Helicobacter Genotyping and Detection in Peroperative Lavage Fluid in Patients with Perforated Peptic Ulcer

Introduction and Objectives Certain Helicobacter pylori genotypes are associated with peptic ulcer disease; however, little is known about associations between the H. pylori genotype and perforated peptic ulcer (PPU). The primary aim of this study was to evaluate which genotypes are present in patients with PPU and which genotype is dominant in this population. The secondary aim was to study the possibility of determining the H. pylori status in a way other than by biopsy.
Introduction
Over the past decades the incidence of perforated peptic ulcer (PPU) has declined in the western world. However, with an incidence varying between two and 10 per 100,000, it still is a problem in modern society.1 Moreover, mortality rates caused by gastric and duodenal ulcer perforation vary between 10 and 40% and zero and 10% respectively, and is higher among elderly patients.2,3 Several risk factors for PPU have been described such as smoking, alcohol abuse, and history of peptic ulcer disease (PUD).2 However, the main pathogenic factors are considered to be the use of non-steroidal anti-inflammatory drugs (NSAID) and the presence of H. pylori.2
Helicobacter pylori are widespread bacteria, with a prevalence ranging from 25% in the industrialized world to more than 70% in developing countries.4,5 Most infected people remain asymptomatic; however, a small group of carriers will develop PUD.
Of patients who have developed PPU, 70% will test positive for H. pylori,2 suggesting the pathogenesis of perforation is associated with the presence of H. pylori. In addition, it is shown that different genotypes of H. pylori are associated with different clinical manifestations like PUD and gastric cancer.6,7 Two well-known H. pylori genes that have been associated with PUD are the cytotoxin-associated gene (cagA) and the vacuolating cytotoxin gene (vacA).6,8–10
VacA is present in all H. pylori strains and is associated with gastritis, PUD, and gastric carcinoma.10–12 It encodes for a vacuolating cytotoxin that causes epithelial cell injury and interferes with the immune system.13,14VacA contains at least two variable regions, the signal peptide (s)-region and the middle (m)-region. The s-region contains two allelic types, s1 and s2. The s1 strain has several subtypes, being s1a, s1b, and s1c.15 Two allelic types exist for the m-region, m1 and m2. The latter has two subtypes, m2a and m2b.16
CagA is considered a marker for a genomic pathogenicity (cag) island that is associated with enhanced virulence.17
If PPU is associated with a specific H. pylori genotype it may be feasible to limit the patients undergoing antibiotic therapy to those who have this genotype. When this specific type is not present, another cause of PPU should be looked for and antibiotic therapy should not be started. This would mean cost reduction and, probably, a reduction in the speed of the development of antibiotic resistance.
Currently, gastric biopsy during endoscopy is a generally accepted method to diagnose H. pylori infection. However, patients with PPU will not undergo endoscopy but will generally be operated upon immediately. Taking a biopsy intraoperatively implicates a higher risk of bleeding and more difficult closure of the defect. Therefore, surgeons are reluctant to take a biopsy.
The primary aim of this study was to evaluate which genotypes are present in patients with PPU and if a genotype is dominant in this population. The secondary aim was to study the possibility of determining the H. pylori status in a manner other than by gastric tissue biopsy.
Methods
From 30 consecutive patients operated on for PPU serum samples, gastric tissue biopsies, lavage fluid, and fluid from the nasogastric tube were collected. These patients were treated in five different medical centers throughout the Netherlands. In each of these centers approval of the medical ethical committee was obtained. Immediately after collection, the materials were frozen at −20°C. One researcher performed the analysis and genotyping. For H. pylori genotyping, the presence of cytotoxin-associated gene (cagA) and the s- and m-region genotypes of the vacuolating cytotoxin gene (vacA) were determined.
DNA was isolated according to Boom’s method as described previously.18 A guanidine thiocyanate (GuSCN) solution was added to the collected material to induce lysis of the bacteria, releasing their DNA. After addition of the silica particles (Celite) the suspension was centrifuged. The silica particles, with the attached DNA, were washed with subsequently GuSCN-containing washing buffer, ethanol 70% and acetone. After drying, the DNA was eluted in an aqueous low salt buffer. The isolated DNA was amplified by means of polymerase chain reaction (PCR) and subsequently the presence of cagA and different types of vacA were analyzed by means of reverse hybridization on a strip (32). This assay consists of a nitrocellulose strip that contains dT-tailed oligonucleotide probes immobilized as parallel lines. For each strain, 10 μl of each PCR product (containing biotin at the 5′ end of each primer) was denatured by the addition of an equal amount of 400 mM NaOH and 10 mM EDTA in a plastic trough. After 5 min, 1 ml of prewarmed hybridization solution (2× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 50 mM Tris–HCl [pH 7.5], 0.1% SDS) was added, and a strip was submerged and incubated in a shaking water bath at 50°C for 1 h. The strips were washed with 2 ml of 2× SSC-0.1% SDS for 30 min at 50°C. Subsequently, the strips were rinsed three times in phosphate buffer, and conjugate (streptavidin–alkaline phosphatase) was added. After incubation at room temperature for 30 min, the strips were rinsed again and 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate substrate was added. Hybrids are visible as purple probe lines. Interpretation of the hybridization patterns was performed visually. As a control, a β-globin PCR was performed. Patient related factors were obtained prospectively. Statistical analysis was performed with SPSS for Windows, version 11.0.
Results
A total of 30 patients were included of whom nine were women. The average age was 65 years, varying between 40 and 87. Ten patients (33.3%) were operated laparoscopically. The perforation was found prepyloric in 11 patients, at the site of the pylorus in eight patients and postpyloric in 11 patients.
A total of five (16.7%) patients had a history of PUD. Ten patients (33.3%) used NSAID’s, two patients (6.7%) used steroids, three patients (10.0%) used acid reducers, and one patient (3.3%) used a proton pump inhibitor (PPI) before admission to the hospital. The average hospital stay was 11.9 days, varying between 3 and 37 days.
Fluid from the nasogastric tube was obtained from 25 patients, lavage fluid from 26 patients, serum samples from 20 patients, and ulcer biopsies from 18 patients. The results of the genotyping are depicted in Table 1.
Table 1Helicobacter pylori Status and GenotypeThe colors represent the β-globin and H. pylori status of the patient.
The β-globin determination was performed as a control. In nine samples of nasogastric tube fluid and in two samples of lavage fluid it was negative, rendering these results as unreliable. Therefore, these results were excluded from further analysis.
Table 2 represents the frequency of the individual genes and the allelic types found in the different samples by means of PCR and LiPA.
Table 2Frequencies of Individual Genes and Allelic TypesGenotypeFluid from Naso-Gastric TubeLavage FluidGastric Tissue BiopsyControl Non-UlcerNo.%No.%No.%%VacA s11090.91477.8777.846.9VacA s20015.5111.138.4VacA multiple19.1316.7111.114.7Total11100181009100100VacA m1654.5950.0555.629.4VacA m2436.4844.4333.355.9VacA incomplete genotype19.115.6111.10 (14.7 % incomplete)Total11100181009100100CagA positive981.81477.8555.647.1Total11100181009100100“VacA multiple” means that more than one allelic type or subtype has been found in one sample.In each different type of sample one incomplete genotype occurred, which is indicated as “vacA incomplete”. The “Control non ulcer” column represents the frequencies, found by van Doorn et al., in a population without PUD and is added to allow easy comparison.
These tables show that for vacA the allelic type s1 is predominantly present in all three types of samples. In the s1 positive strains, subtype s1a is predominant as depicted in Table 3.
Table 3Distribution of the vacA s1 SubtypesVacA SubtypeFluid from Naso-Gastric TubeLavage FluidGastric Tissue BiopsyControl Non-UlcerNo.%No.%No.%%S1a880.01392.9571.481.3S1b220.017.1228.618.7S1c0000000Total10100141007100100The s1a subtype is predominant in all sample types.The “Control non ulcer” column represents the frequencies, found by van Doorn et al., in a population without PUD and is added to allow easy comparison.
With regard to the middle region of vacA the incidence of m1 allelic type is slightly higher; however, the difference is less outspoken compared to s1. The m2a was the only subtype that was found in the samples. In three samples, the genotyping was incomplete (Tables 1 and 2), meaning that determination of the middle region was not possible. This was most likely caused by the small number of bacteria present in those samples.
With regard to the secondary aim of this study, analyzing possibilities to diagnose H. pylori presence in another fashion than through biopsy, the H. pylori status found in each type of sample was compared. A correlation was found between the H. pylori presence in biopsy and its presence in lavage fluid (Fisher’s exact test, p = 0.015), indicating lavage fluid is a valid alternative for determination of H. pylori infection.
The sensitivity and specificity of the lavage fluid analysis was calculated, considering biopsy as a golden standard. Fourteen patients, of which the lavage fluid as well as the biopsy was analyzed, were included into this calculation (patients 2, 4, 5, 6, 8, 10, 15, 23–25, 27–30, Table 1), which is shown in Table 4. Of the remaining patients, either the biopsy or the lavage fluid was missing; therefore, these data cannot be used in the sensitivity/specificity calculation.
Table 4Calculation of Sensitivity and Specificity of Lavage Fluid Analysis BiopsyLavage fluid+–Total+8210–044Total8614Sens 8/8 = 1Spec 4/6 = 0.67
The sensitivity was 100%, which means that in case of the presence of H. pylori in the biopsy specimen, the lavage fluid analysis detected it in 100% of cases. The specificity of lavage fluid analysis was 66.7%, which means the chance for false-positives is over 30%. With regard to gender, age, BMI, history of PUD, location of perforation, complications after procedure, and use of steroids, PPI, or antihistaminic medication, no statistically significant correlation was found.
Discussion
Concerning the role of H. pylori in the pathogenesis of PPU, some studies have been reported comparing the prevalence of H. pylori infection in patients with PPU to the prevalence in controls. They appear to be similar, suggesting that other factors like NSAID use play a role.19–21 However, the substantial genetic heterogeneity of H. pylori that has been revealed over the years leads to the hypothesis of a specific genotype causing PPU.5 Controls might test positive for H. pylori, but not develop PPU because it would not be this specific genotype that is isolated. This study of a selected population of patients, all with PPU, shows a limited diversity of H. pylori genotypes as represented by Table 1.
