Evangelia Razis
email: e.razis@hygeia.gr.

M Bobos

V Kotoula

A G Eleftheraki

H P Kalofonos

K Pavlakis

P Papakostas

G Aravantinos

G Rigakos

I Efstratiou

K Petraki

D Bafaloukos

I Kostopoulos

D Pectasides

K T Kalogeras

D Skarlos

G Fountzilas

Evaluation of the association of PIK3CA mutations and PTEN loss with efficacy of trastuzumab therapy in metastatic breast cancer

Keywords: breast cancer, HER2, trastuzumab, predictive, PTEN, PIK3CA

Evaluation of the association of PIK3CA mutations and

PTEN loss with efficacy of trastuzumab therapy in metastatic breast cancer

Introduction

The cell surface growth factor receptor HER2 is over-expressed in 15-20% of breast tumors usually due to HER2 gene amplification. This tumor characteristic is associated with worse prognosis and increased rates of disease recurrence [START_REF] Slamon | Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene[END_REF]. In the last decade the use of the humanized monoclonal antibody, trastuzumab, that selectively targets the extracellular domain of the HER2 receptor, has proved to significantly improve objective response, time to disease progression and overall survival in patients with metastatic HER2 over-expressing and/or amplified tumors [START_REF] Slamon | Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2[END_REF]. Unfortunately, a significant number of patients with HER2 amplified metastatic disease never respond to trastuzumab treatment, while resistance to treatment develops in the majority of responding patients within the first year of treatment [START_REF] Vogel | Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer[END_REF][START_REF] Marty | Randomized phase II trial of the efficacy and safety of trastuzumab combined with docetaxel in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer administered as first-line treatment: the M77001 study group[END_REF].

Notably, it is relatively easy to rule out patients that are unlikely to benefit from trastuzumab treatment by evaluating HER2 overexpression or HER2 amplification, while on the other hand beneficial predictive factors to further separate HER2 positive patients to those who will benefit from trastuzumab from those who will not, have not been integrated into clinical practice.

Understanding the mechanisms of primary or secondary resistance to trastuzumab treatment is critical to predicting trastuzumab efficacy. Activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway has been reported to confer resistance to trastuzumab treatment in breast tumors [START_REF] Nahta | Mechanisms of disease: understanding resistance to HER2-targeted therapy in human breast cancer[END_REF]. Oncogenic mutations of PIK3CA and low expression or loss of the phosphatase and tensine homologue gene (PTEN), that antagonizes the action of PIK3CA, have both been described as factors that activate the PI3K/AKT pathway and contribute to trastuzumab resistance in breast tumors [START_REF] Berns | A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer[END_REF][START_REF] Nagata | PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients[END_REF]. Investigation in cell lines and clinical studies have suggested the possible use of the PIK3CA and PTEN status as predictive factors of trastuzumab efficacy in breast cancer [START_REF] Nagata | PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients[END_REF][START_REF] Fujita | PTEN activity could be a predictive marker of trastuzumab efficacy in the treatment of ErbB2-overexpressing breast cancer[END_REF][START_REF] Fabi | Clinical significance of PTEN and p-Akt co-expression in HER2positive metastatic breast cancer patients treated with trastuzumab-based therapies[END_REF][START_REF] Gori | EGFR, pMAPK, pAkt and PTEN status by immunohistochemistry: correlation with clinical outcome in HER2-positive metastatic breast cancer patients treated with trastuzumab[END_REF][START_REF] Pandolfi | Breast cancer--loss of PTEN predicts resistance to treatment[END_REF] and in one study PIK3CA mutations were found to be mutually exclusive with PTEN loss [START_REF] Saal | PIK3CA mutations correlate with hormone receptors, node metastasis, and ERBB2, and are mutually exclusive with PTEN loss in human breast carcinoma[END_REF]. In the present study we analyzed samples from patients with presumed HER2-positive breast tumors that were treated with trastuzumab, after local evaluation for HER2 by IHC. We then evaluated HER2, PTEN and PIK3CA status as well as their correlation with patient outcome.

Patients, Materials and Methods

The medical records of all patients with metastatic breast cancer (MBC) treated with trastuzumab-based combinations, between December 1998 and January 2010, were retrospectively reviewed. Eligibility criteria for this study were a: histologically All tumor samples were re-examined centrally for HER2 status by fluorescent in situ hybridization (FISH), or by IHC when FISH could not be performed. Additionally, estrogen receptor (ER), progesterone receptor (PgR) and Ki67 status were examined centrally by IHC.

For the purpose of this study Ki67, PTEN and PIK3CA were evaluated using the methods outlined below and patient outcome was correlated with the biomarkers in accordance with the latest guidelines concerning tumor marker prognostic studies (REMARK) [START_REF] Mcshane | REporting recommendations for tumor MARKer prognostic studies (REMARK)[END_REF].

Tissue micro-arrays (TMAs) FFPE tumor tissue samples were retrospectively collected from 256 patients with MBC treated with trastuzumab. Nineteen cases were excluded for inadequate FFPE tumor tissue and 10 for being treated with trastuzumab in the neoadjuvant setting, thus decreasing the number of eligible/evaluable patients to 227. Seventeen TMA blocks were constructed using a manual tissue microarrayer (Beecher Instruments, Sun Prairie, WI). For the construction of the TMA blocks, 2 core samples (1.5 mm in diameter) were obtained from representative regions of each of the tumor blocks. All markers were assessed by FISH or IHC, in sections cut from the TMA blocks or in whole tissue sections from the original blocks.

Immunohistochemistry (IHC)

TMA sections or whole tissue sections (3 μm thick) were stained for vimentin (clone V9, Dako, Glostrup, Denmark), cytokeratin 8/18 (clone 5D3, Leica Biosystems, Newcastle, U.K.), ER (clone 6F11, Leica Biosystems), PgR (clone 1A6, Leica Biosystems), HER2 (A0482 polyclonal Ab, Dako), Ki67 (clone MIB-1, Dako), and PTEN (clone 6H2.1, Dako) as previously described [START_REF] Fountzilas | Gemcitabine combined with gefitinib in patients with inoperable or metastatic pancreatic cancer: a phase II Study of the Hellenic Cooperative Oncology Group with biomarker evaluation[END_REF][START_REF] Gogas | Paclitaxel and carboplatin as neoadjuvant chemotherapy in patients with locally advanced breast cancer: A phase II Trial of the Hellenic Cooperative Oncology Group[END_REF][START_REF] Bamias | A randomized phase III study of adjuvant platinum/docetaxel chemotherapy with or without radiation therapy in patients with gastric cancer[END_REF]. The evaluation of all IHC sections was done by pathologists, experienced in breast cancer and blinded as to the patients' clinical characteristics and survival data.

Interpretation of the IHC results

The ER and PgR immunostaining was scored using the Histoscore method. Tumors were classified as ER-or PgR-positive if staining was present in 1% or more of tumor nuclei [START_REF] Hugh | Breast cancer subtypes and response to docetaxel in node-positive breast cancer: use of an immunohistochemical definition in the BCIRG 001 trial[END_REF]. HER2 protein expression was scored according to the recent guideline recommendations (scores from 0 to 3+) [START_REF] Wolff | American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer[END_REF]. PTEN protein expression (cytoplasmic, nuclear or both) was evaluated according to a staining intensity scale from 0 (negative, no staining) to 3 (intense staining). Tumors with PTEN scores of 0 or 1 were considered to have PTEN loss. For Ki67, the expression was defined as low (<14%) and high (≥14%) based on the percentage of stained/unstained nuclei from the tumor areas [START_REF] Cheang | Ki67 index, HER2 status, and prognosis of patients with luminal B breast cancer[END_REF].

