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High-Dose Chemotherapy in Poor-Prognosis Adult Small Round-Cell Tumors: Clinical and Molecular Results From a Prospective Study

From the Departments of Medical Oncology and Hematology, General Surgery, and Thoracic Surgery, Istituto Clinico Humanitas, Rozzano, Milan, Italy.

Address reprint requests to Alexia Bertuzzi, MD, Department of Medical Oncology and Hematology, Istituto Clinico Humanitas, Rozzano, Milan, Italy; email: alexia.bertuzzi{at}humanitas.it

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: The prognosis of metastatic/high-risk localized small round-cell tumors (SRCTs) treated conventionally is dismal. In this phase II study, we explored a high-dose chemotherapy (HD-CT) approach and analyzed the clinical significance of fusion transcripts detection.

PATIENTS AND METHODS: From June 1997 to November 1999, 28 SRCT patients (median age, 26 years; 14 peripheral primitive neuroectodermal tumors [pPNETs], seven rhabdomyosarcomas [RMSs], and seven desmoplastic small round-cell tumors [DSRCTs]) received induction chemotherapy with ifosfamide, epirubicin, and vincristine followed by HD-CT. Local treatment (radiotherapy and/or surgery) was performed when possible. Molecular analysis was performed on peripheral-blood and leukapheresis products by reverse-transcriptase polymerase chain reaction.

RESULTS: Overall response (OR) was 65% (18 of 28), with 40% complete response and 25% partial response. According to histology, the OR rate was 86% in pPNET and 43% in both RMS DSRCT. With a median follow-up of 35 months, median overall survival was 16 months and median progression-free survival (PFS) was 10 months. PFS was statistically better in pPNET than other histologic types (P = .0045). No correlation was found between the fusion transcript and clinical outcome during follow-up. Furthermore, transcript detection in leukapheretic products was not of prognostic significance.

CONCLUSION: Intensive HD-CT seems to enhance the response rate and survival when compared with conventional treatment in poor-prognosis pPNET. The poor results of this treatment in RMS and DSRCT do not support the inclusion of such an approach in these patient subsets. No definitive conclusions can currently be drawn concerning the clinical implications of the detection of fusion transcripts during treatment or follow-up.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
SMALL ROUND-CELL tumors (SRCTs) are rare tumors with a common morphologic histology, highly specific chromosomal translocations, and different responses to multimodal treatment. The family includes Ewing’s sarcomas, peripheral primitive neuroectodermal tumors (pPNETs), rhabdomyosarcomas (RMSs), and desmo-plastic small round-cell tumors (DSRCTs).

It has been suggested that SRCTs arise from a multipotent neural crest stem cell outside the central and sympathetic nervous system and have similar morphologic features. Their spectrum of neural differentiation varies between peripheral neuroepitheliomas with well-developed neural features to poorly differentiated Ewing’s sarcomas.

Globally, multimodality treatment programs achieve event-free survival rates of 60% in pPNET/Ewing and RMS patients, whereas the results in DSRCT are dismal, indicating that desmoplastic tumors are generally refractory to conventional treatment modalities.¹ Recently, it has been possible to identify a subgroup of SRCTs with a very poor prognosis, with an overall 2-year survival rate of only 2% to 10%.² These subsets are characterized by either the presence of metastatic disease other than lung metastases at diagnosis, localized disease with a tumor volume of more than 100 mL,³ axial site involvement,^4,5 a lack of response to first-line chemotherapy,⁶ or relapsing disease.

Several studies have therefore been conducted to define the place of high-dose chemotherapy in improving clinical results. To date, the results are not conclusive, although in nonrandomized studies, high-dose chemotherapy seems to produce better results.

Recurrent and specific chromosomal translocations characterize every histologic subtype of SRCT (Table 1). The prototype rearrangement is the joining of the EWS gene on chromosome 22 with ets gene family members (FLI-1, ERG, or ETV1) on chromosome 11 in two closely related tumors, Ewing’s sarcoma and pPNET. The unique chromosomal rearrangement identified in DSRCT, t(11;22)(p13;q12), juxtaposes two genes, the EWS gene and WT1, the Wilm’s tumor suppressor gene, at 11p13.⁷ In both instances, the transcripts comprise the NH2-terminal effector region of EWS, whereas the COOH-terminal RNA binding region is replaced by the fusion partner. The chimeric product, which has been shown in many cases to be transforming in vitro and in vivo,^8,9 presumably functions through this novel combination of effector and binding regions to dysregulate a critical group of the target gene.¹⁰ Alveolar rhabdomyosarcomas are associated with unique chromosomal translocations t(2;13)(75%) and t(1;13)(10%) that arise from fusion of PAX3 on chromosome 2 or PAX7 on chromosome 1, respectively, to the FKHR gene on chromosome 13. The resulting fusion proteins contain the N-terminal region of the PAX proteins and the C-terminal transactivation domain of FKHR. This fusion transcript exerts its carcinogenic role through dysregulation of PAX3-specific target genes.¹¹