VacA s1 strains are predominantly present in the three sample types of which s1a is the predominant subtype. Concerning the vacA m-region, the m1 strains are found in a majority of cases; however, the difference is less convincing than for vacA s1. Except for the biopsy samples, the cagA positive strains were predominantly present is this population. In the biopsy samples, the frequency of cagA-positive strains seemed to be low; however, this number is distorted because in two of nine positive biopsies, a decent comparison with the other samples was not possible. In patient 5, genotyping of the lavage fluid and nasogastric tube fluid was incomplete, and for patient 15, the opposite was the case. This means that the actual incidence should be 71.4 % (5/7).
Summarising, these results shows that the vacA s1, cagA-positive strains were predominant in this population of patients with PPU. This finding is in accordance with literature reporting correlations between the presence of vacA s1, cagA-positive strains and PUD.6,10 Therefore, detection of the genotype vacA s1 does not specifically predict PPU; nevertheless, clinicians should be aware of this association.
In Tables 2 and 3 the genetic distribution in a Dutch population without PUD, as found by van Doorn et al., are added for comparison. The frequencies found in this study for vacA s1, m1, and cagA-positive strains are clearly higher than in the non-PUD group, confirming the aforementioned hypothesis. However, with regard to the subtypes, Table 3 shows an almost similar distribution of frequencies, suggesting that determination of the allelic subtype is of less importance.
In only 60% of patients biopsies could be analyzed. The reason for missing 40% is the restraint of the surgeon to take a biopsy when risk of bleeding and more difficult closure of the defect was estimated to be too high, which emphasizes the importance of finding an alternative. To do so, the H. pylori status of the patient as determined by biopsy was compared to the status as determined by analysis of nasogastric tube fluid, lavage fluid, and serum. A statistically significant correlation was found between the H. pylori status in biopsy and its status in lavage fluid (Fisher’s exact test, p = 0.015). This finding suggests that determination of the H. pylori status can be done with lavage fluid as well, obviously without any risk of bleeding and closure related difficulties. The sensitivity is 100%, but the specificity is 66.7%. This could mean the chance for false-positives is over 30%, which is not optimal and could lead to therapy overshoot. However, considering the fact that with the lavage a larger area is sampled, rendering the chance of positive test results higher than in biopsy, it is more likely to find false negative biopsy results. This could lead to a therapy undershoot, which obviates the importance having an alternative for a biopsy.
In only two samples, both nasogastric tube fluids, a H. pylori genotype was isolated, while β-globin tested negative. In nine samples (seven nasogastric tube fluid, two lavage fluid) both β-globin and H. pylori tested negative. This means that either no humane cells were present in the samples, which is unlikely, or that an error in the PCR procedure had occurred. Because this was unclear these results were considered unreliable. Therefore, it still could be possible that nasogastric tube fluid is a good alternative for determining the H. pylori status as well.
Overall, these results are positive, however they should be confirmed in a larger population.
Conclusion
This study shows that in a population of 30 patients with PPU, vacA s1, cagA positive strains are predominant. This finding is in accordance with literature reporting correlations between the presence of vacA s1, cagA-positive strains, and PUD. Therefore, detection of this genotype does not specifically predict PPU. Nevertheless, clinicians should be aware of this association.
This study shows as well that it is feasible to use intraoperative lavage fluid to determine the H. pylori status of the patient, implicating that biopsies, with a risk of bleeding and more difficult closure of the defect, are not necessary anymore. In addition, considering the fact that a larger area is sampled with lavaging, biopsies may result in more false negative results leading to insufficient therapy.

genotype
peroperative lavage fluid
perforated peptic ulcer
peptic ulcer disease
h. pylori


Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference

The use of RNA interference (RNAi) to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick, Boophilus microplus, has not been reported. We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens: Bm86, Bm91 and subolesin. Gene-specific double-stranded (dsRNA) was injected into two tick stages, freshly molted unfed and engorged females, and specific gene silencing was confirmed by real time PCR. Gene silencing occurred in injected unfed females after they were allowed to feed. Injection of dsRNA into engorged females caused gene silencing in the subsequently oviposited eggs and larvae that hatched from these eggs, but not in adults that developed from these larvae. dsRNA injected into engorged females could be detected by quantitative real-time RT-PCR in eggs 14 days from the beginning of oviposition, demonstrating that unprocessed dsRNA was incorporated in the eggs. Eggs produced by engorged females injected with subolesin dsRNA were abnormal, suggesting that subolesin may play a role in embryonic development. The injection of dsRNA into engorged females to obtain gene-specific silencing in eggs and larvae is a novel method which can be used to study gene function in tick embryogenesis.
1
Introduction
The cattle tick Boophilus microplus is an important pest of cattle in subtropical and tropical regions of the world (Estrada-Pena et al., 2006). Although all Boophilus species including B. microplus have been reclassified to the genus Rhipicephalus (Murrell and Barker, 2003), we maintain use of the previous genus assignment for the purpose of biological clarity. Besides causing direct production losses and leather damage, B. microplus transmits several cattle pathogens, including Babesia bovis, Babesia bigemina and Anaplasma marginale. Control of B. microplus depends primarily on the use of acaricides or genetically resistant animals. Both approaches have limitations, including development of acaricide resistance, environmental contamination, pesticide residues in food products, the expense of developing new pesticides and the difficulty of producing tick-resistant cattle while maintaining desirable production characteristics (Willadsen, 2004). Other tick control approaches which show promise are the use of biological control agents (reviewed by Samish et al., 2004) and anti-tick vaccines (reviewed by de la Fuente and Kocan, 2003; Willadsen, 2004).
Two commercial vaccines have been developed for control of tick infestations on cattle, TickGARD Plus® in Australia and Gavac® in Cuba. Both are based on the same recombinant antigen named Bm86, a glycoprotein of unknown function which is located predominantly on the surface of midgut digest cells (Gough and Kemp, 1993). This ‘concealed’ antigen is not naturally exposed to the host’s immune system. Lysis of midgut digest cells occurs in ticks that feed on vaccinated cattle, resulting in leakage of blood meal into the tick hemocoel. The overall effect of the vaccine is on engorging female ticks and includes a decrease in the number and weight of replete ticks and oviposition. While Bm86-based vaccines were effective against several other tick species, including Boophilus annulatus (Fragoso et al., 1998; Pipano et al., 2003), Boophilus decoloratus, Hyalomma anatolicum anatolicum and Hyalomma dromedarii, they were not effective against Amblyomma variegatum, Amblyomma cajennense and Rhipicephalus appendiculatus (de Vos et al., 2001; Rodriquez and Jongejan, unpublished data).
Another ‘concealed’ antigen, Bm91, was shown to increase the efficacy of the Bm86 vaccine for B. microplus when co-administered (Willadsen et al., 1996). Bm91 is a low-abundance glycoprotein located in the salivary glands and midgut of B. microplus (Riding et al., 1994). The protein, a homologue of carboxydipeptidase, shares many biochemical and enzymatic properties with mammalian angiotensin converting enzyme, but its natural substrate has not been identified (Jarmey et al., 1995).
More recently, a protein first labeled 4D8 and now called subolesin was identified through Ixodes scapularis cDNA expression library immunisation as a potential tick-protective antigen. Immunisation trials using recombinant subolesin caused reductions of larval, nymphal and adult I. scapularis infestations (Almazan et al., 2003, 2005a,b). The protein was later found to be conserved among ixodid tick species. Characterisation of its function by RNA interference (RNAi) in I. scapularis, Amblyomma americanum, Rhipicephalus sanguineus, Dermacentor variabilis and Dermacentor marginatus suggested involvement of this protein in the modulation of blood ingestion and reproduction. Therefore, the generic name “subolesin” was introduced for the 4D8 proteins and “subA” for the subolesin-encoding gene (de la Fuente et al., 2006a). Gene silencing by RNAi of subA and Rs86, the homologue of Bm86, in R. sanguineus, revealed a synergistic effect in which the expression of both genes was silenced and resulted in decreased tick attachment, feeding and oviposition (de la Fuente et al., 2006c).
RNAi is a conserved post-transcriptional gene-silencing mechanism present in ticks and a wide range of eukaryotes in which double-stranded RNA (dsRNA) triggers a sequence-specific degradation of cognate mRNAs. It has been an effective tool to study the function of tick proteins at the tick–pathogen interface in a number of three-host tick species such as in I. scapularis which transmits Anaplasma phagocytophilum and Borrelia burgdorferi (Pal et al., 2004; Ramamoorthi et al., 2005; Sukumaran et al., 2006). RNAi was used to study the function of several tick salivary gland proteins involved in feeding of A. americanum (Aljamali et al., 2003; Karim et al., 2004), Haemaphysalis longicornis (Miyoshi et al., 2004) and I. scapularis (Narasimhan et al., 2004). The inducer of RNAi, dsRNA, is injected into nymphal or adult ticks which are then allowed to feed normally. Capillary feeding of dsRNA (Soares et al., 2005) or incubation of isolated tick tissues with dsRNA (Aljamali et al., 2003; Karim et al., 2005) are other methods used successfully to silence genes in ticks. These studies suggest that RNAi is systemic and effects gene silencing throughout the tick.
In one-host Boophilus ticks, with all life stages feeding and molting on the same host, alternative strategies are required to conduct gene silencing by RNAi as compared with three-host tick species which spend their non-parasitic life stages off-host. Herein, we examined two methods of dsRNA delivery and its effect on the one-host tick B. microplus: (i) injection of dsRNA into freshly molted females and (ii) injection of dsRNA into engorged females. The latter method caused gene-specific silencing in the oviposited eggs and larvae that hatched from these eggs. We believe this is the first report of the silencing of the expression of Bm86, Bm91 and subolesin in Boophilus ticks as quantified by real-time RT-PCR using two routes of dsRNA delivery.
2
Materials and methods
2.1
Experimental animals
Three Holstein–Friesian calves, 5 months of age (#7793, #7794 and #7799), were used. All animals had no previous exposure to ticks. All tick feedings were approved by the Animal Experiments Committee (DEC) of the Faculty of Veterinary Medicine, Utrecht University (DEC No. 0111.0807).
