Dr Jolanta Majcher-Peszynska
email: jolanta.majcher-peszynska@uni-rostock.de

Marko Sass

Sora Schipper

Viktor Czaika

Andreas Gussmann

Ralf Lobmann

Ralf G Mundkowski

Christoph Luebbert

Peter Kujath

Bernhard R Ruf

Horst Koch

Wolfgang Schareck

Ernst Klar

Bernd Drewelow

Pharmacokinetics and penetration of moxifloxacin into infected diabetic foot tissue in a large diabetic patient cohort

Keywords: pharmacokinetics in diabetes, tissue penetration, moxifloxacin, diabetic foot, skin and skin structure infection

Objectives:

Physiological changes occurring in patients with diabetes may affect pharmacokinetics and penetration of antimicrobial agents into peripheral tissue. We examined the pharmacokinetics and the penetration of moxifloxacin into perinecrotic tissue of diabetic foot lesions in patients with diabetic foot infections (DFI).

Patients and methods:

Adult patients suffering from type 2 diabetes mellitus and hospitalized for DFI (Texas classification of at least B2) were treated with 400 mg moxifloxacin intravenously (IV) or orally (PO) once daily. Pharmacokinetics of moxifloxacin and its concentration 3 hours after administration in samples of perinecrotic tissue resected from infected diabetic foot wounds were determined at steady state (day 4 to 8).

Results:

A total of 53 patients with diabetes mellitus type 2 (mean age of 69.4 ± 10.8 years) were included in the study, of whom 28 received PO and 25 IV moxifloxacin therapy for a median of 8 days. In the PO and IV subgroups mean C max in plasma was 2.69 mg/L and 4.77 mg/L at a median of 2.0 h (T max range of 1.0-8.0 h) and 1h after administration, respectively. An AUC 0-24h with a mean of 29.36 mg/L (PO) and 27.09 mg•h/L (IV) was achieved. Mean moxifloxacin concentrations in perinecrotic tissue of infected diabetic foot wounds following PO or IV administration were 1.79 ± 0.82 µg/g and 2.20 ± 1.54 µg/g, thus exceeding the MIC 90 for Staphylococcus aureus (0.25 mg/L) 7-fold and 8.5-fold and the MIC 90 for E. coli (0.06 mg/L) 29-fold and 36-fold, respectively. The mean tissue-to-plasma ratios of moxifloxacin concentration 3 h after administration were 1.01 ± 0.57 (PO) and 1.09 ± 0.69 (IV), respectively. Significant differences between the routes of administration were observed

for T max and C max , (p<0.01), but not for other clinically relevant parameters (AUC 0-24 , moxifloxacin DFI tissue concentration).

Conclusions:

The plasma concentration time curve of moxifloxacin in diabetic patients is similar to that of healthy volunteers. We also observed a good penetration of moxifloxacin into inflamed DFI tissue, which taken together with the possibility of sequential therapy IV / PO designate moxifloxacin 400 mg once daily as a therapeutic option in the treatment of DFI caused by susceptible organisms.

Foot complications are among the most common sequelae of diabetes mellitus and the most important cause for hospitalization of diabetic patients [START_REF] Lipsky | Diagnosis and treatment of diabetic foot infections[END_REF]. Up to one in five patients with diabetes will develop a foot ulcer during the course of disease and about 60% of these will be clinically infected [START_REF] Lipsky | Treating diabetic foot infections with sequential intravenous to oral moxifloxacin compared with piperacillintazobactam/amoxicillin-clavulanate[END_REF]. Diabetic foot infections (DFI) may lead to amputation of the involved limb and are actually responsible for 85% of diabetes-related lower-extremity amputations.

Though DFI are a complex problem and require multidisciplinary management [START_REF] Lipsky | Diagnosis and treatment of diabetic foot infections[END_REF], a prompt and appropriate antibacterial therapy plays a key role and may contribute to the prevention of amputations [START_REF] Lipsky | Diagnosis and treatment of diabetic foot infections[END_REF][START_REF] Lipsky | Treating diabetic foot infections with sequential intravenous to oral moxifloxacin compared with piperacillintazobactam/amoxicillin-clavulanate[END_REF][START_REF] Stengel | Antimicrobial treatment of diabetic foot infections. Effects of primary and secondary prevention measures on amputation rates[END_REF]. Complicated skin and skin structure infections (cSSSIs), including diabetic foot infections, are often polymicrobial, requiring broad-spectrum combination therapy.

For an effective therapy, the antimicrobial agent has to reach an adequate concentration in the involved peripheral tissue [START_REF] Lipsky | Diagnosis and treatment of diabetic foot infections[END_REF][START_REF] Oberdorfer | Tissue and serum levofloxacin concentrations in diabetic foot infection patients[END_REF][START_REF] Majcher-Peszynska | Pharmacokinetics and penetration of linezolid[END_REF]. However, this goal may not be reached in this patient population and for this site of infection. Physiological changes resulting from the underlying diabetic conditions such as acidotic metabolic status, reduced blood flow and altered microenvironment may interfere with the distribution of antibiotics in plasma and tissue.

Local inflammatory processes and fibrotic boundaries in the diabetic foot can additionally impair tissue penetration at the site of infection [START_REF] Oberdorfer | Tissue and serum levofloxacin concentrations in diabetic foot infection patients[END_REF][START_REF] Majcher-Peszynska | Pharmacokinetics and penetration of linezolid[END_REF].

As the fluoroquinolone moxifloxacin is a broad-spectrum antibiotic with high in vitro activity against gram-positive and gram-negative aerobes and anaerobes including the pathogens commonly found in DFI [START_REF] Edmiston | In vitro activities of moxifloxacin against 900 aerobic and anaerobic surgical isolates from patients with intra-abdominal and diabetic foot infections[END_REF], it may be potentially useful for initial empirical therapy.

Moxifloxacin's once daily dose regimen with the possibility of sequential IV/PO therapy offers an advantage to the other current antibiotic regimens used in the treatment of DFI.

Sequential therapy of moxifloxacin is approved for the treatment of adults with complicated skin and skin-structure infections (cSSSIs), including DFI, though its efficacy in osteomyelitis has not been investigated yet.

An earlier pharmacokinetic study in five diabetic patients with peripheral arterial occlusive disease and soft tissue infections has shown that after a single 400-mg dose of moxifloxacin, effective concentrations are attained in the interstitia of healthy and inflamed subcutaneous adipose tissue of the thigh and in plasma [START_REF] Joukhadar | Penetration of moxifloxacin into healthy and inflamed subcutaneous adipose tissues in humans[END_REF].

The present study was set up to investigate the penetration of moxifloxacin into perinecrotic tissue of infected diabetic foot lesions in a large patient cohort and to evaluate its pharmacokinetic profile in a pharmacological risk population of diabetic patients.

Patients and methods

The study population of this prospective, open, multicenter study consisted of male and female patients with type 2 diabetes mellitus hospitalized for DFI graded with a Wagner score of 2 or 3 (equivalent to Texas Diabetic Wound classification of at least B2) and requiring antibacterial therapy.

The following main exclusion criteria were applied: Patients below 18 years of age, with hypersensitivity against quinolones, severe liver dysfunction (Child Pugh class C), heart failure with reduced left ventricular ejection fraction (NYHA III-IV), arrhythmia requiring medical treatment, signs of severe arterial occlusive disease of the lower limbs (Fontaine stages III-IV) or chronic renal insufficiency requiring dialysis.

Written informed consent was obtained from all patients, and the study protocol was approved by the responsible Ethics Committees.

All patients were monitored for adverse events.

Sampling and analytical method

Between days 4 and 8 after start of moxifloxacin treatment the plasma levels were determined (steady-state). The venous blood samples were taken immediately before (baseline) and at 1, 2, 3, 4, 6, 8, 10, 12, and 24 hours after start of 1h-infusion of 400 mg moxifloxacin (following once daily IV administration) or after once daily administration of moxifloxacin 400 mg PO.

Blood samples (2.7 mL) were collected in EDTA containing tubes and immediately centrifuged at 4°C and 2,000 x g for 5 min. Then, plasma was separated, snap frozen at -20ºC and stored at -80°C until analysis.

The concentration of moxifloxacin was determined by means of high performance liquid chromatography (HPLC).