On the basis of these considerations, we carried out a combined clinical and biologic study during an intensive chemotherapeutic program to investigate the effects of high-dose chemotherapy on overall survival (OS) and time to disease progression. We also verified the chemosensitivity of the individual tumor types and explored the prognostic significance of the fusion transcripts detected in peripheral blood, in bone marrow, and in leukapheresis products.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Eligibility Criteria
Patients aged 15 to 60 years with poor prognosis SRCT, as defined above, were considered eligible for the study. The extent of the disease was determined using chest x-rays, technetium-99m bone scans, computed tomography or magnetic resonance imaging, and the evaluation of bone marrow specimens. The diagnosis was established on the basis of histochemical findings as previously described .^12-15 Informed consent for the treatment was obtained in accordance with our institutional review board guidelines.

Treatment Protocol
The study treatment program (Fig 1) included chemotherapy and local treatment (surgery and/or radiotherapy), followed by myeloablative therapy with stem-cell rescue. The chemotherapeutic program consisted of a four-course induction phase with epirubicin 30 mg/m² days 1 to 3, ifosfamide 3 g/m² days 1 to 3, and vincristine 2 mg on day 1 (IVE) every 3 weeks, followed by a mobilization phase with high-dose etoposide 2.4 g/m² or cyclophosphamide 7 g/m², followed by granulocyte colony-stimulating factor (G-CSF). Peripheral-blood stem cells were collected using a Cobe Spectra CS 3000 (Cobe, BCT, Inc, Lakewood, CO). Finally, patients received a high-dose chemotherapy phase with melphalan 160 mg/m² plus mitoxantrone 60 mg/m² or thiotepa 600 mg/m², followed by re-infusion of the peripheral-blood stem cells. G-CSF 300 µg/d starting on day +5 was administered when the number of re-infused CD34⁺ cells was less than 5 x 10⁶/kg. Only patients in complete response (CR) or partial response (PR) proceeded to high-dose chemotherapy.

View larger version (27K): [in this window] [in a new window]   Fig 1. Treatment program. Abbreviations: CY, cyclophosphamide; L-PAM, melphalan; PB, peripheral blood; BM, bone marrow; RT-PCR, reverse transcriptase polymerase chain reaction; PBSC-T, peripheral-blood stem-cell transplantation; MITOX, mitoxantrone; VP-16, etoposide.

Response Evaluation
Tumor response was evaluated after four IVE courses, before the myeloablative regimen, and at the end of all therapies. CR was defined as no detectable SRCT; PR was defined as a more than 50% reduction in measurable tumors; stable disease (SD) was defined as no tumor growth, no new lesions, and a less than 50% reduction in any measurable tumor; and progressive disease (PD) was defined as tumor growth of more than 25% in volume or any new lesion. Side effects were graded using the toxicity criteria of the National Cancer Institute.

Molecular Study
The molecular analyses were made on peripheral blood and bone marrow at the time of diagnosis, on peripheral blood alone after the induction phase, on the leukapheresis products, and during the follow-up.

Sample Preparation and RNA Extraction
RNA was extracted from peripheral blood and bone marrow samples and isolated by TriZol reagent (Gibco BRL, Gaithersburg, MD) according to the manufacturer’s directions and resuspended in diethylpyrocarbonate-treated water.

Reverse Transcriptase Polymerase Chain Reaction
One microgram of total RNA was reverse-transcribed using 200 U of Maloney murine leukemia virus RT (SuperScript II, Gibco BRL) in a 50-µL reaction volume containing 20 mmol/L Tris-HCl, 50 mmol/L KCl pH 8.3, 0.5 mmol/L dNTPs, 10 mmol/L DTT, and 300 ng of random primers. Templates and random hexamers were first incubated at 65°C for 10 minutes, and reverse-transcription was performed at 42°C for 90 seconds; the procedure was stopped by enzyme inactivation at 70°C for 10 minutes.

