Clinical Significance of Occult Central Nervous System Localization in Adult Patients With Acute Lymphoblastic Leukemia. Prospective, Multicenter Study.
Conditions: Acute Lymphoblastic Leukemia
Sponsor: University of Rome Tor Vergata

Version no. 1 dated 1.04.2020 
 
 
 
Clinical significance of occult Central Nervous System localization in adult patients with 
acute lymphoblastic leukemia. Prospective, multicenter study. 
Version no. 1 dated 1.04.2020 
 
Principal Investigator 
Maria Ilaria Del Principe 
UOSD Pathologies Myeloproliferative Syndromes, Fondazione Policlinico Tor Vergata, Rome 
 
Writing comitee 
Maria Ilaria Del Principe 
Anna Rita Guarini 
Sabina Chiaretti 
Irene Della Starza 
Loredana Elia 
Adriano Venditti 
Robin Foà 
 
1.0 Introduction. 
In recent decades, several clinical trials have led to increased responses in adult patients with acute 
lymphoblastic leukemia (ALL).1-3 In this context of improved control of systemic disease, leukemic 
involvement of the central nervous system (CNS) remains a major factor in limiting the achievement 
of cure and a major cause of mortality. Leukemic CNS involvement can occur at onset or at relapse. 
At onset, about 5% of adult patients with ALS have CNS localization, and the overall survival of these 
patients is significantly shorter than those without such involvement.4,5 
Currently, the gold standard for diagnosing CNS involvement in ALS is morphologic examination of 
the cerebrospinal ﬂuid (CSF). Conventional cytology (CC) is estimated to have >95% specificity for 
the diagnosis of leukemic localization in the CNS. Unfortunately, however, this method is burdened 
with low sensitivity (<50%) and consequently is often falsely negative. The low sensitivity of CC is 
due to the paucity of cells in the CSF and morphological similarities that make it difficult to 
distinguish benign from neoplastic cells.6,7 
Phenotypic study with ﬂow cytoﬂuorimetry (FCM) is a valuable tool for the diagnosis and staging of 
hematologic disorders involving lymph nodes, peripheral blood, and bone marrow. Over the years, 
FCM has been implemented in such a way as to allow the detection of abnormal cells representing 
0.01% of events (1 cell in 104), the so-called "rare events," and is therefore a useful tool for 
monitoring minimal residual disease in acute leukemia. 
Several recently published studies focused on determining involvement in the CNS during LLA and 
aggressive early-onset non-Hodgkin lymphomas have demonstrated the superior sensitivity of FCM 
compared with CC.9,10 In contrast, it is not yet clear whether FCM positivity alone, which can be 
defined as occult CNS localization, has a real clinical impact or not. 
In our previous retrospective study, we evaluated the incidence of occult CNS localization and the 
impact of such localization on the prognosis of adult patients with ALS at onset. We collected data, 
between 2007 and 2017, from 240 patients, 103 women and 137 men, median age 44 years (range 
17-80). One hundred eighty-four patients (77%) had a B phenotype. Patients were treated according 
to GIMEMA/NILG protocols or according to the Hyper-CVAD program. One hundred and eight 

Version no. 1 dated 1.04.2020 
 
 
 