2.2
Ticks and tick feeding
Boophilus microplus ticks originating from Mozambique were provided by ClinVet International (Pty), Bloemfontein, South Africa. The ticks were subsequently maintained on cattle at our tick rearing facility. Larvae were kept off-host at 20 °C with 95% relative humidity. Patches used for tick feeding with inner dimensions of 60 × 85 mm and sewn to an open cotton bag were glued to the shaved back of calf #7793 and #7794 using Pattex® contact glue (Henkel Nederland, Nieuwegein, The Netherlands). A batch of larvae eclosed from 1500 mg of pooled eggs oviposited by 25 females (approximately 24,000 larvae), was divided on day 0 between two patches on calf #7794. Since males appear earlier from the nymphal stage than females, approximately 500 unfed males were collected on days 13 and 14 and 600 unfed females on days 14 and 15 and incubated at 27 °C with 95% relative humidity. Freshly molted females were subjected to injection of dsRNA on day 15 as described below. For gene silencing in engorged females and their progeny, 25 engorged females with an average weight of 261 mg (248–272 mg) fed on calf #7794 were collected on day 21. Larvae which hatched from eggs laid by mock-injected, Bm86- and Bm91-dsRNA injected engorged females were fed in three patches on calf #7799.
2.3
RNA extraction and synthesis of tick cDNA for dsRNA preparations
The viscera of five partially fed B. microplus females were dissected in ice-cold PBS and immediately stored in 1 ml Tri reagent (Sigma–Aldrich, Zwijndrecht, The Netherlands) at −80 °C. Total RNA was isolated and subsequently purified using the Nucleospin RNA II kit (Macherey-Nagel, Düren, Germany) in accordance with the reagent and kit manufacturer’s directions. Total RNA concentration was determined spectrophotometrically and the material was stored at −80 °C before use. Complementary DNA was made with the Revertaid first strand cDNA synthesis kit (Fermentas, St. Leon-Rot, Germany) in accordance with the manufacturer’s protocol using random hexamer primers. Control reactions were performed using the same procedures but without RT as a control for DNA contamination in the RNA preparations.
2.4
Cloning and sequencing of the B. microplus subolesin gene
Cloning and sequencing of the subA gene from the Mozambiquan B. microplus strain was performed as described elsewhere (de la Fuente et al., 2006a). The sequence has been submitted to GenBank and can be retrieved under Accession No. DQ923495.
2.5
dsRNA synthesis
Oligonucleotide primers containing T7 promotor sequences at the 5′-end for in vitro transcription and synthesis of dsRNA were used to PCR-amplify cDNA encoding B. microplus Bm86 (421 bp), Bm91 (417 bp) and subolesin (381 bp). All oligonucleotide primers used in this study were synthesised by Isogen Life Science, IJsselstein, The Netherlands and their sequences are shown in Table 1. PCR products were purified using the GfX PCR purification kit (Amersham) and used as templates to produce dsRNA using the T7 Ribomax Express RNAi system (Promega, Leiden, The Netherlands). dsRNA aliquots were stored at −80 °C until used.
2.6
Injection of ticks with dsRNA
Freshly molted females were placed on double-sided sticky tape with the ventral sides upwards and injected into the anal aperture with 0.5 μl Bm86, Bm91 or subolesin dsRNA alone or a combination of Bm86 and subolesin dsRNA (6–9 × 1011 molecules/μl) using a 10 μl syringe with a 33 G needle (Hamilton, Bonaduz, Switzerland) mounted on a MM3301-M3 micromanipulator (World Precision Instruments (WPI), Berlin, Germany) and connected to an UMPII syringe pump (WPI). The tip of a 27 G needle was used to slightly pierce the anal aperture before the 33 G needle was inserted. The dsRNA was dissolved in injection buffer (10 mM Tris–HCl, pH 7 and 1 mM EDTA). A control group was injected with injection buffer alone. The ticks were placed in an incubator at 27 °C with 95% relative humidity for 3–10 h following injection, before they were examined for mortality and placed in five separate patches, one for each group, on calf #7793. One hundred male ticks were placed in each patch simultaneously with the injected females. The ticks were checked twice daily and collected when they dropped from the host. Ticks still attached 14 days after the dsRNA-injection (day 29 after application of the larvae) were removed manually. All ticks were weighed separately within 1 h after collection and stored individually in 1.5 ml Eppendorf tubes with pierced lids at 27 °C and 95% relative humidity for oviposition. For the second experiment, engorged B. microplus females were injected with 5 μl of Bm86, Bm91 or subolesin dsRNA (1–2 × 1012 molecules/μl) or injection buffer alone in the right spiracular plate within 6 h after dropping off the host, using the same methods as described above, or left uninjected. They were stored individually in 2 ml Eppendorf tubes with pierced lids in an incubator at 27 °C and 95% relative humidity. Eggs were removed daily and each daily egg batch was stored separately under the same conditions.
2.7
Analysis to confirm gene silencing by quantitative RT-PCR
Viscera was dissected from five females of each dsRNA-injected or mock-injected group after 6 days of feeding. Total RNA was isolated from these samples using Tri reagent and subsequently purified using the Nucleospin RNA II kit in accordance with the reagent and kit manufacturer’s directions. Total RNA was isolated from 100 mg eggs (14 days after injection), 50 mg larvae (at 6 days and 5 weeks after hatching) laid by/eclosed from the dsRNA- and mock-injected engorged females, 50 mg larvae at 10 weeks after hatching laid by the dsRNA- and mock-injected unfed females and from the dissected viscera of five females and five males which developed from 7-week-old larvae fed on animal #7799 using the same methods. cDNA from 1 μg of RNA (adults, eggs and 6-day-old larvae) and 0.3 μg of RNA (5-week-old larvae) was prepared using the Revertaid first strand cDNA synthesis kit (Fermentas) using random hexamer primers in accordance with the manufacturer’s protocol. All samples were analyzed for transcription of target genes by quantitative real-time RT-PCR using primers Bm86h-F6 and Bm86h-R4, amplifying a 117 bp section of the Bm86 gene; Bm91-F2 and Bm91-R3, amplifying a 129 bp section of the Bm91 gene and Bm-subA-F2 and Bm-subA-R2, amplifying a 166 bp section of the subolesin gene. Tick β-actin was included as a control and used for normalisation. A 126 bp fragment was amplified using primers Actin-F2 and Actin-R. All primer combinations amplified a different part of the targeted genes than the sections which were used for dsRNA synthesis, circumventing re-amplification of any unprocessed dsRNA. Twenty-five microlitres of real-time PCRs were performed using the Quantitect SYBR green PCR kit in accordance with the manufacturer’s protocol (Qiagen, Venlo, The Netherlands) on an iCycler real-time detection system (Bio-Rad Laboratories, Veenendaal, The Netherlands). Real-time PCR data were analyzed by iCycler IQ software version 1.0.
2.8
Analysis to check for the presence of dsRNA in eggs
cDNA from 1 μg of total RNA of eggs 14 days post-oviposition was screened by quantitative real-time RT-PCR for the presence of non-processed Bm86, Bm91 and subolesin dsRNA to see whether the injected dsRNA was incorporated into the eggs and could be re-amplified. Oligonucleotide primers located within the region used for dsRNA synthesis of the Bm86, Bm91 and subolesin genes were used for this purpose. The following primer combinations were used: Bm86-F7 and Bm86h-R3, amplifying a 121 bp section of the Bm86 gene, primers Bm91-F3 and Bm91-R1, amplifying a 128 bp region of the Bm91 gene, primers BmsubA-F2 and BmsubA-R1, amplifying a 121 bp section of the subolesin gene. Real-time RT-PCR conditions were identical to those used to confirm gene silencing.
2.9
Statistical analysis
Statistical analysis of data from two quantitative RT-PCR experiments, the weights of ticks after feeding and oviposited egg masses, was performed using Microsoft Excel and consisted of an unpaired t-test with unequal variances. Tick mortality was compared between the dsRNA- and mock-injected ticks by χ2-test. P values of 0.05 or less were considered statistically significant.
3
Results
3.1
Cloning and sequencing of the subolesin gene from B. microplus
The subolesin gene (subA) from the Mozambiquan B. microplus strain was cloned and sequenced. This gene was found to be 99–100% identical to subA from B. microplus strains from Mexico and Brazil (de la Fuente et al., 2006a; our unpublished data).
3.2
RNAi in freshly molted B. microplus females
Five groups consisting of 120 freshly molted B. microplus females were each injected with 0.5 μl of injection buffer in the following groups: (i) injection buffer alone, (ii) Bm86, (iii) Bm91, (iv) subolesin and (v) both Bm86 and subolesin-dsRNA. An average of 81 (76–82; 32.8% overall mortality) females were alive in each group 3–10 h following injection. These ticks were subsequently fed together on a calf with an excess of B. microplus males until the females became replete or for a maximum of 14 days. Tick weight after engorgement or manual removal, mortality rate, egg mass and hatching rate is presented in Table 2. A significant decrease in tick weight and oviposited egg mass, together with a higher mortality rate, was observed in the subolesin dsRNA injected groups compared with the control group (P < 0.01). Hatching rates were uniformly constant in the control, Bm86- and Bm91-dsRNA-injected groups (>90%), while in the ticks injected with subolesin dsRNA the hatching rate was lower (<20%). Gene silencing was confirmed by quantitative real-time RT-PCR (Fig. 1). The normalised transcript level of Bm86 was reduced with 86% in the Bm86-dsRNA-injected ticks and with 79% in the combined Bm86/subolesin-dsRNA-injected ticks, compared with the normalised transcript level of the mock-injected group (Fig. 1A). For Bm91, the normalised transcript level was reduced with 90% in the Bm91 RNAi-silenced ticks compared with the control ticks. A significant decrease in the Bm91 transcript level of 58% was observed in the subolesin dsRNA-injected ticks (P < 0.01) as well, but this reduction was not observed in the combined Bm86/subolesin-dsRNA-injected ticks (Fig. 1B). Normalised transcript levels of subolesin in the subolesin dsRNA- and combined Bm86/subolesin dsRNA-injected ticks compared with the control group were reduced with 90% and 80%, respectively (Fig. 1C). No differences were observed in the normalised Bm86 and Bm91 transcript levels of 10-week-old larvae which hatched from the Bm86- and Bm91-dsRNA-injected ticks compared with the mock-injected ticks (results not shown).