Ten microliters of the cDNA was used as a template for a nested polymerase chain reaction (PCR) in the presence of 10 pmol of both EWS-1 and FLI1.2 primers. PCR was performed in a total volume of 50 µL and included 1 minute of denaturation at 95°C and 40 cycles of 30 seconds denaturation at 95°C, 60 seconds annealing at 62°C, and 60 seconds extension at 72°C, followed by 10 minutes elongation. The PCR conditions were 30 mmol/L Tris-HCl, 20 mmol/L KCl pH 8.3, 1 mmol/L dNTPs, 1.5 mmol/L MgCl₂, and 2.5 U GoldTaq (Perkin Elmer, Norwalk, CT), and PCR was performed using the Gene Amp Gold RNA Reagent Kit (Gibco, BRL).

The primers were designed to amplify all of the published EWS/ETS-related gene rearrangements and could detect both EWS/FLI1 or EWS/ERG transcripts.

Two microliters of the outer products were amplified in the inner reaction in the presence of 10 pmol of EWS-2 and FLI1.1 primers in the same PCR conditions. The products of the inner amplification were loaded on a 2% agarose-ethidium bromide stained gel and visualized under ultraviolet rays.

TC71 and RD-ES (DSMZ collection) were used as positive controls for type 1 and type 2 pPNET, respectively. DOHH2 cell line (DSMZ collection) was used as negative control.

The type 1 EWS/FLI1 translocation (EWS exon 7/FLI1 exon 6) is 272 base pairs (bp) in length, whereas type 2 is 338 bp long. The two positive controls and DOHH2 negative control were included in each amplification.

Preparation of cDNA from DSRCT samples was similarly performed. Templates were amplified in standard conditions by a single reaction in the presence of 10 pmol of both WT.1 and EWS22.3 primers in the following cycling conditions: 94°C for 10 minutes, 35 cycles at 94°C for 30 seconds, 65°C for 30 seconds, 72°C for 60 seconds, and a final extension at 72°C for 10 minutes. The expected fragment lengths vary according to the translocation type from 259 to 522 bp (Table 2).

Statistical Methods
The survival analysis was determined using the Kaplan-Meier product-limit method by computing the number of days elapsing between the date of the start of treatment and either the date of death or last contact for OS or disease progression for progression-free survival. The differences between the curves were evaluated using the log-rank test.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patient Characteristics
Since June 1997, we have treated 48 SRCT patients. Fifteen (31%) were ineligible for the study because of either previous treatment with an anthracycline-containing regimen (n = 9), age of more than 60 years (n = 2), postsurgical death (n = 1), good-prognosis localized disease (n = 2), or poor performance status (n = 1). Of the 28 enrolled patients (21 men and seven women) with a median age of 26 years (range, 15 to 45 years), 14 (50%) had pPNET, seven (25%) had RMS, and seven (25%) had DSRCT. All of them had a poor prognosis: 15 (53%) had advanced localized disease, nine (32%) had metastatic disease, three (11%) were in relapse, and one (4%) with localized lower-extremity disease had stable disease after induction chemotherapy (Table 3). Five patients are on treatment and were not considered for analysis.

The overall response rate was 65% (18 of 28), with 11 CRs (40%) and seven PRs (25%); all except one of the patients in CR had pPNET. The remaining 10 patients (35%) had PD (Table 5).

View larger version (31K): [in this window] [in a new window]   Fig 2. Responses related to histologic type at the end of the treatment program.

View larger version (10K): [in this window] [in a new window]   Fig 3. OS and PFS for all 28 patients.

View larger version (10K): [in this window] [in a new window]   Fig 4. OS by histologic type.

View larger version (8K): [in this window] [in a new window]   Fig 5. OS in patients receiving (—) or not receiving (---) high-dose chemotherapy (HD-CT).

View larger version (12K): [in this window] [in a new window]   Fig 6. PFS by histologic type.

View larger version (20K): [in this window] [in a new window]   Fig 7. RT-PCR results in pPNET patients in peripheral blood after treatment. Abbreviations: NA, not available; FU, follow-up.

View larger version (20K): [in this window] [in a new window]   Fig 8. RT-PCR results in pPNET patients in leukapheretic products. LK, leukapheresis.