categories of patients were distinguished: patients with overt CNS localization(CC+FCM+), patients 
with occult CNS localization (CC-FCM+), and patients with absence of disease in the CNS (SNCneg, 
CC-FCM-). Overall 179 (75%) patients were SNCneg, 43 (18%) had occult CNS localization and finally 
18 (7%) had manifest disease. No cases of morphological positivity and cytoﬂuorimetric negativity 
were observed. Patients with occult or manifest CNS localization presented higher recurrence rates 
(systemic and meningeal) than SNCneg patients. In addition, disease-free survival and three-year 
overall survival were significantly longer in patients with no CNS disease than in patients with occult 
or manifest CNS localization. On the other hand, no significant diﬀerences in survivals were observed 
between patients with occult or manifest CNS localization, confirming that even a small proportion 
of leukemic cells at this level can inﬂuence patients' prognosis. In multivariate analysis, status at 
diagnosis of occult or manifest CNS localization and age over 45 years were found to be 
independently associated with shorter overall survival. In conclusion, our study suggests that in 
adult patients with LLA, FCM allows detection of occult CNS localization, especially under conditions 
of leukemic cell scarcity in the CRL, and that the presence of such localization appears to be 
associated with poor prognosis. 
However, given the heterogeneity of the treatments our patients had received and the long 
observation period, a prospective validation study is needed to confirm these data. 
The need for further studies is confirmed by the fact that current international guidelines11-13 do 
not recommend the use of FCM in the assessment of CSF at onset in adult ALS patients. In addition 
to confirming the need to perform FCM of CSF at onset to identify patients at higher risk of 
recurrence, large-scale prospective studies will help to clarify whether or not patients with occult 
CNS localization should undergo CNS-directed therapy. Understanding this seems particularly 
important nowadays considering that with the introduction of new drugs (monoclonal antibodies, 
next-generation tyrosine kinase inhibitors, CAR-T) the therapeutic approach of patients with ALS is 
increasingly "chemo-free" 
Clinical trial proposal and objectives 
In light of this, we propose a multicenter prospective study to evaluate the incidence of occult CNS 
localization and the impact of such localization on clinical outcome. 
2.0 Experimental design 
Adult ALS patients routinely undergo diagnostic lumbar puncture (PL); CSF samples will be studied 
by investigation of CC and FCM at the time of the first and subsequent diagnostic PLs. 
3.1 Objectives 
3.1.1 Primary objective. 
To assess the incidence of occult CNS localization in adult patients with ALL. 
3.1.2 Secondary objectives 
a. 
To examine associations between occult CNS localization and: 
b1 overall survival (OS) 
b2 recurrence-free survival (DFS) 
b3 overall and medullary or extramedullary recurrence rates 
b4 cumulative incidence of recurrence (CIR) 
b. 
To evaluate the correlation between occult CNS localization and minimum residual disease 
(MRD) levels. 
c. 
Determine the status of CNS occult localization also by molecular biology, comparing the 
results with those of FCM. 
 
4.0 Study population. 

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4.1 Sample size and duration of the study. 

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4.1.1. 
All patients with Acute Lymphoblastic Leukemia (ALL) who underwent diagnostic lumbar puncture 
(PL) in the planned 12 months of enrollment will be included in the study. The sample size was 
calculated to assess the incidence of occult CNS localization; based on clinical experience14 in order 
to observe an incidence at diagnosis of 20%, with a 95% confidence interval (two-tailed test) and a 
width of 0.20, a number of 70 subjects was calculated as the minimum sample size15. Calculations 
were performed using PASS software. 
4.1.2 
The duration of the study will be 12 months of enrollment of the first patient and 24 months of 
follow-up. 
4.2 Patient selection. 
4.2.1 Inclusion criteria. 
- Patients aged ?18 years with diagnosis of ALL at onset undergoing diagnostic-therapeutic PL. 
- Signed written informed consent in accordance with ICH/EU/GCP guidelines and national and local 
laws. 
4.2.2 Exclusion criteria. 
- Patients <18 years of age 
- Diagnosis other than ALL 
- Inability to perform PL 
 
5.0 Materials and methods 
5.1. Methods 
5.1.1 CC 
Physical chemical examination and cell count and morphological examination will be performed for 
each CSF sample as per common clinical practice. Morphological examination of the CSF will be 
performed by preparation of cytocentrifugate (CC) stained with May-Grunwald-Giemsa. Manifest 
CNS localization will be defined in the unequivocal presence of leukemic cells in the CSF and/or a 
mononuclear cell count ≥5/cml. 
5.1.2 FCM 
For the FCM examination, which is also performed as part of common clinical practice, CSF samples 
of sufficient volume will have to be taken via PL. The CSF sample to be stored on ice will be processed 
within 
1 
hour 
of 
collection 
to 
avoid 
cell 
deterioration 
or 
a 
specific 
fixative 
(TransFix/ethylenediaminetetraacetic acid EDTA; Immunostep SL Salamanca, Spain) should be used. 
The CSF sample will be concentrated by centrifuging it at 300 g for 4 min. The supernatant will be 
gently aspirated and the pellet resuspended in PBS+BSA 0.2% in varying amounts depending on the 
number of tubes to be prepared (100?l/tube). One hundred ?l of the suspension will be incubated 
with monoclonal antibody mix of interest for 20 min in the dark. A wash with 2 ml PBS+BSA 0.2% 
will then be performed, centrifuge at 300 g for 3 min. The pellet will be resuspended in 700?l of 
PBS+BSA 0.2%. The phenotypic study will be performed using a panel of 6-8 monoclonal antibodies. 
The antibody panel will include: CD20-FITC, CD10-PE, CD34-PE, CD19-PerCP, CD45-APC, (Becton 
Dickinson, Mountain View, CA), for line B; CD3-FITC, CD2-FITC, CD7-PE, CD5-APC, CD8-PE, CD4-FITC, 
CD45-PerCP (Becton Dickinson, Mountain View, CA) for line T. All samples will be acquired until 
depletion to obtain the highest number of events. FCM positivity will be defined in the presence of 
≥10 cluster-forming events and leukemic phenotype. FCM acquisition will be done locally, but 3 
independent, experienced operators will review each case. 