Plasma aliquots of 100 µl were spiked with 10 of aqueous ofloxacin (final concentration 200 µg/L) as internal standard and, in case of the calibrators, appropriate concentrations (100 -1600 µg/L) of moxifloxacin. Then, 20 µl of 50% TFA was added, the mixture was vortexed and centrifuged at 6000xg for 4 min. Of the supernatant 80 µl was added to 21 µl of 5.0 M NH 4 OAc, and 20 µl of the mixture subjected to HPLC/fluorescence analysis. The chromatographic system consisted of a guarded YMC Pro C 18 120/5 column, 150 x 2 mm i.d. maintained at 20°C, and a gradient mobile phase comprising of components A (MeOH/1.0 M NH 4 OAc/H 2 O 10:5:85 v/v/v) and B (40:5:55) pumped at a rate of 250 µl/min, with 22% initial B changed to stages of 25%, and 32% after 1 and 2 min, respectively. Detection was performed by the emission at 504 nm at an excitation wavelength of 296 nm. The retention times were 4.5 and 12.5 min for ofloxacin and moxifloxacin, respectively. The assay was validated intra-and inter-daily according to standard procedures with precisions (CV) and accuracies (RE) better than 15%; the lower limit of quantification (LLOQ) was calculated 50 µg/L allowing for a deviation of CV and RE < 20%. Recoveries ranged between 85 and 90% based on spiking solution. Specimens of at least 100 mg of perinecrotic wound tissue of infected diabetic foot lesions were collected during second wound debridement between days 4 and 8 of antimicrobial treatment and approximately three hours after administration of moxifloxacin. The tissue samples were gently blotted with absorbent paper to remove excrescent blood, snap frozen in liquid nitrogen and stored at -80°C until analysis. Moxifloxacin concentration was determined in the homogenized tissue samples by high-performance liquid chromatography with fluorescence detection as described in detail previously [START_REF] Wacke | Penetration of moxifloxacin into the human pancreas following a single intravenous or oral dose[END_REF].

Pharmacokinetic calculations and statistics

Pharmacokinetic parameters were calculated by noncompartmental analysis with Kinetika 4.4 software (Thermo Scientific, Dreieich, Germany). The maximum observed plasma concentrations (C max ) and time to reach C max (T max ) after moxifloxacin administration were determined from the concentration-time curves. The area under the plasma concentration-time curve from time 0 until the last quantifiable plasma concentration (AUC 0-24h ) was calculated by using the linear trapezoidal rule. DFI tissue/plasma ratio was estimated using corresponding concentration of moxifloxacin in plasma (three hours after administration of moxifloxacin).

All data were analyzed descriptively. Calculations were performed using the SPSS software release 15.0 (SPSS GmbH, Munich, Germany). For all variables, arithmetic mean values, standard deviations, ranges and the 95% confidence intervals (CI 95%) were calculated, with the exception of T max , for which only median and minimum-maximum ranges are given.

Relationships were evaluated using nonparametric Spearman-Rho correlation coefficient.

A two-sided P value of <0.05 was considered the level of significance.

Results

Fifty three patients with DFI were enrolled into the study at nine centres in Germany. At study entry, the patients had a mean age of 69.4 ± 10.8 years, a mean body weight of 82.9 ± 17.9 kg and a mean body mass index (BMI) of 28.2 ± 4.5 kg/m 2 . Mean C-reactive protein (CRP) was 76.9 ± 80.4 mg/L, mean leukocyte count was 11.0 ± 3.5 Gpt/L and average serum creatinine was 121.6 ± 97.5 mol/L. Moxifloxacin 400 mg once daily was administered PO to 28 patients and IV to 25 patients for a median of 8 days (range: 4-19). Patients' assignment to IV or PO administration of moxifloxacin followed clinical considerations. As shown in Table 1, both cohorts were similar in their demographic characteristics and laboratory variables at baseline.

In one patient of the IV cohort the amount of tissue collected was too small to measure tissue concentration.

Study drug treatment was well tolerated. No serious adverse events were observed and none of the patients had to be excluded due to adverse events.

The most frequent treatment-emergent adverse event was diarrhea, occurring in 6 patients (10.5%).

The reduction of body temperature by 2.8 degrees C (p<0.01) and C-reactive protein by 1.4 mg/L (p>0.05) at the end of the therapy can indicate an antiinfective effect of moxifloxacin.

Steady-state concentrations of moxifloxacin in plasma (median for both cohorts: day 6) are shown in Figures 1 and2 and summed-up in Table 2. Significant differences between the routes of administration (IV vs PO) were only observed for C max and T max in plasma (p<0.01), but not for other relevant pharmacokinetic parameters.

Mean moxifloxacin concentrations at steady state (median for both cohorts: day 6) in perinecrotic tissue of infected diabetic foot wounds are shown in Table 3. The mean tissue concentrations for PO or IV administration did not differ significantly (p>0.05). The concentrations of moxifloxacin achieved in DFI tissue correlated more strongly with the AUC 0-24 (r=0.659; p<0.01) than with the corresponding (3h) plasma values (r=0.492, p<0.01).

Mean tissue concentration of moxifloxacin exceeded the in vitro MIC 90 for Staphylococcus aureus (0.25 mg/L) [6, 9, 10] 7-fold and 8-fold and the MIC 90 for E. coli (0.06 mg/L) [START_REF] Edmiston | In vitro activities of moxifloxacin against 900 aerobic and anaerobic surgical isolates from patients with intra-abdominal and diabetic foot infections[END_REF] 29fold and 36-fold, respectively.

Based on these in vitro MIC 90 for Staphylococcus aureus and E. coli fluoroquinolone-relevant PK/PD-parameters of moxifloxacin were calculated (see Table 4). Taking into account the predictive PK/PD parameters for moxifloxacin a therapeutic success can be expected in both administration routes.

Discussion

For diabetic foot infection as well as for necrotizing fasciitis the lowest clinical cure rates of skin and skin-structure infections are reported [START_REF] Vick-Fragoso | Efficacy and safety of sequential intravenous/oral moxifloxacin vs intravenous/oral amoxicillin/clavulanate for complicated skin and skin structure infections[END_REF]. Of the fluoroquinolones licensed, moxifloxacin is the only one demonstrating activity against anaerobe pathogens. This activity, along with its rapid bactericidal effect and its high in vitro activity against gram-positive and gram-negative bacteria, make moxifloxacin especially attractive as a potential single-drug regimen for initial antibacterial therapy of mostly polymicrobial DFI, except for methicillinresistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa [START_REF] Citron | Bacteriology of moderate-to-severe diabetic foot infections and in vitro activity of antimicrobial agents[END_REF]. Similar to other fluoroquinolones, moxifloxacin penetrates well into peripheral compartments and achieves high concentrations in many tissues, including skin and soft tissue [START_REF] Lipsky | Treating diabetic foot infections with sequential intravenous to oral moxifloxacin compared with piperacillintazobactam/amoxicillin-clavulanate[END_REF][START_REF] Burkhard | 10 years' experience with the pneumococcal quinolone moxifloxacin[END_REF].

However, the present study is the first to investigate moxifloxacin penetration into perinecrotic tissue (i.e. the transition zone between healthy and necrotic tissue) of patients with an inflamed DFI.

To the best of our knowledge, it is also the largest pharmacokinetic study of antibiotic penetration into tissue of infected diabetic feet.

We observed significant differences between the routes of administration (IV vs PO) only for C max and T max, but not for AUC 0-24h . In general, the plasma pharmacokinetics of moxifloxacin including total exposition were similar to those in healthy volunteers and patients after multiple doses, with the exception of T max which was higher and more variable in our patient population [START_REF] Stass | Pharmacokinetics and elimination of moxifloxacin after oral and intravenous administration in man[END_REF]. Physiological changes occurring in patients with diabetes, e.g. diabetic gastroenteropathy with delayed gastric emptying and intestinal transit times or diabetic nephropathy can be a reason for the extended time to reach C max (T max ) in our study [START_REF] Oberdorfer | Tissue and serum levofloxacin concentrations in diabetic foot infection patients[END_REF][START_REF] Majcher-Peszynska | Pharmacokinetics and penetration of linezolid[END_REF][START_REF] Kim | In vivo microdialysis study of the penetration of daptomycin into soft tissues in diabetic versus healthy volunteers[END_REF].

High variability of pharmacokinetic parameters in patients with diabetes was also seen in further studies with other antibiotics, e.g. linezolid or daptomycin [START_REF] Majcher-Peszynska | Pharmacokinetics and penetration of linezolid[END_REF][START_REF] Kim | In vivo microdialysis study of the penetration of daptomycin into soft tissues in diabetic versus healthy volunteers[END_REF].