In the complete pPNET group of patients in whom multiple molecular tests were able to be performed on peripheral-blood samples, CR was seen where the test became negative after previous positive results (n = 3) and where the reverse was found (n = 2) (Fig 7).

Five of 11 patients had type 2 transcripts detected. Two patients are still alive in CR after 18 and 21 months of follow-up, one patient died in PR as a result of transplantation-related toxicity, one patient died 10 days after mini-allogeneic transplantation, and one patient with metastatic disease at diagnosis experienced PD.

At diagnosis and during treatment, all of the biologic samples taken from the patients with desmoplastic tumors were positive for the EWS/WT1 fusion transcript. In only one of these patients, who had a PR after mini-allogeneic transplantation, was the fusion transcript no longer detectable in peripheral blood after cyclosporine withdrawal.

Toxicity
The treatment program was well tolerated. No toxic deaths were observed after the IVE courses. One pPNET patient died as a result of pulmonary toxicity 4 days after the peripheral-blood stem-cell re-infusion, and one pPNET patient who was allocated to mini-allogeneic transplantation died during the aplasia period as a result of Pseudomonas aeruginosa sepsis. Nonhematologic toxicity after high-dose chemotherapy consisted of oral mucositis: grade 4 in 63% of the treated patients (12 of 19), grade 3 in 21% (four of 19), and grade 2 in 16% (three of 19). No severe infections were observed. The median number of re-infused CD34⁺ cells was 8.8 x 10⁶/kg. All of the patients required platelet and blood transfusions. The median time from commencement of chemotherapy to hospital discharge was 17 days.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
A number of studies have been published on the role of high-dose chemotherapy in patients with poor-prognosis SRCTs, as the 2-year survival rate after conventional treatment is only approximately 10%, with no patient surviving 5 years.² Miser et al¹⁶ described the clinical results in 17 patients with advanced pPNET/Ewing’s sarcoma treated with vincristine, doxorubicin, and cyclophosphamide plus radiotherapy, followed by high-dose chemoradiotherapy supported by autologous bone marrow transplantation: 94% achieved a CR and 58% were still in CR after 18 months. Burdach et al² treated 17 poor-prognosis Ewing’s sarcoma patients with high-dose chemoradiotherapy (melphalan and etoposide ± carboplatin and total-body irradiation) and autologous or allogeneic bone marrow stem-cell support. The 6-year event-free survival rate was 45% as compared with 2% after conventional chemotherapy, and the relapse rate was lower (52% v 98%). However, only the chemosensitive patients were allocated to intensive treatment. Recently, Burdach et al¹⁷ published a more than 5 years follow-up update on 36 patients with primary multifocal or relapsed Ewing’s tumors treated with melphalan and etoposide ± carboplatin and total-body irradiation. Approximately one fourth of patients achieved long-term survival with consolidation by high-dose chemotherapy.

These data have been confirmed by other groups. The data from the European Blood and Marrow Transplantation Registry confirm that better results can be obtained in poor-prognosis patients in first or second CR by administering high-dose consolidation chemotherapy: the 5-year event-free survival rates were, respectively, 21% and 32%, with no difference between the conditioning regimens in terms of survival.¹⁸ Atra et al¹⁹ treated 18 high-risk patients with high-dose busulphan and melphalan followed by peripheral-blood stem-cell re-infusion, and after 2 years, 72% were in continuous CR. However, in this study, the majority of patients had pulmonary metastases alone.

Recently, Kushner and Meyers²⁰ published their results and a literature review about the efficacy of high-dose chemotherapy in bone and/or bone marrow metastatic Ewing’s sarcoma/pPNET. In their experience, although a good response to induction therapy (90%) was observed, only 11 patients (52%) were allocated to high-dose chemotherapy, and only one patient became a long-term event-free survivor (7 years of follow-up). The results of large and small series of patients treated with myeloablative therapy in first remission demonstrate that 20% to 25% may become long-term survivors.^18,21

In our study, 72% of the pPNET/Ewing’s sarcoma patients achieved CR at the end of treatment program: after a median follow-up of 32 months, the median OS was 15 months, and 2-year OS was 55%. Although this was not a randomized study, these findings are in agreement with those previously reported and make it legitimate to consider high-dose chemotherapy as the best treatment option in this particular patient setting. The percentage of complete responders at the end of treatment was also considerably higher than that seen at the end of the induction phase (72% v 25%), and only two (14%) of the patients had PD, both of whom had metastatic disease at diagnosis. The long-term survivors, however, were all patients with locally advanced disease or disease at a primary axial site, in whom local treatment (surgery or radiotherapy) could be radical.

