Diffuse large B-cell lymphoma (DLBCL) represents 30–40% of newly diagnosed adult Non-Hodgkin lymphomas (NHL), and is a heterogeneous group of disorders with variable histological and clinical behaviour (The Non-Hodgkin's Lymphoma Classification Project's, 1997; De Paepe & De Wolf-Peeters, 2007; Jaffe, 2009; Flowers et al, 2010; Nogai et al, 2011; Shenoy et al, 2011). Strong prognostic indicators of outcomes include the International Prognostic Index (IPI) and molecular and genomic markers (Rosenwald et al, 2002; Shipp et al, 2002; Gascoyne et al, 2010; Hong et al, 2011). Although marrow involvement by lymphoma is common in advanced stage DLBCL (Morra et al, 1989; Sieniawski et al, 2009), presentation with circulating lymphoma cells is relatively rare (Come et al, 1980; Morra et al, 1989; Bain & Catovsky, 1995). The presence of circulating malignant lymphoma cells in the blood is well-recognized in mantle cell lymphoma, follicular lymphoma, anaplastic large cell lymphoma, and in terminal phases of all refractory lymphomas (Morra et al, 1984; Berinstein et al, 1993; Criel et al, 1996; Villamor et al, 1999). Clinical and pathological description of DLBCL presenting with circulating lymphoma cells or in ‘leukaemic phase’ has been previously reported as a subgroup of the lymphosarcoma cell leukaemia (Schwartz et al, 1965; Mintzer & Hauptman, 1983) and only in small cohorts focusing primarily on the pathological findings (Come et al, 1980). We herein report the clinical characteristics, response to treatment and outcomes of 29 patients with DLBCL presenting in leukaemic phase at two institutions.
Diffuse large B-cell lymphoma (DLBCL) occasionally presents with circulating malignant cells. The clinical characteristics and long-term outcomes of these patients have not been described. Twenty-nine newly diagnosed DLBCL presenting in leukaemic phase were identified between 1996 and 2010, at two institutions. Median age was 48 years, and patients presented with leucocytosis, high lactate dehydrogenase levels, B symptoms, and high International Prognostic Index score. Extra nodal site involvement was observed in all patients and affected the bone marrow (100%), spleen (62%), pleura/lung (41%), liver (21%), bone (17%), bowels (7%) and cerebrospinal fluid (14%). Blood lymphomatous cells co-expressed CD19, CD20, CD22, CD38, CD45, HLA-DR and FMC7 in >90%, and kappa or lambda light chain restriction in >50%. Ninety per cent received rituximab and anthracycline-based chemotherapy. Overall, remission was complete in 54% and partial in 31%; 15% had resistant disease. Median follow-up was 47 months; 13 (45%) patients remain alive in complete remission. Median progression-free and overall survivals were 11·5 and 46·7 months, respectively. In summary, patients with DLBCL in leukaemic phase present with high tumour burden and frequent involvement of extra nodal sites. In this uncommon DLBCL subgroup, anthracycline-based regimens with rituximab are associated with early morbidity and mortality, but yield approximately 50% 4-year survival.
Methods and materials
A flow cytometry database search identified patients with NHL presenting with circulating lymphoma cells between 1996 and 2010 at Emory University and the University of Nebraska at Omaha. Patients with mantle cell, anaplastic large cell, Burkitt, intravascular, and follicular lymphoma were excluded. Patient's basic demographics (age, gender, race), performance status, Ann Arbor stage, presence of extra nodal sites, bulky disease (nodes >10 cm in diameter), bone marrow and/or cerebrospinal fluid (CSF) involvement, haematological parameters, and lactate dehydrogenase (LDH) levels, as well as treatment, complications, and outcomes were captured onto study-specific case report forms. Pathological characteristics including immunophenotype of the circulating cells, histo-morphology, immunohistochemistry, molecular tests, and marrow cytogenetics or fluorescence in situ hybridization (FISH) when available, were collected. Response to therapy was assessed by computerized tomography and/or positron emission tomography scans, and classified according to the International Working Group criteria (Cheson et al, 1999). Briefly, complete remission (CR) was defined as complete disappearance of signs and symptoms of disease after induction chemotherapy, and partial remission (PR) as a reduction of more than 50% decrease in total of the product of the bidimensional measurement of up to six largest dominant masses; no increase in size of other nodes. This study was approved by the Emory University and University of Nebraska Institutional Review Boards.
