MD Jalid Sehouli
email: sehouli@aol.com

Oumar Camara

Sven Mahner

Thomas Bauknecht

Werner Lichtenegger

Ingo Runnebaum

Katherine Look

Fritz Jaenicke

Guelten Oskay-Oezcelik

A phase-I trial of pemetrexed plus carboplatin in recurrent ovarian cancer

Keywords: recurrent ovarian cancer, pemetrexed, carboplatin, second-line treatment

Background

Epithelial ovarian cancer is the leading cause of death from gynecological malignancies world-wide [START_REF] Jemal | Cancer statistics, 2007[END_REF]. Most patients were present with advanced disease at the initial diagnosis.

Current standard management of primary ovarian cancer includes radical cytoreductive surgery followed by systemic chemotherapy with carboplatin plus paclitaxel. More than 65% of the patients with advanced ovarian cancer will relapse and require additional treatment [START_REF] Ozols | Focus on epithelial ovarian cancer[END_REF].

Retrospective studies have identified two subgroups of patients with recurrent ovarian cancer, termed platinum-sensitive and platinum-resistant, according to the best response to first-line platinum-based chemotherapy and consecutive progression free interval (more or less than 6 months) [3; 4]. A large randomized trial (ICON4/AGO-Ovar 2.2) demonstrated the superiority of a combined carboplatin and paclitaxel regimen over platinum monotherapy in platinum-sensitive patients [START_REF] Parmar | Paclitaxel plus platinum-based chemotherapy versus conventional platinum-based chemotherapy in women with relapsed ovarian cancer: the ICON4/AGO-OVAR-2.2 trial[END_REF]. Today, most of the patients will receive first-line chemotherapy with paclitaxel and a platinum compound, so that the cumulative neurotoxicity of platinum and paclitaxel compromises the retreatment with these agents [6; 7]. Furthermore, many patients prefer to avoid alopecia in this palliative therapeutic setting. Therefore, various study groups are currently exploring other non-taxane containing platinum combinations in order to optimize the therapeutic index for patients with platinum-sensitive recurrent ovarian cancer [7; 8].

Pemetrexed is a multi-targeted antifolate inhibitor of thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT) [START_REF] Shih | LY231514 a pyrrolo[2,3-d] pyrimidine base antifolate that inhibits multiple folate requiring enzyames[END_REF] and is given in combination with vitamin supplementation. It is currently approved for the treatment of malignant pleural mesothelioma and non-small cell lung cancer in conjunction with cisplatin [10; 11] and for the second-line monotherapy of non-small cell lung cancer [START_REF] Hanna | Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small-cell lung cancer previously treated with chemotherapy[END_REF].

Various preclinical studies indicate significant activity of pemetrexed against ovarian xenograft tumors and ovarian carcinoma cells [START_REF] Miotti | Simultaneous activity of two different mechanisms of folate transport in ovarian carcinoma cell lines[END_REF][START_REF] Corona | Role of folate receptor and reduced folate carrier in the transport of 5-methyltetrahydrofolic acid in human ovarian carcinoma cells[END_REF][START_REF] Takimoto | Phase I and pharmacokinetic study of pemetrexed with high-dose folic acid supplementation or multivitamin supplementation in patients with locally advanced or metastatic cancer[END_REF]. The toxicity profile of pemetrexed is generally favorable without severe hematologic or non-hematologic side effects including a very low rate of neurotoxicity and alopecia [START_REF] Vogelzang | Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma[END_REF][START_REF] Scagliotti | Phase III study comparing cisplatin plus gemcitabine with cisplatin plus pemetrexed in chemotherapy-naive patients with advanced-stage non-small-cell lung cancer[END_REF]. Therefore, pemetrexed is a good candidate for novel platinum-based combination chemotherapy in recurrent ovarian cancer.

Based on these results, a phase I study was initiated to further evaluate the toxicity profile and dose-limiting toxicity of the combination of pemetrexed plus carboplatin in patients with platinum-sensitive recurrent ovarian cancer.

Patients and methods

Design

Patients were enrolled at two German centers (Charité: 18 patients, Jena 2 patients). The trial was performed in accordance with the principles of Good Clinical Practice (GCP) and the Declaration of Helsinki. Protocol approval was gained from the institutional review board or the local ethics committee of each participating institution. An independent monitoring institute was responsible for data control.

A prospective, separate phase-I and II was planned to investigate the safety and efficacy of this new chemotherapy combination. As in the study protocol, primary defined individual clinical study reports from the phase I and phase II were planned. Here, we present the data from the dose finding study.

The primary objective of the phase I study was to determine the maximum tolerated dose (MTD) of the combination therapy of pemetrexed and carboplatin when administered to patients with platinum-sensitive recurrent ovarian cancer.

The secondary objectives of this study were: to determine the dose limiting toxicities (DLT) of the combination therapy of pemetrexed and carboplatin, to determine the quantitative and qualitative toxicities of pemetrexed in combination with carboplatin, to determine a recommended dose of the combination therapy of pemetrexed and carboplatin for future phase 2 studies, and to document the antitumor activity of pemetrexed and carboplatin in patients with platinum-sensitive recurrent ovarian cancer through tumor response assessment.

Patients

Women ≥ 18 years of age were eligible who had platinum-sensitive recurrent cancer of the ovary, fallopian tube or peritoneum, at least six months after completion of primary standard therapy with surgery and first-line combination therapy with platinum and paclitaxel. Patients were required to have measurable lesions or non-measurable disease as defined by Response Criteria in Solid Tumors (RECIST) [START_REF] Therasse | New guidelines to evaluate the response to treatment in solid tumors[END_REF] or had to be evaluable for CA-125 response evaluation according to the GCIG-criteria [START_REF] Vergote | Re: new guidelines to evaluate the response to treatment in solid tumors [ovarian cancer]. Gynecologic Cancer Intergroup[END_REF]. Eastern Cooperative Oncology Group (ECOG) performance status had to be ≤ 2, patients were required to have normal hematologic, liver and renal function with laboratory parameters within the normal range, including a glomerular filtration rate ≥ 45 ml/min based on the standard Cockcroft and Gault formula or on measured GFR using an appropriate radio-labeled method (51-CrEDTA or Tc99m-DTPA), serum creatinine levels ≤ 1.5 mg/dl and an adequate bone marrow function (absolute neutrophil count [ANC] ≥ 1.5 x 10 9 /l and platelets ≥ 100 x 10 9 /l). Patients suffering from a secondary malignancy or serious concomitant systemic disorders or psychiatric disease were excluded from the study, as were subjects receiving other cytotoxic, immunological, hormonal or targeted therapy. All patients provided written informed consent. Patients were excluded if they had more than 2 previous lines of therapy for ovarian cancer or a serious concomitant systemic disorder that would compromise the ability to complete the study.

Treatment plan

Intravenous premedication consisting of a 5-HT3-antagonist was administered 15 minutes prior to chemotherapy infusion. Grade 3 or 4 nausea and/or vomiting should be managed with appropriate changes in an anti-emetic regimen and in regard to the discretion of the investigator.

Patients received pemetrexed IV over a 5 to 10 min bolus, followed by carboplatin IV (calculated via Cockcroft-Gault Equation) over 30 to 60 minutes, given every 21 days for 6 cycles (up to 8 cycles could be administered at the investigators discretion if clinical benefit was expected).

Both study drugs were dissolved in 250 ml of 0.9% saline. Patients were required to receive oral folic acid, 350-1000 µg daily at least 1 to 2 weeks before cycle 1 until 3 weeks after the last pemetrexed dose and 1000 µg vitamin B 12 intramuscularly every 9 weeks, starting at least 1 to 2 weeks before cycle 1, and then every 9 weeks until 3 weeks after the last pemetrexed dose [START_REF] Chattopadhyay | Commentary: A Case for Minimizing Folate Supplementation in Clinical Regimens with Pemetrexed Based on the Marked Sensitivity of the Drug to Folate Availability[END_REF]. To prevent skin rash, dexamethasone 4 mg orally (or equivalent) was given twice daily for 3 days, starting on day 1 prior to pemetrexed administration.

Seven dose-levels were defined upfront (Table 1). Patient accrual and dose escalation depended upon the observed pattern of DLTs. According to the protocol, a cohort of 3-6 patients for each dose level had to be enrolled.

