Introduction {#S0001}
============

High-dose therapy (HDT) followed by autologous haematopoietic stem cell transplantation (ASCT) is considered the treatment of choice for patients with relapsed Hodgkin lymphoma (HL) and an effective treatment option for patients who did not adequately respond to conventional therapy.  Approximately half of patients treated with ASCT are likely to be long-term survivors, since this treatment provides long-term disease-free survival in over 50--60% of patients \[[@CIT0001], [@CIT0002]\]. 

Second malignant neoplasms are a well recognized and serious late complication in HL survivors.  Several studies have been reported which proved the increased incidence of second malignancies for patients with HL treated with conventional therapy, especially those who were exposed to alkylating agents, such as mechlorethamine and procarbazine in the MOPP (mechlorethamine, vincristine, procarbazine, prednisone) regimen \[[@CIT0003]--[@CIT0006]\].  Moreover, there are available data indicating that lymphoma patients treated with HDT and ASCT are at increased risk for second neoplasms including therapy-related acute myeloid leukaemia/myelodysplastic syndrome (t-AML/MDS) \[[@CIT0007]--[@CIT0010]\].  However, the potential contribution of ASCT to second malignancy development remains controversial \[[@CIT0011]--[@CIT0013]\].  Studies investigating the contribution of pre-transplant therapy and transplant procedures in the development of second malignancies in HL patients have yielded inconsistent results \[[@CIT0007], [@CIT0011], [@CIT0014], [@CIT0015]\].  Since deaths due to second malignancies are the most common cause of non-relapse mortality among long-term survivors of HL, better characterization of second malignancies and the identification of Hodgkin lymphoma patients with high risk of developing second malignancy after ASCT may be useful for clinicians who care for these patients. 

To enhance the published experience, we conducted a retrospective review of patients who underwent ASCT following a modified BEAM preparative regimen for refractory or relapsed HL at our centre.  We intended to determine the incidence of second malignancies and associated pre-transplant and transplant-related risk factors.  Herein, we report the results of this analysis. 

Material and methods {#S0002}
====================

Study population {#S20003}
----------------

We retrospectively reviewed the data of all patients with refractory or relapsed HL who were treated with a modified BEAM regimen followed by ASCT between January 1992 and January 2012 at our centre.  Patients records were reviewed to obtain patient characteristics and treatment details (sex, clinical stage according to the Ann Arbor system, presence of B symptoms, the type of first line chemotherapy, the number and type of salvage chemotherapy lines, radiotherapy, disease status at transplant, age at transplant).  All patients were treated initially with chemotherapy with or without radiotherapy.  The treatment practices varied during the study period, including type of first line and salvage regimens, and the indications for radiotherapy.  The disease status at transplant was defined using standard criteria of response \[[@CIT0016]\].  With regard to transplant procedures, patients underwent haematopoietic cell collection either by bone marrow harvest or leukapheresis following stem cell mobilization.  Stem cell mobilization was performed using salvage chemotherapy or cyclophosphamide (4 g/m^2^) ± etoposide (600 mg/m^2^) with G-CSF stimulation.  The stem cells were cryopreserved without further manipulation.  The modified BEAM conditioning consisted of carmustine (300 mg/m^2^), etoposide (800 mg/m^2^), cytarabine (6000 mg/m^2^), melphalan (140 mg/m^2^) and dexamethasone (168 mg/m^2^). 

After ASCT, all patients were routinely observed by a haematologist in the outpatient clinic of our institution.  Recommended evaluation included physical examination, cell blood count and biochemistry every 3 months during the first year, and every 6 months thereafter.  Computed tomography, or X-ray of chest and abdomen ultrasonography was performed once per year. 