Outcomes included response, complications of therapy, early mortality, relapse, progression-free survival (PFS) and overall survival (OS). OS was calculated as time from diagnosis to death or date of last follow up. Patients alive at last follow-up were censored for OS. PFS was calculated as time from diagnosis to date of first relapse, progression or death. Patients transplanted in first CR (CR1), and patients alive at last follow-up were censored for PFS. The survivor functions for PFS or OS were estimated by the Kaplan and Meier method (Kaplan & Meier, 1958). Point estimates and 95% confidence interval (CI) estimates of PFS and OS probabilities were calculated. The SAS statistical package (SAS Institute Inc., Cary, NC, USA) was used for all data managements and analyses.
Characteristics of the 29 patients with newly diagnosed DLBCL presenting in leukaemic phase are summarized in Table 1. Median age was 48 (range, 34–80) years, and the male to female ratio was 1·4:1. All patients had Ann Arbor stage IV with bone marrow involvement, and 21% had bulky lymph nodes. B symptoms were present in 62%. Extra nodal sites of involvement were observed in all patients and included spleen (62%), pleura/lung (41%), liver (21%), bone (17%), CSF (14%), and bowel (7%). Four patients had a history of a previously untreated low-grade NHL. Median presenting white blood cell (WBC) count was 12 × 109/l (range, 5·5–127·4 × 109/l), haemoglobin 96 g/l (range, 75–148 g/l) and platelet count 65 × 109/l (range, 4·0–579 × 109/l). The median percent of circulating lymphomatous cells was 34% (range, 1–92). LDH levels were increased in 96%, with a median value of 6·4 times the upper limit of normal (ULN) (range, 0·4–120). IPI was 3 in 59%, 4 in 38% and 5 in 3%.
|Age, median (range)||48 (34–80)|
|Ann Arbor Stage IV, n (%)||29 (100%)|
|B symptoms, n (%)||18 (62%)|
|Extra nodal sites, n (%)||29 (100%)|
|Bulky lymph nodes (>10 cm), n (%)||6 (21%)|
|Elevated LDH, n (%)||28 (96%)|
|Median WBC × 109/l (range)||12 (5·5–127·4)|
|Median percent blood lymphoma cells (range)||34 (1–92)|
|Median Hb g/l (range)||96 (75–148)|
|Median PLT × 109/l (range)||65 (4·0–579)|
|History of low grade NHL, n (%)||4 (13%)|
|International Prognostic Index, n (%)|
The characteristics of the four patients who presented at diagnosis with CSF involvement (n = 4) were analysed and compared to the 25 patients without CSF involvement. Patients presenting with lymphomatous meningitis were younger (median age 44 vs. 58 years; P = 0·18), did not present with bulky lymphadenopathy, had a higher median WBC count (14 × 109/l vs. 11·4 × 109/l; P = 0·78) and percent of circulating lymphomatous cells (65% vs. 37%; P = 0·27), had lower median platelet count (25·500 × 109/l vs. 73 × 109/l; P = 0·08), no prior history of low-grade NHL, and a comparable IPI. Interestingly the median LDH level was lower (3·4 × ULN [2·8–9·4] vs. 6·6 × ULN [04–120]; P = 0·35).
Blood and marrow aspirate smears generally showed large atypical lymphoid cells with pale blue cytoplasm, round irregular nuclear contour with 1–2 distinct nucleoli. The median percentage of marrow involvement by these cells was 50% (range, 1–88%). Immunophenotypic analyses showed distinct cells with intermediate light scatter that co-expressed CD19, CD20, CD22, CD38, CD45, HLA-DR and FMC7 in >90%, and kappa or lambda light chain restriction in >50% of cases. The following surface antigens were less often co-expressed: CD5 (17%), CD10 (21%), CD23 (21%), and CD25 (14%). For the 25 patients who had flow cytometry analyses performed simultaneously on blood and marrow samples, the determined immunophenotypes were identical. Marrow cytogenetic analyses were performed in 14 patients (Table 2). Results showed normal karyotypes in seven patients, complex karyotypes in 6, and no results were available in one case due to the absence of cell growth. Of interest, t(8;22) was detected in one patient as part of a complex karyotype; however, histology findings did not support a diagnosis of Burkitt lymphoma in this case.