If none of the 3 initial patients of a given level experienced a DLT in cycle 1, enrolment proceeded to the next dose level. If 1 of the initial 3 patients of a given level experienced a DLT in cycle 1, additional patients had to be enrolled up to a maximum of 6 patients at that level. If no more than one patient experienced a DLT in cycle 1, patient enrolment proceeded to the next level. If 2 of the 3 initial patients of a given level experienced a DLT in cycle 1, that level had to be considered the maximum tolerated dose (MTD). Dose escalation to the next level was not allowed until the last patient of a given dose level (that is, either the third or sixth patient) had completed the entire first cycle.

A 5-HT3-antagonist was given intravenously 15 minutes prior to every chemotherapy cycle.

Use of erythropoietin stimulating agents was allowed; however, no primary prophylactic use of granulocyte colony-stimulating factor (G-CSF) was allowed.

Toxicity

Toxicity was assessed after each cycle, graded according to the NCI-Common Terminology Criteria for Adverse Events (CTCAE) v3.0, August 9, 2006 [START_REF]Cancer Therapy Evaluation Program: Common Terminology Criteria for Adverse Events v3.0 (CTCAE)[END_REF]. The following toxicities, if observed during cycle 1 of treatment, were considered a dose limiting toxicity (DLT): CTCAE Grade 4 neutropenia (ANC <0.5 x 10 9 /l) lasting ≥7 days, febrile neutropenia (ANC <1.0 x 10 9 /l, fever 38.5 °C, no clinically or microbiologically documented infection), CTCAE Grade 4 thrombocytopenia (platelets <25.0 x10 9 /l), any grade hemorrhage with CTCAE Grade ≥3 thrombocytopenia (50.0 x 10 9 /l), CTCAE Grade ≥3 non hematologic toxicity (excluding nausea, vomiting, or CTCAE Grade 3 ALT or AST that returned to baseline prior to next treatment), treatment delay of more than 1 week due to unresolved Grade 3 or 4 toxicity.

Blood count and blood chemistry were carried out regularly once a week. Subsequent chemotherapy cycles were allowed if the absolute neutrophil count was > 1.5 x 10 9 /l and platelet count was ≥ 100 x 10 9 /l.

Treatment could be postponed up to one week in case of toxicity. Reduction to dose level -1 was required for treatment delays exceeding one week. Protocol treatment was discontinued if the patient required cycle delay longer than one week due to unresolved grade 3 or greater toxicity.

Response

Patients were assessed before treatment, during treatment and every 3 months after treatment for 2 years, then every 6 months for 3 years. Physical examination, CA-125 (according to the criteria established by Vergote et al. [START_REF] Vergote | Re: new guidelines to evaluate the response to treatment in solid tumors [ovarian cancer]. Gynecologic Cancer Intergroup[END_REF]), and radiological evaluation of measurable tumor lesions were performed as baseline assessment. Evaluation of response was performed every other cycle (within 7 days) using the same method that was employed at baseline and in any case of suspected progression of disease. Clinical response was determined by physical examination and ultrasound, computer tomography (CT) or magnetic resonance imaging (MRI) scans. Response was measured according to RECIST criteria [START_REF] Therasse | New guidelines to evaluate the response to treatment in solid tumors[END_REF].

Statistical Analysis

There were 20 patients enrolled in this phase 1 study. This number depended on the dose levels studied in determining the primary endpoint of this study. The primary endpoint, determination of the MTD was not achieved. All secondary endpoints are summarized in tabular form or provided as listings. All confidence intervals for parameters provided were constructed using a two-sided, 5% significance level. Descriptive statistics were conducted on patient demographics and patient characteristics. All statistical analyses were conducted using SAS Version 8.2 (SAS Institute Inc, Cary, NC, USA) .

Results

Patient characteristics

From July 2005 to September 2006, 20 patients with platinum sensitive relapse were enrolled.

Median age was 57.4 years (range 37.3-75.0 years), 15 patients had primary ovarian cancer and 5 patients primary peritoneal cancer. The median platinum free interval was 26.2 months (range 7.2-124.4 months).

Patient characteristics are displayed in Table 2.

Treatment profile

The treatment profile is summarized in Table 3. The total number of administered cycles was 115 for pemetrexed and 112 for carboplatin. The median number of cycles was 6 for pemetrexed (range 1-8) and 6 for carboplatin (range 1-8). In dose level 1, four patients were treated, three patients in dose level 2, another four patients in dose level 3, three patients in dose level 4 and three patients in dose levels 5 and 6, each.

For pemetrexed, 95.4% (range 90.0% -99.7%) of the planned dose intensity (DI) could be administered and 95.4% (range 87.3-102.8%) of carboplatin were applied. Detailed dose intensities are listed in Table 4.

Dose reduction

There were three dose reductions (two at dose level 3 and one at dose level 4) necessary in the 115 cycles of pemetrexed. For carboplatin, there were three dose reductions in 112 cycles (two at dose level 3 and one at dose level 4). There was in one patient a further additional intrapatient dose reduction necessary.

Treatment discontinuation

No patient required treatment discontinuation due to unacceptable toxicity. Prior to protocol completion, seven patients discontinued treatment (five due to progressive disease, and two due to patient choice).

Dose-limiting toxicity

We observed only one DLT: in dose level 3: one patient experienced grade 4 neutropenia for 7 days without clinical squeal. No other dose limiting toxicities were observed. There were no treatment related deaths in this phase I study.

Two serious adverse events were observed, but none of them was considered to be related to study treatment: One patient suffered from gastrointestinal bleeding (at dose level 1). This 60 year-old patient had a diffuse peritoneal carcinomatosis and diffuse liver metastasis. At the time of the gastrointestinal bleeding, the blood parameters were within the normal range with thrombocytes of 193/nl and Hb of 10.5g/dl. The bleeding was managed conservatively. The second patient suffered from subileus (at dose level 6). This event was managed conservatively and was considered to be related to the underlying diffuse peritoneal carcinomatosis and not due to the study drug itself. Five patients required dose reduction due to toxicity.

Haematological toxicity

No neutropenic fever, sepsis or chemotherapy-related deaths occurred during the trial.

Overall, haematological side effects were manageable and without sequelae. Grade 4 neutropenia occurred in one patient in dose level 3. No other grade 4 hematologic toxicities were observed, Details are listed in Table 5.

A prophylactic treatment with Erythropoietin was permitted, but in no patient erythropoietin was administered.

Non-hematological toxicity

Non-hematological side effects were mild and rarely greater than grade 2. Three patients suffered from grade 3 toxicity: 1 patient with nausea (dose level 3), 1 patient with dyspnea (dose level 4) and 1 patient with allergic reaction to carboplatin (dose level 1). No grade 4 non-hematological side effects occurred. Patients did not experience neurotoxicity higher than grade 2. Seven patients suffered from grade 1 and one patient from grade 2 neurotoxicity, both were attributed to the previous chemotherapy with carboplatin and paclitaxel. Grade 1 alopecia was observed in six patients. Allergic reaction to carboplatin occurred in five patients with grade 1 and in one patient with grade 3. Seventeen patients experienced grade 1 or 2 nausea, 14 grades 1 or 2 vomiting. Grade 1 diarrhea occurred in 5 patients. No unexpected non-hematological toxicity was observed; details are listed in Table 6.

Response and survival

Nineteen patients could be assessed for response, of which 17 were analyzed according to the CA-125 response criteria; two patients were analyzed by RECIST criteria. Twelve patients achieved complete remission (63.2%, 95% CI: 41.5-84.9), four patients achieved partial response (21.1%, 95% CI: 2.8-39.4%), one patient had stable disease (5.3%, 95% CI: 0-15.3%) and two patients had progressive disease (10.5%, 95% CI: 0-24.3%).

By intent-to-treat, the response rate was estimated to be 80% (95% CI: 62.5-97.5%). The median follow-up was 9.8 months (95% CI 6.2-14 months). At the time of final data analysis, 18 of the 20 enrolled patients had progressive disease, 17 patients were alive.