It is recognized that effective concentrations of antibiotics must be achieved in tissues for successful clinical outcomes of antimicrobial therapy. The fast decline of the plasma concentrations after the end of infusion in our study indicates a rapid distribution of moxifloxacin, V ss was very high in agreement with previous reports [START_REF] Joukhadar | Penetration of moxifloxacin into healthy and inflamed subcutaneous adipose tissues in humans[END_REF][START_REF] Stass | Pharmacokinetics and elimination of moxifloxacin after oral and intravenous administration in man[END_REF]. Plasma protein binding, which as long been considered one of the important physicochemical characteristics of antibacterials has been shown to substantially affect tissue penetration and the volume of distribution of antimicrobial agents. Independent of the drug concentration, only 40-42% of moxifloxacin is bound to plasma proteins, mainly to serum albumin, leaving most of the drug in an unbound, active form [START_REF] Burkhard | 10 years' experience with the pneumococcal quinolone moxifloxacin[END_REF]. Many factors other than plasma protein binding play a role in extravascular transfer and tissue distribution of drugs, such as lipid solubility, size of the molecule and its degree of ionization. A low molecular weight (437), a zwitterionic nature and a low protein binding of moxifloxacin may be a reasons for its high tissue penetration.

Host-related factors such as site of infection, presence of biological barriers, local pH and bacterial inoculum size may contribute to the inhibition of antibacterial diffusion and antibacterial activities [START_REF] Bergogne-Berezin | Clinical role of protein binding of quinolones[END_REF]. In diabetic patients conditions such as acidotic metabolic status, reduced blood flow, altered microenvironment, local inflammatory processes and fibrotic boundaries can impair penetration of antimicrobial agents in the diabetic foot tissue [START_REF] Oberdorfer | Tissue and serum levofloxacin concentrations in diabetic foot infection patients[END_REF][START_REF] Majcher-Peszynska | Pharmacokinetics and penetration of linezolid[END_REF].

The mean inflamed DFI tissue to plasma concentration ratio in the present study was about 100% for both PO and IV administration. The measured concentrations were lower than those in other tissues, e.g. gastrointestinal or bronchial tissue [START_REF] Wirtz | Moxifloxacin penetration into human gastrointestinal tissues[END_REF][START_REF] Soman | Concentrations of moxifloxacin in serum and pulmonary compartments following a single 400 mg oral dose in patients undergoing fibre-optic bronchoscopy[END_REF]. This difference might be explained by a lower perfusion in DFI tissue due to impaired macro-and/or microcirculation. Another reason could be the perinecrotic nature of the resected DFI tissue.

Our results confirm previous data of an in vivo microdialysis study, in which an enrichment of moxifloxacin was found in well-perfused inflamed subcutaneous tissue compared to plasma [START_REF] Joukhadar | Penetration of moxifloxacin into healthy and inflamed subcutaneous adipose tissues in humans[END_REF]. In the same study diabetes mellitus patients with peripheral arterial occlusive disease were investigated. In this patient population, the tissue to plasma ratio was 0.5 underlining the implication of circulatory disturbances.

The data of the presented study can be analyzed using various pharmacodynamic and pharmacokinetic indices, which are applied to predict clinical outcome. Pharmacokinetic (PK)/pharmacodynamic (PD) considerations are important in optimizing both antibacterial activity and the development of resistance. Optimal PK/PD indices have been described in plasma for the antimicrobial efficacy of moxifloxacin (area under the concentration-time curve over 24 h at steady state divided by the MIC (AUIC) >30 [h] as the PK/PD surrogate parameter, which is predictive for a positive clinical outcome) [START_REF] Stass | The integrated use of pharmacokinetic and pharmacodynamic models for the definition of breakpoints[END_REF]. Assuming that the PK/PD concept is valid for DFI, the indices calculated from these study data (mean AUIC of 108.4-117.4 for Staphylococcus aureus) account for a high efficacy.

The present study might be limited by the fact that tissue concentration was measured at one single time point (3 h after administration), what certainly must not correspond to the T max , so that higher concentrations of moxifloxacin in DFI tissue during a whole dosage interval are possible.

Another limitation might be that moxifloxacin concentration was determined in homogenized tissue samples which do not allow to separate between different compartments. In biopsy samples, the drug is extracted from homogenized tissue comprising cells, extracellular matrix and extracellular space fluid. For drugs that accumulate intracellularly (e.g. fluoroquinolones), the biopsy method can overestimate effective drug concentrations in the interstitial space fluid, which represents the anatomical site for most bacterial infections. Moxifloxacin penetrates phagocytic and nonphagocytic cells (polymorphonuclear leukocytes and epithelial cells), reaching intracellular concentrations several times higher than extracellular ones. The intracellular penetration of moxifloxacin seem to be rapid, reversible, and affected by environmental temperature, pH and metabolic inhibitors. Moxifloxacin seem partially to require an active process, which allow easier penetration of cell membranes. Tissue inactivation of antibacterials can occur at infection sites by binding to interstitial fluid proteins, soluble intracellular proteins or subcellular structures (nucleic acids, leucocytic chromatin, cell membranes and mucopolysaccharides) [START_REF] Pascual | Uptake and intracellular activity of moxifloxacin in human neutrophils and tissue-cultured epithelial cells[END_REF]. The microdialysis technique, as opposed to the biopsy, determines exclusively free concentrations of antibiotics in the interstitial space fluid. This technique is based on the principle of diffusion of solutes through a semipermeable membrane due to a concentration gradient between the interstitial microenvironment and the fluids within a microdialysis probe inserted into a tissue of interest.

Even if the method of microdialysis has many advantages over a biopsy, the authors consider the biopsy with a lower invasivity and a higher feasibility (equipments, resources) as more suitable to conduct a study at such a large population of patients. The tissue concentrations of moxifloxacin found in the present study support the good clinical results observed with this fluoroquinolone in DFI [START_REF] Lipsky | Treating diabetic foot infections with sequential intravenous to oral moxifloxacin compared with piperacillintazobactam/amoxicillin-clavulanate[END_REF][START_REF] Vick-Fragoso | Efficacy and safety of sequential intravenous/oral moxifloxacin vs intravenous/oral amoxicillin/clavulanate for complicated skin and skin structure infections[END_REF][START_REF] Gyssens | Efficacy of IV/oral moxifloxacin in the treatment of complicated skin and skin-structure infections: results of the RELIEF study[END_REF]. In a recent prospective, randomized, multinational clinical study treatment with sequential IV/PO moxifloxacin, 400 mg once daily was clinically comparable to that with IV amoxicillin/clavulanate 1,000 mg/ 200 mg three times daily followed by PO amoxicillin/clavulanate 500 mg/125 mg three times daily for 7-21 days in hospitalized patients with cSSSIs [START_REF] Vick-Fragoso | Efficacy and safety of sequential intravenous/oral moxifloxacin vs intravenous/oral amoxicillin/clavulanate for complicated skin and skin structure infections[END_REF]. Staphylococcus aureus and Escherichia coli were the most frequently isolated pathogens. In a subgroup analysis of 127 patients with DFI from a prospective double-blind study in patients with complicated skin and skin-structure infections, moxifloxacin was shown to be at least as effective as piperacillin-tazobactam (IV) followed by amoxicillin-clavulanate (PO) in the treatment of moderate-to-severe DFI [START_REF] Lipsky | Treating diabetic foot infections with sequential intravenous to oral moxifloxacin compared with piperacillintazobactam/amoxicillin-clavulanate[END_REF]. In the RELIEF study (a prospective, randomised, double-dummy, double-blind, multinational, multicentre study), IV/PO moxifloxacin was clinically non-inferior to IV piperacillin/tazobactam 4.0/0.5 g thrice daily followed by PO amoxicillin-clavulanic acid in the treatment of patients with complicated skin and skin structure infections including those with DFI [START_REF] Gyssens | Efficacy of IV/oral moxifloxacin in the treatment of complicated skin and skin-structure infections: results of the RELIEF study[END_REF][START_REF] Schaper | Efficacy of IV/PO moxifloxacin and IV piperacillin/tazobactam followed by PO amoxicillin/clavulanate in the treatment of diabetic foot infections: results of the RELIEF study[END_REF].