Fluorescence in situ hybridization (FISH)

TMA sections or whole tissue sections (5 μm thick) were used for FISH analysis using the ZytoLight® SPEC HER2/TOP2A/CEN17 triple color probe kit (ZytoVision, Bremerhaven, Germany) and the LSI PTEN/CEP10 Dual Color Probe (Abbott Molecular, Des Plaines, IL). For all probes, sequential (5 planes at 1.0 μm) digital images were captured using the Plan Apo VC x100/1.40 objective (Nikon, Japan) using specific filters for each probe. The resulting images were reconstructed using specially developed software for cytogenetics (XCyto-Gen, ALPHELYS, Plaisir, France). Four carcinoma cell lines (MDA-MB-231, MDA-MB-175, MDA-MB-453 and SK-BR-3) from the Oracle HER2 Control Slide (Leica) with a known HER2 gene status were also used as a control of the FISH assays and analyzed for HER2, topoisomerase II alpha and PTEN genomic status.

FISH evaluation

For the evaluation of HER2 gene status, 20 non-overlapping nuclei from the invasive part of the tumor were selected randomly, according to morphological criteria using DAPI staining, and scored. The HER2 gene was considered amplified when the ratio of the respective gene probe/centromere probe was ≥2.2 [START_REF] Wolff | American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer[END_REF] and deleted when the ratio was ≤0.75. In cases at or near the cut-off (1.8-2.2 for amplifications and 0.6-0.9 for deletions), additional 20 to 40 nuclei were counted and the ratio was recalculated.

In cases with a borderline ratio from 60 nuclei, an additional FISH assay was performed in a whole section. For the PTEN gene, sixty non-overlapping nuclei from the invasive part of the tumor were randomly selected and scored. Deletion was considered to occur when the ratio of the gene probe/centromere probe was <0.80 [START_REF] Christodoulou | Trastuzumab combined with pegylated liposomal doxorubicin in patients with metastatic breast cancer. phase II Study of the Hellenic Cooperative Oncology Group (HeCOG) with biomarker evaluation[END_REF].

Single nucleotide polymorphism (SNP) genotyping for PIK3CA mutations DNA was extracted from 182 FFPE whole tissue sections or macrodissected tissue fragments containing >70% tumor cells, using a fully automated isolation method based on silica-coated magnetic beads (Versant Tissue Preparation Reagents, Siemens Healthcare Diagnostics, Tarrytown, NY) in combination with a liquid handling robot, as previously described [START_REF] Bohmann | RNA extraction from archival formalin-fixed paraffin-embedded tissue: a comparison of manual, semiautomated, and fully automated purification methods[END_REF]. Mutation testing for PIK3CA E542K and E545K (exon 9) and H1047R (exon 20) was accomplished with custom Taqman-MGB-SNP genotyping assays (duplex qRT-PCR for the detection of control DNA and mutant target in the same reaction). Samples were normalized at 50ng/10ul reaction and were run in duplicates under default conditions for Allelic Discrimination in an ABI7500 sequence detection system equipped with the SDS v1.4 software (Applied Biosystems). Sequencing validation was performed in selected cases (n=4 for each assay).

Statistical Analysis

Data on selected patient and tumor characteristics, previous and subsequent lines of treatment, disease progression events and survival were obtained from medical records. Data were entered into a central database, and follow-up information for all patients was updated in February 2010. All examined biomarkers are presented as frequency and corresponding percentages, while associations with HER2 status were examined using the chi-square test or Fisher's exact test where appropriate. The majority of patients received trastuzumab in the 1 st line of chemotherapy for metastatic disease and thus time to progression (TTP) was defined as the time from trastuzumab initiation in the 1 st line treatment (with or without concurrent chemo/hormonal therapy) to the date of documented disease progression. Survival was measured (a) from diagnosis of first metastasis in all patients (survivalM) or (b) from initiation of trastuzumab treatment in the patients receiving trastuzumab as a 1 st line treatment to the date of death (survivalT). Patients alive were censored at the date of the last follow-up contact. Survival probabilities were estimated by the Kaplan-Meier method. For the univariate and multivariate analyses, Cox proportional hazards models were used. Model choice was performed using backward selection criteria with p<0.10, including in the initial step clinical parameters such as: age, menopausal status, performance status, receptor status (ER/PgR), Ki67 expression, number of metastatic sites, and HER2 status. Univariate analyses were performed in the centrally identified HER2 groups and interaction tests for each marker and HER2 were performed. Multivariate analysis was performed in the total cohort and in the population of clinical interest, i.e. HER2-positive patients. All tests are two-sided at the α=0.05 level of significance. No adjustment for multiple comparisons was performed. Analyses were conducted using SPSS 15.0.

Results

Among the 256 registered patients, 227 were evaluated centrally for HER2 and 139 were found to have HER2 gene amplification by FISH and/or 3+ HER2 protein overexpression. We identified only one case with HER2 protein expression 3+ and non-amplified HER2 gene status. Eighty-eight patients had thus received trastuzumab-based therapy despite being HER2-negative, probably because of inexperience with HER2 IHC evaluation at the local laboratories in the early years of trastuzumab use. Selected patient and tumor characteristics of both groups are seen in Table 1. Median follow-up for all patients was 66 months, while for patients treated with trastuzumab in 1 st line, median follow-up was 51 months. 135 patients (59%) received more than one line of therapy with trastuzumab. 192 patients (85% of the 227 analyzed) were treated with trastuzumab in the 1 st line of therapy for metastatic disease and of those 186 received concurrent chemo/hormonal therapy. Six patients received trastuzumab monotherapy, while the rest received trastuzumab in combination with chemotherapy agents, such as taxanes (61%), platinum (12%), gemcitabine (18%), and vinorelbine (11%). In the entire cohort, one patient had been treated with lapatinib before trastuzumab, whereas 35 received lapatinib after trastuzumab. Nine patients had received trastuzumab in the adjuvant setting as well, 6 of them relapsing within a year and 2 within six months. Only 2 of those patients' tumors carried a PIK3CA mutation. Among the 192 patients treated in 1 st line, 151 (79%) progressed and 108 (56%) died. Median TTP was 13.7 months (95% Confidence Interval [CI] 10.9-16.5), while median survivalT was 42 months (95% CI 34.3-49.5). Of 227 patients analyzed, 138 (61%) died (median survivalM 47.5 months, 95% CI 40.9-54.1). HER2-positive patients had significantly longer survival than HER2-negative patients (median survivalM 39.1 months in HER2-negative vs 54.1 months in HER2-positive, p=0.036; median survivalT 35.3 in HER2-negative vs 50.4 months in HER2-positive, p=0.006), but TTP of those who received trastuzumab as 1 st line treatment was not different between the two groups (10.3 months in HER2negative vs 14.4 in HER2-positive, p=0.22). This is probably because in 1 st line treatment a strong chemotherapy effect seen in both groups, dilutes any possible trastuzumab effect.

Association of markers with HER2 status ER positivity was seen more frequently in HER2-negative patients (82% in HER2negative vs. 59% in HER2-positive, p=0.001). PTEN loss by IHC was more common in HER2-negative patients (65% vs 50% in HER2-positive, p=0.048), as were PIK3CA mutations (30% vs 16% in HER2-positive, p=0.039) (Table 2). PIK3CA mutations were more common in ER-positive patients (27% in ER-positive vs 11% in ER-negative, p=0.017), while PTEN loss was more frequent in low Ki67 tumors (90% in low Ki67 vs 53% in high Ki67, p=0.024).