Discussion

Platinum-based combination therapy with paclitaxel or gemcitabine is currently considered to be the standard second-line treatment for patients with platinum-sensitive relapsed ovarian cancer [START_REF] Parmar | Paclitaxel plus platinum-based chemotherapy versus conventional platinum-based chemotherapy in women with relapsed ovarian cancer: the ICON4/AGO-OVAR-2.2 trial[END_REF][START_REF] Pfisterer | Gemcitabine plus carboplatin compared with carboplatin in patients with platinum-sensitive recurrent ovarian cancer: an intergroup trial of the AGO-OVAR, the NCIC CTG, and the EORTC GCG[END_REF]. The incidence of neurotoxicity in patients receiving conventional paclitaxel and carboplatin as first-line treatment was estimated to be 54% of all patients [START_REF] Pignata | Residual neurotoxicity in ovarian cancer patients in clinical remission after first-line chemotherapy with carboplatin and paclitaxel: The Multicenter Italian Trial in Ovarian cancer (MITO-4) retrospective study[END_REF], while approximately 13% suffer from grade 2 to 3 neurotoxicity. In a large phase-III trial of the ICON-AGO intergroup evaluating treatment with carboplatin and paclitaxel as second-line therapy, a significantly higher incidence of grade 2 to 4 neurotoxicity (20%) was observed in comparison with single agent platinum (1%) [START_REF] Parmar | Paclitaxel plus platinum-based chemotherapy versus conventional platinum-based chemotherapy in women with relapsed ovarian cancer: the ICON4/AGO-OVAR-2.2 trial[END_REF]. This observation is notable, especially because in this trial, only 43% of the patients had received platinum-and taxane-based combination chemotherapy during first-line therapy. Since at the present time most patients will be treated with carboplatin and paclitaxel as first-line therapy, the incidence of neurotoxicity will probably be significantly higher and consequently might have substantial impact on the quality of life of these patients.

Thus, in a situation where cure is generally not achieved, there is high clinical need for alternative platinum-based combinations to improve the therapeutic index with special attention to patients' quality of life. Pemetrexed seems to be an attractive candidate for a platinum combination. It has proven activity in mesothelioma and non-small cell lung cancer, and neurotoxicity is rare [START_REF] Vogelzang | Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma[END_REF][START_REF] Scagliotti | Phase III study comparing cisplatin plus gemcitabine with cisplatin plus pemetrexed in chemotherapy-naive patients with advanced-stage non-small-cell lung cancer[END_REF][START_REF] Hanna | Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small-cell lung cancer previously treated with chemotherapy[END_REF]. In the present study, eight patients suffered from neurotoxicity, which pre-existed from prior taxane-based first-line chemotherapy and was not attributed to pemetrexed.

To our knowledge, this is the first dose escalating study of the combination pemetrexed and carboplatin in ovarian cancer. We observed only one DLT: in dose level 3, one patient experienced grade 4 neutropenia for 7 days without clinical squeal. Despite the fact that we applied pemetrexed up to a dose of 900 mg/m² and carboplatin up to an AUC of 6 we did not reach the maximum tolerated dose. Matulonis et al. combined pemetrexed at 500 mg/m² and carboplatin at AUC 5 in a phase-II study [START_REF] Matulonis | Phase II Study of Carboplatin and Pemetrexed for the Treatment of Platinum-Sensitive Recurrent Ovarian Cancer[END_REF]. Grade 3 and 4 hematologic toxicities included neutropenia (41%), thrombocytopenia (23%), and anemia (9%). In their study, the response rate was 51.1% (95% CI: 35.8-66.3%) without any patients achieving a complete response.

Despite the methodical limitation of cross-trial comparison, response rate was substantially higher in the current trial (80%) with 12 patients achieving CR. In contrast to our study, the study by Matulonis included more than 50% of patients with more than two prior chemotherapies.

These differences in the inclusion criteria or a potentially effect of the different applied dose may explain the different toxicity profiles and response rates of both trials. Another explanation might be the difference in response evaluation, as Matulonis et al. used RECIST criteria in contrast to GCIG CA-125 criteria utilized in the current trial.

Regarding vitamin supplementation, the current trial is to our knowledge also the first dose escalating study of the combination of pemetrexed and carboplatin. Until today, one phase-I study with pemetrexed and carboplatin in 27 patients with malignant pleural mesothelioma has been conducted, but this study was near completion when supplementation with folic acid and vitamin B 12 was instituted; therefore, these patients were not supplemented. The MTD for pemetrexed was 500 mg/m 2 and for carboplatin AUC 6 and was defined by two episodes of grade 4 neutropenia (data on file). Vitamin B 12 supplementation causes a statistically significant reduction in plasma homocysteine concentrations over time and a significant reduction in the frequencies of severe hematologic toxicities and non-hematological toxicities in patients receiving pemetrexed therapy [START_REF] Niyikiza | Homocysteine and methylmalonic acid: markers to predict and avoid toxicity from pemetrexed therapy[END_REF]. In contrast to the phase-I study in mesothelioma, in the present study all patients received vitamins. This might be an explanation for the improved dose tolerance we observed.

Despite the favorable hematological and non-hematological toxicity profile in the present study, the optimal dose of pemetrexed in a combination with carboplatin is not yet defined.

Recently, Vergote et al. presented the results of a randomized study comparing pemetrexed 500 mg/m 2 with 900 mg/m 2 in patients with platinum-resistant ovarian cancer [START_REF] Vergote | A Randomized, Double-Blind, Phase 2 Study of Two Doses of Pemetrexed in the Treatment of Platinum-Resistant, Epithelial Ovarian or Primary Peritoneal Cancer[END_REF]. There were no differences in response rate or clinical outcome, but the lower dose had a significantly better toxicity profile.

Additionally, there is now growing evidence that doses higher than the currently approved 500 mg/m² of pemetrexed do not improve the efficacy in several tumor types including ovarian, breast and non-small cell lung cancer [START_REF] Cullen | A randomized phase III trial comparing standard and high-dose pemetrexed as second-line treatment in patients with locally advanced or metastatic non-small-cell lung cancer[END_REF][START_REF] Llombart-Cussac | A randomized, double-blind, phase II study of two doses of pemetrexed as first-line chemotherapy for advanced breast cancer[END_REF][START_REF] Ohe | Efficacy and safety of two doses of pemetrexed supplemented with folic acid and vitamin B12 in previously treated patients with non-small cell lung cancer[END_REF].

Despite the fact that it is unclear whether or not these results can be translated into the platinum-pemetrexed combination, it is very unlikely that a higher dose of pemetrexed could yield a higher efficacy.

Nevertheless it can be criticized that in the present study further dose escalation were may possible because the MTD was not reached. But it should be mentioned that different study groups [START_REF] Pfisterer | Gemcitabine plus carboplatin compared with carboplatin in patients with platinum-sensitive recurrent ovarian cancer: an intergroup trial of the AGO-OVAR, the NCIC CTG, and the EORTC GCG[END_REF][START_REF] Pujade-Lauraine | A randomized, phase III study of carboplatin and pegylated liposomal doxorubicin versus carboplatin and paclitaxel in relapsed platinum-sensitive ovarian cancer (OC): CALYPSO study of the Gynecologic Cancer Intergroup (GCIG)[END_REF] demonstrated the efficacy of specific carboplatin-based combinations such as pegylated doxorubin/carboplatin or gemcitabine/carboplatin without using the highest possible dosages and there is no evidence showing a clear effect of the dose on progression free survival or overall survival in relapsed ovarian cancer.

Therefore the committee of the present study has decided to usefor the subsequent phase-II triala dose of 500 mg/m 2 for pemetrexed in combination with carboplatin according AUC 6.

The primary objective of this study is to define the clinical response rate; secondary objectives are the identification of acute and late toxicities as well as the time up to progression and overall survival. Furthermore, various potential biomarkers will be explored in the phase-II trial. It is only when validated biochemical response markers are available, that patients who will benefit from this new agent can be identified [START_REF] Lederman | Positioning pemetrexed in the treatment of ovarian cancer[END_REF].

Tables

Table 1 :

 1 Dose levels of the study

	Level	Pemetrexed	Carboplatin
	-1	400 mg/m²	AUC 5
	1	500 mg/m²	AUC 5
	2	600 mg/m²	AUC 5
	3	600 mg/m²	AUC 6
	4	700 mg/m²	AUC 6
	5	800 mg/m²	AUC 6
	6	900 mg/m²	AUC 6

Table 2 :

 2 Baseline patient characteristics (n=20)

	Median age, years (range)		57.4 (37.3 -75)
	ECOG performance status	0	3
		1	17
	FIGO stage at primary diagnosis		I 1
		II	1
		III	16
		IV	2
	Median platinum free interval, months (range)	26.2 (7.2 -124.4)

Table 3 : Treatment profile Pemetrexed Carboplatin Dose Level Number of Patients Median Cycles (Range) Total Cycles Median Cycles (Range)

 3

	Total
	Cycles

Acknowledgements

We wish to thank all participating nurses and patients. Without their enthusiastic collaboration, this work would not have been possible.

Conflict of interest

Thomas Bauknecht and Katherine Look are employees of Eli Lilly and Company. For all other authors there is no conflict of interest involved with the data presented here.


