MD Clara Cesana
email: clara.cesana@ospedaleniguarda.it

Catherine Klersy

Barbara Scarpati

Bruno Brando

Maurizio Faleri

Giambattista Bertani

Arianna Gatti

Elisabetta Volpato

Claudia Barba

Ursula Ferri

Linda Scampini

Giovanni Grillo

Giuliana Lando

Annamaria Nosari

Enrica Morra

Roberto Cairoli

Flow cytometry and cytomorphology evaluation of hematologic malignancy in cerebrospinal fluids: comparison with retrospective clinical outcome

Keywords: Cerebrospinal Fluid, Cytomorphology, Flow Cytometry, Hematologic Malignancy, Lymphoid Neoplasm

Introduction

Cerebrospinal fluid (CSF) analysis by flow cytometry (FCM) and cytomorphology (CM) is currently requested to explore central nervous system (CNS) involvement by hematologic malignancy, both at diagnosis and during follow-up [START_REF] Schinstine | Detection of malignant hematopoietic cells in cerebral spinal fluid previously diagnosed as atypical or suspicious[END_REF][START_REF] Finn | Enhanced detection of malignant lymphoma in cerebrospinal fluid by multiparameter flow cytometry[END_REF][START_REF] French | Diagnosing lymphoproliferative disorders involving the cerebrospinal fluid. Increased sensitivity using flow cytometric analysis[END_REF][START_REF] Sayed | Immunophenotyping and immunoglobulin heavy chain gene rearrangement analysis in cerebrospinal fluid of pediatric patients with acute lymphoblastic leukaemia[END_REF][START_REF] Bromberg | CSF flow cytometry greatly improves diagnostic accuracy in CNS hematologic malignancies[END_REF][START_REF] Hegde | High incidence of occult leptomenigeal disease detected by flow cytometry in newly diagnosed aggressive B-cell lyphomas at risk for central nervous system involvement. The role of cytometry versus cytology[END_REF][START_REF] Noto | Critical role of multidimensional flow cytometry in detecting occult leptomeningeal disease in newly diagnosed aggressive B-cell lymphomas[END_REF][START_REF] Quijano | Identification of leptomeningeal disease in aggressive B-cell non-Hodgkin' s lymphoma: improved sensitivity of flow cytometry[END_REF][START_REF] Subirà | Flow cytometry and the study of central nervous disease in patients with acute leukaemia[END_REF][START_REF] Roma | Lymphoid and myeloid neoplasm involving cerebrospinal fluid: comparison of morphologic examination and immunophenotyping by flow cytometry[END_REF][START_REF] Urbanits | FACS analysis -A new and accurate tool in the diagnosis of lymphoma in the cerebrospinal fluid[END_REF]. To date, the two methods have been mostly studied on CSF from newly diagnosed cases, i.e., those optimally evaluable samples not liable to intrathecal treatment-related damage [START_REF] Finn | Enhanced detection of malignant lymphoma in cerebrospinal fluid by multiparameter flow cytometry[END_REF][START_REF] French | Diagnosing lymphoproliferative disorders involving the cerebrospinal fluid. Increased sensitivity using flow cytometric analysis[END_REF][START_REF] Sayed | Immunophenotyping and immunoglobulin heavy chain gene rearrangement analysis in cerebrospinal fluid of pediatric patients with acute lymphoblastic leukaemia[END_REF][START_REF] Bromberg | CSF flow cytometry greatly improves diagnostic accuracy in CNS hematologic malignancies[END_REF][START_REF] Hegde | High incidence of occult leptomenigeal disease detected by flow cytometry in newly diagnosed aggressive B-cell lyphomas at risk for central nervous system involvement. The role of cytometry versus cytology[END_REF][START_REF] Noto | Critical role of multidimensional flow cytometry in detecting occult leptomeningeal disease in newly diagnosed aggressive B-cell lymphomas[END_REF][START_REF] Quijano | Identification of leptomeningeal disease in aggressive B-cell non-Hodgkin' s lymphoma: improved sensitivity of flow cytometry[END_REF][START_REF] Subirà | Flow cytometry and the study of central nervous disease in patients with acute leukaemia[END_REF]. In this setting, immunophenotype analysis is well known to guarantee the highest accuracy, morphologic exam still retaining an additional diagnostic value according to some authors [START_REF] Finn | Enhanced detection of malignant lymphoma in cerebrospinal fluid by multiparameter flow cytometry[END_REF][START_REF] French | Diagnosing lymphoproliferative disorders involving the cerebrospinal fluid. Increased sensitivity using flow cytometric analysis[END_REF][START_REF] Bromberg | CSF flow cytometry greatly improves diagnostic accuracy in CNS hematologic malignancies[END_REF][START_REF] Hegde | High incidence of occult leptomenigeal disease detected by flow cytometry in newly diagnosed aggressive B-cell lyphomas at risk for central nervous system involvement. The role of cytometry versus cytology[END_REF][START_REF] Noto | Critical role of multidimensional flow cytometry in detecting occult leptomeningeal disease in newly diagnosed aggressive B-cell lymphomas[END_REF][START_REF] Quijano | Identification of leptomeningeal disease in aggressive B-cell non-Hodgkin' s lymphoma: improved sensitivity of flow cytometry[END_REF][START_REF] Urbanits | FACS analysis -A new and accurate tool in the diagnosis of lymphoma in the cerebrospinal fluid[END_REF][START_REF] Wu | Lymphoma, leukemia, and pleiocytosis in cerebrospinal fluid. Is accurate cytopathologic diagnosis possible based on morphology alone?[END_REF]. The point at issue related to CSF follow-up evaluations is possible lack of method accuracy due to poor cellularity and/or therapy-related cell changes [START_REF] Urbanits | FACS analysis -A new and accurate tool in the diagnosis of lymphoma in the cerebrospinal fluid[END_REF][START_REF] Peter | Cytological changes in the cerebrospinal fluid following intrathecal methotrexate treatment[END_REF].

Indeed, variable proportions of samples with the mentioned characteristics were arbitrarily excluded from the analysis as quantity not sufficient in previous reports [START_REF] Finn | Enhanced detection of malignant lymphoma in cerebrospinal fluid by multiparameter flow cytometry[END_REF][START_REF] French | Diagnosing lymphoproliferative disorders involving the cerebrospinal fluid. Increased sensitivity using flow cytometric analysis[END_REF][START_REF] Sayed | Immunophenotyping and immunoglobulin heavy chain gene rearrangement analysis in cerebrospinal fluid of pediatric patients with acute lymphoblastic leukaemia[END_REF][START_REF] Subirà | Flow cytometry and the study of central nervous disease in patients with acute leukaemia[END_REF][START_REF] Roma | Lymphoid and myeloid neoplasm involving cerebrospinal fluid: comparison of morphologic examination and immunophenotyping by flow cytometry[END_REF][START_REF] Urbanits | FACS analysis -A new and accurate tool in the diagnosis of lymphoma in the cerebrospinal fluid[END_REF].

Although CSF analysis is essential for the management of intra-thecal treatment schedules, the diagnostic performance of either FCM or CM in the follow-up setting is mostly unknown [START_REF] Hegde | High incidence of occult leptomenigeal disease detected by flow cytometry in newly diagnosed aggressive B-cell lyphomas at risk for central nervous system involvement. The role of cytometry versus cytology[END_REF][START_REF] Roma | Lymphoid and myeloid neoplasm involving cerebrospinal fluid: comparison of morphologic examination and immunophenotyping by flow cytometry[END_REF][START_REF] Urbanits | FACS analysis -A new and accurate tool in the diagnosis of lymphoma in the cerebrospinal fluid[END_REF]. Moreover, whether the two methods retain differential accuracy in different diagnostic subgroups is largely undetermined [START_REF] Finn | Enhanced detection of malignant lymphoma in cerebrospinal fluid by multiparameter flow cytometry[END_REF][START_REF] French | Diagnosing lymphoproliferative disorders involving the cerebrospinal fluid. Increased sensitivity using flow cytometric analysis[END_REF][START_REF] Sayed | Immunophenotyping and immunoglobulin heavy chain gene rearrangement analysis in cerebrospinal fluid of pediatric patients with acute lymphoblastic leukaemia[END_REF][START_REF] Hegde | High incidence of occult leptomenigeal disease detected by flow cytometry in newly diagnosed aggressive B-cell lyphomas at risk for central nervous system involvement. The role of cytometry versus cytology[END_REF][START_REF] Noto | Critical role of multidimensional flow cytometry in detecting occult leptomeningeal disease in newly diagnosed aggressive B-cell lymphomas[END_REF][START_REF] Quijano | Identification of leptomeningeal disease in aggressive B-cell non-Hodgkin' s lymphoma: improved sensitivity of flow cytometry[END_REF][START_REF] Subirà | Flow cytometry and the study of central nervous disease in patients with acute leukaemia[END_REF].