PTEN loss and PIK3CA mutation are thought to be mutually exclusive [START_REF] Saal | PIK3CA mutations correlate with hormone receptors[END_REF]. However, we identified 17 cases (among 153 tumors evaluated by both methods) where PIK3CA was mutated in the presence of PTEN loss, 10 of which were HER2negative. PTEN status was also evaluated by FISH, and was not found to correlate with PTEN status by IHC.

Association of markers with clinical outcome

In the univariate analysis performed in the HER2-positive patients, PIK3CA mutations were associated with increased risk of progression (Hazard Ratio

[HR]=2.50, 95% CI 1.35-4.61, Wald-p=0.003) (Figure 1), but not with survival (survivalT, Wald-p=0.27; survivaM, Wald-p=0.29). PTEN loss was associated with increased risk of death (HR=1.92, 95% CI 1.11-3.31, Wald-p=0.019; HR=1.73, 95% CI 1.04-2.89, Wald-p=0.035) (Figure 2), but not with TTP (Wald-p=0.16). Of note, PTEN status by FISH was not associated with TTP (Wald-p=0.66) or survival (Wald-p=0.51; Wald-p=0.91) in the univariate analysis. In the HER2-negative patients there was no significant association of PIK3CA mutations or PTEN loss with TTP, survivalT or survivalM. Tests for interaction of PIK3CA mutations or PTEN protein expression with HER2 status were not significant (Wald-p>0.05 for all survival parameters).

In the multivariate analysis performed in the entire population (Table 3), HER2positive status was associated with a lower risk of disease progression (HR=0.60, 95% CI 0.38-0.95, Wald-p=0.028) and PIK3CA mutations with a higher risk of progression (HR=1.86, 95% CI 1.13-3.05, Wald-p=0.014). The association of risk of relapse with PTEN status did not reach statistical significance (HR=0.67, 95% CI 0.44-1.02, Wald-p=0.062). In terms of survival, PIK3CA mutation status was not associated with risk of death. However, PTEN loss was associated with a higher risk of death (HR=1.85, 95% CI 1.24-2.76, Wald-p=0.003 for survivalM; HR=1.64, 95% CI 1.05-2.57, Wald-p=0.029 for survivalT). Survival from the initiation of trastuzumab was associated with HER2 status (HER2-positive vs negative, HR=0.51, Wald-p=0.004) and adjuvant chemotherapy use (HR=1.64, Wald-p=0.038), whereas survival measured from either time point (survivalT vs survivalM) was associated with the number of metastatic sites (HR=1.71, Wald-p=0.029 and HR=1.56, Wald-p=0.036, respectively) and patients' performance status (HR=2.31 and HR=2.51, respectively, both Wald-p<0.001).

We also performed a multivariate analysis on the HER2-positive population, since this is ultimately the clinically relevant population (final model not shown). In this analysis, PIK3CA mutations were the only parameter significantly associated with worse TTP (HR=2.50, 95% CI 1.35-4.61, Wald-p=0.003). In terms of survival, poor performance status was associated with worse survivalM (HR=1.99, 95% CI 1.17-3.42, Wald-p=0.012). Increased number of metastatic sites at trastuzumab initiation was associated with worse survivalT (HR=2.20, 95% CI 1.15-4.23, Wald-p=0.017) but not significantly so with survivalM, while PTEN loss with shorter survival, as measured from either time point (HR=1.92, Wald-p=0.017, survivalT; HR=1.69, Wald-p=0.047, survivalM).

PIK3CA mutations and/or PTEN loss were also evaluated together, as a single parameter, in the multivariate analysis, to estimate the impact of the activation of the molecular pathway. In the HER2-positive population, it was found to be significantly associated with both worse TTP (HR=2.16, 95% CI 1.27-3.66, Wald-p=0.004) and survival (HR=2.12, Wald-p=0.041, survivalT; HR=2.51, Wald-p=0.006, survivalM) (Table 4). Kaplan-Meier curves for PIK3CA mutations and/or PTEN loss, as one variable, in terms of TTP and survivalT are given in Figure 3. Results were similar in the whole population.

Discussion

Our study is among the first to evaluate both PIK3CA mutations and PTEN protein expression status in trastuzumab-treated patients, and it confirms the results of previous investigators that these two markers are involved in the development of resistance to trastuzumab [START_REF] Nahta | Mechanisms of disease: understanding resistance to HER2-targeted therapy in human breast cancer[END_REF][START_REF] Berns | A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer[END_REF][START_REF] Nagata | PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients[END_REF][START_REF] Fujita | PTEN activity could be a predictive marker of trastuzumab efficacy in the treatment of ErbB2-overexpressing breast cancer[END_REF][START_REF] Fabi | Clinical significance of PTEN and p-Akt co-expression in HER2positive metastatic breast cancer patients treated with trastuzumab-based therapies[END_REF][START_REF] Gori | EGFR, pMAPK, pAkt and PTEN status by immunohistochemistry: correlation with clinical outcome in HER2-positive metastatic breast cancer patients treated with trastuzumab[END_REF][START_REF] Pandolfi | Breast cancer--loss of PTEN predicts resistance to treatment[END_REF][START_REF] Saal | PIK3CA mutations correlate with hormone receptors, node metastasis, and ERBB2, and are mutually exclusive with PTEN loss in human breast carcinoma[END_REF]23,24].

PTEN loss was associated with shorter survival, whether measured from first development of metastasis or from trastuzumab initiation, both in the entire population and, more importantly, in HER2-positive patients. Additionally, there is a trend for longer TTP in total population without PTEN loss that doesn't quite reach statistical significance. These findings are consistent with the prognostic role of PTEN in HER2-positive, trastuzumab-treated patients. On the other hand the effect of PIK3CA activating mutations was mostly an effect on the immediate action of trastuzumab and was thus seen as a prolongation of TTP. Additionally, PIK3CA mutations appear to correlate with good prognostic indicators, such as ER and PgR expression [START_REF] Saal | PIK3CA mutations correlate with hormone receptors, node metastasis, and ERBB2, and are mutually exclusive with PTEN loss in human breast carcinoma[END_REF][START_REF] Christodoulou | Trastuzumab combined with pegylated liposomal doxorubicin in patients with metastatic breast cancer. phase II Study of the Hellenic Cooperative Oncology Group (HeCOG) with biomarker evaluation[END_REF]. Another interesting finding is the difference in the "areas of influence" of PI3K and PTEN. Why should PI3K, though it is a critical molecule in the AKT pathway, influence TTP more than survival and PTEN the reverse, is something that needs to be explored further. Could very small, statistically insignificant, incremental benefits in TTP, in several lines of treatment with trastuzumab, lead to a cumulative survival gain?

Clearly, what is most relevant is the activation of the entire pathway. Hence the combined analysis of both markers yielded a strong association with both survival and TTP, thus confirming the significance of this pathway in trastuzumab resistance.

A recent study evaluated the clinical significance of PTEN and pAKT in HER2positive disease and found that when both PTEN and pAKT are overexpressed patients enjoy longer progression-free survival (PFS) on trastuzumab [START_REF] Fabi | Clinical significance of PTEN and p-Akt co-expression in HER2positive metastatic breast cancer patients treated with trastuzumab-based therapies[END_REF]. In this prospective 1 st line trastuzumab/chemotherapy study, PI3K was also evaluated by IHC, but its overexpression did not seem to be associated with PFS. The authors, however, note that IHC is probably not the optimal way to assess PI3K activity and that PIK3CA mutations are much more relevant in this setting [START_REF] Fabi | Clinical significance of PTEN and p-Akt co-expression in HER2positive metastatic breast cancer patients treated with trastuzumab-based therapies[END_REF]. PTEN status on the other hand is best evaluated by IHC.