MD János Gaál

Tamás Bender

Ph.D József Varga

Irén Horváth

Judit Kiss

MD. Péter Somogyi

correspondent), Irén HORVÁTH MD, Judit KISS MD Péter Surányi

Overcoming resistance to bisphosphonates through the administration of alfacalcidol: results of a 1-year, open follow-up study

Introduction

Osteoporosis is a disorder that leads to enhanced bone fragility through a decrease in the mass as well as deterioration of the microarchitecture of bones. Owing to its high prevalence and negative impact on quality of life, osteoporosis is the third most important public health disorder (after malignancies and cardiovascular disease), afflicting 200 million people worldwide [1]. Oral bisphosphonates are routinely prescribed for treatment for patients with osteoporosis. Adequate calcium and vitamin D sufficiency of the body is essential for antiresorptive treatment to be effective. According to HORVÁTH et al., serum 25hydroxyvitamin D 3 level is lower than normal in 40% of elderly nursing home residents, whereas data from BHATTOA et al. reflect the same in 56.7% of postmenopausal women [START_REF] Cs | A D-vitamin-hiány szerepe a gondozóintézetben ápolt betegek osteopeniájában[END_REF][START_REF] Bhattoa | Prevalence and seasonal variation of hypovitaminosis D and its relationship to bone metabolism in community dwelling postmenopausal Hungarian women[END_REF]. Some studies demonstrated statistically significant reduction of fractures during five years of high dose (100 000 E every four months) treatment with vitamin D 3 [START_REF] Trivedi | Effect of four monthly oral vitamin D 3 (cholecalciferol) supplementation on fractures and mortality in men and women living in the community: randomised double blind controlled trial[END_REF], but others not [START_REF] Law | Vitamin D supplementation and the prevention of fractures and falls: results of a randomised trial in elderly people in residential accommodation[END_REF][START_REF] Lyons | Preventing fractures among older people living in institutional care: a pragmatic randomised double blind placebo controlled trial of vitamin D supplementation[END_REF][START_REF] Smith | Effect of annual intramuscular vitamin D on fracture risk in elderly men and womena population-based, randomised, double-blind, placebo-controlled trial[END_REF]. According to a recent study, in 8 to 25 per cent of osteoporotic patients, bone loss continues despite adequate treatment with bisphosphonates, administered in combination calcium and vitamin D 3 supplementation [START_REF] Shebba | Significance of a decline in bone mineral density while receiving oral bisphosphonate treatment[END_REF]. A proportion of these patients may have functional hypovitaminosis D potentially resulting from insufficient intake (despite supplementation), impaired activation, genuine resistance to vitamin D3 or secondary hyperparathyroidism [START_REF] Barone | Secondary Hyperparathyroidism Due to Hypovitaminosis D Affects Bone Mineral Density Response to Alendronate in Elderly Women with Osteoporosis: A Randomised Controlled Trial[END_REF]. In the remainder, the cause underlying the lack of bisphosphonate effects is unknown.

In osteoporosis, genuine resistance to bisphosphonates is probably non-existent or may be considered extremely uncommon, at the least. In Paget's disease, by contrast, where therapy is aimed at achieving complete biochemical remission, resistance to bisphosphonates has been demonstrated by several researchers. The lack of a complete biochemical remission is regarded by some as the criterion for bisphosphonate resistance [START_REF] Joshua | Bisphosphonate resistance in Paget's disease of bone[END_REF], whereas according to others [START_REF] Gutteridge | Paget's disease: acquired resistance to one aminobisphosphonate with retained response to another[END_REF], the latter is established by a lower than 50-per-cent decrease in serum alkaline phosphatase (AP) level. Notwithstanding the criteria, the prevalence of resistance varies with different bisphosphonates in the range between 83% (etidronate [START_REF] Joshua | Bisphosphonate resistance in Paget's disease of bone[END_REF]) and 11% (zolendronate [START_REF] Siris | Medical management of Paget's disease of bone: indications for treatment and review of current therapies[END_REF]).

According to additional, relevant evidence, the efficacy of activated analogues of vitamin D (alfacalcidol, calcitriol) in reducing bone fractures and increasing bone density is superior to that of native vitamin D. This has been demonstrated by several studies in various disease forms, i.e. in postmenopausal osteoporosis, as well as in osteoporosis related to inflammatory joint disease, or glucocorticoid therapy [START_REF] Richy | Vitamin D analogs versus native vitamin D in preventing bone loss and osteoporosis-related fractures: a comparative meta-analysis[END_REF][START_REF] Ringe | Alfacalcidol versus plain vitamin D in the treatment of glucocorticoid/inflammation-induced osteoporosis[END_REF][START_REF] Shiraishi | The advantage of alfacalcidol over vitamin D in the treatment of osteoporosis[END_REF][START_REF] Ringe | Superiority of a combined treatment of Alendronate and Alfacalcidol compared to the combination of Alendronate and plain vitamin D or Alfacalcidol alone in established postmenopausal or male osteoporosis (AAC-Trial)[END_REF].

This study was intended to ascertain:

1. whether replacing vitamin D 3 with alfacalcidol is followed by an increase in BMD (i.e. bisphosphonate resistance can be overcome) in patients not responding to treatment with a bisphosphonate administered in combination with supplemental calcium and conventional vitamin D 3 ; 2. the incidence of hypovitaminosis D 3 in the study population;

3. the changes occurring in biochemical markers of bone turnover during one year of treatment with alfacalcidol, 4. the frequency of adverse events associated with the use of alfacalcidol.

Patients and methods

Patients

Seventy-six patients were enrolled into the study conducted between January 2006 and March 2007. The duration of follow-up was 12 months on average. Inclusion criteria were: diagnosed postmenopausal or senile osteoporosis (in females) or idiopathic osteoporosis in males, and greater than 3% decrease in BMDas demonstrated by axial DEXAdespite adequate treatment (vitamin D 3 400-1000 U/day, calcium 1000 mg/day and alendronate 70 mg/week) for a year or longer. Exclusion criteria comprised established secondary osteoporosis, other forms of calcipenic osteopathy, hypercalcaemia, and history of renal calculosis. The male-to-female ratio was 4:72; mean age of the study population was 70.5 (±8.2) years. Ten patients had a history of at least one prevalent vertebral or low-trauma peripheral fracture. The average serum 25-hydroxyvitamin D3 level was 72,2± 36,2 nmol/l at the starting and 74,1 ± 34,0 nmol/l at the end of the study (the difference was not significant statistically). Below normal serum 25-hydroxyvitamin D 3 level (<75 nmol/l) was found in 38 patients, along with elevated (>72 pg/ml) serum PTH level in 23 of these subjects. The mean glomerular filtration rate (GFR) was 85,3 ml/min/1,73m 2 at baseline and 4 patients had GFR lower than 60 ml/min/1,73 m 2 at starting of the study, 2 of them had elevated serum PTH level. In the included patients, the average administered dose of D3 vitamin was 530 U/day along with 1000 mg of calcium supplementation before the starting of the study.

Study design

During the follow-up period of one-year on average, control visits were scheduled at baseline and at 3-month intervals thereafter. At baseline, a detailed history (concomitant diseases, fractures, osteoporosis and risk factors) was recorded along with appraisal of clinical status by obtaining a lateral x-ray of the lumbosacral spine and performing a laboratory screen (ESR, CRP, CBC, serum calcium, phosphorus, BUN, creatinine, GOT, GPT, AP, albumin, osteocalcin, 25-hydroxyvitamin-D 3 , parathormon [PTH] levels, urinary calcium/creatinine [UCa/Cr] and deoxypyridinoline crosslinks/creatinine [D-Pyr/creatinine] ratios). Bone density was measured in both peripheral bones (DTX200) and in the axial skeleton (DEXA, Lunar DPX Pro) and BMD values obtained from the radius distal segment, as well as the means of the values measured in the 1 st through 4 th lumbar vertebrae were taken into account. Serum calcium level and urinary calcium/creatinine ratio were determined at control visits repeated at 3-month intervals. After one year, information was gathered on fractures and renal calculosis that had occurred during the follow-up period, as well as osteodensitometry, lumbosacral x-ray, and laboratory screen were repeated.

Treatment protocol

The pre-existing regimen of alendronate 70 mg once per week was left unchanged, but conventional vitamin D 3 and calcium treatment was replaced with 0.5 µg/day alfacalcidol (1αhydroxvitamin D 3 ). The calcium supplementation was stopped also.