In the present study, we matched immunophenotype and morphology results obtained from the analysis of 227 CSF, mostly drawn during follow-up, with an independent clinical outcome in a retrospective longitudinal study, in order to compare the accuracy of the two methods, and to explore a combined diagnostic approach assuring the best sensitivity in detecting hematologic malignancy, both in all conditions and in selected clinical settings.

Materials And Methods

Selection Criteria

Among CSF analyzed by four-color FCM between 2002 and 2010, we selected those fulfilling each of the following criteria: (i) suspected/known hematologic malignancy at the time of withdrawal, (ii) CM performed on the same sample, and (iii) available follow-up findings for reliable retrospective clinical outcome. According to these criteria, 227 samples from 110 patients (69 males, 41 females) were included in a database and retrospectively examined (Table 1). The study was carried out according with the ethical standards of the Institutional Committee on Human Experimentation. Each patient gave informed consense to lumbar puncture (LP) in the context of his/her clinical management.

Sample Analysis

FCM and CM were carried out in two different laboratories, in a double blind manner. A sample volume of whatever size was accepted. Fresh specimens were processed within 3 hours from collection.

Immunophenotype

Samples were processed for four color analysis as previously described for effusions [START_REF] Cesana | Flow cytometry vs cytomorphology for the detection of hematologic malignancy in body cavity fluids[END_REF].

Monoclonal antibodies were dependent on patient's diagnosis and/or clinical history. All measurements were performed on a dual-laser FACSCalibur flow cytometer (Becton Dickinson, San José, CA, USA). Data were acquired with the CellQUEST software (Becton Dickinson), and analyzed with either CellQUEST or Paint-a-Gate softwares. Cell subpopulations, evaluated by means of multiparameter analysis including logical gates on forward scatter, side scatter, and FL1-FL4, were expressed both as number of acquired events and as percentages of CD45 + events.

Cytomorphology

Samples were prepared by cytocentrifugation, using a Shandon cytospin4® (Thermo Shandon, Loughborough UK) at 250 rev/min for 15 min, loading every funnel with 250 µl of fluid. Slides were air-dried and stained with May Grünwald-Giemsa stain. When an opportunistic disease was clinically suspected, extra slides were alcohol-fixed for further appropriate stainings.

Assessment Criteria

Retrospective clinical outcome was expressed as positive or negative for neoplastic cells. It was formulated by the clinician regardless of the results obtained by FCM and CM, and was based on clinical follow-up retrospectively collected for each case from the electronic hospital patient data management system. Retrospective clinical outcome positive for hematologic malignancy was defined as follows: (i) evidence of monoclonal cell population by molecular biology on the sample itself, or (ii) presence of CNS symptoms attributable to the disease at the time of LP and/or signs of CNS involvement at imaging data available within one moth after LP and/or malignant hematopoietic cells identified by CM and/or FCM in CSF samples drawn within one month after LP, with/without subsequent response to intrathecal treatment and/or to parenteral specific treatments. Retrospective clinical outcome negative for hematologic malignancy was defined as follows: (i) evidence of polyclonal cell population by molecular biology on the sample itself, or (ii) absence of CNS signs/symptoms as documented by clinical/imaging data, plus negative FCM/CM on CSF samples drawn in the aftermath, during a follow-up of at least 3 months after LP, or (iii) diagnosis of non-hematologic malignancy disease by means of laboratory and coltural tests plus no evidence of hematologic malignancy as documented by clinical and/or imaging data for at least three months after LP. FCM results were expressed as positive or negative for neoplastic cells (see below). CM results were expressed as positive (positive or uncertain results) or negative for neoplastic cells. A combined method assessment (CMA) was also established (positive if at least one method gave positive results, negative in the remaining cases). Samples with blood contamination were retrospectively considered not evaluable.

Statistical Analysis

Data were described as median and interquartile range (IQR) when continuous, and as absolute and relative frequency when categorical. The optimal cut-off of phenotypically abnormal event number for discriminating 'positive' FCM samples was found by constructing receiver operating characteristic (ROC) curves, which were generated by calculating sensitivities and specificities of FCM data at several predetermined cut-off points on 2/3 of samples, randomly selected (testing population); the best cut-off was then confirmed on the remaining samples (validating population). ROC curve analysis showed that finding ≥ 5 phenotypically abnormal events by FCM identified samples with higher risk of being positive at retrospective clinical outcome. Sensitivity, specificity, positive predictive value and negative predictive value were calculated for each method, together with their 95% confidence intervals (95%CI). Methods accuracy comparisons (intersample and inter-method) was assessed by means of a paired test (with Chi2 distribution) comparing the area under the ROC curves of sensitivity and specificity [START_REF] Delong | Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach[END_REF]. The Bonferroni correction was used for post-hoc comparisons of methods. Analyses were performed with the use of Stata 11 (StataCorp, College Station, TX, USA).

Results

Specimens and Clinical Settings

In 6 cases with suspected hematologic malignancy, LP was performed in the presence of CNS symptoms with/without lymphoadenopathies; in the remaining cases, a hematologic malignancy was screened for disease localization. Sample distribution according to clinical setting is reported in Table 1. Fifty-five samples were drawn from newly diagnosed hematologic malignancy. In this setting, 14 samples (25%) were evaluated in the presence of CNS signs and/or symptoms, while 41 samples (75%) were evaluated in the context of prophylactic purposes according to the risk of CNS involvement/relapse [START_REF] Tomita | Prophylactic intrathecal methotrexate and hydrocortisone reduces central nervous system recurrence and improves survival in aggressive non-Hodgkin lymphoma[END_REF][START_REF] Mcmillan | Central nervous system-directed preventative therapy in adults with lymphoma[END_REF][START_REF] Hill | CNS prophylaxis in lymphoma: who to target and what therapy to use[END_REF]. One-hundred sixty-six samples were drawn during follow-up. In this setting, 42 samples (25.5%) were evaluated for suspected CNS relapse/progression in the presence of CNS signs/symptoms, while 124 samples (74.5%) were drawn according to diseasespecific schedule [CNS signs/symptoms were present in 15 (12%) and absent in 109

(88%) cases].
Thirty-two samples from newly diagnosed hematologic malignancy (58%) were evaluated only at diagnosis; in the remaining 23 cases (42%), a median of 2 (IQR 1-3) follow-up samples per patient was also available. One-hundred twenty-three samples were instead available only during follow-up (median per patient 1, IQR 1-2, range 1-17).

CSF Assessment according to

Matching between FCM and CM according to retrospective clinical outcome

The matching between FCM and CM could be performed in 213 cases; in the remaining 14, the results by each or both methods were retrospectively considered not evaluable for blood contamination. Matching of method assessments according to retrospective clinical outcome is reported in Table 3. Concordant results between FCM and CM were observed in 181 evaluable cases (85%). One hundred fourty-five cases (68%) were true negative by both methods, 26 cases (12.5%) were true positive by both methods, 1 case (0.5%) was false positive by both methods, and 9 cases (4%) were false negative by both methods. The latter were samples from acute monoblastic leukemia (n=1), acute promyelocytic leukemia (n=1), DLBCL NOS (n=5), peripheral T-cell lymphoma NOS (n=1), and T lymphoblastic L/L (n=1). Discordant results between FCM and CM were observed in 32 cases (15%). FCM gave false negative results in 5 cases (2.5%) with true positive CM. The latter were samples from acute promyelocytic leukemia (n=2), DLBCL NOS (n=2), and B lymphoblastic L/L with recurrent genetic abnormalities (n=1). FCM gave false positive results in 9 cases (4%) with true negative CM. Clinical settings and immunophenotype of the latter are detailed in Table 4. Four out of 9 false positive results by FCM were related to either occult CNS disease at diagnosis (#1,2,3) or minimal residual leptomeningeal disease under effective intrathecal treatment (#8). FCM gave true negative results in 1 case (0.5%) with false positive CM, and true positive results in 17 cases (8%) with false negative CM. Clinical settings and immunophenotype of the latter are detailed in Table 5.

Inter-Method Comparisons of Accuracy

Comparison with retrospective clinical outcome could be evaluated in 208 cases for CM, in 221 cases for FCM and in 224 cases for CMA. Sensitivity, specificity, positive predictive value, negative predictive value, and the results of inter-method comparisons of accuracy, both in all samples and in subsets of samples selected according to clinical interest, are reported in Table 6. The accuracy of CMA was significantly higher than that of CM in the analysis of all samples (p<.0001), follow-up samples (p<.0001), samples from patients with mature B-/T-cell neoplasm (p=.0021), and samples drawn after at least one intrathecal treatment (p=.0001). In the analysis of these samples, CM accuracy was lower than that of FCM. Combining methods led to higher accuracy than either CM or FCM in the analysis of screening samples (p=.2036), samples from patients with precursor lymphoid neoplasm (p=.0656) and samples from patients with acute myeloid leukemia and related precursor neoplasm (p=.0574). FCM accuracy was lower than CM accuracy when acquiring ≤ 220 (corresponding to the third tertile of distribution of) CD45 + cell events. In this setting, combining methods added to FCM a statistically significant advantage in accuracy (p=.0043). On the contrary, when acquiring > 220 CD45 + cell events FCM retained significantly higher accuracy than CM (p=.0065). In this setting, combining methods did not add to FCM any diagnostic advantage. Of note, both the positive and negative predictive values were high for all methods, overall and in each subgroup. Exceptions were acute myeloid leukemia and related precursor neoplasm, where the negative predictive values were low, and acquired CD45 + Cells 220, where the positive predictive values of FCM and CMA were low.