|1||47,X,add(X)(p22·1),+3,der(4)t(4;18)(?p14;?q22),del(6)(q22),−8,del(9)(q34),−13, +14,add(14)(q32) × 2,der(18)t(?4;18;?22),der(22)t(18;22)(q22;q11·2), +mar[cp13]/46,XX|
|3||Failed, No growth|
|5||51,XY,+3,del(3)(q27),der(3)del(3)(p25)del(3)(q27),+6,add(6)(q21) × 2,+7,del(7)(q36) × 2,+8,del(13)(q14q21),add(19)(q13·3),+21/46,XY|
|10||44,XY,add(6)(q21),add(11)(q25),del(13)(q22q32),del(15)(q21), −17, −18, −19,+mar[cp6]/46,XX|
One patient died within 6 d of presentation from sepsis complicated by acute renal failure without receiving induction chemotherapy. One patient received a single dose of rituximab, but died within 26 d from sepsis, respiratory failure and acute renal failure. One patient received one cycle of rituximab with a combination of cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP) and died within 30 d from complications of tumour lysis syndrome and sepsis. The characteristics of these three patients that expired in the first 30 d from diagnosis were analysed. When compared to the other patients, these patients were older (median age 70 vs. 50 years; P = 0·16), had a comparable WBC count (median 11 × 109/l vs. 12 × 109/l; P = NS), fewer circulating lymphomatous cells (5% vs. 43%; P = 0·79), lower platelet counts (50 × 109/l vs. 75 × 109/l; P = 0·31), and had high IPI (IPI of 4–5 100% vs. 35%; P = 0·05). The Ann Arbor stage, the number and sites of extra-nodal involvement, and LDH levels were otherwise comparable.
Two patients received a combination of cyclophosphamide, vincristine, doxorubicin, methotrexate (CODOX-M) alternating with ifosfamide, etoposide, and cytarabine (IVAC) (Maruyama et al, 2010). One received six cycles of rituximab, cyclophosphamide, mitoxantrone, vincristine and prednisone (RCNOP), and one received six cycles of combination of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP). Fifteen (52%) received up to eight cycles of RCHOP. Six patients received rituximab and hyperfractionated cyclophosphamide, vincristine, adriamycin, dexamethasone alternating with high-dose methotrexate and cytarabine (RHCVAD) for 2 (n = 1), 4 (n = 2) or 8 (n = 3) cycles. One patient was switched after one cycle of RCHOP to RHCVAD due to primary resistant disease. Of note, patients with very high WBC counts were cytoreduced prior to initiation of therapy, especially rituximab, with a combination of cyclophosphamide and prednisone (pre-phase). All patients received allopurinol and hydration with sodium bicarbonate to prevent tumour lysis. The four patients (14%) with CSF involvement by lymphoma at diagnosis received therapeutic intrathecal injections of methotrexate and/or cytarabine. Five patients (17%) received haematopoietic stem cell transplantation (HSCT). Four were autografted: one in CR1, two in second CR (CR2), and one in PR. One patient was allografted from a matched sibling in PR following salvage therapy for relapsed disease after initial CR.
The first course of chemotherapy was complicated by acute renal failure in eight patients (28%), of whom two required haemodialysis. Five patients (17%) developed tumour lysis syndrome, 6 (21%) bacterial sepsis, and 3 (10%) respiratory failure that required intubation and mechanical ventilation. Other complications included mucositis in 3 (10%) patients and disseminated intravascular coagulopathy in one patient.
Response and survival
Response was assessed after at least two cycles of chemotherapy. Three patients (10%) were not evaluable due to death within 30 d. Following induction ± intrathecal chemotherapy, all patients cleared lymphomatous cells from both blood and CSF. Overall, of the 26 evaluable patients, 14 (54%) achieved CR (8/15 with RCHOP, 4/6 with RHCVAD, 1 with RCNOP, and 1 with RHCVAD following progression with RCHOP), eight patients (31%) achieved PR (6/15 with RCHOP, 1 CHOP, and 1/6 RHCVAD), and 4 (15%) had primary resistant disease (1 RCHOP, 2 CODOX/M/IVAC, and 1 RHCVAD). Median follow-up was 47 months. Of the 14 patients who achieved CR, 6 (42%) relapsed a median of 10·9 months (range, 3·5–45 months) after diagnosis: two died from progressive disease and four achieved CR2 (of who two were autografted) and are still alive with a median follow-up of 57·5 months. Of the eight patients who achieved PR: one received a salvage autologous transplant and is still alive 54 months post-transplant, five progressed and died from resistant disease, and two were lost to follow-up. All four patients with primary resistant disease died a median of 5·6 months (range, 3·7–18) from diagnosis. Among the six patients with complex karyotype, two had resistant disease, three achieved CR, and one achieved PR. One of the four patients who presented with leukaemic meningitis relapsed with CSF disease following four cycles of RHCVAD and eight intrathecal injections of methotrexate/cytarabine. Of the 25 patients without CSF involvement at diagnosis, three patients (all treated with RCHOP) relapsed with lymphomatous meningitis. Table 3 summarizes the treatment, complications, response to therapy, relapse and survival for all patients. PFS and OS curves are depicted in the Fig 1. Median PFS and OS was 11·5 and 46·7 months, respectively.