Endpoints

The primary endpoint of the study was defined as the change measurable (by DEXA) in bone mineral density (BMD) of the forearm and of axial bones one year after the introduction of alfacalcidol treatment. Secondary endpoints included changes in clinical chemistry parameters (serum calcium, phosphorus, and alkaline phosphatase levels), biochemical markers of bone turnover (serum osteocalcin, PTH, urinary D-Pyr/creatinine), UCa/Cr ratio determined in first-voided morning urine, treatment-emergent adverse reactions, and osteoporotic fractures that occur during follow-up.

Statistical analysis

The differences between the values of study parameters (BMD; serum Ca, P, AP, osteocalcin, PTH levels; urinary D-Pyr/creatinine and UCa/Cr ratios) determined at two time points were analysed using the SPSS 15.0 software package. First, the normality of differences was checked with the Kolmogorov-Smirnov test (normal distribution would allow performing a parametric [t or d] test). The differences between the paired values of all nine variables were significantly different from the normal distribution (p<0.01 for UCa/Cr and p<0.001 for the rest). As the data were unsuitable for analysis with the t-test, non-parametric Wilcoxon signed-rank test was performed to check the uniformity of repeated measurements. (This method is a non-parametric alternative to performing a paired t-test.)

Results

a) Changes in BMD during follow-up

The median of changes as well as the minimal and maximal changes are summarised in 2). The decline of serum alkaline phosphatase level over the follow-up period by a median of 13 U/l was also significant, but clinically not relevant (see Figure 3).

c) Changes in biochemical markers of bone turnover

Changes in serum parathormone (PTH) level were as expected. At baseline, elevated PTH was ascertained in 32 patients; this number decreased to 16 during the one-year follow-up.

The median change in serum PTH level was significantly lower one year later (median change: -10.7 pg/ml) (Figure 3). Serum osteocalcin level decreased by 0.4 ng/ml (median) after treatment; this was accompanied by a decrease in the D-Pyr/creatinine ratio by 0.2 nmol/mmol (p<0,001) (Figure 3). Although the changes showed high scatter, they proved to be highly significant (Wilcoxon's signed rank test, p<0.001). The ranges of relative changes expressed as percentages of the base values for all parameters (Figure 4).

d) Adverse events

Mild hypercalcaemia (serum calcium level <3 mmol/l) was observed in 3 patients; no other clinically relevant adverse reactions were seen. There was no significant increase in urinary calcium based on alendronate therapy.

e) New fractures

As stated above, ten patients had a history of at least one prevalent vertebral or low-trauma peripheral fracture at starting of the study. Four new vertebral and two peripheral (wrist) fractures were recorded during follow up. Two of the vertebral fractures were sustained by patients with a prevalent vertebral fracture. of the femoral neck 1.2% vs. 0.8% (p<0.006), respectively. The proportion of patients who have sustained at least one new vertebral fracture was 9.7% in the alfacalcidol and 24.8% in the control group (risk reduction: 0.61, p=0.005), whereas the proportions of patients with at least one new non-vertebral fracture were 15% vs. 25% (risk reduction: 0.41, p=0.08). Taking the occurrence of any type of incident fractures into account, the above proportions were 19.4% vs. 40.6%, respectively (risk reduction: 0.52, p<0.001) [START_REF] Ringe | Alfacalcidol versus plain vitamin D in the treatment of glucocorticoid/inflammation-induced osteoporosis[END_REF].

Discussion

During the AAC study published in 2007, 30-30 patients were randomised into either of the following 3 treatment arms: a) alfacalcidol 1 µg + calcium 500 mg/day; b) alendronate 70 mg/week + calcium 1000 mg/day + vitamin D 3 1000 IU/day; c) alendronate 70 mg/week + calcium 500 mg/day + alfacalcidol 1 µg/day. During the 2 years of follow up, bone density of the lumbar spine and of the total hip increased by 3%/1.5%, 5.4%/2.4%, and 9.6%/3.8%, respectively. The magnitude of changes observed in patients treated with alendronate + alfacalcidol + calcium was significantly greater than of those seen in the other two groups (p<0.0001 for lumbar BMD and p<0.0002 for femur BMD). The number of new osteoporotic (vertebral + non-vertebral) fractures occurring over 2 years of treatment in the three treatment groups was 9, 10, and 2, respectivelythis finding is also in support of the favourable effect of alfacalcidol and especially of the combination of alendronate + alfacalcidol on fracture prevention [START_REF] Ringe | Superiority of a combined treatment of Alendronate and Alfacalcidol compared to the combination of Alendronate and plain vitamin D or Alfacalcidol alone in established postmenopausal or male osteoporosis (AAC-Trial)[END_REF].

As suggested by several studies, administering antiresorptive agents (e.g. bisphosphonates)

with activated vitamin D 3 derivatives may increase the success of therapy. The beneficial effects of alfacalcidol on intestinal calcium absorption, osteoid mineralization, muscle function and motor coordination, serum PTH level, and the risk of falls may substantially enhance the efficacy of treatment with bisphosphonates [START_REF] Ringe | Improving the outcome of established therapies for osteoporosis by adding the active D-hormone analog alfacalcidol[END_REF][START_REF] Schacht | The therapeutic effects of alfacalcidol on bone strength, muscle metabolism and prevention of falls and fractures[END_REF].

The anabolic effect of alfacalcidol on bone is supported also by in vivo and in vitro data.

Under the effect of alfacalcidol, osteoblasts have been shown to release various growth factors (TGF-β, IGF-1 and -2, bone morphogenetic proteins [BMPs], bone matrix proteins [collagen I, osteocalcin, osteopontin]) and thereby counterbalance the reduction of bone turnover during treatment with bisphosphonates [START_REF] Schacht | The therapeutic effects of alfacalcidol on bone strength, muscle metabolism and prevention of falls and fractures[END_REF][START_REF] Iwamoto | Comparison of the effect of alendronate on lumbar bone mineral density and bone turnover in men and postmenopausal women with osteoporosis[END_REF][START_REF] Greenspan | Early changes in biochemical markers of bone turnover are associated with long-term changes in bone mineral density in elderly women on alendronate, hormone replacement therapy, or combination therapy: a threeyear, double-blind, placebo-controlled, randomized clinical trial[END_REF][START_REF] Reszka | Bone anabolic effects of 1,25(OH)2 Vitamin D3 are detected only in the presence of a powerful antiresorptive[END_REF]. This osteoanabolic effect of alfacalcidol administered in combination with alendronate has been demonstrated by several studies [START_REF] Reszka | Bone anabolic effects of 1,25(OH)2 Vitamin D3 are detected only in the presence of a powerful antiresorptive[END_REF][START_REF] Erben | Prevention of bone loss in ovariectomized rats by combined treatment with risedronate and 1•,25-dihydroxyvitamin D3[END_REF][START_REF] Ito | Curative effect of combined treatment with alendronate and 1-α--Hydroxyvitamin D3 on bone loss by ovariectomy in aged rats[END_REF][START_REF] Schacht | Combined therapies in osteoporosis: bisphosphonates and vitamin D-hormone analogs[END_REF].