Discussion

Serial CSF studies represent an important criterion for the management of active CNS disease. However, few data are available on FCM/CM diagnostic performance in followup evaluations, since a definitive categorization has been reported difficult to obtain in the presence of hypocellular and/or little-sized samples [START_REF] Hegde | High incidence of occult leptomenigeal disease detected by flow cytometry in newly diagnosed aggressive B-cell lyphomas at risk for central nervous system involvement. The role of cytometry versus cytology[END_REF][START_REF] Noto | Critical role of multidimensional flow cytometry in detecting occult leptomeningeal disease in newly diagnosed aggressive B-cell lymphomas[END_REF][START_REF] Quijano | Identification of leptomeningeal disease in aggressive B-cell non-Hodgkin' s lymphoma: improved sensitivity of flow cytometry[END_REF][START_REF] Roma | Lymphoid and myeloid neoplasm involving cerebrospinal fluid: comparison of morphologic examination and immunophenotyping by flow cytometry[END_REF][START_REF] Urbanits | FACS analysis -A new and accurate tool in the diagnosis of lymphoma in the cerebrospinal fluid[END_REF]. In our study the majority of samples were drawn during follow-up. Neither cellularity, nor previous intrathecal therapy, nor sample size (CSF volumes ranged from 200 to 3400 L) represented an exclusion criterion for the analysis. Instead, an independent clinical assessment provided by the planning of a retrospective longitudinal study was used, in order to test a diagnostic approach assuring the best sensitivity. To this end, (i) ROC curves of accuracy were used to establish the more appropriate number of FCM events for positive FCM, (ii) uncertain morphologic results were classified as positive CM, and (iii) a combined method assessment (i.e., a positive assessment if at least one method gave positive results) was sampled.

The number of phenotypically abnormal events defining positive screening samples by FCM have been so far arbitrarily chosen, cut-offs of 10 to 30 events allowing FCM to retain higher sensitivity than CM in both prospective and retrospective studies [START_REF] Bromberg | CSF flow cytometry greatly improves diagnostic accuracy in CNS hematologic malignancies[END_REF][START_REF] Hegde | High incidence of occult leptomenigeal disease detected by flow cytometry in newly diagnosed aggressive B-cell lyphomas at risk for central nervous system involvement. The role of cytometry versus cytology[END_REF][START_REF] Noto | Critical role of multidimensional flow cytometry in detecting occult leptomeningeal disease in newly diagnosed aggressive B-cell lymphomas[END_REF][START_REF] Quijano | Identification of leptomeningeal disease in aggressive B-cell non-Hodgkin' s lymphoma: improved sensitivity of flow cytometry[END_REF]. By combining immunophenotype results with an all over independent clinical assessment, we identified the 5 cell event cut-off as the most accurate. In the screening setting, true positive FCM results, as well as false positive ones likely related to an occult CNS disease, displayed ≥ 20 phenotypically abnormal events, according to previous observations 6,19,20 . However, in one case of newly diagnosed, clinically positive acute monoblastic leukemia, only six CD13 + CD14 -CD34 + CD56 + CD45 ± events were observed. This is likely to suggest that, in view of a disease with frankly abnormal phenotype, primary neoplastic contamination should be suspected even in the presence of very few pathological cells. Immunophenotype diagnostic performance was confirmed to be higher than morphologic one regardless the clinical setting analyzed, and found to reach 100% sensitivity when at least 220 leukocyte events could be analyzed [START_REF] Finn | Enhanced detection of malignant lymphoma in cerebrospinal fluid by multiparameter flow cytometry[END_REF][START_REF] French | Diagnosing lymphoproliferative disorders involving the cerebrospinal fluid. Increased sensitivity using flow cytometric analysis[END_REF][START_REF] Bromberg | CSF flow cytometry greatly improves diagnostic accuracy in CNS hematologic malignancies[END_REF][START_REF] Hegde | High incidence of occult leptomenigeal disease detected by flow cytometry in newly diagnosed aggressive B-cell lyphomas at risk for central nervous system involvement. The role of cytometry versus cytology[END_REF][START_REF] Noto | Critical role of multidimensional flow cytometry in detecting occult leptomeningeal disease in newly diagnosed aggressive B-cell lymphomas[END_REF][START_REF] Quijano | Identification of leptomeningeal disease in aggressive B-cell non-Hodgkin' s lymphoma: improved sensitivity of flow cytometry[END_REF][START_REF] Urbanits | FACS analysis -A new and accurate tool in the diagnosis of lymphoma in the cerebrospinal fluid[END_REF][START_REF] Wu | Lymphoma, leukemia, and pleiocytosis in cerebrospinal fluid. Is accurate cytopathologic diagnosis possible based on morphology alone?[END_REF]. Moreover, follow-up FCM evaluations resulted as sensitive as screening ones, while follow-up morphologic evaluations did not. This is likely to indicate that intrathecal treatment-related cell changes do not affect immunophenotype diagnostic performance as much as morphologic one [START_REF] Urbanits | FACS analysis -A new and accurate tool in the diagnosis of lymphoma in the cerebrospinal fluid[END_REF][START_REF] Peter | Cytological changes in the cerebrospinal fluid following intrathecal methotrexate treatment[END_REF].

Highest accuracy of an assessment combining immunophenotype and morphologic data has been so far displayed in previous studies [START_REF] Finn | Enhanced detection of malignant lymphoma in cerebrospinal fluid by multiparameter flow cytometry[END_REF][START_REF] French | Diagnosing lymphoproliferative disorders involving the cerebrospinal fluid. Increased sensitivity using flow cytometric analysis[END_REF][START_REF] Bromberg | CSF flow cytometry greatly improves diagnostic accuracy in CNS hematologic malignancies[END_REF][START_REF] Hegde | High incidence of occult leptomenigeal disease detected by flow cytometry in newly diagnosed aggressive B-cell lyphomas at risk for central nervous system involvement. The role of cytometry versus cytology[END_REF][START_REF] Noto | Critical role of multidimensional flow cytometry in detecting occult leptomeningeal disease in newly diagnosed aggressive B-cell lymphomas[END_REF][START_REF] Quijano | Identification of leptomeningeal disease in aggressive B-cell non-Hodgkin' s lymphoma: improved sensitivity of flow cytometry[END_REF][START_REF] Roma | Lymphoid and myeloid neoplasm involving cerebrospinal fluid: comparison of morphologic examination and immunophenotyping by flow cytometry[END_REF][START_REF] Urbanits | FACS analysis -A new and accurate tool in the diagnosis of lymphoma in the cerebrospinal fluid[END_REF]. According to our combined method assessment, 2.5% of total samples, resulting false negative by FCM, were rescued by true positive CM, and 8% of total samples, resulting false negative by CM, were rescued by true positive FCM. CMA sensitivity was higher than that of each method, predominantly (although not exclusively) due to FCM contribution. In particular, FCM allowed CMA to reach significantly higher sensitivity than CM in the setting of mature B-/T-cell neoplasms, in which lymphocytes frequently display typical pathological phenotype patterns while looking reactive at morphology. Instead, in the setting of acute myeloid leukemia and related precursor neoplasms, where typical morphology of blast cells is much more identifiable, FCM and CM equally contributed to CMA sensitivity.

Evidence has been provided that CM evaluation of CSF is still essential in selected cases [START_REF] Finn | Enhanced detection of malignant lymphoma in cerebrospinal fluid by multiparameter flow cytometry[END_REF][START_REF] French | Diagnosing lymphoproliferative disorders involving the cerebrospinal fluid. Increased sensitivity using flow cytometric analysis[END_REF][START_REF] Bromberg | CSF flow cytometry greatly improves diagnostic accuracy in CNS hematologic malignancies[END_REF][START_REF] Hegde | High incidence of occult leptomenigeal disease detected by flow cytometry in newly diagnosed aggressive B-cell lyphomas at risk for central nervous system involvement. The role of cytometry versus cytology[END_REF][START_REF] Noto | Critical role of multidimensional flow cytometry in detecting occult leptomeningeal disease in newly diagnosed aggressive B-cell lymphomas[END_REF][START_REF] Quijano | Identification of leptomeningeal disease in aggressive B-cell non-Hodgkin' s lymphoma: improved sensitivity of flow cytometry[END_REF][START_REF] Roma | Lymphoid and myeloid neoplasm involving cerebrospinal fluid: comparison of morphologic examination and immunophenotyping by flow cytometry[END_REF][START_REF] Urbanits | FACS analysis -A new and accurate tool in the diagnosis of lymphoma in the cerebrospinal fluid[END_REF]. In our study, the setting in which CM retained a diagnostic advantage as compared to FCM was represented by very hypocellular samples, i.e. those samples in which FCM acquired less than 220 leukocyte events. In this case, CMA retained significantly higher sensitivity than FCM due to CM contribution. Only positive morphologic assessment reliably predicted CNS disease, as suggested by high positive predictive value. On the basis of previous data, in our study population the eight cases with uncertain CM, each of them drawn in the follow-up setting, were considered positive for hematologic malignancy localization [START_REF] Schinstine | Detection of malignant hematopoietic cells in cerebral spinal fluid previously diagnosed as atypical or suspicious[END_REF]. Comparison with retrospective clinical outcome demonstrated seven out of these eight to be true positive cases, so that such diagnostic approach increased CM sensitivity to the detritment of a mild impairment in specificity. Interestingly, in six out of these seven true positive cases immunophenotype could evaluate up to 300 leukocyte events. Therefore, although uncertain morphologic findings have been demonstrated to be definitively negative in the screening setting, they are likely to retain a more incisive diagnostic role in the evaluation of samples drawn in the aftermath [START_REF] Bromberg | CSF flow cytometry greatly improves diagnostic accuracy in CNS hematologic malignancies[END_REF].