Additional interest in our study derives from the evaluation of the HER2-negative, trastuzumab-treated patients. In this subgroup of patients, survival from the initiation of trastuzumab and from first diagnosis of metastatic disease was shorter than in the HER2-positive patients. This finding underlines the dramatic effect of trastuzumab on the survival of HER2-positive patients, who without trastuzumab, would be expected to have more aggressive disease and worse prognosis [START_REF] Slamon | Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene[END_REF].

In HER2-negative patients the effect of treatment was not influenced by the presence of PIK3CA mutations and/or PTEN loss. It is particularly interesting to examine this HER2-negative subgroup, since it is unlikely that one will ever come across such a population in the future, as HER2 testing methods have improved and cost regulation and established treatment guidelines would prevent the use of trastuzumab in HER2negative patients. However, one could have postulated that in the setting of wild type PIK3CA or PTEN overexpression, trastuzumab could have exerted a therapeutic effect in a "HER2-borderline" population, or even that trastuzumab could have an effect that would possibly be unrelated to HER2 and more dependent on downstream molecules. There was a recent review suggesting that, as HER2 positivity is more significant in stem cells, in the adjuvant setting, trastuzumab may be active in HER2 1+ or 2+ cases, as well [25]. Our results however, render such hypotheses unlikely, at least in the metastatic setting, and reinforce the importance of HER2 overexpression or amplification in predicting benefit from trastuzumab.

Our study is fraught with the disadvantages of all retrospective analyses, especially those that come from a non-trial population. Our results should thus be considered as hypothesis generating, while results of prospective studies, such as the NEO-ALTO and ALTO studies, are eagerly awaited to clarify the field. Meanwhile the scientific and clinical community has to establish optimal ways to evaluate PI3K and PTEN expression and standardize the methods and cut-offs.

In conclusion, the efficacy of trastuzumab therapy in patients with HER2-positive MBC is highly dependent on the activation of the PI3K

  confirmed MBC, b: adequacy of clinical data on patient's history, demographics, tumor characteristics, treatment details (drug dosages, schedule of administration, serious toxicities), and clinical outcome, c: availability of adequate formalin-fixed paraffin-embedded (FFPE) tumor tissue for biological marker evaluation, and d: trastuzumab-based treatment for metastatic disease. The translational research protocol was approved by the Bioethics Committee of the Aristotle University of Thessaloniki School of Medicine (4283; Jan 14 2008) under the general title "Investigation of major mechanisms of resistance to treatment with trastuzumab in patients with metastatic breast cancer". Waiver of consent was obtained from the Bioethics Committee for patients included in the study before 2005. All patients included in the study after 2005, provided written informed consent for the provision of biological material for future research studies, before receiving any treatment. We evaluated clinical data and tumor samples from patients with MBC, and what was thought to be HER2-positive disease by locally performed immunohistochemistry (IHC), that were treated off protocol with trastuzumab, with or without chemotherapy.

Table 1 .

 1 /AKT pathway, induced by activating PIK3CA mutations or PTEN loss. Further studies are necessary to validate the predictive value of PIK3CA mutations and PTEN loss especially in light of the new therapies targeting the PI3K/AKT pathway. Selected patient and tumor characteristics (at trastuzumab initiation) according to HER2 status (HER2-positive: HER2-amplified and/or HER2 IHC 3+)

	node metastasis, and
	ERBB2,	and	are
	mutually	exclusive
	with PTEN loss in
	human	breast
	carcinoma.	Cancer
	research 65 (7):2554-
			2559.
	doi:10.1158/0008-
	5472-CAN-04-3913
	23. Perez-Tenorio G,
	Alkhori L, Olsson B,
	Waltersson	MA,
	Nordenskjold	B,
	Rutqvist LE, Skoog L,
	Stal O (2007) PIK3CA
	mutations and PTEN
	loss correlate with
	similar	prognostic
	factors and are not
	mutually exclusive in
	breast cancer. Clin
	Cancer	Res	13
		(12):3577-3584.
	doi:13/12/3577 [pii]
		10.1158/1078-
	0432.CCR-06-1609
	24. Pohlmann PR,
	Mayer IA, Mernaugh
	R (2009) Resistance to
	Trastuzumab in Breast
	Cancer. Clin Cancer
	Res 15 (24):7479-
	7491. doi:15/24/7479
	[pii]
		10.1158/1078-
	0432.CCR-09-0636
	25.	Kakarala	M,
	Wicha MS (2008)
	Implications of the
	cancer		stem-cell
	hypothesis for breast
	cancer prevention and
	therapy. J Clin Oncol
	26	(17):2813-2820.
	doi:26/17/2813 [pii]
	10.1200/JCO.2008.16.
	3931

Table 2 .

 2 Association of examined markers with HER2 status

		HER2 status
	Negative			Positive
	N	%	N	%	Fisher's exact p

Table 3 .

 3 Multivariate Cox regression analysis in the whole population treated with trastuzumab (T)

		TTP			SurvivalT			Surviva
	HR	95% CI	Wald-p	HR	95% CI	Wald-p	HR	95% C
	1			1			1
	0.60	0.38-0.95	0.028	0.51	0.32-0.80	0.004	0.73	0.49-1.0
	1
	0.67	0.42-1.06	0.086
	0.98	0.97-1.00	0.084
	us at T initiation
				1			1
				2.32	1.47-3.65	<0.001	2.51	1.66-3.7
	tatic sites at T
				1			1
				1.71	1.06-2.78	0.029	1.56	1.03-2.3
				1
				1.64	1.03-2.61	0.038
	1
	1.86	1.13-3.05	0.014
	1			1			1
	0.67	0.44-1.02	0.062	0.61	0.39-0.95	0.029	0.54	0.36-0.8

, chemotherapy; SurvivalT and SurvivalM, See text

Table 4 .

 4 Multivariate Cox regression analysis in the HER2-positive population : When the PTEN and PIK3CA parameters were entered seperately in the analysis, PIK3CA mutations was the only parameter significantly associated with worse TTP (HR=2.50, 95% CI 1.35-4.61, p=0.003), while PTEN loss was associated with shorter survival as measured from either time point (HR=1.92, p=0.017, survivalT; HR=1.69, p=0.047,

		TTP			SurvivalT
	HR	95% CI	Wald-p	HR	95% CI	Wald-p

Note

T, trastuzumab; SurvivalT and SurvivalM, See text

p=0,018; CT, chemotherapy; HT, hormonal therapy; RT, radiation therapy

Acknowledgements

We would like to thank Ms. Thalia Spinari for tissue collection, Ms. Dimitra Katsala for monitoring the study, Ms. Maria Moschoni for coordinating the data management, and Ms. Stella Dallidou for secretarial assistance.

The study was supported by a Hellenic Cooperative Oncology Group grant HER_11P/05.


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Oxidative stress in fungal fermentation processes: the roles of alternative respiration

Keywords: alternative NADH dehydrogenases, alternative oxidase, alternative respiratory pathway, fungi, metabolism, oxidative stress

Filamentous fungi are arguably the most industrially important group of microorganisms.

Production processes involving these simple eukaryotes are often highly aerobic in nature, which implies these cultures are routinely subject to oxidative stress. Despite this, little is known about how filamentous fungi cope with high levels of oxidative stress as experienced in fermenter systems. More surprisingly, much of our knowledge of oxidative stress responses in fungi comes from environmental or medical studies. Here, the current understanding of oxidative stress effects and cellular responses in filamentous fungi is critically discussed. In particular the role of alternative respiration is evaluated, and the contributions of the alternative oxidase and alternative dehydrogenases in defence against oxidative stress, and their profound influence on fungal metabolism is critically examined. Finally, the importance of further research which would underpin a less empirical approach to optimising fungal strains for the fermenter environment is emphasised.