3.3
RNAi in engorged B. microplus females
Five groups of five engorged females each with an average weight of 261 mg (248–272 mg) were injected with 5 μl of Bm86 dsRNA, Bm91 dsRNA, subolesin dsRNA, injection buffer alone or left uninjected within 6 h after dropping from the host. No reflux of the injected solution or hemolymph was observed from the puncture when the needle was gently withdrawn. Oviposition began in all groups within 3 days, except for one tick from the control-injected group and another tick from the Bm91 dsRNA-injected group which did not lay any eggs. The course of oviposition was not significantly influenced by the injection of dsRNA (Table 3), and dried or shriveled eggs were not observed, indicating that all eggs were successfully coated with a secretion from Gené’s organ. Interestingly, nearly all (>99.4%) eggs oviposited by engorged females injected with subolesin dsRNA showed an aberrant phenotype compared with those from the other groups. A typical example is shown in Fig. 2. Development of embryos in these eggs was not observed while many undifferentiated cells with some yolk cells were seen in Giemsa-stained egg crush smears. Most eggs did not hatch and eventually dried up and shriveled after 6–7 weeks of incubation at 27 °C/95% relative humidity. The few eggs from this group which did develop and hatched normally (<0.6%) were all laid during the first day of oviposition.
A 64% decrease of subolesin transcript levels was found in eggs from the subolesin dsRNA-injected engorged females (Fig. 3C). Interestingly, a 30-fold increase in the Bm86 transcript level and a 56% decrease in Bm91 transcript level were also found in these aberrant eggs (Figs. 3A and B). An 84% decrease in the number of Bm86 copies was observed in eggs laid by the engorged females injected with Bm86 dsRNA (Fig. 3A) and the transcript level of Bm91 was reduced by 97% in eggs from the Bm91 dsRNA-injected engorged females compared with the mock-injected control group (Fig. 3B), confirming gene-specific silencing in the eggs of dsRNA-injected engorged females.
Injected dsRNA could be re-amplified from the eggs of dsRNA-injected engorged females when primers located within the dsRNA sections were used, instead of primers located downstream of the dsRNA region which were used to demonstrate gene silencing (Table 1). Results of quantitative real-time RT-PCR performed with these primers showed levels of Bm86, Bm91 and subolesin which were 10.5, 4.3 and 4.5 times higher, respectively, in the Bm86, Bm91 and subolesin injected groups than levels found in the mock-injected group (Fig. 4).
Gene silencing was observed in larvae 6 days after hatching; a decrease of 86% in Bm86 transcript level and of 91% in Bm91 transcript level in the Bm86- and Bm91 dsRNA-injected groups, respectively (Figs. 5A and B, grey bars). Subolesin transcript levels were not measured in the few larvae which hatched from the subolesin dsRNA-injected females due to their small number. Quantitative real-time RT-PCR analysis of RNA extracted from larvae 5 weeks after hatching (9 weeks after the initial injection of engorged females with dsRNA) revealed that genes remained silenced in both Bm86- and Bm91-silenced groups (Figs. 5A and B, black bars). This effect diminished over time, in particular for the Bm86-silenced group, in which transcript levels were now 67% lower compared with the control group. Bm91 transcript levels were still 90% lower in the Bm91-silenced group compared with the control group. When 7-week-old larvae from these three groups were fed in separate patches on a calf and total RNA from the viscera of adults which had developed from these larvae was analyzed by quantitative real-time RT-PCR, gene silencing was not observed (results not shown).
4
Discussion
The production characteristics of the subolesin-silenced female B. microplus ticks corresponded with previous results from subolesin RNAi studies in other ixodid tick species, and typically resulted in decreased tick and egg mass weights and high mortality (de la Fuente et al., 2005, 2006a,c). The synergistic effect of combined silencing of the Bm86 and subolesin gene reported previously in R. sanguineus (de la Fuente et al., 2006c) could not be confirmed for B. microplus (Table 2).
Significant changes in the production characteristics of the Bm86- and Bm91-silenced females were not observed, suggesting that the deleterious effect on ticks feeding on Bm86- or Bm91-vaccinated cattle is not caused by a loss of function of the Bm86 or Bm91 protein alone. In fact, the protective effect of Bm86-based vaccines is through gut damage mediated by anti-Bm86 antibodies during tick feeding (Willadsen et al., 1989). These results suggest that the protection mechanisms of Bm86 and subolesin-based vaccines are different and may contribute to the increased efficacy of Bm86 and subolesin combined vaccines.
Quantitative real-time RT-PCR performed on samples taken 6 days after dsRNA injection and feeding resulted in a decrease of 79–90% of the targeted gene transcript level compared with the mock-injected group. This result was comparable with previous semi-quantitative measurements of the gene silencing effect by dsRNA-injection in A. americanum. In this RNAi study, a decrease of ∼90% to 50% in cystatin transcript level was observed from 24 h to 9 days of feeding (Karim et al., 2005). In the present study, although gene silencing was specific in the Bm86 and Bm91 dsRNA-injected groups, subolesin dsRNA injection resulted in both significantly decreased subolesin as well as Bm91 expression levels. This effect was not observed in the combined Bm86/subolesin silenced group, which may be explained by a slight difference in midgut:salivary gland ratio in the dissected viscera between the groups. Since Bm91 is present in relatively high concentrations in salivary glands compared with midgut (Riding et al., 1994), a shift in the midgut:salivary gland ratio of the dissected viscera in favor of midgut tissue could result in the lower Bm91 expression levels we found in this specific sample. Alternatively, the effect of silencing subolesin expression may affect the expression of other genes. The pleiotropic effect on tick tissues in which subolesin expression has been silenced suggests that this gene may be involved in the regulation of multiple pathways in ticks (de la Fuente et al., 2006a).
Injection of pilocarpine solution into the hemocoel via the spiracular plate of engorged ticks has been described for inducement of salivation in engorged B. microplus ticks (Bechara et al., 1988). These openings of the tracheae are sclerotised structures located posterior to the fourth pair of legs. Their rigid structure allows for puncturing and injection of small quantities of fluid by a fine needle without subsequent reflux of the injected solution, hemolymph or tissue. When we injected Bm86, Bm91 or subolesin dsRNA into the hemocoel of engorged females, the course of oviposition appeared to be unaffected. Only one Bm91 dsRNA-injected female and one mock-injected female tick died before ovipositing. Interestingly, embryogenesis was undisturbed in eggs oviposited by the Bm86 dsRNA-, Bm91 dsRNA- and mock-injected engorged females, but an aberrant development was seen in the majority of egg masses oviposited by the subolesin dsRNA-injected engorged females. This egg phenotype has not been described previously and suggests that subolesin plays a role in embryonic development. When total RNA isolated from these aberrant eggs was analyzed by real-time RT-PCR, significantly higher Bm86 levels and decreased Bm91 and subolesin levels were found. Again, these results suggest that subolesin may be a regulator of transcription in ticks.
Injected dsRNA was detected in eggs from dsRNA-injected engorged females by real-time RT-PCR using primers located within the dsRNA section, indicating that unprocessed dsRNA is incorporated in the eggs. The suggested route of incorporation of exogenously produced yolk directly from the hemolymph into oocytes (Saito et al., 2005) may be followed for the incorporation of dsRNA into oocytes as well. Further experiments are needed to determine whether this dsRNA forms a reservoir of mobile silencing signals inducing gene-specific silencing in eggs, or whether small interfering RNAs are responsible for this effect. Other possible routes for the mobile silencing signal to enter the oocyte are through the pedicel in the ovary or, once the oocyte has ovulated, by contact with cells from the genital tract.
Some eggs (<0.6%) found in the batches laid during the first day of oviposition by subolesin dsRNA-injected engorged females, hatched normally. It is likely that these eggs developed to a stage which was not accessible by the dsRNA prior to injection of the dsRNA. They may have been ovulated eggs which were not in direct contact with the hemolymph or ones in which the shell was hardened during the oocyte passage through the oviduct and thus became impermeable to dsRNA during this process (Diehl et al., 1982).
After dsRNA was injected into the body cavity of B. microplus, the gene silencing effect spread throughout the organism and its progeny. This systemic RNAi has been associated with the sid-1 protein, a transmembrane protein which enables passive cellular uptake of dsRNA (Winston et al., 2002; Feinberg and Hunter, 2003). Unfortunately, our attempts to detect a B. microplus sid-1 homologue, as described previously in studies on presence of a sid-1 homologue in grasshopper species Schistocerca americana, were not successful (data not shown) (Dong and Friedrich, 2005). The only protein currently present in the B. microplus expressed sequence tag (EST) database (Guerrero et al., 2005) which is associated with the RNAi machinery is the nuclease Argonaute-2 (Ago-2), the central catalytic component of the RNA-induced silencing complex (RISC) in mammals and Drosophila (Liu et al., 2004; Miyoshi et al., 2005). Homologues of other RNAi-associated proteins such as Dicer, which is responsible for the cleavage of exogenous long dsRNA into short interfering RNA (siRNA), remain to be identified in B. microplus and other tick species as well.
RNAi described herein provides an important tool to screen for tick-protective antigens in this one-host tick species, B. microplus (de la Fuente et al., 2005) and allowed for characterisation of the effect and function of tick protective antigens, as well as the role of genes involved in tick–host–pathogen interactions and the transmission of tick-borne pathogens (de la Fuente et al., 2006b). Initiation and completion of the B. microplus genome sequencing project would greatly enhance these kinds of studies (Guerrero et al., 2006), as well as the availability of the genomes from cattle and the pathogens transmitted by B. microplus, most notably: A. marginale (Brayton et al., 2005), B. bigemina and B. bovis, which are currently being sequenced. Although sequences of the dsRNAs used in this study do not contain any significant overlap with other known B. microplus genes, the possibility of off-target gene silencing effects cannot be excluded due to the limited amount of sequence data available. Availability of the complete B. microplus genome sequence data will facilitate screening for potential off-target effects. These can subsequently be minimised by avoiding the use of dsRNAs or siRNAs containing sequences which are present in multiple genes.