Our study was conducted on patients whose bone density was declining despite adequate treatment with a bisphosphonate and supplementation of calcium plus conventional vitamin D 3 since a year at least. Patients with renal impairment or secondary osteoporosis were not allowed to participate in the study. Notwithstanding this, the study population included a substantial number of patients with decreased serum 25-hydroxyvitamin D 3 levels (n=38), accompanied by higher than normal serum PTH level (n=23). This might suggest a potential relationship between resistance to bisphosphonates and the level of vitamin D 3 insufficiency and the increase of serum PTH. Persistence of secondary hyperparathyroidism reduces BMD response to alendronate in older women with osteoporosis [START_REF] Barone | Secondary Hyperparathyroidism Due to Hypovitaminosis D Affects Bone Mineral Density Response to Alendronate in Elderly Women with Osteoporosis: A Randomised Controlled Trial[END_REF]. The improvement of the biochemical markers of bone turnover and increasing BMD indicate that patients apparently resistant to treatment with a bisphosphonate plus supplementation with calcium and conventional vitamin D 3 are more likely to benefit from combination therapy with a bisphosphonate and alfacalcidol [START_REF] Barone | Secondary Hyperparathyroidism Due to Hypovitaminosis D Affects Bone Mineral Density Response to Alendronate in Elderly Women with Osteoporosis: A Randomised Controlled Trial[END_REF]. The enhanced efficacy of alendronate and alfacalcidol in combination is probably related to the synergism between the considerably different modes of action of the two components. In particular, the antiresorptive effect of alendronate is favourably supplemented by the mitigation of osteoclastogenesis [START_REF] Ito | Curative effect of combined treatment with alendronate and 1-α--Hydroxyvitamin D3 on bone loss by ovariectomy in aged rats[END_REF][START_REF] Shibata | Vitamin D hormone inhibits osteoclastogenesis in vivo by decreasing the pool of osteoclast precursors in bone marrow[END_REF] and reduction of serum PTH level, along with the stimulation of osteoblast activity [START_REF] Reszka | Bone anabolic effects of 1,25(OH)2 Vitamin D3 are detected only in the presence of a powerful antiresorptive[END_REF], improvement of bone quality [START_REF] Shiraishi | The advantage of alfacalcidol over vitamin D in the treatment of osteoporosis[END_REF][START_REF] Ito | Curative effect of combined treatment with alendronate and 1-α--Hydroxyvitamin D3 on bone loss by ovariectomy in aged rats[END_REF] and the microarchitecture of trabecular bone by alfacalcidol [START_REF] Ito | Curative effect of combined treatment with alendronate and 1-α--Hydroxyvitamin D3 on bone loss by ovariectomy in aged rats[END_REF]-which also enhances muscle strength and thereby activates the 'mechanostat' function of the skeleton [START_REF] Schacht | The therapeutic effects of alfacalcidol on bone strength, muscle metabolism and prevention of falls and fractures[END_REF]. The whole array of these effects might explain the outstanding effect on bones [START_REF] Ringe | Improving the outcome of established therapies for osteoporosis by adding the active D-hormone analog alfacalcidol[END_REF][START_REF] Schacht | The therapeutic effects of alfacalcidol on bone strength, muscle metabolism and prevention of falls and fractures[END_REF][START_REF] Schacht | Combined therapies in osteoporosis: bisphosphonates and vitamin D-hormone analogs[END_REF]. Further elucidation of these mechanisms would require large-scale studies with fracture endpoints, defined in addition to monitoring changes in bone density.

Although the lack of a control group may be considered a potential flaw of this study, the inclusion of untreated patients losing more than 3 per-cent of their bone density each year would have been ethically questionable at the least.

Conclusion

As shown by the results of this study, patients whose bone density decreases inexorably despite adequate treatment with alendronate and supplemental calcium and vitamin D 3 might benefit from a combined treatment of alendronate with alfacalcidol. The latter combination accomplished significant increase of BMD along with improvement of the biochemical markers of bone turnover, but without any substantial increase in the incidence of adverse effects.

  Some data from the literature suggest that activated vitamin D derivatives (alfacalcidol, calcitriol), also called D-hormone analogs, are superior to conventional vitamin D, as regards the mitigation of fracture risk. In their meta-analysis of 33 clinical studies, RICHY et al. found a 13.4-per-cent (delta RD, 95% CI 7.7-19.8) reduction of fracture risk during the use of activated vitamin D 3 derivatives, compared to a mere 6% (delta RDi, 95% CI[1][START_REF] Cs | A D-vitamin-hiány szerepe a gondozóintézetben ápolt betegek osteopeniájában[END_REF][START_REF] Bhattoa | Prevalence and seasonal variation of hypovitaminosis D and its relationship to bone metabolism in community dwelling postmenopausal Hungarian women[END_REF][START_REF] Trivedi | Effect of four monthly oral vitamin D 3 (cholecalciferol) supplementation on fractures and mortality in men and women living in the community: randomised double blind controlled trial[END_REF][START_REF] Law | Vitamin D supplementation and the prevention of fractures and falls: results of a randomised trial in elderly people in residential accommodation[END_REF][START_REF] Lyons | Preventing fractures among older people living in institutional care: a pragmatic randomised double blind placebo controlled trial of vitamin D supplementation[END_REF][START_REF] Smith | Effect of annual intramuscular vitamin D on fracture risk in elderly men and womena population-based, randomised, double-blind, placebo-controlled trial[END_REF][START_REF] Shebba | Significance of a decline in bone mineral density while receiving oral bisphosphonate treatment[END_REF][START_REF] Barone | Secondary Hyperparathyroidism Due to Hypovitaminosis D Affects Bone Mineral Density Response to Alendronate in Elderly Women with Osteoporosis: A Randomised Controlled Trial[END_REF][START_REF] Joshua | Bisphosphonate resistance in Paget's disease of bone[END_REF][START_REF] Gutteridge | Paget's disease: acquired resistance to one aminobisphosphonate with retained response to another[END_REF][START_REF] Siris | Medical management of Paget's disease of bone: indications for treatment and review of current therapies[END_REF] accomplished by treatment with conventional vitamin D3[START_REF] Richy | Vitamin D analogs versus native vitamin D in preventing bone loss and osteoporosis-related fractures: a comparative meta-analysis[END_REF]. The rate difference (RD) was statistically significant (ANOVA-1; P< 0.001) RINGE et al. studied the changes of BMD and bone fractures in patients with glucocorticoid-induced osteoporosis, over 3 years of treatment with either alfacalcidol or vitamin D 3both administered with calcium supplementation of 500 mg daily. Changes in the BMD of the lumbar spine were 2.4% vs. -0.8% (p<0.0001), and

Figure 1 .

 1 Figure 1. The changes of BMD during follow-up. Box and whiskers plots are summarizing the median, quartiles, and extreme values.

Figure 2 .

 2 Figure 2. Changes of serum calcium and phosphorus levels, as well as of UCa/creatinine ratio during the study.

Figure 3 .

 3 Figure 3. The change of serum alkaline phosphatase activity (U/l), parathormone level (pg/ml), osteocalcin level (ng/ml ) and of urinary deoxypirydinoline crosslinks/creatinine (D-Pyr/creatinine, nmol/mmol) by the end of the follow-up.

Figure 4 .

 4 Figure 4. The ranges of relative changes expressed as percentages of the base values for all parameters.

Figure 2 .

 2 Figure 2. Changes of serum calcium and phosphorus levels (mmol/l), as well as of UCa/creatinine ratio during the study.

Figure 3 .

 3 Figure 3. The change of serum alkaline phosphatase activity (U/l), parathormone level (pg/ml), osteocalcin level (ng/ml ) and of urinary deoxypirydinoline crosslinks/creatinine (D-Pyr/creatinine, nmol/mmol) by the end of the follow-up.

Figure 6 .

 6 Figure 6. The ranges of relative changes expressed as percentages of the base values for all parameters.

Table 1 .

 1

		Unit	Median	Minimum	Maximum
	Se Calcium	mmol/l	0.06	-0.09	1.13
	Se Phosphate	mmol/l	-0.05	-0.49	0.3
	Alkaline phosphatase	U/l	-13	-46	287
	Parathormone	pg/ml	-10.7	-150	22.5
	Urinary Ca/creatinine		0.1	-0.32	0.49
	Osteocalcin	ng/ml	-0.4	-5.8	4.2
	BMD of forearm	g/cm²	0.007	-0.011	0.039
	BMD of lumbar 1-4 vertebrae	g/cm²	0.012	-0.998	0.116
	Relative change of forearm BMD	%	2.18	-3.5	16.3
	Relative change of lumbar BMD	%	1.38	-11.3	15.8
	Deoxypyridinoline crosslinks/creatinine	nmol/mmol	-0.2	-15.69	0.12

Table 1 :

 1 Median, minimal and maximal changes in various parameters as a result of treatment After treatment for 396,5 ± 21,3 days with alendronatewith no change in the original dosage regimen of 70 mg/week, butin combination with 0.5 µg/day alfacalcidol instead of The whiskers are lines that extend from the box to the highest and lowest values, excluding outliers. A line across the box indicates the median.

	the previously prescribed conventional vitamin D 3 (administered with calcium), we observed
	the following changes (median values): forearm BMD increased by 0.007 g/cm², and lumbar
	(L 1-4 ) BMD increased by 0.012 g/cm². The changes in T-scores showed rather high scatter.
	The relative changes in BMD values were 2.18% and 1.38% for the forearm and lumbar
	vertebrae, respectively. BMD changes that have occurred during one-year follow-up are

shown in Figure

1

. Box and whiskers plots are summarizing the median, quartiles, and extreme values. The box represents the interquartile range which contains the 50% of values.