Introduction

Fungi, which consist of both the yeasts and the filamentous fungi, are among the most widely used microorganisms in traditional and advanced bio-industries. In the past decade, novel yeast strains and filamentous fungi have been progressively invading the field of bio-pharmaceutical protein expression dominated by Escherichia coli and mammalian cell expression systems (Sodoyer 2004).

In terms of synthesis of heterologous proteins, filamentous fungi have a complex posttranslational protein modification system, unlike the simpler bacterial systems which tend to be used for production of non-glycosylated proteins [START_REF] Schmidt | Recombinant expression systems in the pharmaceutical industry[END_REF]). The fungal species preferred for recombinant protein expression are those widely employed in traditional processes for manufacturing enzymes, acids and antibiotics, and these include species from a few key genera including Aspergillus, Penicillium, Acremonium, Fusarium and Trichoderma. Since filamentous fungi typically obtain the nutrients they require by secretion of digestive enzymes and subsequent absorption of the digested materials, the innate protein productivity and secretion potential of these filamentous fungal hosts are often far superior to other expression systems.

However, even some yeasts, in particular Saccharomyces cerevisiae and Pichia pastoris, have been used successfully to express a multitude of recombinant proteins, such as Fab (fragment antibody) and ScFv (single chain variable fragment) therapeutic antibodies [START_REF] Schoonooghe | Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris[END_REF][START_REF] Yamawaki | Production of single-chain variable fragment antibody (scFv) in fed-batch and continuous culture of Pichia pastoris by two different methanol feeding methods[END_REF]) and protein vaccines [START_REF] Avril | Optimizing expression of the pregnancy malaria vaccine candidate, VAR2CSA in Pichia pastoris[END_REF]), since they posses many of the bioprocessing advantages of E. coli. For example, short doubling times and readily manipulated genomes, but also have the additional benefits of eukaryotes such as improved protein folding and many post-translational modifications [START_REF] Durocher | Expression systems for therapeutic glycoprotein production[END_REF]Yurimoto & Sakai 2009). Furthermore, filamentous fungi and yeasts are more robust than mammalian cells and can produce some enzymes in excess of 100g/l in fermenters [START_REF] Cherry | Directed evolution of industrial enzymes: an update[END_REF] and are easily adapted to fermentation processes in simple and cost-effective culture media [START_REF] Gasser | Antibody production with yeasts and filamentous fungi: on the road to large scale?[END_REF][START_REF] Durocher | Expression systems for therapeutic glycoprotein production[END_REF]. Even though filamentous fungi and yeasts have now been used industrially for decades, and are now used for production of some of our most complex and potent drugs, they are still not adequately characterized at the physiological and genetic level, particularly in terms of protein modification and secretion metabolism [START_REF] Durocher | Expression systems for therapeutic glycoprotein production[END_REF] and especially in relation to their physiological responses to the stressful growing environment in bioreactors [START_REF] Li | Oxidative stress in industrial fungi[END_REF]Bai et al. 2003a).

The term "stress" is widely used, and is somewhat controversial, but it is also very difficult to define in objective terms. "Oxidative stress" is a very frequently encountered term, so at the outset this term should be clearly defined. Oxidative stress can be simply defined as a state where there is a relative imbalance within cells between generation of reactive oxygen species (ROS) and the intracellular anti-oxidant defences in favour of the former [START_REF] Li | Oxidative stress in industrial fungi[END_REF]. Most fungal processes are highly aerobic in nature, and thus sufficient O 2 supply and the maintenance of O 2 transfer rate in the bioreactor is always a challenge during production processes, and especially in those processes involving high cell density. As a solution, the use of O 2 -enriched air is frequently applied in many industrial fungal processes (Bai et al. 2003a). However, O 2 can be a two-sided sword for microbial cells inside bioreactors, mainly due to the generation of ROS, which is of particular relevance to the cultivation of industrial fungi. A clear understanding of how fungal cells cope with the challenges of toxic ROS is, thus, critical for improved insight into fungal physiology. To a large extent, this is consistent with the regulatory view that we should seek to better understand our bio-manufacturing processes. Unfortunately our knowledge on many aspects of oxidative stress in fungal processes is still deficient in many areas. To fully exploit the potential of filamentous fungi and yeast, their physiology and particularly the mechanisms of defensive responses to such a highly aerobic growing environment has to be investigated and clarified. Recent advances in this important field are discussed here, with an emphasis on the implications of the findings of these studies to fungal bio-processing.

Oxidative stress is the unavoidable consequence of an aerobic lifestyle. We, as biotechnologists, operate fungal bioproduction processes with very high O 2 transfer rates, and occasionally O 2 enrichment in order to achieve high productivities. Thus, as an industry we take for granted a set of conditions which impose, at some process time points, intense oxidative stress on the fungal production and expression systems we employ. Despite this, it is astounding that we have spent little time or effort trying to understand clearly how the fungi we use in bioreactors deal with the conditions we impose. This leaves us in the realm of empiricism in process definition and operation. Much of what knowledge we possess about fungal defence against oxidative stress comes not from fermenter studies, but from environmental and medical mycology, and often uses relatively convenient chemical stressors, e.g. simple addition of exogenous hydrogen peroxide to cultures, to simulate actual oxygen induced stress (as in fermenters), thus, the relevance to aerobic fermentations of fungi is questionable. Our first need is, therefore, for more carefully though-out studies focused upon fungal bioproduction systems and the process consequences of oxidative stress.

General responses to oxidative stress in fungi

There is some evidence suggesting that oxidative stress imposes significant effects on fungal cultures in bioproduction systems, in terms of morphology, growth rate, metabolism, and protein secretion. This review will briefly summarise the findings of such studies and then discuss an increasingly important but generally neglected area, i.e. the role of alternative respiratory enzymes in oxidative stress. For more comprehensive discussions, readers are encouraged to refer to a recent review by our group [START_REF] Li | Oxidative stress in industrial fungi[END_REF].

The effects of oxidative stress on fungal morphology

Filamentous fungi exhibit two extreme types of morphology in submerged culture, pelleted and filamentous forms. Gassing batch cultures of A. niger with O 2 -enriched air decreases the size of hyphal elements and increases the proportion of active cytoplasm, but hyphal adhesion occurs at the highest level of O 2 enrichment (50% v/v) [START_REF] Wongwicharn | Effect of oxygen enrichment on morphology, growth, and heterologous protein production in chemostat cultures of Aspergillus niger B1-D[END_REF]). Similar results have been found where O 2 enrichment results in branched and shorter hyphae [START_REF] Kreiner | Morphological and enzymatic responses of a recombinant Aspergillus niger to oxidative stressors in chemostat cultures[END_REF][START_REF] Bai | Effects of oxidative stress on production of heterologous and native protein, and culture morphology in batch and chemostat cultures of Aspergillus niger B1-D[END_REF]). Addition of exogenous H 2 O 2 leads to a decrease of the hyphal growth unit [START_REF] Kreiner | Morphological and enzymatic responses of a recombinant Aspergillus niger to oxidative stressors in chemostat cultures[END_REF]. In yeast, a study showed morphological changes from unicellular to pseudomycelium in the yeast Ustilago maydis when treated with a pro-oxidant compound, octyl gallate (Sierra-Campos et al. 2009). Substantial evidence therefore exists to indicate that morphological change may be a direct consequence of oxidatively stressful conditions in the fungi.