Conclusions

Adequate drug concentrations were achieved in plasma of diabetic patients and in perinecrotic areas of diabetic foot wounds following IV as well as PO administration of moxifloxacin 400 mg once daily in a large cohort of diabetic patients with foot infections. This finding, taken together with the good clinical data, support a role for moxifloxacin in the initial therapy of patients with DFI.

Figure 1 .

 1 Figure 1. Mean steady-state concentrations of moxifloxacin (400 mg once daily PO) in

Figure 2 .

 2 Figure 2. Mean steady-state concentrations of moxifloxacin (400 mg once daily IV) in

Table 1 .

 1 Patients' demographic and clinical characteristics at baseline (n=53)

	Parameter	PO cohort (n = 28)	IV cohort (n = 25)
	Age (years)	68.8 ± 9.8 (47-86)	70.0 ± 11.9 (42-93)
	Male/Female ratio	2.1	1.8
	Male n (%)	19 (67.9)	16 (64)
	Female n (%)	9 (32.1)	9 (36)
	Body weight (kg)	87.2 ± 17.4 (52-123)	77.9 ± 17.5 (51-110)
	Body mass index (kg/m²) 29.5 ± 4.3 (22.9-40.6)	26.7 ± 4.3 (16.0-37.2)
	CRP (mg/L)	66.9 ± 76.3 (0.2-329.0)	91.9 ± 89.3 (1.7-345.4)
	Leukocyte count (Gpt/L)	10.2 ± 2.1 (7.0-14.5)	11.4 ± 4.4 (4.9-26.4)
	Serum creatinine ( mol/L) 118.7 ± 47.7 (52-245)	129.1 ± 139.4 (51.0-755.0)
	values as mean ± SD (range)

Table 2 .

 2 Steady-state pharmacokinetic parameters of moxifloxacin in plasma following PO or IV CI, confidence interval; C max , maximal plasma concentration; AUC 0-24h , area under concentration time curve from time 0 to 24 hours; V ss , volume of distribution at steady state; Cl tot , total clearance a median; * p<0.01 vs. PO

	administration of 400 mg once daily to diabetic patients with DFI (n=53)
	Route of administration PK parameter	Mean (SD)	95% CI	Range
	PO (n=28)	C max,ss (mg/L)	2.69 (0.94)	2.4; 3.0	0.9 -4.8
		T max,ss (h)	2.0 a	-	1.0 -8.0
		AUC 0-24h,ss (mg•h/L) 27.09 (10.86) 23.1; 31.1 10.4-52.7
		V ss (L/kg)	2.34 (0.96)	2.0;2.7	1.2 -4.7
		Cl tot,ss (L/h)	14.13 (7.5)	11.4; 16.9 4.0 -35.4
	IV (n=25)	C max,ss (mg/L)	4.77* (1.87)	4.0; 5.5	2.2 -8.2
		T max,ss (h)	1.0*	-	-
		AUC 0-24h,ss (mg•h/L) 29.36 (12.47) 24.3; 34.4 16.2-76.1
		V ss (L/kg)	1.81 (0.47)	1.6; 2.0	1.0 -2.8
		Cl tot,ss (L/h)	13.38 (4.79)	11.4; 15.3 3.0 -22.2

Table 3 .

 3 Steady-state concentration of moxifloxacin in inflamed DFI tissue and tissue/plasma ratio 3 hours following PO (n=28) or IV(n=25) administration of 400 mg moxifloxacin.

		PO (n=28)	IV (n=25)
		Mean (SD) Range	Mean (SD) Range
	DFI-tissue concentration (µg/g) 1.79 (0.82) 0.53 -3.5 2.2 (1.54) 0.56 -6.4
	DFI tissue/plasma ratio	1.01 (0.57) 0.36 -2.55 1.09 (0.69) 0.26 -2.84

Table 4 .

 4 Predictive PK/PD parameters of moxifloxacin following once daily PO or IV administration of 400 mg for 4 to 8 days in 53 patients with DFI PK/PD Parameters MIC 90 = 0.25 mg/L6,9,10 MIC 90 = 0.06 mg/L 6 This study was supported in part by an unrestricted grant by Bayer Vital GmbH, Leverkusen, Germany. .M.P. has received speaking fee from Bayer Vital GmbH M.S. no conflict S.S. no conflict V.C. has received research support from Bayer Vital GmbH A.G. has received research support from Bayer Vital GmbH R.L. has received speaking fee from Bayer Vital GmbH R.G.M.has received research grants from Bayer Vital GmbH C.L. no conflict P.K. has received speaking fee from Bayer Vital GmbH B.R. has received research grants from Bayer Vital GmbH H.K. has received speeking fee from Bayer Vital GmbH W.S. no conflict E.K. has received research grants from Bayer Vital GmbH B.D. has served as a consultant and received research grants from Bayer Vital GmbH

		PO	IV	PO	IV
	C max / MIC 90	10.8 (3.8)	19.1 (7.5)	44.8 (15.7)	79.5 (31.2)
	AUC 0-24 / MIC 90	108.4 (43.4)	117.4 (49.9)	451.5 (180.9)	489.3 (207.8)

J

Acknowledgements

The authors express their thanks for the excellent technical assistance provided by Katrin Kroesche and Andrea Bruss. We thank Klaus A. Schmidt, Aachen, for his assistance in the preparation of the manuscript.

Results of an interim analysis of this study were presented at the 16 th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), Nice, France, April 2006; abstract P1516. [http://www.blackwellpublishing.com/eccmid16/abstract.asp?id=50311]

Funding


L Vallier

M Hiller

S Iddawela

K Hilborne

P Taylor

H M Britton

R Earl

Sinnatamby

Dr Ioannis Gounaris
email: ioannis.gounaris@cancer.org.uk

E Provenzano

A L Vallier

L Hiller

M Iddawela

S Hilborne

K Taylor

P Britton

M Earl

R Sinnatamby

ACCURACY OF UNIDIMENSIONAL AND VOLUMETRIC ULTRASOUND MEASUREMENTS IN PREDICTING GOOD PATHOLOGICAL RESPONSE TO NEOADJUVANT CHEMOTHERAPY IN BREAST CANCER PATIENTS

Keywords: Breast cancer, neoadjuvant chemotherapy, volumetric ultrasound, pathological response

Accuracy of unidimensional and volumetric ultrasound measurements in predicting good pathological response to neoadjuvant chemotherapy in breast cancer patients

I.

INTRODUCTION

Despite recent decreases in incidence [START_REF] Li | Changes in breast cancer incidence rates in the United States by histologic subtype and race/ethnicity, 1995 to 2004[END_REF][START_REF] Ravdin | The decrease in breast-cancer incidence in 2003 in the United States[END_REF], breast cancer remains the commonest cancer in women and the second most common cause of death due to cancer after lung cancer. In the United States it was estimated that more than 40000 women died of breast cancer in 2009 [START_REF] Jemal | Cancer Statistics 2009[END_REF].

Neoadjuvant chemotherapy (NAC) is an established treatment option for breast cancer patients presenting with large primary tumours or locally advanced disease. Numerous randomised clinical trials as well as a meta-analysis [START_REF] Mauri | Neoadjuvant versus adjuvant systemic treatment in breast cancer: a meta-analysis[END_REF] have confirmed the equivalence of neoadjuvant and adjuvant chemotherapy in terms of disease-free and overall survival.

Pathological complete response (pCRthe complete absence of invasive tumour in both the breast and the axillary lymph nodes) has emerged as a powerful prognostic marker for overall and disease-free survival following neoadjuvant chemotherapy [START_REF] Sataloff | Pathologic response to induction chemotherapy in locally advanced carcinoma of the breast: a determinant of outcome[END_REF][START_REF] Kuerer | Clinical course of breast cancer patients with complete pathologic primary tumour and axillary lymph node response to doxorubicin-based neoadjuvant chemotherapy[END_REF].

However, tumour response to chemotherapy is a continuum from frank resistance to pCR. Ogston et al [START_REF] Ogston | A new histological grading system to assess response of breast cancers to primary chemotherapy: prognostic significance and survival[END_REF] showed that the Miller-Payne classification system that is based on residual tumour cellularity is an independent predictor of survival in multivariate analysis. Symmans et al [START_REF] Symmans | Measurement of residual breast cancer burden to predict survival after neoadjuvant chemotherapy[END_REF] also reported that the Residual Cancer Burden (RCB) Index that takes into account residual tumour size and cellularity as well as the number and size of nodal metastases is also predictive of outcome, with minimal residual disease carrying the same excellent prognosis as pCR.

