priola AM, et al. Phase II study of weekly paclitaxel and sorafenib as second/third-line therapy in patients with adrenocortical carcinoma. Eur J Endocrinol. 2012;166:451-8.

The VEGF inhibitor axitinib has limited effectiveness as a therapy for adrenocortical cancer. C O'sullivan, M Edgerly, M Velarde, J Wilkerson, A M Venkatesan, S Pittaluga, J Clin Endocrinol Metab. 99O'Sullivan C, Edgerly M, Velarde M, Wilkerson J, Venkatesan AM, Pittaluga S, et al. The VEGF inhibitor axitinib has limited effectiveness as a therapy for adrenocortical cancer. J Clin Endocrinol Metab. 2014;99:1291-7.

Sunitinib in refractory adrenocortical carcinoma: a phase II, single-arm, open-label trial. M Kroiss, M Quinkler, S Johanssen, N P Van Erp, N Lankheet, A Pöllinger, J Clin Endocrinol Metab. 97Kroiss M, Quinkler M, Johanssen S, van Erp NP, Lankheet N, Pöllinger A, et al. Sunitinib in refractory adrenocortical carcinoma: a phase II, single-arm, open-label trial. J Clin Endocrinol Metab. 2012;97:3495-503.

Insulin growth factor receptor (IGF-1R) antibody cixutumumab combined with the mTOR inhibitor temsirolimus in patients with metastatic adrenocortical carcinoma. A Naing, P Lorusso, S Fu, D Hong, H X Chen, L A Doyle, Br J Cancer. 108Naing A, Lorusso P, Fu S, Hong D, Chen HX, Doyle LA, et al. Insulin growth factor receptor (IGF-1R) antibody cixutumumab combined with the mTOR inhibitor temsirolimus in patients with metastatic adrenocortical carcinoma. Br J Cancer. 2013;108:826-30.