In conclusion, FCM is confirmed to be the best diagnostic tool for detecting neoplastic cells in CSF from patients with hematologic malignancy, regardless the clinical setting.

However, diagnostic advantage is obtained by combining FCM results with morphologic ones. The latter might be essential especially in evaluating clinically positive, nearly acellular follow-up samples.

  Clinical Follow-Up During a a median follow-up of 11 months (IQR, 6-20 months) from sample withdrawal, 64/227 CSF samples (28%) were assessed as positive and 163/227 CSF samples (72%) were assessed as negative for hematologic malignancy localization by retrospective clinical outcome. The distribution of positive samples according to clinical setting is reported in Table 2. Eleven out of 55 CSF submitted in newly diagnosed hematologic malignancy were retrospectively assessed as positive for acute monoblastic leukemia (n=1), diffuse large B-cell lymphoma (DLBCL) not otherwise specified (NOS) (n=5), follicular lymphoma (n=1), and peripheral T-cell lymphoma NOS (n=4). Fifty-three out of 166 CSF submitted during hematologic malignancy follow-up were assessed as positive for acute monoblastic leukemia (n=2), acute promyelocytic leukemia (n=9), B lymphoblastic leukemia/lymphoma (L/L) with recurrent genetic abnormalities (n=4), B lymphoblastic L/L NOS (n=10), B cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma (n=5), DLBCL NOS (n=17), lymphoplasmacytic lymphoma (n=1), nodal marginal zone lymphoma (n=1), peripheral Tcell lymphoma NOS (n=2), and T lymphoblastic L/L (n=2).

Table 1 . Distribution of 227 Cerebrospinal Fluid Samples according to Clinical Setting Number of Samples (%)

 1

	Suspected Hematological Malignancy

BL: Burkitt Lymphoma; DLBCL: Diffuse Large B Cell Lymphoma; NOS: Not Otherwise Specified

Table 2 . Cerebrospinal Fluids Positive for Hematological Malignancy according to Retrospective Clinical Outcome Number of Samples (%)

 2

	Suspected Hematological Malignancy (n=6)

Table 4 . Immunophenotype Findings in 9 Cases of False Positive Fow Cytometry (and True Negative Cytomorphology) according Retrospective Clinical Outcome

 4

	#	Clinical Setting							Immunophenotype	N° of + Events (% of CD45 + Cells)
	1	CLL/SLL, Screening	CD5	+ CD19	+ CD23	+ Cells	82 (17.4)	Negative MRI; CNS sympt
	2	CLL/SLL, Screening	CD5	+ CD19	+ CD23	+ Cells	24 (10.9)	Negative MRI; CNS sympt
	3	DLBCL NOS, Screening	CD10	+ CD19	+ CD20	+ SSC ++ Cells	20 (10.5)	No CNS Signs/Symptoms
	4	T Lymphoblastic L/L,	CD2	-CD3	-CD5	-CD7	± CD34	± CD45	± Cells	10 (20)	No CNS Signs/Symptoms
		Screening
	5	DLBCL NOS, Relapse	CD19	+ CD20	+ CD79a	+ Cells	8 (5)	No CNS Signs/Symptoms
	6	B Lymphoblastic L/L with	CD10	+ CD20	+ CD34	+ CD45	-Cells	7 (2.1)	No CNS Signs/Symptoms
		Recurrent Genetic
		Abnormalities, Follow-up
		According to Schedule
	7	B Lymphoblastic L/L NOS,	CD10	+ CD20	+ CD34	+ CD45	± Cells	7 (5.5)	No CNS Signs/Symptoms
		Follow-up According to
		Schedule
	8	DLBCL NOS, Follow-up	CD10	+ CD19	+ CD20	+ SSC ++ Cells	9.5 (12.8)	No CNS Signs/Symptoms
		According to Schedule
	9	PTC Lymphoma NOS,	CD2	+ CD3	+ CD4	+ CD5	+ CD7	+ CD8	+ Cells	7 (5)	Polyclonal TCR by Molecu
		Follow-up According to
		Schedule
	CLL/SLL: Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma; CNS: Central Nervous System; DLBCL: Diffuse Large B-Cell Lymphoma
	L/L: Leukemia/Lymphoma; MRI: Magnetic Resonance Imaging; NHL: Non-Hodgkin Lymphoma; NOS: Not Otherwise Specified; PTC: Peripheral T
	Cell; SSC: Side Scatter; TCR: T-Cell Receptor

CSF Samples in Which Flow Cytometry Led to True Positive Results in the Presence of Benign Cytomorphologic Diagnosis

	Clinical Setting			Immunophenotype	N° of + Events (% of CD45 + Cells)	Definitive Retrospective Clinical Assessment
	ute Monoblastic Leukemia	CD13	+ CD14	-CD34	+ CD56	+ CD45	±	6 (5)	CNS localization; Positive MRI;
	aging)	Blasts							CNS Symptoms
	ute Monoblastic Leukemia	CD13	+ CD14	-CD34	+ CD56	+ CD45	±	12 (4)	Residual Meningeal Disease;
	llow-up)	Blasts							Positive MRI; CNS Symptoms
	ell Lymphoblastic L/L	CD10	+ CD19	+ CD20	+ CD45	± Blasts	76 (10)	Residual Meningeal Disease;
	S (Follow-up)										Positive MRI; CNS Symptoms
	ell Lymphoblastic L/L,	CD10	+ CD19	+ CD20	+ CD45	± Blasts	7 (3.2)	Residual Meningeal Disease;
	S (Follow-up)										Positive MRI; CNS Symptoms
	ell Lymphoblastic L/L,	CD10	+ CD19	+ CD20	+ CD34	-CD45	±	667 (57)	Residual Meningeal Disease;
	S (Follow-up)	Blasts							Positive MRI; CNS Symptoms
	ell Lymphoblastic L/L,	CD10	+ CD19	+ CD20	+ CD34	-CD45	±	13 (2)	Residual Meningeal Disease;
	S (Follow-up)	Blasts							Positive MRI
	ell Lymphoma,	CD10	+ CD19	+ HLA-DR + CD45	±	144 (89.4)	CNS Relapse; Positive MRI
	classifiable, with Features	Lymphocytes
	ermediate between
	BCL and BL (Follow-up)
	ell Lymphoma,	CD10	+ CD19	+ HLA-DR + CD45	±	100 (62)	Residual Meningeal Disease;
	classifiable, with Features	Lymphocytes					CNS Symptoms
	ermediate between
	BCL and BL (Follow-up)
	ell Lymphoma,	CD10	+ CD19	+ HLA-DR + CD45	±	45 (17.2)	CNS Progression; Positive MRI;
	classifiable, with Features	Lymphocytes					CNS Symptoms
	ermediate between
	BCL and BL (Follow-up)
	BCL, NOS (Staging)	CD10	+ CD19	+ Lymphocytes	70 (8.5)	CNS localization; Positive MRI
	BCL, NOS (Follow-up)	CD10	+ CD19	+ CD34	-Lymphocytes	315 (73.5)	Residual Meningeal Disease;
											Positive MRI; CNS Symptoms
	BCL, NOS (Follow-up)	CD10	+ CD19	+ HLA-DR + Lymphocytes	6 (1.6)	Residual Meningeal Disease;
											Positive MRI; CNS Symptoms
	BCL, NOS (Follow-up)	CD10	+ CD19	+ HLA-DR + Lymphocytes	180 (28)	Residual Meningeal Disease;
											Positive MRI; CNS Symptoms
	BCL, NOS (Follow-up)	CD10	+ CD19	+ HLA-DR + Lymphocytes	19 (15)	Residual Meningeal Disease;
											Positive MRI; CNS Symptoms
	llicular Lymphoma	CD10	+ CD19	+ CD20	+ k	+ Lymphocytes	111 (5)	Progressive Disease; Deceased
	aging)
	ripheral T Cell	CD2	+ CD3	+ CD4	-CD5	+ CD7	+ CD8	+	13 (2)	CNS Localization; CNS
	phoma, NOS (Staging)	Lymphocytes					Symptoms
	ripheral T Cell	CD2	+ CD3	-CD5	+ CD7	+ Lymphocytes	297 (100)	CNS Localization; Monoclonal
	phoma, NOS (Follow-										TCR by Molecular Biology
	)

Burkitt Lymphoma; CNS: Central Nervous System; CSF: Cerebrospinal Fluid; DLBCL: Diffuse Large B Cell Lymphoma; L/L: Leukemia/Lymphoma : Magnetic Resonance Imaging; NOS: Not Otherwise Specified; TCR: T-Cell Receptor.