Patients electing to undergo NAC are closely monitored. In most centres in Europe and North America the monitoring schedule includes clinical breast examination at each chemotherapy visit as well as imaging of the breast with mammography, ultrasound (US) and, in selected patients, MRI at diagnosis, mid-way through chemotherapy, and after completion of chemotherapy, prior to surgery. This enables timely identification of patients whose tumours do not respond or frankly progress during NAC, although the latter is a rare occurrence [START_REF] Von Minckwitz | Intensified neoadjuvant chemotherapy in early-responding breast cancer: Phase III randomised GeparTrio study[END_REF]. It is also important for final surgical planning, identifying or confirming those patients suitable for breast conserving surgery.

Although routinely performed in clinical practice, the literature regarding the utility of mammography and US in assessing response to NAC and predicting pCR is surprisingly sparse and consists mostly of relatively small retrospective case series. Von Minckwitz et al [START_REF] Von Minckwitz | Intensified neoadjuvant chemotherapy in early-responding breast cancer: Phase III randomised GeparTrio study[END_REF] reported that the sensitivity of US after 2 cycles of chemotherapy for predicting eventual pCR was only 7.6% in the GeparTrio study. Chapgar et al [START_REF] Chapgar | Accuracy of physical examination, mammography and ultrasonography in predicting residual pathologic tumour size in patients treated with neoadjuvant chemotherapy[END_REF] noted that, in 189 patients treated with NAC at the MD Anderson Cancer Center (Houston, TX), the correlation of physical examination, mammography and US with pathological tumour size was weak to moderate with correlation coefficients of 0.42, 0.41 and 0.42 respectively. Small case series however, have reported much higher accuracy of US in predicting pCR [START_REF] Kanazawa | Diagnosis of complete response to neoadjuvant chemotherapy using diagnostic imaging in primary breast cancer patients[END_REF][START_REF] Schott | Clinical and radiologic assessments to predict breast cancer pathologic complete response to neoadjuvant chemotherapy[END_REF] and there is increasing interest in employing advanced ultrasound-based technologies, for example assessing vascularity, to further improve performance [START_REF] Zhu | Noninvasive monitoring of breast cancer during neoadjuvant chemotherapy using optical tomography with ultrasound localization[END_REF][START_REF] Singh | Role of color doppler indices in predicting disease-free survival of breast cancer patients during neoadjuvant chemotherapy[END_REF].

The ability to accurately predict which patients are destined to achieve a good response to NAC early in the course of treatment would be a major advance in the field. It would greatly facilitate clinical trial designs of early switch to alternative cytotoxic or molecularly targeted agents (or even early recourse to surgery) in an attempt to improve pCR rates and ultimately survival from breast cancer.

In this study, utilising data collected prospectively in the context of a randomised, phase III trial of NAC (Neo-tAnGo), we attempted to correlate changes in either tumour longest diameter (LD) or volume as determined by conventional and 3D US with final pathology at surgery. We investigated the performance of proportional changes in tumour LD and volume in predicting good response to chemotherapy. We also examined the ability of absolute residual tumour size, in terms of either LD or volume, to predict the same.

METHODS

The Neo-tAnGo study

The Neo-tAnGo study is a randomized phase III study of NAC that enrolled 831 patients with high-risk early breast cancer from 57 UK centres between January 2005 and September 2007. In a 2-by-2 factorial design these patients were randomised to 4 cycles of epirubicin-cyclophosphamide (EC) followed by 4 cycles of paclitaxel with or without gemcitabine, or the reverse sequence (i.e. the taxane with or without gemcitabine first, followed by the anthracycline component). The primary endpoint was pCR and preliminary results have been reported [START_REF] Earl | Neo-tAnGo: a neoadjuvant randomized phase III trial of epirubicin/cyclophosphamide and paclitaxel+/-gemcitabine in the treatment of women with high-risk early breast cancer (EBC): first report of the primary endpoint, pathological complete response (pCR)[END_REF].

Imaging Substudy

As per the main study protocol, all enrolled patients underwent tumour assessment, usually by mammography and/or US at baseline, after 4 cycles of chemotherapy (mid-treatment) and after finishing chemotherapy, just prior to surgery (end-treatment). In addition, patients enrolled at Cambridge were offered 3D volumetric US and breast MRI at the same time-points (evaluation of MRI performance will be reported separately). US data were acquired using a combination of conventional B-mode US (Aplio 80; Toshiba Medical Systems, Tochigiken, Japan) with a 12-MHz linear transducer, followed by a freehand 3D US data set, using a system developed in collaboration with our University's Department of Engineering. For the 3D acquisition, an optical position sensor (Polaris; Northern Digital, Waterloo, Ontario, Canada) was used, to continually track the orientation of 15 infrared-emitting diodes attached to the transducer. High spatial resolution US images (0.1 X 0.1mm pixel size) together with their spatial orientation data, were simultaneously recorded to an 800-MHz personal computer (World of Computers, Cambridge, England) equipped with research software (Stradwin; Department of Engineering, Cambridge University, Cambridge, England; freely available from: http://mi.eng.cam.ac.uk/~rwp/stradwin). This non-commercial research software was also used to analyze and display the 3D data and has been described in detail elsewhere [START_REF] Prager | Freehand 3D ultrasound without voxels: volume measurement and visualisation using the Stradx system[END_REF][START_REF] Treece | High definition freehand 3D ultrasound[END_REF]. All 3D US data acquisition was performed by 1 of 2 experienced Consultant radiologists. The 3D US dataset was reviewed at the personal computer, the tumour was manually segmented and a series of outlines representing the tumour was produced. Volume measurements were produced from the target outlines for each dataset using the Stradwin software [START_REF] Prager | Freehand 3D ultrasound without voxels: volume measurement and visualisation using the Stradx system[END_REF][START_REF] Treece | High definition freehand 3D ultrasound[END_REF].

Histopathology Review

Following surgery, all pathology reports were reviewed by an experienced breast histopathologist (EP) in conjunction with the study's chief investigator (HME). Each one separately assessed the response of the primary tumour using 5 categories: pCR, minimal residual disease (MRD, >90% response), chemotherapy response present but not gradable, no evidence of response or unable to comment. In case of disagreement, the case was reviewed jointly and final agreement reached by consensus. It should be noted that the categories of MRD and pCR that were used as outcome measures in this project correspond exactly to Grades 4 and 5 of the "Miller and Payne" system [START_REF] Ogston | A new histological grading system to assess response of breast cancers to primary chemotherapy: prognostic significance and survival[END_REF].

After the analysis of the imaging substudy data had been completed, an independent pathology review of all specimens was undertaken and the RCB Index scores were calculated for the patients enrolled at Cambridge. We elected not to repeat the analysis using the RCB Index, as this is invalid in patients with a positive sentinel lymph node (SLN) biopsy [START_REF] Symmans | Measurement of residual breast cancer burden to predict survival after neoadjuvant chemotherapy[END_REF], a fact that would further limit the sample size.

Substudy Endpoints

The primary objective of this substudy was to compare the performance of tumour LD and volume changes, as well as size of residual disease as measured by US in predicting good pathological response to chemotherapy. Two alternative definitions of good response were used: pCR only and pCR or MRD (pCR/MRD). pCR was defined as the absence of any invasive component at examination of both the primary tumour site and any axillary lymph nodes. Samples with in situ disease alone could still be classified as showing pCR if there was no associated invasive component. MRD was defined as evidence of >90% tumour necrosis at surgery, without any macroscopic residual axillary lymph node involvement.

In order to assess US performance, radiological response to chemotherapy was classified on the basis of either proportional change in tumour size or absolute residual tumour size. For LD proportional changes the following threshold values were used: 30% (corresponding to partial response by RECIST criteria), 50%, 75% and 100% (complete response [CR]) LD reduction. Proportional volume changes were examined at the following thresholds: 50%, 65% (corresponding to 30% LD change assuming a spherical volume), 75%, 90% and 100% (CR) reduction. The following absolute residual size thresholds were also used: residual LD less than 10, 20 and 30mm and residual volume less than 1, 2 and 3cm 3 . For patients with multifocal tumours, US measurements were summed and therefore all the reported values are per patient and not per lesion.

Fifty-five patients had at least two available unidemensional and 3D US assessments.

This study focused on the 48 patients that had available measurements at baseline and mid-treatment. We elected to do so as the utility of being able to predict good pathological response early is greater compared to just prior to surgery since, in the latter case, the tumour soon becomes available for detailed histopathological assessment. The outcomes for the 38 patients with available measurements and baseline and end-treatment are mentioned briefly.