MD Josef Finsterer

MD Stefan Höger

Multi-system mitochondrial disorder with recurrent steroid-responsive eosinophilia

Keywords: respiratory chain, central nervous system, brain, neuromuscular, myopathy, multi-system disease

Objectives: Though mitochondrial disorders due to a respiratory chain defect may manifest with haematological abnormalities such as anaemia, neutropenia, or thrombocytopenia, recurrent steroid-responsive eosinophilia has not been reported as a manifestation of a mitochondrial disorder.

Clinical presentation and intervention: In a polymorbid 65yo female recurrent erythema, asthma, chronic bronchitis, gastritis with eosinophilic granuloma, recurrent episodes hypereosinophilia, and polyarthralgia suggested Churg-Strauss syndrome, which was excluded upon absence of eosinophilic vasculitis on any of the biopsies. However, corticosteroids were effective for hypereosinophilia. In addition to the hypereosinophilic syndrome a mitochondrial disorder with multi-system affection was diagnosed upon short stature, basal ganglia calcification, pituitary adenoma, hypoacusis, tinnitus, atrial fibrillation, diffuse weakness of all four limbs, diffuse wasting, tetraspasticity, muscle aching, restless-leg syndrome, axonal neuropathy, polyarthralgia, osteoporosis, arterial hypertension, occasional thrombocytopenia, hypercholesterolaemia, hyponatriaemia, elevated liver function parameters, and a muscle biopsy indicative of a mitochondrial disorder. Hypereosinophilia was interpreted as a manifestation of the mitochondrial disorder after exclusion of various differentials.

Conclusion: Mitochondrial disease may go along with marked eosinophilia mimicking

Churg-Strauss syndrome. Steroids may be useful to resolve episodic eosinophilia but may be ineffective and contraindicated for mitochondrial myopathy.

Introduction

Mitochondrial disorders due to a respiratory chain defect frequently manifest as multisystem disorders, affecting the central nervous system, peripheral nervous system, endocrinological system, heart, eyes, ears, gastrointestinal tract, kidneys, dermis, or the bone marrow [START_REF] Zeviani | Mitochondrial disorders[END_REF]. Haematological manifestations of mitochondrial disorders include anaemia, neutropenia, or thrombocytopenia [START_REF] Finsterer | Hematological manifestations of primary mitochondrial disorders[END_REF]. Recurrent steroid-responsive eosinophilia has not been reported as a manifestation of a mitochondrial disorder.

Case report

The patient is a 65-yo, HIV-negative, Caucasian female (height: 161cm, weight: 59kg), with the following history: recurrent herpetic keratitis since age 14y; recurrent erythema at changing skin locations since about age 40y; coughing and chronic bronchitis due to suspected asthma bronchiale after discontinuation of smoking since age 50y; bronchoscopy at age 53y and 54y to treat recurrent atelectases; resection of the left lower lobe of the lung because of bronchiectasias and atelectasis at age 54y; recurrent eosinophilia since at least age 54y, for which she received steroids during two months with success.

At age 60y she was hospitalised for chest pain. Acute myocardial infarction was excluded but echocardiography showed mitral insufficiency with dilation of the left atrium and tricuspid insufficiency. There was slight leucocytosis, extensive eosinophilia, and slight thrombocytopenia (table 1). IgE were elevated to 834U/l (n, <120U/l). Upon gastroscopy a chronic gastritis and an eosinophilic granuloma were detected. Colonoscopy revealed a non-specific colitis. Skin biopsy for the extensive exanthema of the skin revealed alterations initially being interpreted as eosinophilic vasculitis. Churg-Strauss syndrome (CSS) was suspected and corticosteroids were restarted. After dismissal hypercholesterolaemia with a maximum value of 318mg/dl (n <200mg/dl) and hyperhomocysteinaemia with a maximum value of 18.6 μmol/l were found. A statin therapy was initiated. Since about age 60y she complained about arthralgias in the distal Linking powered by eXtyles interphalangeal joints being attributed to rhizarthrosis at age 64y. Tinnitus was noted since age 62y. After discontinuation of corticosteroids at age 62y nasal congestion and running nose occurred, being attributed to chronic sinusitis. Also at age 62y discontinuation of steroids during three months resulted in a relative eosinophilia count of 30% (table 1), why corticosteroids were restarted with immediate benefit. At age 64y bilateral coxarthrosis with left-sided coxitis and moderate osteoporosis were diagnosed.

Since age 64y she complained about muscle aching in the thighs after exercise. At the same age a pituitary adenoma was detected on cerebral MRI and successfully removed by surgery at age 65y. Postoperatively, temporary diabetes insipidus and hyponatriaemia required adequate substitution. Since surgery arterial hypertension occurred and was sufficiently treated with bisoprolol. At age 64y cyclophosphamide was tried but had to be discontinued after 2 weeks because of gastrointestinal side-effects. At age 64y interferone-α2a was given three times without discontinuation of corticosteroids. At age 66y atrial fibrillation was documented for the first time and oral anticoagulation started.

Occasionally, glutamate-oxalate transaminase (GOT), glutamate-pyruvate transaminase (GPT), and α-amylase were elevated, but creatine-kinase was normal throughout the observational period (table 1). Echocardiography at age 64y. carried out to assess the degree of cardiac involvement, revealed normal thickness of the left ventricular myocardium, slight mitral insufficiency, dilation of the left atrium, and a minimal tricuspid insufficiency. At age 65y corticosteroids were discontinued and methotrexate was tried but had to be discontinued because of elevated liver function parameters.

Corticosteroids were restarted. At age 65y she developed permanent muscle aching and cramps and transient muscle tenderness in all muscles of the left upper limb, carpopedal spasms, and hypaesthesia of the fingers upon voluntary contractions. Recurrent erythema, was lastly interpreted as atopic dermatitis after exclusion of a vasculitis by several skin biopsies. Concerning the family history her mother experienced a myocardial infarction twice and her father suffered from diabetes and a stroke. A neuromuscular disorder had not been reported. Linking powered by eXtyles Clinical neurologic examination revealed hypoacusis with left-sided predominance, a positive Brudzinski sign, postural tremor, discrete diffuse weakness of the upper limbs with left-sided predominance, weakness for hip flexion (M5-) bilaterally, diffuse wasting on the upper and lower limbs, an exaggerated biceps tendon reflex on the right side, and exaggerated patella tendon reflexes bilaterally. Nerve conduction studies at age 64y revealed reduced amplitude of the compound muscle action potentials in the left median, left ulnar, and right median nerves, and reduced nerve conduction velocity of the left median nerve. Needle electromyography of the biceps, triceps, and brachioradialis muscles on the left side at age 64y revealed abnormal spontaneous activity in form of positive sharp waves and extensive pseudomyotonic discharges, normal motor unit architecture, and a dense interference pattern with an amplitude of 0.5mV. Biopsy of the left deltoid muscle at age 64y showed mild myopathic changes with single ragged-red fibres (figure 1a and1b), COX negative fibres (figure 1c) and slight glycogen increase, suggestive of metabolic/mitochondrial myopathy. Ultrastructurally, no intramitochondrial paracristalline inclusions were demonstrable. In addition, a myoadenylate-deaminasedeficiency was observed (figure 1c). The MRI of the cerebrum at age 64y revealed cortical atrophy and the pituitary adenoma. Antibodies against Filaria, Trichinella spiralis, Toxocara, Taenia solium, Fasciola hepatica, Toxoplasma gondii, Entamoeba histolytica were negative. After discontinuation of statins, because of the mitochondrial disorder, a statin and ezetimibe had to be restarted for recurring hyperlipidemia. The patient refused to consent for investigations of the CHIC2 deletion, FIP1L1 FDGFR fusion, or for lymphocyte-variant eosinophilia by PCR for T-cell receptor gene rearrangement, by IL-5 level determination, or by flow on the PB or marrow to look for clonal populations. The aspergillus IgE level was not determined.

Discussion

The described patient was presented for the unusual occurrence of eosinophilia together with a mitochondrial disorder. The mitochondrial disorder was diagnosed upon the clinical findings, electromyography, and the muscle biopsy findings. The patient presented with short stature, had a history of pituitary adenoma, basal ganglia calcification, tetraspasticity, restless legs, hypoacusis and tinnitus, paroxysmal atrial fibrillation, myopathy, polyneuropathy, polyarthralgia, osteoporosis, recurrent thrombocytopenia, occasionally hyponatriaemia, slightly increased GOT and GPT, and hyperlipidemia [START_REF] Walker | Respiratory chain encephalomyopathies: a diagnostic classification[END_REF]. Haematologic abnormalities are a frequent finding in patients with a mitochondrial disorder and include anaemia, neutropenia, thrombocytopenia, pancytopenia, or thrombocytosis [START_REF] Finsterer | Hematological manifestations of primary mitochondrial disorders[END_REF], but eosinophilia has not been reported [START_REF] Jongpiputvanich | Mitochondrial respiratory chain dysfunction in various neuromuscular diseases[END_REF].