Statistical analysis {#S20004}
--------------------

Survival curves were estimated according to the method of Kaplan and Meier.  Overall survival (OS) was measured from the time of transplantation until death of any cause and progression-free survival (PFS) was measured from the time of transplantation until documented progression or relapse or death.  Non-relapse mortality (NRM) was defined as death of other causes than lymphoma relapse/progression.  The probabilities of non-relapse mortality, relapse and second malignancy were calculated with the cumulative incidence estimator.  The cumulative incidence of NRM and relapse were calculated with either relapse or non-relapse related mortality treated as competing risk.  The cumulative incidence of second malignancy was calculated in the survivors' group, with death from any reason other than second neoplasm treated as competing risk.  The time at risk for the development of a second malignancy was calculated from the date of ASCT. 

Cox regression analysis for second malignancy incidence included potential prognostic factors, age at transplant, sex, clinical stage, presence of B symptoms, total number of chemotherapy lines before ASCT, MOPP or MOPP-type chemotherapy prior to transplant, radiotherapy, and disease status at transplant. * P*-values \< 0.05 were considered significant. 

SPSS version 14.0 (SPSS, Chicago, IL) was used for all statistical analyses except for cumulative incidence curve analyses, which were calculated using the statistical package NCSS version 2007 (NCSS, Kaysville, Utah). 

Results {#S0005}
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Patients' characteristics, prior treatment and transplantation procedure details {#S20006}
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From January 1992 to January 2012, the 170 patients (90 men and 80 women) with refractory (*n* = 111) or relapsed (*n* = 59) HL underwent ASCT following a modified BEAM conditioning regimen. 

In our study group 73.5% (125/170) of patients were treated according to the ABVD (adriamycin, bleomycin, vinblastine, dacarbazine) regimen as a frontline chemotherapy.  The majority of patients (76%) received a cisplatin-based regimen, DHAP (dexamethasone, cytarabine, cisplatin) or ESHAP (etoposide, methylprednisolone, cytarabine, cisplatin), as second line chemotherapy.  Twenty patients (12%) received MOPP or MOPP-type regimen as either an upfront or second line chemotherapy.  Subsequent lines of salvage treatment included IVE (ifosfamide, etoposide, epirubicin), ICE (ifosfamide, carboplatin, etoposide), dexaBEAM (dexamethasone, carmustine, etoposide, cytarabine, melphalan) or gemcitabine-based regimens.  The patients received a median of 2 (range 1--5) chemotherapy lines prior to ASCT.  Seventy-five patients (44%) received radiotherapy prior to transplant.  Finally, 72 patients were in CR and 81 in PR at ASCT, respectively.  Seventeen patients did not respond to the salvage chemotherapy and they underwent ASCT in less than PR. 

The mobilized peripheral blood in 63%, and bone marrow in 31% of all cases were used as the autologous graft source.  Six percent of patients received both bone marrow and mobilized peripheral blood as a source of stem cells. 

Patients' baseline characteristics with treatment details are presented in [Table 1](#T0001){ref-type="table"}. 

###### 

Patient characteristics and treatment details

  Characteristics                                     Number (%)
  --------------------------------------------------- ------------
  Total number of patients                            170 (100)
  Age (years) at transplant median 31, range 17--61   
   \< 40 years                                        136 (80)
   ≥ 40 years                                         34 (20)
  Gender                                              
   male                                               90 (53)
   female                                             80 (47)
  Clinical stage                                      
   II                                                 32 (19)
   III                                                54 (32)
   IV                                                 79 (46)
   Iunknown                                           5 (3)
  Constitutional symptoms                             
   absent                                             45 (26.5)
   present                                            118 (69.5)
   unknown                                            7 (4)
  Induction chemotherapy                              
   ABVD                                               125 (73.5)
   BEACOPP or escalated BEACOPP                       16 (9.5)
   MOPP or MOPP-type                                  15 (9)
   other regimens                                     14 (8)
  Second line chemotherapy                            
   ESHAP or DHAP                                      130 (76)
   Escalated BEACOPP                                  11 (7)
   ABVD                                               9 (5)
   MOPP or MOPP-type                                  5 (3)
   other regimens                                     15 (9)
  Number of pre-transplant chemotherapy regimens      
    2                                                 102 (60)
   \> 2                                               59 (35)
   unknown                                            9 (5)
  Radiotherapy prior to ASCT                          
   yes                                                75 (44)
   no                                                 86 (51)
   unknown                                            9 (5)
  Disease status at ASCT                              
   CR                                                 72 (42)
   PR                                                 81 (48)
   less than PR                                       17 (10)