Table 6 . Inter-Method Comparisons of Accuracy in the Analysis of Cerebrospinal Fluids and their Subsets

 6 Acute Myeloid Leukemia; CI: Confidence Interval; NPV: Negative Predictive value; PPV: Positive Predictive Value. * Test (with Chi2 Distribution) Comparing the Receiver Operating Characteristic curves of Sensitivity and Specificity. 0,1,2 Post-hoc Comparisons: Significant Differences after Bonferroni Correction (p<.017):

	N°	% Sensitivity (95% CI)	% Specificity (95% CI)	% PPV


Jean-Christophe Ianotto

Christian C Berthou

Dr Tempescul Adrian
email: adrian.tempescul@chu-brest.fr

and local radiotherapy into the treatment of primary intraocular lymphoma

Keywords: primary ocular lymphoma, treatment, chemotherapy, radiotherapy

Combined therapy associating systemic platinum-based chemotherapy and local radiotherapy into the treatment of primary intraocular lymphoma

Primary intraocular lymphoma (PIOL) is a very rare form of non-Hodgkin's lymphoma that is characterised by localisation in various parts of the eye, a low rate of response to treatment and a high risk of relapse, both in the eye and in the central nervous system. The diagnosis of PIOL is very difficult because of its localisation and the ways it initially manifests itself. This type of lymphoma is sometimes misdiagnosed as inflammatory uveitis and thus treated with steroids, resulting in a delay of definitive diagnosis and proper therapy. The treatment of PIOL is not standardised. Multiple regimens including chemotherapy, monoclonal antibodies and radiotherapy have been proposed, but the results thus far have been disappointing. Here, we present a group of 4 patients in which a treatment regimen combining systemic chemotherapy and local radiotherapy resulted in persistent complete remission.

Over a period of three years, we identified and treated 4 patients with PIOL. The characteristics of these patients are presented in Table 1.

The median age of patients was 73.2 years. Two patients were in relapse after a first line of treatment consisting of high dose methotrexate (i.e., GOELAMS's MBVP protocol), and two patients were undergoing their first round of this treatment. In all patients, PIOL was diagnosed following an anatomopathological examination of a biopsy sample obtained by vitrectomy. Cytological analyses found diffuse large B cell lymphomas that were CD20 positive in all patients. The vitreous level of interleukin 10 was strongly elevated, more than 10 times the normal values, in three of four analyses. All patients underwent cerebral computed tomography with negative results, confirming that the lymphoma was localised to the eye. The patients underwent DHAOx chemotherapy, which consisted of the combined administration of dexamethasone, cytarabine and oxaliplatin. The chemotherapy was administrated every 21 days, with primary prophylaxis for neutropenia provided by administration of the GCSF analogue filgrastim (5 μg/kg) from day 8 to day 12. After three cycles of chemotherapy, clinical and ophthalmologic examinations were made, and computed tomography performed; after the CT scan, consolidation therapy consisting of DHAOx chemotherapy followed by localised 3D radiotherapy was administered.

In the four patients we treated in this manner, we obtained four complete remissions. The principal adverse events due to the chemotherapy were grade 2-3 anaemia and grade 3-4 thrombopenia. No infectious or thrombotic complications were noted. The principal adverse events due to the radiotherapy were sicca syndrome and blepharitis, both of which occurred in two patients. The symptoms rapidly regressed upon treatment with local antibiotics and artificial tears. At the time of diagnosis, the patients had received artificial implants; therefore, there was no risk of cataract due to the radiotherapy. The follow-up examination of the patients was composed of both ophthalmological and haematological examinations. The median follow-up time for the patients was 19.25 months, with a range of 10-28 months. At the time of writing, all four patients were alive and in complete remission.

Diagnosis of primary ocular lymphoma (PIOL) is a challenge because this disease is a very rare type of lymphoma that can arise in different parts of the eye and features a wide variety of symptoms. PIOL is considered to be a rare subtype of central nervous system lymphoma with a high risk of relapse that is characterised by either intraocular relapse or metastasis into the central nervous system [START_REF] Grimm | Primary intraocular lymphoma: an International Primary Central Nervous System Lymphoma Collaborative Group Report[END_REF][START_REF] Coupland | Understanding intraocular lymphomas[END_REF]. This type of lymphoma is mainly found in elderly and immunocompromised patients and rarely in young patients [START_REF] Levy-Clarke | Diagnosis and management of primary intraocular lymphoma[END_REF][START_REF] Grimm | Primary CNS lymphoma with intraocular involvement: International PCNSL Collaborative Group Report[END_REF]. The true incidence of PIOL is not known, but its occurrence seems to be increasing [START_REF] Levy-Clarke | Diagnosis and management of primary intraocular lymphoma[END_REF][START_REF] Grimm | Primary CNS lymphoma with intraocular involvement: International PCNSL Collaborative Group Report[END_REF]. The treatment of PIOL is not standardised. Several therapeutic approaches have been proposed, but the results are not very encouraging. Low-dose external radiotherapy results in regression of the tumour with some complications, such as retinopathy and cataract (4). Intraocular methotrexate administration could prove to be an effective treatment; however, it is an invasive procedure that can lead to complications such as keratopathy and macular oedema [START_REF] Sen | Intravitreal methotrexate resistance in a patient with primary intraocular lymphoma[END_REF][START_REF] Frenkel | Intravitreal methotrexate for treating vitreoretinal lymphoma: 10 years of experience[END_REF]. Other proposed strategies include intraocular and systemic administration of rituximab [START_REF] Kitzmann | Intraocular use of rituximab -Eye[END_REF] and classical CNS lymphoma treatment with drugs designed to cross the haemato-encephalic barrier, such as methotrexate and cytarabine [START_REF] Grimm | Primary CNS lymphoma with intraocular involvement: International PCNSL Collaborative Group Report[END_REF][START_REF] Chan | Intraocular lymphoma[END_REF]. Despite this large panel of approaches, the results have been disappointing. In a very interesting study, SA Grim and colleagues identified and followed 83 patients with PIOL who were treated with various regimens. Irrespective of the type of treatment, 56% of the patients relapsed, with a time-torelapse of 19 months [START_REF] Grimm | Primary CNS lymphoma with intraocular involvement: International PCNSL Collaborative Group Report[END_REF]. In our study, the treatment consisted of induction chemotherapy consisting of cytarabine, dexamethasone and oxaliplatin (according to the DHAOx protocol) [START_REF] Machover | Dexamethasone, high-dose cytarabine, and oxaliplatin (DHAOx) as salvage treatment for patients with initially refractory or relapsed non-Hodgkin's lymphoma[END_REF] followed by consolidation radiotherapy. We obtained four persistent complete remissions. Adverse events due to the chemotherapy and/or radiotherapy were tolerable. We suggest that induction therapy according to the DHAOx protocol followed by localised consolidation radiotherapy is a potentially useful treatment for primary intraocular lymphoma.