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5.1.3 Ancillary study. Molecular biology. 

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Given the scarce data in the literature regarding the role of molecular diagnostics in the diagnosis of 
occult localization in the CNS, the study will include an ancillary part to evaluate this role by 
comparing molecular findings with FCM. The success of the molecular study will be related to the 
number of leukemic blasts present in the CSF, so a morphological study will be needed on the 
starting material that will be able to give accurate information about the material being processed. 
All cases will need to be processed within one hour of collection of the material to avoid degradation. 
It will then be necessary to centrifuge the sample immediately upon arrival for 10' at 1500g. A 
portion of the supernatant will be discarded but the portion of the liquid closest to the bottom will 
be retained. Given that at the time of performing PL in most patients the positivity or non-positivity 
for a molecular marker is known, one may proceed in the following ways. 
If the patient has a known molecular marker, the pellet will be resuspended in trizol for preservation 
and RNA extraction. If, on the other hand, the patient does not have a known molecular marker, the 
pellet will be stored at -20°C. 
The ancillary study will be performed by the standardized molecular biology laboratories that will 
be involved in the study (Rome-Policlinico Tor Vergata, Rome-Policlinico Umberto I, Bologna-Istituto 
Seragnoli, Bergamo- Giovanni XIII Hospital, Palermo-Ospedali Riuniti) 
5.2 Data collection sheet. 
Cases will be documented using data collection sheets. A sequential identification number will be 
assigned to each patient registered in the study. This number will identify the patient and will be 
included in the data collection sheet thus allowing the patient's anonymity to be maintained. 
Variables collected will be age, gender, date of ALS diagnosis, immunophenotype at onset, genetic 
and cytogenetic features of ALS, stratification by risk group, extramedullary localizations, type of 
chemotherapy, date of first PL, white blood cells at onset and at the time of PL, lacticodehydrogenase 
levels at onset and at the time of PL, CRL cell count, CRL protein count, CRL cytospin outcome, date 
of complete remission. Subsequent data collection sheets will be provided for patient follow-up, at 
12 and 24 months after enrollment, in which the number of PLs performed, MMR evaluation, MMR 
assessment, outcome, date of any recurrence, any SNC radiotherapy, salvage chemotherapy, date of 
last follow-up, date of death will be reported. 
 
5.3 Statistical considerations. 
Patient characteristics will be described by frequency tables for qualitative variables and position 
indicators for quantitative variables. Associations of clinical-biological parameters with CNS location 
will be analyzed by Chi-square or Fisher's exact test for qualitative variables and Wilcoxon or Kruskal- 
Wallis test for quantitative variables. OS will be calculated from the date of treatment initiation to 
the date of death from any cause; subjects alive at the last follow-up will be censored. DFS will be 
calculated from the date of achieving complete remission to the date of relapse or death from any 
cause; subjects alive without progression at last follow-up will be censored. CIR will be estimated via 
via nonparametric method considering death as competitive risk. OS and DFS probabilities will be 
estimated by Kaplan Meier method; log rank test will be used to compare subgroups. Confidence 
intervals will be calculated at 95%, all tests will be two-tailed, and diﬀerences with p < 0.05 will be 
considered statistically significant. R software (A language and environment for statistical computing. 
R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/). 
6.0 Ethical aspects 
The study will be conducted in adherence with Good Clinical Practice guidelines, as described in: 
1. 
ICH Guidelines of Good Clinical Practice, 1996. CPMP/ICH/135/95 
2. 
EU Directives and 2001/20/EC, 2005/28/EC. 

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3. 
Declaration of Helsinki (1964, and its amendments and subsequent clarifications). 

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Approval of the local ethics committee will be required to conduct the study: Ethics Committee of 
the Fondazione Policlinico Tor Vergata in Rome. 
Patients will be required to sign informed consent to process the data. 
Investigators will facilitate monitoring, verification, inspection of regulatory bodies by providing 
direct access to original data/documents. 
 
7.0 Dissemination of results. 
The results obtained will be the subject of one or more publications in leading international scientific 
journals dealing with topics related to hematology and cytoﬂuorimetry. In addition, the results will 
be submitted for presentation at major national and international hematology congresses (American 
Society of Hematology, European Hematology Association, Italian Society of Hematology, Italian 
Society of Experimental Hematology). 
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