ASCT -- autologous haematopoietic stem cell transplantation, CR -- complete response, PR -- partial response

Survival data {#S20007}
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The median follow-up time of surviving patients was 73 months (range 12--242 months).  After 7 years following transplantation, estimated overall survival (OS) and progression-free survival (PFS) were 75.3% (95% CI: 67.6--81.6%) and 63.9% (95% CI: 55.6--71.5%), respectively ([Fig.  1](#F0001){ref-type="fig"}). 

![ Kaplan-Meier estimates of overall survival (OS) and progression-free survival (PFS) for all patients](WO-17-20638-g001){#F0001}

Forty-one (24%) patients in our study died.  The cause of death in 24 patients was relapse/progression of Hodgkin lymphoma.  Three other patients who relapsed after ASCT died from complications after subsequent allogeneic (*n* = 2) or autologous (*n* = 1) haematopoietic stem cell transplantation.  Fourteen patients (8%) died from causes not related to lymphoma relapse/progression.  Eight patients died within 3 months of ASCT from infection (*n* = 7) and primary graft failure (*n* = 1).  Causes of late non-relapse mortality included hepatic veno-occlusive disease (*n* = 1), cardiac disease (*n* = 1), ischaemic stroke (*n* = 1), and second malignancy (*n* = 3).  The 1-year and 5-year cumulative incidence of NRM was 4% (95% CI: 2--8%) and 5% (95% CI: 3--10%), respectively. 

Second malignancy characteristics, incidence and risk factors {#S20008}
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Second malignancy occurred in 7 of the 170 patients, including one myelodysplastic syndrome, two acute myeloid leukaemias, two aggressive non-Hodgkin lymphomas and two solid tumors.  With regard to solid tumors, one bladder cancer was diagnosed 8 years after ASCT, and one thyroid cancer occurred 13.5 years after transplant.  Second haematological malignancy developed at a median of 4.7 years (range, 0.4--8.4 years) from ASCT.  The median time of occurrence any second malignancy was 8 years (range 0.4--13.5).  The 10-year and 15-year cumulative incidence of developing a second malignancy were 7% (95% CI: 3--16%) and 13% (95% CI: 5--33%) ([Fig.  2](#F0002){ref-type="fig"}).  Four of the seven patients with post-transplant diagnosed second neoplasm died.  Three of them died from progression of second malignancy.  One patient diagnosed with non-Hodgkin lymphoma and subsequently treated with chemotherapy died from cardiac disease.  The median survival calculated from the diagnosis of the second malignancy was 32 months.  The 10-year OS estimates for patients with post-transplant diagnosed second neoplasm and without second neoplasm calculated from ASCT were 43% and 73%, respectively ([Fig.  3](#F0003){ref-type="fig"}).  Despite the observed trend for shorter survival of patients with second malignancy, the survival difference did not reach statistical significance (*p* = 0.18). 

![ Cumulative incidence of second malignancy development for all patients](WO-17-20638-g002){#F0002}

![ Kaplan-Meier estimates of overall survival stratified by the post-transplant development of second malignancy](WO-17-20638-g003){#F0003}

The median age of the patients with second malignancy at transplant was 44 years (range 23--50).  Of the seven patients developing second neoplasm, four patients received MOPP-type chemotherapy prior to ASCT, and two patients received both radiotherapy and chemotherapy.  In univariate analysis, age at ASCT (\< 40 vs. ≥ 40 years; *p* = 0.001) and pre-transplant MOPP-type chemotherapy (MOPP vs. other; *p* = 0.056) were the only significant factors for the development of the second malignancy.  Both of those two factors remained statistically significant in multivariate analysis ([Table 2](#T0002){ref-type="table"}). 