The effect of silencing tick genes suggested to be involved in embryogenesis such as vitellin degrading cysteine endopeptidase (Seixas et al., 2003), in the transovarial transmission of Babesia spp. or genes associated with acaricide resistance, which is measured in larvae by the Larval Packet Test (Li et al., 2003), could be studied using our method to silence genes in B. microplus embryos and larvae by injecting engorged females with dsRNA. These experimental approaches are more efficient and less labour intensive than the three other dsRNA delivery approaches into oocytes and embryos using microinjection (Wargelius et al., 1999), transgenic RNAi (Tavernarakis et al., 2000) and electroporation (Grabarek et al., 2002).

bm86
bm91
subolesin
one-host tick
boophilus microplus
rnai


Recurrent respiratory papillomatosis: an overview of current thinking and treatment

Human papillomaviruses (HPV) infection in benign laryngeal papillomas is well established. The vast majority of recurrent respiratory papillomatosis lesions are due to HPV types 6 and 11. Human papillomaviruses are small non-enveloped viruses (>8 kb), that replicate within the nuclei of infected host cells. Infected host basal cell keratinocytes and papillomas arise from the disordered proliferation of these differentiating keratinocytes. Surgical debulking of papillomas is currently the treatment of choice; newer surgical approaches utilizing microdebriders are replacing laser ablation. Surgery aims to secure an adequate airway and improve and maintain an acceptable quality of voice. Adjuvant treatments currently used include cidofovir, indole-3-carbinol, ribavirin, mumps vaccine, and photodynamic therapy. The recent licensing of prophylactic HPV vaccines is a most interesting development. The low incidence of RRP does pose significant problems in recruitment of sufficient numbers to show statistical significance. Large multi-centre collaborative clinical trials are therefore required. Even so, sufficient clinical follow-up data would take several years.
Introduction
Sir Morrell Mackenzie (1837–1892) was the first to recognize papillomas as a lesion of the laryngo-pharyngeal system in children in the late 1800s. It is now apparent that these benign tumours may occur at other parts of the upper gastrointestinal and respiratory tracts, and in all age groups. It was not until the 1940s that Chevalier Jackson (1865–1958) first coined the term “juvenile laryngeal papillomatosis”. The prevalence of laryngeal papillomatosis has been estimated at between four to seven cases per million person-years in the Western World [4, 5, 25, 43]. Furthermore, the incidence of recurrent respiratory papillomatosis (RRP) has been estimated at about 2 per 100,000 in adults and 4 per 100,000 in children [9]. The disease can be categorized into adult onset and juvenile onset forms. Age of first presentation of disease is usually in the teens (50%) for the juvenile onset form but can be as early as the first year of life. Initial presentation in the adult form tends to peak in the third and fourth decades.
It is now well established that human papillomaviruses (HPVs) are the aetiological agent of many benign and malignant tumours arising from epidermal tissues. They are a necessary cause of the second most common female cancer worldwide, cancer of the cervix [7, 45], and strongly associated with several other ano-genital cancers such anal, penile, vulval and vaginal carcinomas [17]. Furthermore, there is mounting evidence of at least some head and neck cancers associated with HPV infection [15, 17, 23]. These malignancies are associated with ∼15 high risk (HR) types, in particular HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82. Benign tumours such as common warts, flat warts and genital warts are caused by low risk types such as HPV 1, 2, 3, 4, 6, 10, 11 and others. HPV 6 and 11 have been described as the dominant types found in RRP [10]. Despite the benign nature of these lesions, there is significant morbidity and occasional mortality due to multiple recurrences which necessitate hospital admission for surgical removal. Dissemination or extension of the growths into the lower airways indicates a poorer prognosis. The clinical behaviour is variable and lesions can regress, persist and in rare instances, progress to carcinoma if other environmental factors such as smoking or irradiation are involved.
Epidemiology
Human papillomaviruses infection in benign laryngeal papillomas is well established. One Danish study showed that 95% of solitary laryngeal papillomas were positive for HPV DNA by in situ hybridization [26]. Another study from Hong Kong found that 59% of laryngeal papillomas showed the presence of HPV 6, 11, 16, 18 subtypes, with 6 and 11 the dominant types [10]. Malignant transformation of some lesions has been described in association with HPV 11 integration into the genome and mutation of p53 [32]. Infection with HPV 11 is more likely to be associated with the development of distal airways disease than HPV 6. Clinical manifestation of disease prior to three years of age is a further risk factor associated with distal spread of disease [1, 47].
The vast majority of RRP lesions are due to HPV types 6 and 11, and the reservoir for these types is the human ano-genital tract. Ano-genital HPV is the commonest sexually transmitted viral infection and the prevalence of clinically apparent genital warts is thought to be the “tip of the iceberg” of HPV infection. Koutsky et al. estimates that ∼10–20% of the US population between 15 and 49 years of age have molecular evidence of infection and that another 60% have had prior infection [22]. In the UK, there were well over 81,000 incident genital wart infections reported from genitourinary medicine clinics in 2005 [16] and this has been increasing every year. Such high background prevalence suggests that there is a risk of mother to child transmission at the time of delivery, especially if there are noticeable genital warts. Data supporting this hypothesis showed that a history of maternal condylomata during pregnancy was associated with a 200-fold risk of RRP in the child [37]. An uncomplicated vaginal delivery in a mother with HPV infection has been estimated to carry a risk of transmission of 1:80–1:1,500 (median of 1:400) [34]. In one group of children with juvenile laryngeal papillomatosis, 54% had a maternal history of vulval condylomata at the time of delivery [13]. Another study found that in 77 mothers with condylomata at delivery, 9 children (11.6%) were later diagnosed with juvenile laryngeal papillomatosis [21].
The route of transmission is likely to be different in the juvenile onset and adult onset forms of RRP. Evidence for this has been suggested by a case control study, in which the risk factors for both forms were compared [20]. The authors found that adult onset patients were more likely to have had more sexual partners and oral sex than their controls. Patients with the juvenile form were more likely to have been born to teenage mothers and first-born children compared to their controls.
Aetiological and histopathological features
Papillomatous lesions preferentially occur anatomically at the sites of “transformation zones”, where squamous epithelia abut ciliated columnar epithelia but can infect anywhere in the respiratory tract [28]. The classical sites for recurrent disease in the upper aero-digestive tract would be the nasopharyngeal area of the soft palate, limen vestibuli, midzone of laryngeal area of the epiglottis, upper and lower margins of the ventricle, vocal fold undersurface, carina and bronchial spurs. It is interesting to note that papillomata have been observed at tracheotomy sites and tracts where the iatrogenic induction of change of epithelialization also occurs [19].
Human papillomaviruses are small nonenveloped viruses (∼8 kb), with a double stranded circular DNA genome encapsulated within an icosahedral capsid that replicate within the nuclei of infected host cells. The genome codes for 8–10 genes (median of eight). The late L1 and L2 genes code for the viral capsid proteins, the early proteins E1 and E2 are responsible for viral replication and transcription, and E4 appears to aid virus release from infected cells. The early genes E6 and E7 have transforming ability in in vitro assays for HR types but LR types have little to no ability for this feature [2, 33, 42].
Electron microscopic analysis reveals the virion to be ∼55 nm in diameter and the capsid to be comprised of 72 pentameric capsomers. The predominant protein in the capsid consists of the L1 protein, with a smaller proportion of L2 embedded deep within the protein shell. It is this L1 protein which provides the dominant antigenic epitopes recognized by neutralizing antibodies and forms the basis for the bivalent (GlaxoSmithKline) and quadrivalent (Merck, Hohenbrumn, Germany) vaccines currently available.
The virus is thought to bind to and gain entry to its host cell, the basal keratinocyte, by microtrauma or abrasions to the surface epithelium. The receptor has not been definitively identified but α6-integrin and heparin sulphate may play important roles in viral entry [11, 18, 30].
Following infection and uncoating, the virus is thought to maintain its genome as a low copy number episome in the basal cells. It has been suggested that expression of E1 and possibly E2, may be sufficient for basal maintenance of viral episomes [50]. Viral early proteins E6, E7, E1 and E2 are expressed at low level in early passage cell lines derived from naturally occurring low-grade cervical lesions and the viral genome is maintained at around 10–200 copies per cell [8, 41].
The viral genome is amplified in differentiating keratinocytes via rolling-circle amplification that will synthesize sufficient viral genome for packaging [3, 12]. This requirement for differentiating epithelial cells is a key part of the virus life cycle but the normal restraint on cell cycle progression appears to be abolished by the E6 and E7 proteins and normal terminal differentiation is retarded [36]. These E6 and E7 effects on key apoptotic proteins such as Rb and p53 have been demonstrated in HPV 16 and other high-risk subtypes in in vitro assays [24, 27, 28]. HPV 6 and 11 E6 and E7 proteins do not readily bind to or degrade the p53 or Rb proteins [14, 31, 46]. This suggests that alternative mechanisms of altered cellular growth and proliferation may exist for the low-risk subtypes of HPV. Furthermore, there are little data on HPV 6 and 11 life cycles, replication, maintenance and viral production in respiratory cells. It is not known if these are similar to or different to disease in the ano-genital region.
Treatment
Surgical
Surgical debulking is currently the treatment of choice; newer surgical approaches utilizing microdebriders are replacing laser ablation. Surgical excision aims to secure an adequate airway and improve and maintain an acceptable quality of voice [49].
HPV is present in the normal macroscopically unaffected mucosa and it is currently not possible to distinguish infected cells with a normal appearance from uninfected epithelia. Repeated recurrences are frequent, however, repeated attempts to treat the papillomas may cause serious complications [40]. Current practice in the treatment of RRP was recently evaluated by a questionnaire in the UK [44]. Various lasers such as CO2, KTP, and pulsed dye were found to be the preferred method of surgical removal of RRP in children [49]. Spontaneous ventilation (65.3%) is the preferred method of anaesthesia.