Generally, episodic eosinophilia is attributable to nonparasitic or parasitic (helminthic or protozoan) infection, allergic or atopic disease, to skin disorders, immunologic or rheumatologic diseases, hypoadrenalism, neoplastic or lymphoreticular disorder, hypereosinophilic syndromes, irradiation, or drugs [START_REF] Nutman | Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 1-2002. A 24-year-old woman with paresthesias and muscle cramps after a stay in Africa[END_REF]. Arguments for an allergic reaction are that eosinophilia was first detected during an episode of asthma attacks, that the dermal abnormalities were interpreted as atopic dermatitis, that IgEs were elevated once, and that asthma, dermatitis and eosinophilia responded well to steroids. Arguments against allergy, however, are that no causative allergen could be identified and that eosinophilic infiltrates were also found in the stomach. Infection as the cause of eosinophilia was excluded upon absent signs of inflammation and negative tests for antibodies against the most common parasites. Hypocorticism was excluded upon normal aldosterone and cortisol levels and normal morphology of the suprarenal gland on imaging studies. A haematological malignancy or lymphoreticular disease was excluded upon a normal bone marrow aspirate.

CSS was initially considered responsible for the cutaneous, pulmonary, and hematological abnormalities but was lastly excluded since the findings did not fulfill the criteria of the American College of Rheumatology for CSS [START_REF] Cottin | Churg-Strauss syndrome[END_REF][START_REF] Masi | The American College of Rheumatology 1990 criteria for the classification of Churg-Strauss syndrome (allergic granulomatosis and angiitis)[END_REF]. According to these criteria CSS is diagnosed in the presence of asthma, eosinophilia >10%, mononeuritis multiplex or polyneuropathy, transient, non-fixed, pulmonary infiltrates on chest X-ray, paranasal sinus abnormalities, such as allergic rhinitis, nasal polyposis, recurrent sinusitis, or a biopsy, showing a blood vessel with extravascular, eosinophilic tissue infiltration [START_REF] Cottin | Churg-Strauss syndrome[END_REF][START_REF] Masi | The American College of Rheumatology 1990 criteria for the classification of Churg-Strauss syndrome (allergic granulomatosis and angiitis)[END_REF]. Rare manifestations of CSS comprise eosinophilic gastroenteritis, Linking powered by eXtyles cutaneous vasculitis, nephropathy, or arthromyalgia [START_REF] Noth | Churg-Strauss syndrome[END_REF]. According to these criteria CSS takes a progressive course and follows three stages [START_REF] Lanham | Churg-Strauss syndrome[END_REF]. The first stage, lasting for years, starts with allergic rhinitis followed by nasal polyposis, allergic sinusitis, or asthma [START_REF] Noth | Churg-Strauss syndrome[END_REF].

The second stage is characterised by blood and tissue eosinophilia with eosinophilic infiltrations of the respiratory system and the gastrointestinal tract. In the third stage, symptoms of systemic vasculitis occur with a mean delay of 3y after onset [START_REF] Cottin | Churg-Strauss syndrome[END_REF].

Corticosteroids are the treatment of choice for all stages. In patients with severe lifethreatening manifestations, however, cyclophosphamide may be helpful [START_REF] Hellmich | Recent progress in the pharmacotherapy of Churg-Strauss syndrome[END_REF]. In cases with better prognosis, methotrexate can be given to reduce the cumulative dose of corticosteroids. If corticosteroids or cyclophosphamide are ineffective, immunoglobulins or anti-TNF blocking agents, like infliximab or etanercept, may be tried. A therapeutic effect of cyclosporin, mofetil, azathioprin, or interferon, is unproven.

Arguments for CSS in the presented patient are the occurrence of asthma, eosinophilic gastric granuloma, occasionally eosinophilia >10%, and the prompt response to steroids.

Arguments against CSS are the absent progression of the disease, the absence of allergic rhinitis, nasal polyposis, and recurrent sinusitis, the normal blood sedimentation rate, and the absence of a biopsy showing eosinophilic vasculitis [START_REF] Masi | The American College of Rheumatology 1990 criteria for the classification of Churg-Strauss syndrome (allergic granulomatosis and angiitis)[END_REF]. The initial diagnosis of an eosinophilic vasculitis was lastly turned down by the dermatologists. Furthermore, chest X-ray did not show patchy, non-segmental, peripheral infiltrates, often fleeting or nonspecific in nature [START_REF] Cottin | Churg-Strauss syndrome[END_REF][START_REF] Noth | Churg-Strauss syndrome[END_REF], p-ANCA were not elevated, which is the case in 66% of the cases, and the rheumatoid factor was not elevated, which is the case in >80% of the cases. Furthermore, muscular manifestations did not resolve under corticosteroids, which is usually the case in CSS, but progressed despite long-term administration of steroids.

Additionally, muscle biopsy did not show any eosinophilic infiltrations. Differentials of eosinophilia with neuromuscular disease excluded in addition to CSS were the eosinophilic fasciitis (Shulman syndrome), focal eosinophilic myositis (calpainopathy), eosinophilic polymyositis, eosinophilia-myalgia syndrome due to ingestion of L-tryptophan, and the toxic oil syndrome due to ingestion of adulterated oil containing aniline [START_REF] Pellissier | Neuromuscular diseases with eosinophilia[END_REF]. These differentials were excluded upon the history, and clinical Linking powered by eXtyles and muscle biopsy findings. Eosinophilic polymyositis was additionally excluded upon normal creatine-kinase and absent myopathic pattern of the motor unit action potentials on needle electromyography. Idiopathic hypereosinophilic syndrome, characterised by chronic hypereosinophilia for at least six months leading to tissue damage and polyneuropathy [START_REF] Moles | Idiopathic hypereosinophilic syndrome: toward a new molecular-targeted therapy and a new cytomorphological and molecular definition[END_REF][START_REF] Gotlib | Molecular classification and pathogenesis of eosinophilic disorders: 2005 update[END_REF], was excluded since hypereosinophilia never lasted >6 months and since there was no tissue damage. Other differentials of eosinophilia were excluded upon the history and appropriate laboratory tests [START_REF] Nutman | Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 1-2002. A 24-year-old woman with paresthesias and muscle cramps after a stay in Africa[END_REF]. Corticosteroids were excluded as the cause of the skeletal muscle abnormalities upon the histological findings on muscle biopsy, the eosinophilia, and the electromyographic findings.

In conclusion, this case shows that mitochondrial disease may go along with marked eosinophilia and manifestations mimicking CSS. Steroids may be useful to resolve episodic eosinophilia in mitochondrial disease but may be ineffective and contraindicated for mitochondrial myopathy. Linking powered by eXtyles We thank Dr. Ellen Gelpi for providing the muscle biopsy and interpreting the findings Linking powered by eXtyles

Figure 1 .

 1 Figure 1. Muscle biopsy showing single ragged red fibres on the haematoxylin-eosin stain (a) and the Gomori-trichrome stain (b), and single COX negative fibres (c). Additionally, myoadenilat-deaminase deficiency was observed (d. Inset: normal MAD activity). (a, b, and c: original magnification x400; d and inset: x200)

Table 1 .

 1 Lavboratory findings in the described patient over 13 years Parameter RL 4/94 12/00 12/00 1/01 1/00 2/00 na/02 12/04 1/05 2/05 3/05 5/05 6/05 7/05 8/05 5/07

	Leukocyte count 4-10G/l	na	11.2 9.4	14.9 10.9 16.0 na	9.9	8.0	6.9	9.0	6.2	9.6	5.0	8.5	10.0
	Eosinophil count 2-4%	40.0 8.5	24.2 44.1 32.9 na	30.0 3	1.6	3	0.8	4	1.4	7.7	2.7	na
	Thrombocytes	150-300G/l na	168 na	132 na	na	na	146 206 168	164 133 172 138	164 154
	GOT	<31U7i	na	na	na	na	na	na	na	na	72	45	28	27	27	na	14	na
	GPT	<34U/l	na	na	na	na	na	na	na	na	54	33	10	9	10	na	29	na
	CK	<145U/l	na	na	na	na	na	na	na	na	102 53	90	85	89	na	89	144

GOT: glutamate-oxalate transaminase, GPT: glutamate-pyruvate transaminase, CK: creatine-kinase, na: not available