###### 

Cox regression analysis of cumulative incidence of second malignancy at 7 years after autologous haematopoietic stem cell transplantation

                                 Cumulative incidence% (95% CI)   HR (95% CI)       *P*
  ------------------------------ -------------------------------- ----------------- -------
  Age at transplant                                                                 
   \< 40 years                   1 (1--5)                         1.0               0.008
   ≥ 40 years                    8 (2--31)                        8.8 (1.8--43.6)   
  Prior MOPP-type chemotherapy                                                      
   no                            1 (0--8)                         1.0               0.030
   yes                           10 (3--37)                       5.6 (1.2--26.5)   

CI -- confidence interval, HR -- hazard ratio

Discussion {#S0009}
==========

Despite satisfactory 7-year survival rates of 75% for patients with refractory and relapsed HL treated with HDT followed by ASCT reported in the present retrospective study, our results confirm that second malignancies are one of the major concerns in long-term HL survivors, since deaths of second malignancies were the leading cause of late non-relapse mortality in our analysis.  Four of a total of six late deaths not related to HL relapse/progression were associated with second malignancy. 

Our retrospective analysis reports 13% incidence of second malignancy at 15 years from ASCT.  This incidence rate is comparable to those reported by other authors.  The 5-year cumulative incidence (CI) of second malignancies reported by Sureda was 4.3% \[[@CIT0002]\] and the 15-year CI reported by Forrest and Goodman were 8% and 15.3% \[[@CIT0011], [@CIT0017]\], respectively.  We identified age at ASCT and pre-transplant MOPP-type therapy as risk factors for second malignancy development in univariate and multivariate model.  Available published studies investigating different potential risk factors associated with pre-transplant therapy and transplant procedures have shown inconsistent results.  Therapy-related factors and clinical features that were reported as predictors of second malignancy development after ASCT included patient age, the number of pre-transplant therapy lines, prior extended-field radiation therapy, prior treatment with rituximab, use of the second harvest peripheral blood haematopoietic stem cell for autograft, disease status at ASCT, and post-transplant radiotherapy \[[@CIT0007], [@CIT0011], [@CIT0014], [@CIT0015]\].  Among these factors, age is the best known risk factor for the development of second malignancies in long-term HL survivors.  Our results confirm the strong and independent impact of this factor on the incidence of second neoplasm after ASCT.  We have also shown that pre-transplant MOPP-type chemotherapy contribute to risk of second neoplasm after ASCT.  There have been several reports documenting that patients with HL treated with conventional chemotherapy and exposed to DNA-breaking alkyIating agents such as mechlorethamine and procarbazine in the MOPP regimen, and topoisomerase II inhibitors, such as etoposide are at increased long-term risk of developing t-AML/MDS \[[@CIT0018]--[@CIT0020]\].  Our results confirm that pre-transplant MOPP-type chemotherapy plays a critical role in development of post-transplant second malignancies.  In contrast, we did not find that clinical stage of the disease, disease status at transplant, number of prior chemotherapy lines or radiotherapy significantly affected incidence of second neoplasms.  In conclusion, the results of the present analysis support the previously published evidence that the introduction of the ABVD regimen substantially reduced the risk of second malignancy in HL survivors \[[@CIT0021], [@CIT0022]\] and indicate the major role of pre-transplant chemotherapy with alkylating agents in the post-transplant development of second malignancies after ASCT. 

We believe that the characteristics of second malignancies diagnosed in patients with HL who were treated with ASCT, as well as the results of risk factors analysis reported in our study may be useful in optimization of second malignancy screening in these patients. 

We thank the nurses and physicians of our department for the dedicated patient care. 

The authors declare no conflict of interest.