The frequent recurrence of papillomas has resulted in the use of different adjuvant treatments alongside surgical removal of macroscopically obvious in the attempt to improve outcomes. In the future, advances in the understanding of the immune response to HPV may improve our treatment modalities and prevention strategies.
Adjuvant treatment
Adjuvant treatments currently used include cidofovir, indole-3-carbinol, ribavirin, mumps vaccine, and photodynamic therapy. As with surgical management, viral persistence occurs following treatment with these adjuvant modalities. Intralesional cidofovir may help control papilloma regrowth and reduce disease severity in many children with RRP [39]. In most cases, cidofovir would appear to be less efficacious in producing disease eradication. There appears to be little evidence to support prolonged treatment regimes (i.e. more than eight treatments) [35]. Subcutaneously injected cidofovir has been tested on cartilage in a rabbit model [39]. There was a positive dose-response relationship which existed for gross skin changes; however, there was no dose-response relationship for severity of change in the epithelium. Higher doses of cidofovir than commonly are used in the treatment of RRP may be safe, although the effects of repeat application and long-term complications are not yet known. In animals, cidofovir is carcinogenic (mammary adenocarcinoma in rats), embryotoxic and teratogenic [48]. Care must be taken in humans that the possibility of pregnancy is excluded when usage is considered. In view of the severe nephrotoxicity shown when intravenous cidofovir is administered to animals and humans, caution would be advised for repeated intralesional or subcutaneous applications for RRP treatment. The less common complications of bone marrow toxicity, iritis and uveitis may also arise and vigilance is required from clinicians.
Controlled trials failed to provide sufficient evidence to draw reliable conclusions about the effectiveness of antiviral agents as adjuvant therapy in the management of RRP. Further research is required before any specific antiviral adjuvant therapy can be recommended.
Factors leading to virus activation in RRP have not been recognized, however, extra-oesophageal acid reflux disease (EERD) has been suggested as a possible factor, initially by Borkowski et al. [6], and then by a group from Harvard in 2005 [29]. There is clinical evidence suggesting a link between the presence of EERD and RRP. Inflammation induced by acid exposure may result in the expression of HPV in susceptible tissues. Therefore, treatment of EERD should be considered in all patients with difficult to control RRP with EERD.
Despite currently available surgical and adjuvant management options, tracheotomy may become necessary in selected patients with extensive disease. Decannulation should be performed as early as possible to avoid further spread of viral infection and improve the quality of life. The primary cause of papilloma extension to the lower airways appears to be iatrogenic, i.e. the tracheotomies performed in children with laryngeal papillomatosis (92.5% of cases). This was reported in a case group of 448 children with RRP treated in St. Vladimir Moscow Children’s Hospital between 1988 and 2003 [38].
The recent licensing of prophylactic HPV vaccines is a most interesting development. In particular, the quadrivalent vaccine from Merck & Sanofi-Pasteur (Gardasil®) which shows efficacy against HPV 6, 11, 16, 18 subtypes, may be anticipated to impact upon the incidence of RRP. The low incidence of RRP does pose significant problems in recruitment of sufficient numbers to show statistical significance. Large multi-centre collaborative clinical trials are therefore required. Even so, sufficient clinical follow-up data would take several years.

recurrent respiratory papillomatosis
vaccination
hpv 6 and 11
human papilloma virus


Contrast-enhanced magnetic resonance imaging of the breast: the value of pharmacokinetic parameters derived from fast dynamic imaging during initial enhancement in classifying lesions

The value of pharmacokinetic parameters derived from fast dynamic imaging during initial enhancement in characterizing breast lesions on magnetic resonance imaging (MRI) was evaluated. Sixty-eight malignant and 34 benign lesions were included. In the scanning protocol, high temporal resolution imaging was combined with high spatial resolution imaging. The high temporal resolution images were recorded every 4.1 s during initial enhancement (fast dynamic analysis). The high spatial resolution images were recorded at a temporal resolution of 86 s (slow dynamic analysis). In the fast dynamic evaluation pharmacokinetic parameters (Ktrans, Ve and kep) were evaluated. In the slow dynamic analysis, each lesion was scored according to the BI-RADS classification. Two readers evaluated all data prospectively. ROC and multivariate analysis were performed. The slow dynamic analysis resulted in an AUC of 0.85 and 0.83, respectively. The fast dynamic analysis resulted in an AUC of 0.83 in both readers. The combination of both the slow and fast dynamic analyses resulted in a significant improvement of diagnostic performance with an AUC of 0.93 and 0.90 (P = 0.02). The increased diagnostic performance found when combining both methods demonstrates the additional value of our method in further improving the diagnostic performance of breast MRI.
Introduction
Breast cancer is the most commonly diagnosed cancer in women and the most prevalent cancer worldwide [1]. In breast imaging, mammography is still the most commonly used imaging techniques both in screening for and staging of breast cancer. However, dynamic contrast-enhanced magnetic resonance imaging (MRI) is becoming an increasingly important imaging modality in the detection and staging of breast cancer. Because of its superior sensitivity for the detection of invasive breast cancer, MRI has become a very important modality in breast imaging [2–5].
However, the classification of a lesion detected on MRI as benign or malignant still remains a challenge. Reported specificities in clinical studies range between 20% and 100% [6–15]. The main characteristics used for classification of detected lesions on MRI are the lesion morphology and the enhancement dynamics [4]. Dynamic evaluation is often based on late dynamic characteristics of enhancing lesions. In this approach, the decrease of signal intensity, often referred to as a type 3 curve or washout, is highly suggestive for breast cancer with the likelihood for malignancy of 87% [12]. This dynamic evaluation makes use of high-resolution T1-weighted MRI images with a relatively low time resolution of 42 s or more [3, 12, 16–20]. The high spatial resolution of these sequences is necessary for accurate morphologic evaluation. Irregular lesion contour, inhomogeneous internal enhancement and rim enhancement have been described as features indicating a malignancy [21].
Schnall et al. [4] found focal mass margins and signal intensity to be a highly predictive imaging features.
However, the combination of both dynamic and morphological parameters resulted in the highest diagnostic accuracy in multivariate analysis.
The evaluation of early enhancement using a high temporal resolution has also been a subject of study in breast MRI. Boetes et al. [6] found in a group of 87 lesions a sensitivity of 95%, a specificity of 86% and an overall accuracy of 93% based on early enhancement characteristics. In this study, a temporal resolution of 2.3 s was achieved using a turboFLASH sequence. The value of a high temporal resolution during initial enhancement was confirmed by Sardanelli et al. [22] who used a temporal resolution of 15 s during the first minute of enhancement. The overlap between malignant and benign enhancement curves was only 9% using the fast dynamic evaluation compared with 50% using a lower temporal resolution of 1 min.
The value of first pass high temporal resolution imaging for the differentiation of benign and malignant lesions was studied in a direct comparison of steady-state dynamic MRI (30 s temporal resolution) and first-pass imaging (2 s temporal resolution) of induced mammary tumors in female rats by Helbich et al. [23]. In their study, an estimate of first-pass perfusion using T2*-weighted imaging almost reached a significant difference between benign and malignant tumors. All other methods used, including T1-weighted first-pass imaging, failed to differentiate benign from malignant tumors. Gibbs et al. [24] also used a high temporal resolution (10.5–14.5 s) in the evaluation of small breast lesions and evaluated their data using a pharmacokinetic model. The incorporation of data from pharmacokinetic modeling in the evaluation of lesions improved diagnostic accuracy in their group.
High temporal resolution sequences often cover a limited area of the breast [6, 24]. These imaging protocols are, therefore, less suitable in clinical MRI or screening. For this study, we adjusted the scanning protocol in order to obtain a high temporal resolution during initial enhancement while covering both breasts entirely. The aim of this study is to asses the value of pharmacokinetic parameters derived from fast dynamic contrast enhanced imaging during initial enhancement in differentiating between benign and malignant breast lesions on MRI.
Materials and methods
Patient selection
All lesions detected on clinically performed breast MRI examinations in the period from January 2004 until June 2005 were initially included. All detected lesions were evaluated based on the following inclusion criteria: (1) histological confirmed diagnosis or (2) follow-up based on unchanged MRI morphology and enhancement characteristics during at least 24 months indicating a benign nature of the lesion [25]. Lesions that could not be classified as benign or malignant using these criteria were excluded. The protocol was approved by the institutional review board.
Imaging protocol
All patients were examined using a 1.5-Tesla MRI scanner (Sonata or Symphony, Siemens, Erlangen, Germany) in combination with a double breast coil. In premenopausal women, the MRI examination was performed in the second week of the menstrual cycle to minimize enhancement of normal glandular tissue [26]. Prior to the MRI examination, an intravenous catheter was inserted in the left or right arm. All patients were placed in the prone position with the breasts in the double breast coil and positioned at the isocenter of the magnet. After localizer images were obtained in three directions, low spatial resolution proton-density-weighted images were acquired in the transverse plane covering both breasts completely (TE 1.56, TR 800, FA 8, FOV 320, slices 24, TA 50 s, image resolution 3.9 mm × 1.3 mm × 4.0 mm). Subsequently, a coronally orientated high-resolution three-dimensional fast low-angle shot series (FLASH 3D) was acquired (TE 4, TR 7.5, FA 8, FOV 320, slices 120, TA 86 s, image resolution 1.3 mm × 1.3 mm × 1.3 mm). Thereafter, high temporal resolution T1-weighted images (turboFLASH) were recorded 22 times with identical spatial resolution and orientation as the proton-density-weighted images (TE 1.56, TR 66, FA 20, FOV 320, slices 24, TA 22 × 4.1 s) during an intravenous bolus injection of a paramagnetic gadolinium chelate—0.2 mmol of gadoterate meglumine (Dotarem; Guerbet, The Netherlands) per kilogram of body weight—which was administered with a power injector (Spectris; Medrad, Pittsburg, USA) at 2.5 ml/s and followed by a 15-ml saline flush. Following these series, the FLASH 3D series was repeated four times. Total scan time for this protocol was 9 min 42 s, including the time needed to record localizer images.