Statistical Analysis

Correlation coefficients were calculated between unidimensional and volumetric measurements. Mean US LD and volume measurements at baseline and at the assessment time-point were compared with a paired t-test. The mean tumour diameters and volumes in patients with or without good response were compared using t-tests.

Radiological and pathological responses at the thresholds defined were compared with Fisher's exact test or χ 2 as appropriate. For thresholds significant at this first analysis, sensitivities, specificities, positive and negative predictive values, likelihood ratios as well as accuracy were calculated from the tables. To further examine the trade-offs between sensitivity and specificity, receiver-operating characteristic (ROC) curves were constructed and the corresponding areas under the curve (AUC) were calculated and compared using a modification of Wilcoxon's rank sum test.

Univariate logistic regression models were constructed including age, tumour grade, hormone and HER2 receptor status, clinical T stage, nodal status, chemotherapy sequence, proportional tumour size change thresholds and residual size thresholds at mid-treatment. Threshold values were entered in the model one at a time. The outcome variables were pCR and pCR/MRD and covariates with P<0.10 at univariate analysis were entered into multivariate models. In an unplanned post hoc analysis, the investigations were repeated for the HER2 positive and HER2 negative patients separately.

As this was an exploratory study using a convenience sample, formal sample power calculations were not done. All analyses were conducted using SPSS version 17.0 unless otherwise specified. Significance was determined by p<0.05 and no corrections for multiple testing were performed. The Toronto Centre for Evidence-Based Medicine online calculator was used for calculating confidence intervals for sensitivity, specificity, positive and negative predictive values and likelihood ratios (available from: http://ktclearinghouse.ca/cebm/practise/ca/calculators/statscalc). The freely available MedCalc program (www.medcalc.be) was used to compare different AUC curves.

RESULTS

Patients

Forty-eight women had both conventional and 3D US measurements of their primary tumours at baseline and mid-treatment (Table 1). This was a cohort of young patients (median age 46.5 years; 30 [62.5%] being <50 years old) with high risk breast cancer. Twelve (25%) patients had multifocal disease at presentation and 21 (44%) presented with T3 or T4 primaries. SLN biopsy was not mandated by the trial protocol and was not in routine clinical use at the start of the trial; only 12 (25%) of the patients underwent an SLN biopsy procedure. There were relatively high rates of oestrogen receptor negativity (31%) and HER2 positivity (42%). Five (10%) patients were triple negative (i.e. negative for ER, PR and HER2).

Treatment received and final pathology

Nineteen (40%) women received 4 cycles of EC followed by paclitaxel with or without gemcitabine, whereas 29 (60%) received the reverse sequence. As paclitaxel was administered in a dose dense fashion (every two weeks), the women receiving taxane first had their mid-treatment US scan after 8 weeks of chemotherapy, while the women in the anthracycline-first arm underwent the same evaluation after 12 weeks. All 48 women successfully completed 8 cycles of NAC and underwent surgery (11 had breast conserving surgery and 37 a mastectomy). The 20 women with HER2 positive tumours also received adjuvant trastuzumab for 1 year.

Eleven (23%) of patients achieved pCR (Table 2) with 38% achieving pCR/MRD. The mean residual invasive tumour size was 18.83mm (SE 3.22mm), while the combined mean residual invasive and in situ component was 27.53mm (SE 3.54mm).

Baseline and mid-treatment ultrasound and clinical examination measurements

Tumour sizes by US and clinical examination, using callipers, at baseline and midtreatment are presented in Table 3. US measurements of LD at baseline were on average 6.27mm smaller than the corresponding clinical measurements (95% CI 1.37-11.17mm, p=0.013). At mid-treatment, as assessed by US, the tumours had significantly decreased in size on both uni-dimensional and volumetric criteria (both p<0.001). As would be expected, the LD and volumetric measurements were significantly correlated both at baseline and mid-treatment (r=0.74 at baseline and r=0.57 at mid-treatment, both p<0.001). On a per-patient, proportional basis, tumour LD changes ranged from +19% to -100% (radiological CR) between baseline and mid-treatment. The corresponding volume changes were +118% to -100%. The mean LD and volume changes were 30.2% (S.E. 5.5%) and 51% (S.E. 7.5%) respectively.

Ultrasound measurements and pathological response

The 11 patients with pCR and the 37 patients without pCR had similar mean tumour LD and volume at mid-treatment (Table 4). Mean LD was 27.7mm and 23.92mm (p=0.572) and mean volumes were 2.95cm 3 and 2.49cm 3 respectively (p=0.691). Similarly, at midtreatment, LD decreased by 33.4% in patients with eventual pCR and by 29.2% in patients without pCR (p=0.754). The corresponding volume changes were 62.3% and 47.5% (p=0.415). There is therefore, no evidence in this sample that tumours that have shrunk proportionately more at mid-treatment, as measured by US, are more likely to show pCR. The results were essentially unchanged when combined pCR/MRD (18 patients) was considered as the response variable (Table 4).

Using a threshold of 30% reduction in LD that would be consistent with the RECIST response criteria, 27 (56%) of the 48 women had a radiological response. Six (22%) of these women eventually showed a pCR, an outcome shared with 5 (24%) of the 21 women with less than 30% reduction in tumour LD at mid-treatment (p=1). Using thresholds of 50% and 75% LD reduction did not improve US performance (Table 5).

Even among the 3 patients that had a radiological CR (rCR) at mid-treatment, only 1 (33%) showed eventual pCR, whereas 10 (22%) of the 45 patients with less than rCR did so (p=0.551).

As far as volume changes are concerned, 30 (62.5%) women had a >50% reduction in tumour volume, of which 7 (23%) showed a pCR, a proportion not statistically different from the 4 (22%) of 18 women with less than 50% volume reduction that eventually showed a pCR (p=1). Using a threshold of 65% volume reduction, which corresponds to a 30% LD reduction if a spherical volume is assumed, the corresponding proportions were 6/24 (25%) and 5/24 (21%) respectively (p=1). Thresholds of 75%, 90% and even 100% (rCR) did not improve ultrasound performance (Table 5).

The ability of absolute residual tumour size rather than proportional change at midtreatment to predict response was also examined. None of the thresholds examined could separate patients with or without pCR in a statistically significant way (Table 5).

The same proportional change and absolute size thresholds were analysed for the outcome of pCR/MRD. No statistically significant findings emerged when examining the various proportional change thresholds' ability to separate the patients with good response to chemotherapy (pCR/MRD) from the rest (Table 6). However, examining residual tumour size thresholds (Table 6) revealed that patients with residual tumour volume ≤1cm 3 at mid-treatment were more likely to show pCR/MRD than those with larger residual volumes. Eleven (61%) of the 18 patients with residual volume ≤1cm 3 had pCR/MRD compared with 7 (23%) of the 30 patients with larger residual tumour volumes (uncorrected p=0.014).

Binary classification tests, likelihood ratios and overall accuracy of the 1cm 3 residual volume threshold at mid-treatment

Residual tumour volume ≤1cm 3 had a sensitivity of 61% (95% C. 39-80%), specificity of 77% (95% CI 59-88%) and overall accuracy of 71% for the outcome of pCR/MRD. PPV and NPV were also good at 61% (95% CI 39-80%) and 77% (95% CI 59-88%) respectively. The positive likelihood ratio (LR+) was 2.62 (95% CI 1.24-5.52) and the negative likelihood ratio (LR-) was 0.51 (95% CI 0.28-0.94). A ROC curve was constructed, using residual tumour volume ≤1cm 3 as a binary cut-off point. The AUC value was 0.689 (95% CI 0.528-0.849, p=0.03) implying discrimination significantly different from chance (Fig. 1).

Logistic Regression modelling

Univariate binary logistic regression models, using pCR/MRD as the outcome variable, with age, tumour grade, hormone and HER2 receptor status, clinical T stage, nodal status, chemotherapy sequence, proportional tumour size change thresholds and residual size thresholds at mid-treatment as covariates were constructed. The following three variables were significant at p≤0.1: residual volume at mid-treatment ≤1cm 3 , grade and age (Table 7). The odds ratio (OR) for pCR/MRD was 0.194 for patients with residual volume >1cm 3 compared to ≤1cm 3 (95% CI 0.054-0.69, p=0.011). When all three variables were forced into a multivariate model (Table 8), none retained statistical significance. Residual volume ≤1cm 3 however, still trended towards statistical significance (OR 0.279, 95% CI 0.072-1.082, p=0.065).