DFS -disease free survival


Martin Neumann

Igor W Blau

Thomas Burmeister

Carola Tietze-Buerger

Olga Blau

Armin Gerbitz

Lutz Uharek

M.D. Eckhard Thiel
email: eckhard.thiel@charite.de

Intrathecal application of donor lymphocytes in leukemic meningeosis after allogeneic stem cell transplantation

Keywords: Intrathecal, DLI, leukemic meningeosis, extramedullary relapse, adoptive cell therapy, CNS

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Due to a longer survival after allogeneic stem cell transplantation (allo SCT) as well as by enlarged treatment options, e.g. reduced-intensity conditioning regimens, treatment of evolving relapses after allo SCT is more and more challenging. Thereby, extramedullary (EM) relapses play an important role as they occur more frequently after allo SCT compared to non-transplant leukemia treatments. They can be accompanied by a bone marrow relapse but also occur seperately [START_REF] Au | Extra-medullary relapse of leukemia following allogeneic bone marrow transplantation[END_REF]. The median time to EM relapse is usually longer compared to bone marrow relapse [START_REF] Au | Extra-medullary relapse of leukemia following allogeneic bone marrow transplantation[END_REF]. Allotransplanted patients suffering from graft versus host disease (GvHD) show a significant reduction of bone marrow relapses compared to patients without GvHD, which is attributed to the coexistent graft versus leukemia (GvL) effect [START_REF] Giralt | Leukemia relapse after allogeneic bone marrow transplantation: a review[END_REF]. However, the rate of EM relapses seems to be independent on the rate of GVHD [4;5]. A possible explanation might be the concept of the so-called "sanctuary" sites for chemotherapy. These sites also might represent immunologically privileged organs, where there is only a slight GvL-reaction if any at all [6;7]. This hypothesis is supported by the fact that there is apparently no influence on the EM relapse after systemic infusion of donor lymphocytes (DL) and subsequent occurrence of GvHD [8;9]. Among the EM relapses the affection of the central nervous system (CNS) implicates additional treatment problems resulting in a poor outcome. Limiting factors of an effective treatment are mainly due to the extended neurotoxicity and the restricted disposability of agents acting effectively across the blood-brain-barrier (BBB) [10;11]. To enlarge hitherto used therapeutic options (including local irradiation, systemic cytostatic therapy, second allo SCT [START_REF] Bekassy | Granulocytic sarcoma after allogeneic bone marrow transplantation: a retrospective European multicenter survey. Acute and Chronic Leukemia Working Parties of the European Group for Blood and Marrow Transplantation[END_REF]) application of DL directly into the cerebrospinal fluid (CSF) is an obvious possibility. But so far this procedure has not been performed in the case of a leukemic CNS relapse after allo SCT. This appears to be of special relevance in the situation of isolated CNS relapse with full chimerism in bone marrow and blood. In our study we infused DL intrathecal in three patients suffering from isolated leptomeningeal relapse after allo SCT, having no other established conventional treatment option.

PATIENTS AND METHODS

Patients and treatment

After diagnosis of leukemic meningeosis in three patients (table 1) after allo SCT we started treatment with systemic high dose cytarabine chemotherapy followed by intrathecal applications of DL as an induction therapy. Thereafter, in intervals of one up to three weeks we continued the application of intrathecal DL as consolidation therapy in escalating doses.

Treatment was changed when either no leptomeningeal remission could be achieved or the disease progressed. Time and doses of the intrathecal applications are displayed in figure 1A.

Patients gave written informed consent to this individualized treatment plan including possible life threatening side effects like encephalitis, meningitis, cerebral GvHD or other infectious complications leading to death.

Cell preparation

Under GMP conditions, a monocyte free CD14 depleted T cell product from donor PBMC gathered in steady state was generated by negative immunomagnetic selection using a CliniMACS cell selector device (Miltenyi Biotec, Bergisch Gladbach, Germany). Counted amounts of 1x10 6 cells per vial (containing approximately 60% CD3+cells) were stored in liquid nitrogen for repeated injections and administered by a lumbar puncture.

Analysis of the cerebrospinal fluid

CSF was taken at each time point of DL application and was examined regarding cell count, cell cytology and immunphenotypical investigations performed by flow cytometry. Molecular genetic analysis of the CSF and bone marrow was done for quantitative analysis of the hematopoietic chimerism and in the case of the CML patient additionally for the BCR-ABL fusion transcript using conditions according to the EAC recommendations with the CT method and bGUS as housekeeping gene [START_REF] Beillard | Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using 'real-time' quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) -a Europe against cancer program[END_REF]. Chimerism analysis was performed as previously described [START_REF] Blau | Engraftment kinetics and hematopoietic chimerism after reduced-intensity conditioning with fludarabine and treosulfan before allogeneic stem cell transplantation[END_REF]. At the beginning of the treatment as well as in the follow-up period, cerebral imaging was accomplished by MRI or CT scans. Routine neuroclinical examinations were performed to monitor neurological disorders.

RESULTS AND DISCUSSION

Patient # 1, who was 15 months after the diagnosis of an acute myeloid leukemia (AML) and who underwent allografting from his identical brother one year ago, was admitted to our So far, just one case with suspected refractory lymphomatous meningitis is described in which intrathecal infusions of DL were performed [START_REF] Meuleman | Intrathecal donor lymphocyte infusion for the treatment of suspected refractory lymphomatous meningitis: a case report[END_REF]. Because of the lacking experience with this experimental approach we were not able to estimate possible side effects in advance. First, there were concerns about direct inflammatory reactions after application. To avoid or reduce monocyte derived cytokine reactions we applied a CD14 depletion as described recently [START_REF] Thiel | Treatment of leukemic meningeosis by CD14 depleted adoptive intrathecal cell therapy[END_REF]. Whether this preparation is really necessary remains to be determined in future studies. Secondly, we were afraid of an induction of a cerebral GvHD reaction after allo SCT. Reviewing the literature there is no evident opinion regarding this topic. CNS associated neurologic complications like cerebrovascular events, encephalitis or leukencephalopathy are mainly caused by drug toxicity, infections due to immunosuppression or bleeding in thrombocytopenia. Neurologic symptoms associated with a systemic GVHD are mainly neuromuscular syndromes or lesions of peripheral nerves in the presence of skin changes e.g. due to sclerodermic GVHD [START_REF] Saiz | Neurological complications of hematopoietic cell transplantation[END_REF]. In rare cases a CNS angiitis was associated with a chronic GVHD [START_REF] Padovan | Angiitis of the central nervous system after allogeneic bone marrow transplantation?[END_REF]. These observations are also underlined by data of murine BMT models [START_REF] Padovan | Cerebral involvement in graft-versus-host disease after murine bone marrow transplantation[END_REF]. An onset of the CNS angiitis is reported for up to two years after allo SCT, so that the observation period might not be long enough to evaluate any cerebral GVHD effects in our patients. Thirdly we could not observe any infections like e.g. meningitis or encephalitis caused by the treatment procedure with cells prepared under GMP conditions over a period up to 17 months.

Regarding the efficacy of intrathecal application of DL, no definite conclusion can be drawn.

Additional treatments (including cytarabine, liposomal cytarabine, nilotinib), used in a situation with poor prognosis and no established therapy, makes it difficult to determine exactly the impact of the effect of the DL treatment. Whether the transient absence of CSF cell count is still an effect of initial treatment or if there is a prelongation due to the sequentially used DL transfusions (in patient 1 with a decrease of the donor chimerism in the CSF, in patient 2 with an increase) must remain open. Furthermore, the efficacy of the DL depends on the track of T lymphocytes in and out the CSF. Whether the CSF is a hostile place for T lymphocytes and how many and which subsets of the T lymphocytes could act in the CSF is unsolved up to date [START_REF] Engelhardt | The ins and outs of T-lymphocyte trafficking to the CNS: anatomical sites and molecular mechanisms[END_REF][START_REF] Pedemonte | Mechanisms of the adaptive immune response inside the central nervous system during inflammatory and autoimmune diseases[END_REF][START_REF] Kleine | Immune surveillance of the human central nervous system (CNS): different migration pathways of immune cells through the blood-brain barrier and blood-cerebrospinal fluid barrier in healthy persons[END_REF]. From our data we could only conclude that one week after the application of 6x106 cells less than 2.5% of these cells were detectable in the CSF at the location of infusion (assuming a homogenous distribution in a CSF volume of 150ml).

Of course we could not rule out that a considerable amount of T cells is attached to the meninges.

The intrathecal application of DL still is an experimental approach for patients with poor prognosis. Nevertheless the application of in total 16 doses up to 1x107 cells showed no acute or delayed toxic side effects over an observation period of 17 months. This approach should merit further investigations in a multicenter trial accompanied by studies to track the T lymphocytes and determine still possible side effects to enhance the therapeutic options in the case of a leptomeningeal relapse after allo SCT. If direct applications of T lymphocytes in the CSF remain a feasible option as reported here in three patients, the treatment might not only include hematologic but also neurologic diseases.

  photophobia. At the time point of the leptomeningeal relapse, confirmed by CSF cytology

Fig. 1 A

 1 Fig. 1 A Treatment schedule of the three patients and number of T-cells of intrathecal DL infusions. In Patient #1 the intervals were longer between i.th. applications in order to react on possible side effects. Patient #2 received almost weekly increasing numbers of DL. Patient #3 developed chloroma lesions very soon after onset of DL treatment and the therapy was changed.

Fig. 1 B 3 (

 13 Fig. 1 B demonstrates CSF cell number and quantitative analysis of hematopoietic chimerism in CSF during treatment. In total, Patient #1 showed two decreases of donor chimerism in the CSF. Only the first decrease was paralleled by a significant increase in the CSF cell number. The second decrease was associated with a bone marrow relapse. In patient #2 full donor chimerism of CSF was seen approximately 80 days after diagnosis of leptomeningeal relapse. The relative expression of BCR-ABL in the CSF decreased to a level below 10-2.3 (dashed line) and could not be quantified in the following CSF fluid samples due to low RNA-amounts.

  the characteristics of the three patients. GvHD = Graft-versus-Host-Disease, BM = bone marrow, ITT = intrathecal triple therapy, EM = extramedullary, Flu = fludarabine, Treo = treosulfane, TBI = total body irradiation, Bu = busulfan, Cy = cyclophosphamide, Mel = Melphalane, Outcome: CR = complete remission defined by CSF clearance (cytology, flow cytometry, molecular genetics), no bone marrow relapse and major clinical improvement, PR = partial remission defined by at least 50% CSF clearance, no bone marrow relapse and clinical improvement, PD = progressive disease defined by increase of CSF cell count, BM relapse or new EM relapses.