The effects of oxidative stress on fungal growth rates and metabolism

Oxidative stress can be provoked by addition of exogenous chemical stressors (simulated) or by highly aerobic conditions within a fermenter system, for example, by oxygen enrichment of the gas phase. Oxidative stress generally exerts a negative impact on fungal growth. This was shown by the prolonged lag phase noted in cultures transferred from shake flasks to a highly aerated and well mixed fermenter system (O' Donnell et al. 2007), slow growth rates, low substrate consumption rates (O 'Donnell et al. 2007;Li et al. 2008a), low protein content [START_REF] Bai | Effects of oxidative stress on production of heterologous and native protein, and culture morphology in batch and chemostat cultures of Aspergillus niger B1-D[END_REF] and low ATP content (Li et al. 2008b) in oxidatively-stressed fungal cells. One possible explanation for many of these observations is the engagement of alternative respiration, one of whose putative roles is to diminish ROS production, but at the expense of ATP generation due to their non-proton-pumping nature. Another possible explanation is the specific inhibition of key metabolic enzymes by ROS. Two frequently studied enzymes that are vulnerable to ROS inactivation are glyceraldehyde-3-phosphate dehydrogenase (Li et al. 2008a) and aconitase [START_REF] Murakami | Inactivation of aconitase in yeast exposed to oxidative stress[END_REF]. Both these enzymes are essential for primary metabolism in fungi. Our results indicate that the decreased glucose uptake rate in A. niger correlates closely with deceased glyceraldehyde-3-phosphate dehydrogenase activity when cells are treated with H 2 O 2 , but that the decreased ammonium uptake rate is due to decreased ATP and NAPDH availability rather than oxidative inactivation of the ammonium assimilatory enzymes in cells (Li et al. 2008a).

In order to alleviate oxidative stress that is caused to the cells during bioprocessing, there have been some attempts to generate fungal mutants with increased antioxidant capacity. Such studies have a measure of success, for example, in over-expression of genes coding for antioxidant enzymes such as catalase [START_REF] Abbott | Catalase overexpression reduces lactic acid-induced oxidative stress in Saccharomyces cerevisiae[END_REF]) and thioredoxin [START_REF] Perez-Torrado | Fermentative capacity of dry active wine yeast requires a specific oxidative stress response during industrial biomass growth[END_REF], introduction of genes for synthesis of antioxidants such as vitamin C [START_REF] Branduardi | Biosynthesis of vitamin C by yeast leads to increased stress resistance[END_REF]) and astaxanthin [START_REF] Ukibe | Metabolic engineering of Saccharomyces cerevisiae for astaxanthin production and oxidative stress tolerance[END_REF] all increase yeast cells' resistance to oxidative stress. Provided the genetic alterations made to these strains do not reduce vigour, or lead to altered proteolysis, such mutants could have clear advantages in industrial fermentation where oxidative stress is encountered.

The effects of oxidative stress on enzyme production in fungi

Oxidative stress, caused by over-oxygenation to bioreactors, adversely affects the production of a recombinant protein from A. niger [START_REF] Wongwicharn | Effect of oxygen enrichment on morphology, growth, and heterologous protein production in chemostat cultures of Aspergillus niger B1-D[END_REF][START_REF] Bai | Effects of oxidative stress on production of heterologous and native protein, and culture morphology in batch and chemostat cultures of Aspergillus niger B1-D[END_REF]). Thus, mitigating oxidative damage could ultimately increase protein production by fungi. Overexpression of the Kluyveromyces lactis SOD1 (KlSOD1) gene increased the production of two different heterologous proteins by K. lactis, human serum albumin (HSA) and Arxula adeninivorans glucoamylase [START_REF] Raimondi | SOD1, a new Kluyveromyces lactis helper gene for heterologous protein secretion[END_REF]. This was thought to be due to the alleviation of the oxidative stress caused by sustained endoplasmic reticulum damage during recombinant protein production. Sustained stress of the endoplasmic reticulum (ER) causes oxidative stress both in yeast and mammalian cells (Harding et al. 2003;Haynes et al. 2004)). Overexpression of SOD alleviated oxidative stress, thus promoting recombinant protein production.

Conversely, oxidative stress may contribute to increased enzyme production under certain circumstances, for example, enhanced extracellular laccase activity is found as a part of the response system of white rot fungi, Trametes versicolor and Abortiporus biennis, to paraquatcaused oxidative stress conditions (Jaszek et al. 2006).

Having briefly outlined the general responses of fungi to oxidative stress, the discussion will now centre upon the central role of the alternative respiratory pathways in oxidative stress.

Alternative respiratory pathways

In the fungi, as in other eukaryotes, the cytochrome respiratory chain consists of four protein complexes: Complex I (NADH:ubiquinone oxidoreductase), Complex II (succinate:ubiquinone oxidoreductase), Complex III (ubiquinone:cytochrome c oxidoreductase), Complex IV (cytochrome c oxidase). During electron transfer, energy is conserved by pumping out protons at Complexes I, III, IV across the inner membrane of mitochondria. Each complex has specific inhibitors, such as rotenone and piericidin for complex I, antimycin for complex III and cyanide for complex IV (Joseph-Horne et al. 2000). Complex II does not pump protons due to the low free energy change during respiration.

The alternative respiratory and electron transfer chain

[Figure 1 here]

In addition to Complex I, most plants and many fungi have alternative NADH dehydrogenases which accept electrons from NADH and transfer them to ubiquinone directly ( Fig 1). However, these enzymes do not pump protons out of the matrix across the inner mitochondrial membrane, and the free energy between NADH and ubiquinone (73kJ/2e-) is lost as heat [START_REF] Siedow | Plant mitochondrial electron transfer and molecular biology[END_REF]. Unlike Complex I, alternative NADH dehydrogenases are insensitive to rotenone, a fact that is often used to estimate the alternative respiratory activity by inhibiting the cytochrome pathway using rotenone.

In addition to the alternative dehydrogenase, there is also an alternative oxidase (AOX) which transfers electrons from ubiquinol to the final acceptor, O 2 , bypassing both Complex III and IV (Fig. 1). As with the alternative dehydrogenases, AOX does not translocate protons when electrons flow through this pathway, thus no energy is conserved but is instead again lost as heat [START_REF] Moore | Function of the alternative oxidase: is it still a scavenger?[END_REF]. This enzyme has been found in the inner membrane of mitochondria from all the higher plants, many algae, fungi, yeast and certain protozoa [START_REF] Moore | Function of the alternative oxidase: is it still a scavenger?[END_REF][START_REF] Baurain | Regulation of the alternative oxidase Aox1 gene in Chlamydomonas reinhardtii. Role of the nitrogen source on the expression of a reporter gene under the control of the Aox1 promoter[END_REF][START_REF] Juarez | The physiologic role of alternative oxidase in Ustilago maydis[END_REF]. It is distinguished from the conventional cytochrome pathway by its insensitivity to cyanide, which inhibits cytochrome c oxidase (complex IV), and sensitivity to salicylhydroxamic acid (SHAM) and n-propyl gallate. These characteristics are often used to measure the activity of AOX in intact cells or isolated mitochondria by measuring O 2 uptake rate in the presence of cyanide and/or SHAM.

Regulation of AOX in fungi.