Subgroup analyses

The 20 (42%) patients with HER2-positive tumours had higher pCR rates than the rest (30% vs. 18%) but the difference was not statistically significant (p=0.488). Similarly, 9 (45%) of 20 HER2-positive patients achieved pCR/MRD compared to 9 (32%) of 28 HER2-negative patients (p=0.385). When proportional US threshold changes were tested for their ability to predict pCR and pCR/MRD, separately in the HER2-positive and HER2-negative patients, none reached statistical significance. However, in HER2positive patients, tumour volume ≤1cm 3 was an even more powerful predictor of pCR.

Five (71%) of 7 HER2-positive patients with residual tumour volume ≤1cm 3 achieved pCR compared to 1 (8%) of 13 with larger tumours (p=0.007). When the outcome of pCR/MRD was examined, residual volume ≤1cm 3 for HER2-positive patients retained statistical significance, with 6 (86%) of 7 patients having pCR/MRD compared to 3 (23%) of 13 with larger tumours (p=0.017). Sensitivity, specificity, PPV, NPV, LR+, LR-and overall accuracy were calculated for the outcome of pCR in HER2-positive patients with residual tumour volume ≤1cm 3 at mid-treatment. The results were 83%, 86%, 71%, 92%, 5.83, 0.19 and 85% respectively. The AUC of the ROC curve was 0.845 (Fig. 2), with discrimination statistically significantly different from chance (p=0.017). The 1cm 3 threshold however, was not significant in HER2-negative patients.

Measurements at the end of chemotherapy

Thirty-eight women had available measurements at baseline and end-treatment (of which 31 [82%] had also assessments at mid-treatment and form part of the main analysis). The correlation between the US measurement of residual tumour LD at endtreatment and the size of residual invasive tumour at pathological assessment was weak to moderate, although statistically significant (r=0.359, p=0.027). The correlation was only marginally improved when the size of residual invasive plus in situ tumour was considered (r=0.381, p=0.018). None of the proportional or absolute size thresholds were statistically significant in this cohort (data not shown).

DISCUSSION

The main findings of our analysis can be summarised as follows: i) proportional LD and volume changes showed very poor ability in predicting pCR or pCR/MRD in the current series; ii) irrespective of their initial size, tumours with volumes ≤1cm 3 at midtreatment, were more likely to show eventual pCR/MRD compared to tumours >1cm 3 ; iii) in a post hoc unplanned subgroup analysis, this observation seemed to be limited to

HER2 positive tumours

The use of the traditional thresholds of 30% and 100% reduction in LD, equivalent to partial (PR) and complete (rCR) radiological response by RECIST criteria, could not separate patients into good pathological responders or not, in a statistically significant way. Similarly, proportional volume thresholds did not show an advantage. This is to an extent expected, as there was strong correlation between LD and volume measurements at each time point (r=0.57-0.848), implying that most tumours had a reasonably regular shape, limiting the scope for dramatic differences between the unidimensional and volumetric approaches.

In our series, only 1 of 3 patients with rCR at mid-treatment showed eventual pCR and a further 1 showed MRD. Other authors have also reported poor concordance between US-assessed rCR and pCR [START_REF] Kanazawa | Diagnosis of complete response to neoadjuvant chemotherapy using diagnostic imaging in primary breast cancer patients[END_REF]. As all sonograms were performed by experienced breast radiologists, one must consider the possibility that US is inherently limited in predicting pCR. Breast tumours in patients receiving NAC do not just melt away. They tend to fragment [START_REF] El-Didi | Pathological assessment of the response of locally advanced breast cancer to neoadjuvant chemotherapy and its implications for surgical management[END_REF][START_REF] Huber | Ultrasonographic tissue characterization in monitoring tumour response to neoadjuvant chemotherapy in locally advanced breast cancer (work in progress)[END_REF] and sometimes tumour necrosis is accompanied by a stromal reaction [START_REF] Pinder | Laboratory handling and histology reporting of breast specimens from patients who have received neoadjuvant chemotherapy[END_REF]. Furthermore, US cannot distinguish between in situ and invasive components. The consequence of these is that US will often show a residual mass in cases were pathological examination reveals pCR or MRD. The opposite can also be true as in cases of tumour necrosis without stromal reaction, ultrasound will show rCR whereas pathological examination may reveal residual viable tumour. The correlation coefficient r=0.359 between end-treatment US-assessed LD and pathology in our series similar to the value r=0.29 reported by Fiorentino et al [START_REF] Fiorentino | Accuracy of mammography and echography versus clinical palpation in the assessment of response to primary chemotherapy in breast cancer patients with operable disease[END_REF] but lower than other reports in the literature (Table 9). Of note, the correlation only marginally improved (r=0.381) when residual in situ carcinoma was taken into account.

We tested various other proportional LD and volume thresholds for their ability to predict pCR or pCR/MRD. A similar approach in breast cancer patients receiving NAC has been reported by Akazawa et al [START_REF] Akazawa | Potential of reduction in total tumour volume measured with 3-D MRI as a prognostic factor for locally-advanced breast cancer patients treated with primary chemotherapy[END_REF]. These authors tested various 2D US as well as 2D and 3D MRI thresholds at end-treatment for their ability to predict recurrence in a series of 51 patients. They found that proportional size change thresholds of 50%, 60%, 70%, 75% and 80% as assessed by 2D US were not predictive of disease recurrence.

However, >75% volume reduction as assessed by MRI was predictive with an uncorrected p=0.03. Similarly to the situation with our series, multiple performed comparisons increase the study's vulnerability to type I errors.

The unique approach of our study was to examine absolute residual size thresholds, hypothesising that tumours that had shrunk below a given size would be more likely to show pCR or pCR/MRD, irrespective of their initial size or the proportional size change. Adopting this approach, it was found that residual volume ≤1cm 3 at midtreatment was predictive of pCR/MRD. The AUC of the ROC curve was 0.689, indicating discriminatory ability significantly better than chance alone (p=0.03). Choi et al [START_REF] Choi | The role of PET CT to evaluate the response to neoadjuvant chemotherapy in advanced breast cancer: comparison with ultrasonography and magnetic resonance imaging[END_REF] reported that US-assessed rCR had an AUC of 0.68 in predicting pCR (p>0.05) whereas Keune et al [START_REF] Keune | Accuracy of ultrasonography and mammography in predicting pathologic response after neoadjuvant chemotherapy for breast cancer[END_REF] found it to be 0.71 (p-value not given). The AUC for the 1cm 3 volume threshold compares favourably to those, with the added advantage that it is determined at the midway point rather than the end of chemotherapy. However, the multiple (26) hypothesis tests that were involved at arriving at this threshold raise the possibility of Type I statistical error. Therefore, independent validation is a larger dataset is needed. Subgroup analysis showed that the threshold of volume ≤1cm 3 at midtreatment was particularly informative for HER2-positive tumours for both pCR and pCR/MRD. Again, this may reflect a chance finding, as calculations involved multiple hypothesis tests and the sample sizes were very small (20 HER2-positive and 28 HER2negative patients). It should also be kept in mind that, although pathological responses such as MRD that fall short of pCR may confer a similarly good prognosis, further validation of this outcome measure is required.

US during NAC has a limited ability to predict pCR and, despite some positive findings, volumetric ultrasound in the current study did not result in marked improvements in performance. The limitations of clinical examination [START_REF] Bear | Sequential preoperative or postoperative docetaxel added to preoperative doxorubicin plus cyclophosphamide for operable breast cancer: National Surgical Adjuvant Breast and Bowel Project Protocol B-27[END_REF] and mammography [START_REF] Chapgar | Accuracy of physical examination, mammography and ultrasonography in predicting residual pathologic tumour size in patients treated with neoadjuvant chemotherapy[END_REF] have also been extensively discussed in the literature. Breast MRI [START_REF] Kanazawa | Diagnosis of complete response to neoadjuvant chemotherapy using diagnostic imaging in primary breast cancer patients[END_REF][START_REF] Akazawa | Potential of reduction in total tumour volume measured with 3-D MRI as a prognostic factor for locally-advanced breast cancer patients treated with primary chemotherapy[END_REF] appears to be more accurate than US, but not enough to enable it to be used to predict pCR with certainty.