Table 1 : Patients characteristics

 1

	Patient	1	2	3
	Diagnosis	AML	CML	AML
	Allogeneic Transplantation of
	hematologic stem cells
	Age	37	25	18
	Disease status at transplant	1. CR	Accelerated phase	2. CR
	Donor	Brother	Brother	Unrelated
	HLA match	10/10	10/10	10/10
	Conditioning regimen	Flu/Treo	Cy/TBI	Bu/Cy/Mel
	GvHD prophylaxis	Cyclosporine	Cyclosporine	Unknown
	Acute GvHD	Yes Skin I°	Yes Skin I°	No Skin I°-II°
	Chronic GvHD	No	Yes Skin I°	No
	Leptomeningeal Relapse
	Day after BMT	+302	+3900	+1312
	CNS cell count (cells/µl)	720	300	6 (blasts)
	Bone marrow chimerism	100%	100%	100%
	Immunosuppression	None	None	Cortisone
	Initial Treatment	HD cytarabine	HD cytarabine	FLAG, ITT
	No. of DLI	7	8	2
	Additional Treatment	Liposomal cytarabine	Nilotinib	None
	Outcome	PR	CR	PD
	Follow up
	Time of Observation (mos)	16	17	8
	Relapse	BM&EM (extra-and intracerebral)	EM (intracerebral)	EM (extra-and intracerebral)
	Leptomeningeal progression	Yes	Yes	No

Table 1

 1 The tableshows

Conflict


Dr Sonja Krause
email: sonja.krause@med.uni-heidelberg.de

Jens Hillengass

Hartmut Goldschmidt

Jürgen Debus

Dirk Neuhof

Radiotherapy of solitary plasmacytoma

Purpose: Solitary plasmacytoma of the bone (SBP) or extramedullary plasmacytoma (EP) are rare neoplasms amenable to local radiotherapy. In this retrospective analysis, we report the University Heidelberg experience in the treatment of solitary plasmacytoma. Patients and methods: From 1995 to 2008, 18 patients were treated with local radiotherapy. Ten patients suffered from SBP, eight patients showed a single extramedullary lesion. Results: Local radiotherapy with a median dose of 45 Gy yielded excellent local control with only one patient suffering from local relapse. SBP and EP had significantly different 5-year multiplemyeloma-free survival rates of 36.8% and 86.7%, respectively. However, no significant difference in overall survival could be detected. Conclusions: Radiotherapy can achieve excellent local control of solitary plasmacytoma. Progression to multiple myeloma, especially in the case of SBP, remains to be addressed by further studies.

Solitary plasmacytoma are rare neoplasms originating from plasma cells in bone (solitary plasmacytoma of the bone, SBP) or in soft tissue (extramedullary plasmacytoma, EP). SBP constitute about 10% of plasma cell neoplasms and are mainly found in the axial skeleton [START_REF] Holland | Treatment results and conversion to myeloma[END_REF].

EP are even more rare (about 3%) with most manifestations in the head-and-neck region [START_REF] Bachar | Solitary extramedullary plasmacytoma of the head and neck--long-term outcome analysis of 68 cases[END_REF].

Although both neoplasms originate from the same cell type, distinct features concerning progression to multiple myeloma suggest two different pathologic entities [START_REF] Galieni | Solitary plasmacytoma of bone and extramedullary plasmacytoma: two different entities?[END_REF] Patients typically present with symptoms caused by local tumor mass: pain and neurological deficits in the case of SBP, dysphagia, breathing problems and epistaxis in the case of EP of the head-and-neck region.

SBP and EP being rare malignant disorders, data on treatment options are sparse. However, several single-and multicenter retrospective studies report effective treatment of solitary plasmacytoma with radiotherapy.

This study is a retrospective analysis of the University of Heidelberg experience in the radiooncological treatment of solitary plasmacytoma. 19 patients met the inclusion criteria. One patient was lost to follow-up due to relocation abroad. Ten patients suffered from SBP, with most manifestations in the spine (seven patients), one in the sternal body, one in the skull base and one in the rib cage. Of the eight patients with EP, five had manifestations in the epi-/oropharynx, one in the orbita, one in the paranasal sinuses and one in the penile urethra. For further patient's characteristics, see Table I.

Patients and methods

Clinical

Ten patients were primarily treated with radiotherapy, in eight cases postoperative radiotherapy after partial resection or spinal decompression was performed.

Median age was 60.9 years (range: 35-81 years) and the majority of patients was male.

Radiotherapy was performed using linear accelerators. 2D-planning was used for SBP lesions.

For EP lesions, 2D-planning (three patients), CT-based 3D-plans (three patients) or intensitymodulated radiotherapy (IMRT, two patients) were used.

The median applied dose was 45 Gy (range: 30 -50.4 Gy) in single fractions ranging from 1.8 to 2.5 Gy (for details, see table II) with no difference between SBP and EP.

Clinical follow-up consisted of clinical examination, blood count and chemistry, serum protein electrophoresis and plain film skeletal survey or whole-body-MRI or -CT.

Overall survival (OS) and multiple-myeloma-free survival (MMFS) were calculated using the Kaplan-Meier method. Survival distributions were compared using the log-rank test (Statistica, StatSoft Inc., Tulsa, USA).

Results

Follow-up

Median follow-up was 47 months (range: 8-167 months). At last follow-up, four patients had died (three deaths were myeloma-related). Of the 14 remaining patients, eight presented with no sign of local recurrence or progression to multiple myeloma.

Local control

Local control was very good with only one patient suffering from local relapse (6%). In this case, local relapse occurred seven years after development of multiple myeloma and eight years after initial treatment of vertebral bodies Th1-L2 with a total dose of 38 Gy, so that reirradiation was possible. Yet, radiotherapy had to be stopped due to the development of perforated diverticulitis.

Progression to multiple myeloma

Nine patients (50%) progressed to multiple myeloma. Median time to progression was 34.3 months (range: 2 -154 months). Progression occurred in the majority of cases (six patients) in the first year after initial diagnosis. For all 18 patients, 2-and 5-year MMFS rates were 59.1%, the 10-year MMFS rate was 49.2%. Patients suffering from EP showed a significantly lower progression rate than SBP patients. Only two of eight patients (25%) with EP progressed to multiple myeloma, whereas seven of eight patients (70%) with SBP suffered from progression. The 5-year MMFS rates for SBP and EP were 36.8% and 86.7%, respectively (p=0.015, Figure I). Age, sex and dose applied to the lesion had no influence on multiple-myeloma-free survival on univariate analysis.

Overall survival

Four patients (22.2%) died during follow-up. Three patients died of multiple myeloma, all of them initially had presented with SBP. One patient initially diagnosed with EP died of dementia.

2-year OS was 87.5%, 5-year and 10-year OS were 77.8% with no statistically significant difference between SBP and EP (p=0.234, Figure II).

Toxicity

In general, local radiotherapy was tolerated very well. No grade III-IV toxicity occurred.

Patients with irradiation of the head and neck mainly suffered from mild acute mucositis and skin erythema. Patients treated for bone lesions reported no side effects. None of the patients in our cohort developed secondary malignancies.

Discussion

Local control

In line with other retrospective studies, radiotherapy achieved excellent local control with only one patient suffering from local relapse (94% local control). In a large multicenter retrospective analysis of 258 patients with SBP/EP, Ozsahin et al. saw a 5-year local control rate of 86% [START_REF] Ozsahin | Outcomes and patterns of failure in solitary plasmacytoma: a multicenter Rare Cancer Network study of 258 patients[END_REF]. Along this line, Liebross et al. reported 96% local control in patients with SBP [START_REF] Liebross | Solitary bone plasmacytoma: outcome and prognostic factors following radiotherapy[END_REF] and Bachar et. al observed a 5-year freedom of local recurrence of 81% [START_REF] Bachar | Solitary extramedullary plasmacytoma of the head and neck--long-term outcome analysis of 68 cases[END_REF]. There is no reported significant difference in local control between SBP and EP. For example, Dagan et al. observed local control rates of 88% in their series of 32 patients with SBP or EP with no statistically significance between tumor types [START_REF] Dagan | Solitary plasmacytoma[END_REF].

Progression to multiple myeloma

We found a significantly higher percentage of patients with progression to multiple myeloma in the case of SBP (70%) compared to EP (25%). Again, this is in accord with the data published so far. In the University of Florida series, 68% of SBP patients progressed to multiple myeloma, whereas only 20% of EP patients had progressive disease [START_REF] Dagan | Solitary plasmacytoma[END_REF]. In their series of 46 patients with SBP or EP, Tsang et al. saw significantly different 8-year myelomafree survival rates of 36% and 84%, respectively [START_REF] Tsang | Solitary plasmacytoma treated with radiotherapy: impact of tumor size on outcome[END_REF]. Ozsahin et al. identified bone localization as a predictor for development of multiple myeloma [START_REF] Ozsahin | Outcomes and patterns of failure in solitary plasmacytoma: a multicenter Rare Cancer Network study of 258 patients[END_REF].