Image evaluation
For the evaluation, the MRI data were divided into two sets of dynamic data for each patient. The first dataset contained the high spatial resolution T1-weighted images (FLASH 3D) only. These were used for the evaluation of both lesion morphology as well as signal intensity versus time curves. This method will be further referred to as the ‘slow dynamic’ analysis. The second dataset contained the proton-density-weighted images, the high temporal resolution images as well as the precontrast high spatial resolution sequence. A high-resolution subtraction of the pre- and first postcontrast FLASH3D series prepared on the MRI scanner was also included in this dataset to aid in lesion detection. The proton-density-weighted sequence was necessary for an accurate estimation of the T1 value necessary for the quantitative analyses. The evaluation of this dataset will be further referred to as the ‘fast dynamic’ analysis. In this fast dynamic analysis, the last three postcontrast FLASH 3D series were not used.
All cases were evaluated prospectively by two experienced breast MRI radiologists (reader 1 and reader 2). Both readers had over 5 years of experience in dynamic breast MRI. The evaluation on the two workstations was performed independently in different sessions with at least a 2-month time interval between both sessions.
For the slow dynamic analysis, a dedicated breast MRI workstation was used (Dynacad, Invivo, Germany). This workstation creates subtraction images for all time points after contrast administration, of which the first is automatically displayed together with the precontrast T1 acquisition, both in a coronal orientation. Furthermore, axial reconstructions were displayed for both the subtracted and T1-weighted images with color overlays of wash-in/wash-out enhancement characteristics projected over the T1-weighted images [27]. A maximum intensity projection and signal intensity versus time curves were also displayed. This display protocol resembles the protocol used in the clinical workflow of dynamic breast MRI in our hospital. A BI-RADS classification was assigned for each lesion based on their morphology and enhancement dynamics [28]. No clinical information, mammography or prior MRI data were provided to the readers during the evaluation of the cases.
In the fast dynamic analysis, a workstation, developed in-house for the evaluation of dynamic contrast enhanced MRI, was used [29, 30]. On this workstation, pharmacokinetic parameters derived from the high temporal resolution turboFLASH series were automatically calibrated, calculated and displayed using color overlays. Examples of the recorded high temporal resolution enhancement versus time curves are presented in Fig. 1. In the preparation of this high temporal resolution data, each MRI signal enhancement/time curve was first fitted to a general exponential signal enhancement model, as described previously [31]. This reduces a curve to model with the following five parameters: baseline (s0); start of signal enhancement (t0), which defines the onset of the exponential curve; time-to-peak (ttp), the exponential constant; peak enhancement (sp), the signal amplitude at which the exponential curve levels off; and late wash, defined as the slope of the late part of the exponential curve. The reduced signal enhancement/time curve was converted to a reduced tracer concentration (mmol/ml)/time curve [31, 32], effectively converting sp to concentration tracer after initial rapid wash-in (often at a peak or plateau level) (Cgd,p). The reduced plasma concentration time curve was estimated using the reference tissue method [33]. Deconvolution of the plasma profile and estimation of pharmacokinetic parameters conformed to the theoretical derivations [34], but was implemented in the reduced signal space as: Ve = Cgd,ptissue/Cgd,pplasma; kep = 1/(ttptissue – ttpplasma). Ktrans = Ve × Kep. Where Ve is an estimate of the extracellular volume [%], Ktrans, the volume transfer constant (1/min), and kep, the rate constant (1/min), between extracellular extravascular and plasma space. The subscript ‘tissue’ stands for a measurement in the tissue under investigation and the subscript ‘plasma’ for the reference tissue plasma estimate. The reference tissue was automatically determined by selecting a set of voxels in the whole image volume [relative enhancement, (sp-s0)/s0] larger than 0.2 and smaller than 2.0). This was most often the pectoral muscle, sometimes the liver or spleen. The additionally recorded proton density images were used to correct for the coil profile. The data were presented on the workstation with high-resolution precontrast T1-weighted images in an axial, coronal and sagital reconstruction (FLASH 3D) as background. Color overlays were projected over the images representing Ktrans, kep and Ve parameter values that were based on the high temporal resolution images (turboFLASH). A subtraction image based on the pre- and first postcontrast FLASH 3D series was presented to aid in lesion detection. No criteria for differentiating between benign and malignant lesions were derived from the subtracted images. In this evaluation, the readers selected a region of interest (ROI) within the enhancing lesion. The ROIs were sphere-shaped and placed in an area within the lesion where the parameter values of Ktrans, Ve and kep were highest, based on the color-overlays. The outer limit of the lesion was used as a boundary of the ROI to rule out partial volume effects [35]. This method of ROI selection has previously been referred to as a hotspot method [36]. Each reader placed only one ROI per lesion. From this ROI, the workstation calculated the mean values for each of the pharmacokinetic parameters. Again, no clinical information, mammography or prior MRI data were provided to the readers during the evaluation of the cases. In case of multifocality, the tumor was analyzed as a single lesion.
Fig. 1Relative enhancement versus time curves of a benign (a) and malignant (b) lesion. Note that the slope of enhancement and the level of enhancement is higher for the malignant lesion compared with the benign lesion. These fast dynamic acquisitions were analyzed as described in the Materials and methods section and resulted in the color overlays as presented in Figs. 4 and 5. The data used in this figure were respectively derived from a histopathology proven fibroadenoma and an invasive ductal carcinoma. The same lesions as presented in Figs. 4 and 5
Statistical analysis
Differences in pharmacokinetic parameter values between the malignant and benign group were evaluated using an independent sample t-test. The performance of both methods was compared using a receiver operator characteristic (ROC) analysis. From the slow dynamic analysis, the reader’s final BI-RADS classification of the lesion was used in the ROC evaluation; from the fast dynamic analysis, the mean parameter values calculated from the ROI selected by each reader were used. Multivariate analysis was performed using logistic regression in order to evaluate the possible additional value of both methods to one another. Since the differentiation between benign and malignant lesions is more difficult in smaller lesions a subgroup of all lesions of 2 cm and smaller were also separately evaluated. The comparison of the various results, including the interobserver variability, was done by using the area under the ROC curve (AUC) as an estimate of diagnostic accuracy. A pairwise comparison was performed to evaluate differences in the AUC. P values <0.05 were considered to indicate statistical significance.
Results
A total of 870 consecutive clinical breast MRI examinations in 787 patients were performed. In these studies a total of 188 lesions were detected. Eighty-six lesions could not be included due to lack of histological diagnosis or insufficient follow-up. This resulted in a total of 102 lesions in 96 patients; 34 benign and 68 malignant lesions. The mean age was 51 years (range 28–74 years). Ninety-four lesions were included based on histological evaluation, eight lesions based on follow-up. Mean lesion size on MRI for the malignant group was 32 mm (range 9–90 mm) and this was 15 mm (range 5–50 mm) for the benign lesions. A total of 52 lesions were 2 cm or smaller; 25 malignant (mean lesion size 14 mm, range 6–20 mm) and 27 benign (mean lesion size 11 mm , range 5–20 mm).
The histological evaluation of the malignant lesions was in 14 cases based only on the core biopsy, in 14 cases based on an excision biopsy or breast saving surgery specimen and in 40 cases based on the mastectomy specimen. Histological proven benign lesions were in 19 cases based on a core biopsy and in seven cases based on an excision biopsy. Eight lesions were proven benign based on follow-up, mean follow-up was 37 months (range 24–52 months). The histological composition of the entire group is summarized in Table 1.
Table 1Histological composition of the benign and malignant group of lesions (IDC invasive ductal carcinoma, DCIS ductal carcinoma in situ, ILC invasive lobular carcinoma)Benign (n = 34)Malignant (n = 68)Fibroadenoma11IDC47Fibrosis4DCIS14Adenosis3ILC7Inflammation2Ductal papilloma2Scar tissue1Hyperplasia1Hamartoma1Radial scar1Follow-up8
Imaging results
In the slow dynamic analysis reader 1 classified 25 lesions as a BI-RADS 2 (benign:malignant = 22:3), seven as BI-RADS 3 (4:3), 50 as BI-RADS 4 (6:44) and 20 an BI-RADS 5 (2:18). This was respectively 33 (24:9), 12 (4:8), 41 (5:36) and 16 (1:15) for reader 2. The ROC analysis for the slow dynamic analysis resulted in an AUC of 0.85 (95% CI = 0.773–0.918) and 0.83 (95% CI = 0.74–0.89) for reader 1 and 2, respectively.
The mean volume of the ROIs selected by the readers in the fast dynamic evaluation was 0.51 cm3 for reader 1 (range 0.15–1.94 cm3, SD 0.30) and 0.52 cm3 for reader 2 (range 0.15–1.94 cm3, SD 0.41). No significant difference was found for ROI size (P = 0.72). The pharmacokinetic parameters used in the fast dynamic analysis showed a significant difference between the benign and malignant group for both readers (Table 2). The diagnostic performance of the fast dynamic analysis resulted in an AUC for Ktrans of 0.82 (95% CI = 0.735–0.905) and 0.82 (95% CI = 0.739–0.909) for reader 1 and 2. For Ve the AUC was 0.78 (95% CI = 0.682–0.873) and 0.77 (95% CI = 0.670–0.866) and for the kep parameter 0.72 (95% CI = 0.609–0.828) and 0.74 (95% CI = 0.629–0.841) for reader 1 and 2, respectively. Scatter plots of Ktrans and V displaying the parameter values of benign and malignant lesions found in the two readers are provided in Fig. 2. The comparison of the diagnostic performance from the slow dynamic analysis with the single parameter fast dynamic analysis showed no significant differences for the Ktrans and V parameter. A significant difference was found for reader 1 between the slow dynamic analysis and the kep parameter (P = 0.02) , the slow dynamic analysis showing better results. This was not found for reader 2 (P = 0.08).