It is noteworthy that the median age of our patients was higher (26 years) than that of previously published reports. The unfavorable prognostic role of age^3,17 seems to have been abrogated by high-dose chemotherapy in our study.

The results obtained in the RMS patients were dismal, with only one patient in continuous CR after a follow-up of 31 months. The median duration of response was consequently short (3 months), and the majority of the patients experienced PD. These results are in line with the European Blood and Marrow Transplantation Registry data, showing a 3-year OS rate of 28% in RMS patients in first CR and 12% in those in second CR.²² Furthermore, it has been reported that consolidation with high-dose chemotherapy in responding pediatric patients does not lead to any clinical improvement in comparison with conventional treatment.²³ However, Boulad et al²⁴ reported a 2-year OS rate of 56% and a PFS rate of 53% in 21 pediatric RMS patients treated with high-dose induction chemoradiotherapy followed by autologous bone marrow transplantation. Indeed only patients in CR or with a good PR were considered eligible for high-dose chemotherapy. Further analysis on a larger series of patients is mandatory if definitive conclusions about this histologic subgroup are to be made.

Kushner and Meyers²⁰ treated 12 DSRCT patients with a median age of 14 years according to the P6 protocol, with only four patients (two in CR and two in PR) receiving high-dose consolidation chemotherapy: the patients in CR were alive and disease-free at the time of the report, but both PR patients experienced PD. In our study, three (43%) of seven DSRCT patients achieved PR after induction chemotherapy and were subsequently allocated to high-dose chemotherapy: two of them experienced PD, whereas the third underwent debulking surgery and has been assigned to allogeneic stem-cell transplantation. According to these observations, DSRCT should be considered chemoresistant, and high-dose treatment does not seem to modify the outcome.

We have conducted biologic studies to obtain more informations about cytogenetic characteristics of these tumors and their correlation with clinical outcome. SRCTs are characterized by specific and recurrent translocations that generate unique fusion transcripts.

On the basis of literature data, the fusion transcript is detected in approximately 25% of patients with localized pPNET and in approximately 50% of those with metastatic diseases in peripheral blood and in bone marrow.^25,26 The relationship between the detection of the transcripts and prognosis is unclear,²⁷ although some types of fusion transcript (type 1 v 2) seem to have a favorable prognostic significance in localized disease (regardless of the presence of other known prognostic factors²⁸) and in metastatic disease.²⁹

In the present study, the detection of fusion transcripts in peripheral blood and/or bone marrow was of limited use, as complete tumor response occurred during treatment irrespective of the initial transcript results or the pattern of results observed during the treatment program.

However, these results, as well as the prognostic power of the type of fusion transcripts (type 1 v type 2), should not be considered conclusive for the small number of patients included.

Considering the tumor cell contamination of leukapheretic products, only two patients were positive for the fusion transcript; however, these findings did not seem to have any adverse effect in terms of outcome, as both patients are still alive in CR after 34 and 26 months of follow-up (Fig 7). In other histologic subtypes, the effect of the fusion transcript result on prognosis is unknown. In our experience, all peripheral-blood samples were positive in all DSRCTs at both diagnosis and during treatment. However, the significance of these results is limited by the inefficacy of chemotherapy. Only in the patient allocated to mini-allogeneic transplantation after high-dose chemotherapy did we observe the disappearance of fusion transcript at the response time, while tapering cyclosporine therapy.

In conclusion, our data clearly show that pPNET/Ewing’s sarcoma is highly chemosensitive and that high-dose chemotherapy improves the clinical outcome, mainly in locally advanced disease. On the other hand, the intensive approach does not seem to change the prognosis in patients with metastatic pPNET, RMS, or DSRCT. It therefore is not useful to include these patients in clinical trials using such an approach.

Although the molecular results as well as other studies are not yet conclusive, the genetic characteristics could be exploited to explore new innovative therapies. The fusion transcript may be a tumor-associated neoantigen that could be elicited to induce an immune response, and, if this is true, it may be interesting to test the effects of dendritic cells pulsed with tumor antigen or the use of immunotherapy by means of allogeneic transplantation.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Submitted July 9, 2001; accepted January 2, 2002.

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