Compared to other studies, the median time to progression of 34.3 months we saw in our patients forms the upper end of the reported range of 13 -36 months [START_REF] Holland | Treatment results and conversion to myeloma[END_REF][START_REF] Tsang | Solitary plasmacytoma treated with radiotherapy: impact of tumor size on outcome[END_REF][START_REF] Liebross | Clinical course of solitary extramedullary plasmacytoma[END_REF][START_REF] Mendenhall | Solitary plasmacytoma of bone and soft tissues[END_REF]. Six out of nine patients with progressive disease developed multiple myeloma in the first year after diagnosis. This fact underlines the relevance of thorough pretherapeutic staging. With the introduction of whole-body MRI scans, staging sensitivity could be enhanced significantly. According to Wilder et al., up to 25% of patients initially diagnosed with solitary plasmacytoma are found to have systemic disease [START_REF] Wilder | Persistence of myeloma protein for more than one year after radiotherapy is an adverse prognostic factor in solitary plasmacytoma of bone[END_REF].

Given the high probability of progression to systemic disease, a small prospective study by Aviles et al. applied local radiotherapy followed by low dose prednisone/melphalan over 3 years, resulting in 12% progression to multiple myeloma compared to 54% of patients treated with radiotherapy alone [START_REF] Aviles | Improved outcome in solitary bone plasmacytomata with combined therapy[END_REF]. However, the question of adjuvant chemotherapy for solitary plasmacytoma remains to be addressed by larger prospective studies.

Overall survival

Analysis showed that 2-year-OS in our series was 87.5% and 5-year-and 10-year-OS was 77.8% with no statistically significant difference between SBP and EP. Data published so far are inconsistent: Significantly different OS rates were found by Ozsahin et al., who described 10-year overall survival rates of 72% for EP and 52% for SBP (p=0.04) [START_REF] Ozsahin | Outcomes and patterns of failure in solitary plasmacytoma: a multicenter Rare Cancer Network study of 258 patients[END_REF], and Tsang et al., who reported 8-year overall survival rates of 70% or 33%, respectively (p=0.023). In contrast, neither Bolek et al. [START_REF] Bolek | Solitary plasmacytoma of bone and soft tissue[END_REF] nor Jyothirmay et al. [START_REF] Jyothirmayi | Radiotherapy in the treatment of solitary plasmacytoma[END_REF] could detect a significant difference in OS.

For an overview of all studies cited, see table III.

Influence of dose

In our patient cohort, we could not detect a dose-dependence of local control. In agreement with our findings, Tsang et al. reported no higher risk of local recurrence for doses <35 Gy [START_REF] Tsang | Solitary plasmacytoma treated with radiotherapy: impact of tumor size on outcome[END_REF] and Oszahin et al. described no dose-dependence of local control for doses >30 Gy [START_REF] Ozsahin | Outcomes and patterns of failure in solitary plasmacytoma: a multicenter Rare Cancer Network study of 258 patients[END_REF]. In contrast, Mendenhall et al. found that doses >40 Gy resulted in 94% local control of SBP compared to 69% after <40 Gy [START_REF] Mendenhall | Million RR Solitary plasmacytoma of bone and soft tissue[END_REF]. For EP, significantly better local control was reported for doses >45 Gy (100% 5-year LCR vs. 55% for <45 Gy) [START_REF] Tournier-Rangeard | Radiotherapy for solitary extramedullary plasmacytoma in the head-and-neck region: A dose greater than 45 Gy to the target volume improves the local control[END_REF]. Considering the low treatment toxicity, doses of 40-50 Gy seem to be a general consensus for SBP [START_REF] Holland | Treatment results and conversion to myeloma[END_REF][START_REF] Bolek | Solitary plasmacytoma of bone and soft tissue[END_REF][START_REF] Kumar | Management of multiple myeloma: a systematic review and critical appraisal of published studies[END_REF] and of 40-60 Gy for EP [START_REF] Creach | Radiotherapy for Extramedullary Plasmacytoma of the Head and Neck[END_REF][START_REF] Michalaki | Definitive radiotherapy for extramedullary plasmacytomas of the head and neck[END_REF].

Conclusion

Local radiotherapy of solitary plasmacytoma results in excellent local control with low treatment toxicity. SBP and EP show distinct features concerning progression to multiple myeloma with SBP patients being at a higher risk of progression. Further studies are required to identify risk factors for progression in order to establish more individualized therapeutic strategies.

Legends

Table I.

Patients' and treatment characteristics.

Table II. Prescribed dose and fractionation scheme.

  data of all patients treated for plasmacytoma or multiple myeloma in the Department of Radiation Oncology of the University of Heidelberg from 1995 to 2008 were searched, ruling out all patients with generalised disease. Inclusion criteria were: one solitary lesion confirmed by histology (fine-needle-or open biopsy), verification of localized disease by whole-body-MRI or -CT or plain film skeletal survey, normal serum calcium, blood count and renal function, <10% plasma cells in bone marrow biopsy, normal serum protein electrophoresis and no prior treatment for plasmacytoma.

Table I .

 I Patients' and treatment characteristics.

			No. of	RT dose	Local		MMFS	OS
	Patients' and treatment characteristics patient localisation patients	total dose control	single dose	n
	Sex Bachar 2008	68 (only	Male (Gy) 35.4 Gy	5-y-LCR	(Gy) 5-y-MMFS 77%	12	5 y 76%
	1	sternum EP)	Female 45 median 81%	2.5	6
	Age at diagnosis, median 2 nasopharynx Bolek 1996 37 (SBP 27,	60.9 years (range: 36-81) 50.4 1.8 43.2 Gy LC 97% 10-y-MMFS 46%	5-y-OS EP
	SBP 3	C4 -C6 EP 10)	Vertebral body 40 median	2	SBP, 89% EP	7	100%, SBP
	4	nasopharynx		Skull base 48	2	1	32%
	5 Chao 2005	pharynx 16 (only	45 Gy	Sternum 46 LC 100%	2	1 10-y-MMFS 75%	10-y-OS
	Table III. 6 EP 7 Creach 2008 Th8 -L2 EP) Literature overview. median Ribs 38 Head-and-neck L2 -4 40 18 (only 50.4 Gy LC 100%	2 2	Development of	1 7	54% 10-y-OS
	Figure I. 8 Figure II. Primary radiotherapy nasopharynx EP) Multiple-myeloma-free survival for SBP and EP patients. Urethra 38 median MM or 2 Overall survival for SBP and EP patients. SBP 9 Th1 -3 40 2 plasmacytoma at EP Postoperative radiotherapy SBP EP 10 right orbit and cheek 48 distant sites 33% 2 11 C7 -Th2 50 after median 3.1 y 1 5 5 6 2 2 12 Th5 -9 30 Frassica 1989 46 (only 39.75 Gy LC 89% 10-y-MMFS 25% 3 13 skull base 45 SBP) median 1.8 14 tongue 44 Jyothirmayi 30 (SBP 23, 45 Gy LC EP development of 2 15 8th rib 50 1997 EP 7) median 86%, SBP MM EP 43% after 2 16 L1-4 26 (boost 40) 100% median 30 mo., 2 17 paranasal sinuses 50 SBP 39% after 2 18 penile urethra 46 median 28 mo. 1.8 Liebross 1998 57 (only 50 Gy LC 96% 53% development	55% 10-y-OS 45% 5-y-OS EP 57%, SBP 75% median
			SBP)	median			of MM after	survival
								median 1.8 y	11.0 y
	Liebross 1999 22 (only	50 Gy		LC 95%		5-y-MMFS 68%	Not stated,
			EP)	median			median
								survival 9.5
								y
	Mayr 1990		30 (EP 13,	40-60 Gy	LC EP		Development of	5-y-OS EP
			SBP 17)			92%, SBP		MM EP 23%, SBP	58%, SBP
						88%		53%	47%
	Ozsahin 2006 258 (SBP	40 Gy		5-y-LCR		10-y-MMFS all	10-y-OS all
			206, EP 52)	median	86%		31%, EP 55%, SBP	54%, EP
								25%	72%, SBP
								52%
	Tournier-		17 (only	52.6 Gy	5-y-LCR		5-y-MMFS 64.1 % Not stated
	Rangeard		EP)	median	72.8 Gy
	2006
	Tsang 2001		46 (SBP 32,	35 Gy		8-y-LCR		8-y-MMFS: 50%	8-y-OS
			EP 14)	median	83%		65%
	Wilder 2002	60 (only	46 Gy		10-y-LCR		10-y-MMFS 38%	10-y-OS
			SBP)	median	90%		59%