Table 2Mean pharmacokinetic parameter values categorized for malignant and benign lesions per reader. All parameter values proved significantly higher in the malignant group compared to the benign group (P < 0.01) Benign (n = 34)95% CIMalignant (n = 68)95% CIReader 1Ktransa1.20.9–1.42.32.1–2.6Ve41.634.9–48.363.958.6–69.1kepa3.02.7–3.33.83.5–4.0Reader 2Ktransa1.31.0–1.52.52.2–2.8Ve44.637.2–52.067.162.0–72.3kepa3.02.6–3.33.93.7–4.2a1/min.Fig. 2Scatter plots from the extracellular volume (V) versus the transfer constant (Ktrans) for reader 1 (a) and reader 2 (b). Benign and malignant cases were clustered. Clusters were summarized with an iso-probability contour computed from the cluster mean and covariance at a squared normalized radius of 2. The continuous-line ellipsoid represents the benign subgroup, the dotted-line ellipsoid represents the malignant subgroup
Combining the pharmacokinetic parameters (Ktrans, Kep and V) in a multivariate analyses resulted in an AUC of 0.83 (95% CI = 0.74–0.90) and 0.83 (95% CI = 0.74–0.90) for reader 1 and 2. No significant difference was found between the multivariate fast dynamic and the slow dynamic diagnostic performance (P = 0.49 and P = 0.85).
The multivariate analysis from all pharmacokinetic parameters combined with the slow dynamic analysis (combined analysis) resulted in an AUC of 0.93 (95% CI = 0.85–0.96) and 0.90 (95% CI = 0.83–0.95) for reader 1 and 2, respectively. The results from the combined analysis were significantly higher when compared with the fast dynamic analysis for both readers (P = 0.01 and P = 0.02). This was also found for the slow dynamic analysis (P = 0.02 for both readers). The ROC curves are presented in Fig. 3.
Fig. 3ROC curve for reader 1 (a) and reader 2 (b) displaying the fast dynamic, slow dynamic and combined analysis. No significant differences were found between the fast and slow dynamic analysis in both readers. A significant difference was found between the slow dynamic analysis and the combined analysis for both readers (P = 0.02 for both readers). The comparison between the fast dynamic analysis and the combined analysis also resulted in a significant difference for both readers (P = 0.01 and P = 0.02). No significant differences were found between the two readers
In the group of lesions of 2 cm and smaller, the slow dynamic analyses resulted in an AUC of 0.87 (95% CI = 0.75–0.95) for reader 1 and 0.79 (95% CI = 0.67-0.91) for reader 2. Overall, the fast analyses resulted in this group in an AUC of 0.83 (95% CI = 0.70–0.92) and 0.85 (95% CI = 0.72–0.93), respectively. No significant difference was found between the slow and fast dynamic analyses for both readers (P = 0.54 and P = 0.41). The combined analysis resulted in an AUC of 0.97 (95% CI = 0.88–0.99) and 0.94 (95% CI= 0.84–0.99), respectively. The results from the combined analysis were significantly higher when compared with the fast dynamic analysis for both readers (P < 0.01 and P = 0.04). This was also found when compared to the slow dynamic analysis (P = 0.03 and P < 0.01).
No significant differences were found between the two readers in any of the analyses. An example of a benign and a malignant lesion is presented in Figs. 4 and 5.
Fig. 4a Transverse reconstruction of the high-resolution subtraction sequence of the right breast. b Time versus relative enhancement curve of the slow dynamic series. Ktrans (c) and V (d) color overlay images of the right breast, including a scalar bar, to demonstrate the parameter values. The subtraction image shows a rounded, mostly sharp delineated lesion. The time versus signal intensity curve demonstrates a type 1 curve, indicative for a benign lesions. The readers classified this lesion as benign (BI-RADS 2) or probably benign needing follow up (BI-RADS 3) based on the slow dynamic analysis. The Ktrans and V parameter color overlays demonstrate relatively low values for both parameters (see Table 2 for comparison) indicative for a benign lesion. Histopathology proved this lesion to be a fibroadenomaFig. 5a Transverse reconstruction of the high-resolution subtraction sequence of the right breast. b Time versus relative enhancement curve of the slow dynamic series. Ktrans (c) and V (d) color overlay images of the right breast, including a scalar bar, demonstrates the parameter values. The subtraction image shows a spiculated lesion retromammillar. The time versus signal intensity curve demonstrates a type 3 curve (wash-out) suggestive for a malignancy. Both readers classified this lesion as malignant (BI-RADS 4) based on the slow dynamic analysis. The Ktrans and V parameter color overlays demonstrate high values for both parameters (see Table 2 for comparison), indicative for a malignant lesion. Histopathology proved this lesion to be an invasive ductal carcinoma
Discussion
In this study, we demonstrated that the pharmacokinetic parameters derived from fast dynamic scanning during initial enhancement were a valuable additional tool for the differentiation between benign and malignant breast lesions on MRI. The pharmacokinetic parameters were significantly higher for the malignant group compared with the benign lesions (Table 2). The diagnostic performance of the pharmacokinetic parameters was, compared with the results of the slow dynamic analysis, not significantly different. The combination of both methods, however, did improve the diagnostic performance significantly for both readers. These results were also found in the subgroup analysis of smaller breast lesions.
The slow dynamic analysis resembles the evaluation as routinely performed in the clinical workflow in our hospital. The performance of the workstation used in the slow dynamic evaluation has already been investigated and proven by Wiener et al. [27] in the evaluation of breast lesions prior to surgical treatment. Schnall et al. [4] evaluated the performance of both dynamic and morphological features in 854 women with 995 lesions. The results of their multivariate evaluation based on both morphological and relatively slow dynamic lesion characteristics resulted in a similar diagnostic accuracy (AUC values of 0.87 and 0.88) compared with the results obtained in the slow dynamic analyses of our study (0.85 and 0.83). Our results found in the slow dynamic analysis are, therefore, considered representative for the diagnostic performance of an experienced radiologist in this group of patients.
In the fast dynamic analysis, both the morphologic characteristics and slow dynamic characteristics were not included in the evaluation; instead, a quantitative analysis of pharmacokinetic parameters was used based on manual ROI placement within the lesion. In the literature, both a “hot-spot” and “whole-tumor method for ROI placement are reported [36]. In this study, we used a hot-spot method. The importance of a consistent ROI placing strategy has been described by Liney et al. [36]. In our study, both readers were instructed with a simple ROI-placing strategy placing the ROI in an area with the highest parameter values guided by color overlays. Since no significant differences were found in the ROC analyses in any of the pharmacokinetic parameters used it is assumed that the performance of the fast dynamic analysis was not negatively affected by this manual ROI selection strategy. The optimal strategy of ROI selection within a breast lesion is a subject that still needs to be further investigated; this is beyond the scope of this study.
Gibbs et al. [24] found the use of quantitative pharmacokinetic parameters in the evaluation of sub 1-cm breast lesions to be beneficial. In their study of 43 women, a diagnostic accuracy of 0.92 was found combining the postcontrast images with the dynamic data in a logistic regression analysis. The exchange rate constant was found to be the best individual parameter with a diagnostic accuracy of 0.74. The Ktrans was also found to be the best individual parameter in our study with a diagnostic accuracy of 0.82. Furman-Haran et al. [18] concluded in their study of 141 lesions that the quantitative evaluation of perfusion parameters should be able to improve breast cancer diagnosis on MRI. In their study the Ktrans was also found to be the best discriminating parameter. Their analysis showed results of invasive ductal carcinoma versus fibroadenomas or fibrocystic changes. Unlike our study, the pharmacokinetic parameters used by Furman-Haran and coworkers were derived from high-resolution images with relatively a low temporal resolution of 2 min. Although our analysis used a more diverse histological distribution (Table 1) compared with the results presented by Furman-Haran et al. [18], only a relatively small number of benign lesions could be included. The diverse histological distribution also resulted in the inclusion of benign lesions that do not necessarily cause a diagnostic dilemma in daily practice. This can be seen as a limitation of our study. In the subgroup analysis of smaller lesions, a more equal distribution between benign and malignant lesions was found. The analysis performed in this subgroup also proved the additional value of the fast dynamic analysis in classifying small breast lesions on MRI.
The three-time-point method used by Kelcz et al. [37] provides the reader with a composite image showing contrast uptake and wash-out characteristics related to the product of microvessel surface area and permeability, as well as to the extracellular volume fraction. In their study, the observers correctly diagnosed 27 of 31 malignant and 31 of 37 benign lesions (sensitivity 87%; specificity 84%) using the three-time-point method. The evaluation based on wash-in and wash-out curves in combination with morphology resulted in a sensitivity of 93% and a specificity of 82%. Our results not only demonstrate a similar performance of the pharmacokinetic analysis compared with the evaluation based on morphology and slow dynamics but also demonstrate the potential gain if both methods are combined. The results presented by Kelcz et al. [37] are, like other authors, again derived from high spatial resolution images with a relatively low temporal resolution of 2 min compared with our fast dynamic scanning protocol.
With a scanning protocol using only the fast dynamic evaluation and morphology the scantime could be reduced significantly when compared with a protocol including the evaluation of wash-out. This without loss of diagnostic performance when compared with the results of the slow dynamic analysis in our study and the results presented by other authors [4, 12, 18]. This reduction of scantime can in the future contribute to the cost-effectiveness of MRI screening. However, since the highest diagnostic performance was obtained by combining both the fast and slow dynamic analysis, further studies are needed before the scantime can be reduced.
The results presented in this study are our initial results using this scanning protocol. Therefore, no cut of values for the differentiation between benign and malignant lesions from the pharmacokinetic parameters were used in the evaluation or can be provided at this point. The results presented only show the potential of our method in differentiation between benign and malignant lesions in this group of patients. The value of our method needs to be further studied in a larger group, preferably using a more even distribution between benign and malignant cases and with lesions that can be classified on imaging as a BI-RADS 3 or higher.
Unfortunately, the study design used did not allow a multivariate analysis combining the fast dynamic data with morphological characteristics. Also, the possible trade-off between the pharmacokinetic parameters based on initial enhancement and the wash-out based on late dynamic characteristics cannot be derived from these data. Both analyses will need to be performed in future projects in order to evaluate the full potential of the fast dynamic analysis as used in our study.In conclusion, pharmacokinetic parameters derived from fast dynamic imaging during initial enhancement have great potential in classifying enhancing lesions in the breast. In this study, the diagnostic performance for the fast dynamic analysis proved to be equal to the results of experienced radiologists using more common evaluation methods based on morphologic characteristics and slow dynamic enhancement characteristics. An increased diagnostic performance was found in combining both methods. This shows the additional value of this method in further improving the diagnostic accuracy of breast MRI.