Expression of AOX genes is constitutive

In Magnaporthe grisea (Yukioka et al. 1998), Neurospora crassa [START_REF] Tanton | Alternative oxidase expression in Neurospora crassa[END_REF]A. niger (Hattori et al. 2009), AOX genes were constitutively transcribed at a low basal level even though the protein and enzyme activity were not detectable. The observation that non-induced wild-type cultures occasionally contained similar amounts of aod-1 mRNA to induced cultures but revealed no detectable AOX in mitochondria of N. crassa suggested translational control is also indispensable for the regulation of gene expression [START_REF] Tanton | Alternative oxidase expression in Neurospora crassa[END_REF].

AOX is not stimulated by pyruvate but by purine nucleotides

In contrast to plants, the fungal AOX is not stimulated by pyruvate, but by purine nucleotide monophosphate and diphosphates like AMP, ADP, dAMP and GMP. Evidence for this has been found in a wide range of yeasts and fungi, such as P. stipitis, N. crassa [START_REF] Umbach | The cyanide-resistant alternative oxidases from the fungi Pichia stipitis and Neurospora crassa are monomeric and lack regulatory features of the plant enzyme[END_REF], U. ayclis [START_REF] Juarez | The mitochondrial respiratory chain of Ustilago maydis[END_REF]), U. maydis (Sierra-Campos et al. 2009), Tapesia acuformis (Joseph-Horne et al. 2000) and in the yeasts Hansenula anomala [START_REF] Sakajo | Effects of nucleotides on cyanide-resistant respiratory activity in mitochondria isolated from antimycin A-treated yeast Hansenula anomala[END_REF]) and Yarrowia lipolytica [START_REF] Medentsev | Reactivation of the alternative oxidase of Yarrowia lipolytica by nucleoside monophosphates[END_REF].

AOX is activated in the presence of cytochrome respiration inhibitors

Numerous studies have shown that AOX is activated when antimycin (an inhibitor of Complex III in the main respiratory chain), is added to fungal cultures [START_REF] Minagawa | Possible involvement of superoxide anion in the induction of cyanide-resistant respiration in Hansenula anomala[END_REF][START_REF] Uribe | Identification and characterization of an alternative oxidase in the entomopathogenic fungus Metarhizium anisopliae[END_REF]Sierra-Campos et al. 2009). This property indicates that AOX overflows electrons when the cytochrome respiratory chain is inhibited or saturated. In the pathogenic fungus, U. maydis, AOX is thought to increase the metabolic flexibility of the cell, a role that might be valuable for an organism exposed to variations in temperature, nutrient source and availability, and biotic or abiotic factors that limit the activity of the cytochrome pathway [START_REF] Juarez | The physiologic role of alternative oxidase in Ustilago maydis[END_REF].

The role of AOX in oxidative stress

It is generally accepted that mitochondria are the site for the generation of ROS in fungi due to election leakage during the process of aerobic respiration [START_REF] Li | Oxidative stress in industrial fungi[END_REF]). One of the putative roles of the fungal alternative respiratory pathways may be to diminish ROS generation due to the non-proton pumping nature of many of the components of this pathway. There is some evidence to support this hypothesis. In M. grisea, for example, active oxygen species are thought to be signal mediators to activate transcription of the AOX gene (Yukioka et al. 1998), and the superoxide anion ( O 2

•-) is involved in the induction of cyanide-resistant-respiration (CRR) in the yeast H. anomala [START_REF] Minagawa | Possible involvement of superoxide anion in the induction of cyanide-resistant respiration in Hansenula anomala[END_REF]). The presence of H 2 O 2 and menadione induces CRR in Pichia membranifaciens and Debaryomyces hansenii [START_REF] Veiga | Stress situations induce cyanide-resistant respiration in spoilage yeasts[END_REF]. Karrafa et al. (2001) reported a negative correlation between CRR and intracellular peroxide levels in A. chrysogenum, supporting the protective role of alternative respiration against oxidative damage in cells. The effects of two ROS-generating agents, paraquat and H 2 O 2 were investigated in 12 fungal species, the results of which indicated exposure of fungal spores or mycelia to both agents stimulated CRR [START_REF] Angelova | Oxidative stress response of filamentous fungi induced by hydrogen peroxide and paraquat[END_REF]. In A. fumigatus, AOX activity and mRNA expression were both induced with menadione or paraquat, suggesting an important role of AOX under oxidative stress conditions [START_REF] Magnani | Cloning and functional expression of the mitochondrial alternative oxidase of Aspergillus fumigatus and its induction by oxidative stress[END_REF]. A recent study by our group showed that oxidative stress in batch cultures of A. niger caused by over-oxygenation led to a general decrease of ATP content in the cells (Li et al. 2008b) which corroborates the engagement of alternative respiration under such conditions proposed by Bai et al. (2003b).

Though the role of AOX in oxidative stress has been extensively researched, most of the work is based on "simulated" oxidative stress events, that is using oxidants that are highly pro-oxidative to simulate an oxidative environment. Valuable as those studies undoubtedly are, the unambiguous role (or roles) of AOX in aerobic fungal fermentation processes is still elusive. To address this, a comparison between a wild type and a mutant that is deficient in AOX cultivated under fermentation conditions that are known to induce oxidative stress would be very helpful.

Alternative NADH dehydrogenases

In addition to the alternative oxidase (AOX), many filamentous fungi contain alternative NADH:ubiquinone oxidoreductase (NADH dehydrogenase) enzymes. These alternative NADH dehydrogenase enzymes do not contain characteristic Fe-S clusters and are insensitive to rotenone or amytal [START_REF] Kerscher | Diversity and origin of alternative NADH:ubiquinone oxidoreductases[END_REF]. Likewise, the alternative NADH dehydrogenases do not contain FMN, instead incorporating flavin adenine dinucleotide (FAD) as their sole prosthetic group [START_REF] Prömper | The role of the proton-pumping and alternative respiratory chain NADH:ubiquinone oxidoreductases in overflow catabolism of Aspergillus niger[END_REF], and are, like AOX, unable to translocate protons [START_REF] Videira | On complex I and other NADH:ubiquinone reductases of Neurospora crassa mitochondria[END_REF]. As a result of the non-proton pumping nature of these alternative enzymes, fungi must avoid pairing alternative NADH dehydrogenases with the alternative oxidase, as this would result in complete dissolution of the proton gradient necessary for ATP synthesis [START_REF] Joseph-Horne | Fungal respiration: a fusion of standard and alternative components[END_REF]. Therefore, alternative NADH dehydrogenases must be used synergistically with

Complex III and IV, whereas AOX must only be used alongside Complex I, if the cell is to maintain sufficient ATP synthesis to drive all cellular processes [START_REF] Joseph-Horne | Fungal respiration: a fusion of standard and alternative components[END_REF].

Therefore, no filamentous fungus has been shown to contain an electron transport chain in which AOX is present but Complex I is absent. In many filamentous fungi there are three alternative NADH dehydrogenases, two of which are oriented towards the intermembrane space and hence oxidise cytosolic NADH/NADPH ("external" enzymes), and another which faces the mitochondrial matrix and oxidises internal NADH ("internal" enzyme) [START_REF] Prömper | The role of the proton-pumping and alternative respiratory chain NADH:ubiquinone oxidoreductases in overflow catabolism of Aspergillus niger[END_REF][START_REF] Videira | On complex I and other NADH:ubiquinone reductases of Neurospora crassa mitochondria[END_REF].