For reasons already discussed (tumour fragmentation, stromal reactions, presence of in situ residual carcinoma), it may be that no anatomy-based imaging technique will be able to result in highly accurate predictions.

In conclusion, this study adds to the evidence that proportional size changes, either unidimensional or volumetric, as assessed by US cannot discriminate between patients with and without good pathological responses. However, residual volume ≤1cm 3 The correlation column refers to correlation coefficient r as reported in the original publication. The ultrasound accuracy column refers to the proportion of tumours in which the difference between ultrasound-measured and pathological longest diameter was within the stated limit.

US: ultrasound

  at midtreatment could potentially predict which patients are destined to exhibit good pathological response 8 to 12 weeks before the end of chemotherapy, particularly in the HER2-positive population. Validation in different datasets is now required and, if successful, would facilitate trial designs that assign patients to different treatment arms at mid-treatment based on predicted pathological responses.

Figure 1 .Figure 2 .

 12 FIGURE CAPTIONS:

Table 1 .

 1 Characteristics of patients with baseline and mid-treatment ultrasound

	measurements available
	Characteristic	Patients n=48 (%)
	Age
	Median	46.5
	Range	31-64
	<50	30 (62.5%)
	≥50	18 (37.5%)
	Side
	Left	22 (46%)
	Right	26 (54%)
	Histology
	Ductal	46 (96%)
	Lobular	2 (4%)
	Number of lesions
	1	36 (75%)
	2	9 (19%)
	3	3 (6%)
	Clinical T stage
	T1	2 (4%)
	T2	25 (52%)
	T3	17 (35%)
	T4 (inflammatory)	4 (9%)
	Nodal involvement
	Clinically involved -CB positive 13 (27%)
	Clinically negative -No CB	23 (48%)
	SLNB positive	4 (9%)
	SLNB negative	8 (17%)
	Grade
	1	0
	2	19 (40%)
	3	29 (60%)
	ER status
	Positive	33 (69%)
	Negative	15 (31%)
	HER2 status
	Positive	20 (42%)

Table 2 .

 2 Histopathological findings

	Characteristic	Patients (%)
	Pathological assessment
	pCR	11(23%)
	MRD	7(15%)
	Evidence of response	21(44%)
	Response cannot be classified	9(19%)
	Residual tumour size
	Invasive only	18.83mm (SE 3.22)
	Invasive + in situ	27.53mm (SE 3.54)
	pCR: pathological complete response; MRD: Minimal Residual Disease

Table 3 .

 3 Mean baseline and mid-treatment measurements by ultrasound and clinical examination

			Baseline (SE)	Mid-Treatment (SE)
	Clinical	examination	42.83mm (3.19)	Not available a
	(LD)
	Ultrasound LD	36.76mm ( SE 2.34)	24.79mm (SE 2.11)
	Ultrasound volume	6.43cm 3 (SE 1.08)	2.6cm 3 (SE 0.49)
	a Clinical examination results not consistently documented
	LD: Longest diameter

Table 4 .

 4 Ultrasound measurements at mid-treatment between responders and nonresponders

			Respon	Non-	Differen	95% CI of	P value
			ders	Responders	ce	the
			Mean	Mean		difference
			Response = pCR
			n=11	n=37
	LD (mm)	27.7	23.92	3.78	-10.34 -17.89 0.572
	Volume (cm 3 ) 2.95	2.49	0.47	-1.88 -2.81	0.691
	% LD change -33.4% -29.2%	-4.2%	-30.8 -22.5	0.754
	%	volume	-62.3% -47.5%	-14.8%	-50.9 -21.4	0.415
	change
			Response = pCR plus MRD
			n=18	n=30
	LD (mm)	24.02	25.25	-1.23	-10.07 -7.62 0.781
	Volume (cm 3 ) 2.09	2.9	-0.82	-2.84 -1.21	0.422
	% LD change -31.8% -29.3%	-2.5%	-25.7 -20.7	0.829
	%	volume	-59.7% -45.7%	-14.1%	-45.4 -17.3	0.371
	change

pCR: pathological complete response; LD: Longest Diameter; MRD: Minimal Residual Disease

Table 5 .

 5 Thresholds of ultrasound-assessed proportional changes and residual tumour sizes at mid-treatment, split by eventual pCR

		pCR No pCR %pCR %no pCR	p value
	LD reduction
	<30%	5	16	24%	76%	>0.99
	≥30%	6	21	22%	78%
	<50%	8	26	24%	76%	>0.99
	≥50%	3	11	21%	79%
	<75%	10	35	22%	78%	0.551
	≥75%	1	2	33%	67%
	<100%	10	35	22%	78%	0.551
	100%	1	2	33%	67%
	Volume reduction
	<50%	4	14	22%	78%	>0.99
	≥50%	7	23	23%	77%
	<65%	5	19	21%	79%	>0.99
	≥65%	6	18	25%	75%
	<75%	6	24	20%	80%	0.724
	≥75%	5	13	28%	72%
	<90%	9	33	21%	79%	0.609
	≥90%	2	4	33%	67%
	<100%	10	35	22%	78%	0.551
	100%	1	2	33%	67%
	Residual LD
	≤10mm	2	4	33%	67%	0.609
	>10mm	9	33	21%	79%

pCR: pathological complete response; LD: longest diameter

Table 6 .

 6 Thresholds of ultrasound-assessed proportional changes and residual tumour sizes at mid-treatment, split by eventual pCR or MRD

		pCR/	No pCR	%pCR	%no pCR	p value
		MRD	/MRD	/MRD	/MRD
	LD reduction
	<30%	7	14	33%	67%	0.765
	≥30%	11	16	41%	59%
	<50%	13	21	38%	62%	>0.99
	≥50%	5	9	36%	64%
	<75%	16	29	36%	64%	0.547
	≥75%	2	1	67%	33%
	<100%	16	29	36%	64%	0.547
	100%	2	1	67%	33%
	Volume reduction
	<50%	7	11	39%	61%	>0.99
	≥50%	11	19	37%	63%
	<65%	8	16	33%	67%	0.766
	≥65%	10	14	42%	58%
	<75%	11	19	37%	63%	>0.99
	≥75%	7	11	39%	61%
	<90%	15	27	36%	64%	0.658
	≥90%	3	3	50%	50%
	<100%	16	29	36%	64%	0.547
	100%	2	1	67%	33%
	Residual LD
	≤10mm	3	3	50%	50%	0.658
	>10mm	15	27	36%	64%

pCR: pathological complete response; MRD: minimal residual disease; LD: longest diameter; vol: volume

Table 7 .

 7 Univariate logistic regression for prediction of pCR or MRD at mid-treatment

	Odds Ratio 95% C.I.	P-value

Table 8 .

 8 Multivariate logistic regression for prediction of pCR or MRD at midtreatment

	Odds Ratio	95% C.I.	P-value

Table 9 .

 9 Correlation between ultrasound and pathological longest tumour diameter

	Study	Number of	Year of	Correlation	Ultrasound
		patients	Publication	US-	Accuracy
				pathology c	(limit) c
	Akazawa et al [26]	38	2006	0.48	64.9% (2cm)
	Balu-Maestro et al [27] 51	2002		43%
	Chapgar et al [10]	189	2006	0.42	75% (1cm)
	Fiorentino et al [21]	141	2001	0.29
	Herrada et al [28]	100	1997	0.60
	Keune et al [24]	104	2010		59.6% (1cm)
	Londero et al [29]	16	2004	0.57
	Peintinger et al [30] a	162	2006	0.66	82.7% (1cm)
	Segara et al [31] Singh et al [32]	51 25	2007 2005	0.61 0.69-0.81 b	66% (1cm)
	Forouhi et al [33]	35	1994	0.94
	Akashi-Tanaka et al	57	2001	0.56
	[34]

a Combined assessment of mammography and ultrasound b Colour Doppler usedvarious indexes with different correlation coefficients reported c

pCR: pathological complete response; MRD: minimal residual disease; LD: longest diameter; vol: volume; ER: oestrogen receptor

ACKNOWLEDGEMENTS

We would like to thank Drs Graham Treece, Richard Prager and Andrew Gee at the Department of Engineering, University of Cambridge who set up the 3D US imaging system and the whole Medical Imaging Group at the department for loaning of the equipment and providing technical support. We would also like to thank Miss Natalie Hicks for assistance with data input. EP, PB, HME and RS are supported with research funding from the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre (BMRC).

CONFLICTS OF INTEREST

The authors declare no relevant conflicts of interest