Thus far, three alternative dehydrogenase enzymes have been characterised in N. crassa, two external and one internal. These are designated NDE1, NDE2, and NDI1, respectively. NDE1 is calcium-dependent and can oxidise NADPH in addition to NADH, as can NDE2, whereas NDI1 can oxidise internal NADH alone [START_REF] Duarte | The internal alternative NADH dehydrogenase of Neurospora crassa mitochondria[END_REF][START_REF] Carneiro | The main external alternative NAD(P)H dehydrogenase of Neurospora crassa mitochondria[END_REF]. Of these, NDI1 is undoubtedly the most important protein complex, since inactivation of the ndi1 gene, which encodes the enzyme, has been shown to result in complete inhibition of respiratory activity in the presence of rotenone [START_REF] Duarte | The internal alternative NADH dehydrogenase of Neurospora crassa mitochondria[END_REF].

There is marked contrast between the alternative NADH dehydrogenases of filamentous fungi and those of yeast, although some homology is conserved. The yeast Yarrowia lipolytica, for instance, contains only one alternative NADH dehydrogenase, in contrast with the numerous alternative respiratory enzymes of filamentous fungi such as N. crassa. This enzyme is designated NDH2, and is encoded by the YLNDH2 gene. It is oriented towards the external face of the mitochondrial inner membrane, and oxidises cytosolic NADH or NADPH [START_REF] Kerscher | A single external enzyme confers alternative NADH:ubiquinone oxidoreductase activity in Yarrowia lipolytica[END_REF]. Conversely, S. cerevisiae contains no Complex I, instead relying on three alternative NADH:ubiquinone oxidoreductases, SCNDE1, SCNDE2, and SCNDI1, the first two of which are external and the latter internal [START_REF] Kerscher | Diversity and origin of alternative NADH:ubiquinone oxidoreductases[END_REF]. No specific inhibitor of the alternative NADH dehydrogenases of fungi has yet been reported, although unspecific inhibitors include flavone, platanetin (6-dimethylallyl-3,5,7,8-tetrahydroxyflavone) [START_REF] Kerscher | Diversity and origin of alternative NADH:ubiquinone oxidoreductases[END_REF], and 7-iodoacridone 4-carboxylic acid (IACA) [START_REF] Roberts | Platanetin and 7-iodo-acridone-4-carboxylic acid are not specific inhibitors of respiratory NAD(P)H dehydrogenases in potato tuber mitochondria[END_REF]. Polymyxin B and nanaomycin A also inhibit alternative NADH dehydrogenases in bacteria [START_REF] Mogi | Polymyxin B identified as an inhibitor of alternative NADH dehydrogenase and malate: quinone oxidoreductase from the Gram-positive bacterium Mycobacterium smegmatis[END_REF]), which provides further scope for investigation of these compounds against fungal NADH dehydrogenases with which the bacterial dehydrogenases share a degree of similarity [START_REF] Duarte | The internal alternative NADH dehydrogenase of Neurospora crassa mitochondria[END_REF].

Although at present the exact physiological role of alternative NADH dehydrogenases in filamentous fungi is obscure, numerous researchers have postulated that they may be involved in prevention of, or perhaps even contribute to, reactive oxygen species generation. As regards the prevention of oxidative stress, Complex I and Complex III are the major sites of ROS generation by the mitochondrial ETC, accounting for some 20% and 80% of total O 2 •-production, respectively [START_REF] Turrens | Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria[END_REF][START_REF] Sigler | Oxidative stress in microorganisms -I: microbial vs. higher cellsdamage and defences in relation to cell aging and death[END_REF]. Therefore, in cultures experiencing oxidatively stressful conditions, for example O 2 enrichment, it would be expected that levels of ROS would rise due to an increased rate of respiration. However, Bai et al. (2003b) proposed that this may be due to enhanced activity of an alternative NADH dehydrogenase enzyme, which could act in place of Complex I. Upon studying the total and rotenone-insensitive respiration of these cultures, those sparged with O 2 -enriched air had markedly higher ratios of rotenone-insensitive:total respiration (RR/TR). The researchers therefore concluded that under oxidatively challenging conditions, filamentous fungi manipulate electron transport to favour flux through the alternative respiratory pathway, thus reducing ROS generation, a theory which has likewise been proposed for other organisms [START_REF] Li | Diphenyleneiodonium, an NAD(P)H oxidase inhibitor, also potently inhibits mitochondrial reactive oxygen species production[END_REF]Guerrero-Castillo et al. 2009). publicly available in 2007 [START_REF] Pel | Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88[END_REF]). The public availability of these genomes brings with it further opportunity to understand the functions of each of the alternative respiratory enzymes much more deeply, and in particular how the genes controlling these currently abstruse proteins are regulated in response to changing environments.

Genome analysis of Aspergillus; alternative respiration pathways

Analysis of the fully sequenced genomes of Aspergillus species (www.aspergillusgenomes.org)

for alternative respiratory pathways shows the presence of homologous genes encoding multiple pathways similar to those used by N. crassa [START_REF] Duarte | The internal alternative NADH dehydrogenase of Neurospora crassa mitochondria[END_REF]. BlastP searching of the -Horne et al. 2000;[START_REF] Duarte | The internal alternative NADH dehydrogenase of Neurospora crassa mitochondria[END_REF][START_REF] Carneiro | The main external alternative NAD(P)H dehydrogenase of Neurospora crassa mitochondria[END_REF].

  [START_REF] Seo | The single subunit NADH dehydrogenase reduces generation of reactive oxygen species from complex I[END_REF] demonstrated that transduction of human neuroblastoma cells with the gene encoding the internal alternative NADH dehydrogenase of S. cerevisiae significantly reduced the generation of superoxide radicals within the cell. Inhibition of fungal NDI1 with IACA also led to a significant increase in superoxide production by the culture, most likely as a result of the high rate of electron leakage from Complex I (O'Donnell 2007).Despite the protective properties of alternative NADH dehydrogenases against oxidative stress, further investigation in A. niger has shown that when these enzymes are enhanced in their activities, intracellular ATP concentrations and cellular viability are decreased (O'Donnell et al. 2007). This was particularly true under conditions of high (50%) oxygen enrichment, which led primarily to a significant decrease in intracellular ATP concentrations, with a concomitant decrease in both intracellular and secreted protein concentrations and decreased respiratory quotients (O'Donnell 2007). Additionally, inhibition of the alternative NADH dehydrogenases by IACA significantly increased the intracellular ATP concentrations, both in control and O 2- enriched batch cultures (O'Donnell 2007). Other investigators have also claimed such alternative NADH dehydrogenase enzymes actually increase ROS production.[START_REF] Fang | External alternative NADH dehydrogenase of Saccharomyces cerevisiae: a potential source of superoxide[END_REF] demonstrated that inhibition of alternative NADH dehydrogenases in yeast (S. cerevisiae) led to a decrease in ROS production by the culture. The apparent contradiction could well be due to the differing responses of yeasts and filamentous fungi to oxidative stress.[START_REF] Li | Oxidative stress in industrial fungi[END_REF].Since 2005, the genomes have been available for A. nidulans, A. fumigatus and A. oryzae (Jones 2007), with the genome for A. niger being determined by DSM (The Netherlands) and made

Fig 1

 1 Fig 1 The respiratory chain for filamentous fungi. The arrows indicate the flow of electrons.

Fig. 2

 2 Fig. 2 Genome Analysis of alternative respiratory pathways.

  described a significantly decreased O 2 •-generation in cultures of A. niger in O 2 -enriched air, and thencefore

potentially rewarding target for metabolic engineering of fungal strains optimised for the fermenter environment.

Guerrero-Castillo S, Vázquez-Acevedo M, González-Halphen D, Uribe-Carvajal S (2009) In Yarrowia lipolytica mitochondria, the alternative NADH dehydrogenase interacts specifically with the cytochrome complexes of the classic respiratory pathway. Biochim Biophys Acta