|1||RCHOP × 4||ARF, sepsis, respiratory failure, DIC||CR||No||Yes||48·7|
|2||RHCVAD × 4||No||CR||No||Yes||51·8|
|3||RCHOP × 1||ARF,TLS, sepsis||NE||N/A||No||0·72|
|4||RCHOP × 7||ARF, TLS||CR||Yes||No||46·7|
|5||RHCVAD × 8||N/A||CR||No||Yes||44·9|
|7||Rituximab × 1||ARF, sepsis, respiratory failure||NE||N/A||No||0·85|
|8||RCHOP × 6||No||PR||Yes||No||7·03|
|9||RHCVAD × 4||ARF, TLS||CR||Yes||No||11·07|
|10||RHCVAD × 2||Sepsis||R||No||No||6·5|
|11||RCHOP × 6||No||CR||No||Yes||44·7|
|12||RCHOP × 8||No||PR||No||LFU||8·6|
|13||RHCAVD × 8||No||CR||No||Yes||58·1|
|14||RCHOP × 1–RHCVAD × 8||No||CR||Yes||Yes||54·3|
|15||RHCVAD × 8||TLS||PR||No||No||8·81|
|16||RCHOP × 8||Mucositis||PR||No||No||5·2|
|17||RCHOP × 4||Mucositis||PR||No||LFU||7·9|
|18||CHOP × 6||No||PR||Yes||No||9·57|
|19||RCNOP × 6||No||CR||Yes||Yes||115|
|20||RCHOP × 6||No||CR||Yes||Yes||98·4|
|21||RCHOP × 4||No||R||NA||No||18|
|22||CODOXM/IVAC × 3||ARF, mucositis, sepsis||R||NA||No||3·7|
|23||RCHOP × 8||No||PR||Yes||Yes||70|
|24||CODOXM/IVAC × 4||ARF, sepsis||R||NA||No||5·6|
|25||RCHOP × 6||No||CR||No||Yes||64·5|
|26||RCHOP × 8||TLS||CR||No||Yes||53·3|
|27||RCHOP × 6||No||CR||No||Yes||43·8|
|28||RCHOP × 6||No||CR||Yes||Yes||12·1|
|29||RCHOP × 5||ARF, respiratory failure||PR||Yes||No||7|
In this collaborative study between two Institutions, 29 patients with DLBCL presenting with circulating lymphoma cells were identified through a flow cytometry database search. It is unclear why patients with DLBCL circulate malignant cells, and the mechanisms leading to the release of these lymphoma cells into the bloodstream remain unknown. A differential expression of adhesion molecules was proposed as a possible mechanism for the migration of lymphocytes from the lymph nodes (Drillenburg & Pals, 2000). In a study where blood flow cytometry was performed in 47 patients with mantle cell lymphoma, chronic lymphocytic leukaemia and splenic marginal B-cell lymphoma presenting in leukaemic phase, expression of CD11a, CD11c, CD18, CD29, CD49c, CD49d and CD54 was reported on the surface of the circulating lymphomatous cells (Matos et al, 2006). However, data on tumour immunophenotype in patients without circulating lymphomatous cells was not available for comparison. In our study, no specific flow cytometry pattern or a differential expression was identified on the surface of simultaneously obtained blood and marrow lymphomatous cells. Specifically, expression of CD11c was noted in only three patients, and CD56 expression was not detected. Of note, CD5 or CD10 expression was not over-represented in this patient population, and was found in 17% and 10%, respectively. Exploration using whole genome sequencing in this unusual subtype of DLBCL may shed more light on pathogenetic pathways.
All patients with DLBCL in leukaemic phase presented with extra nodal involvement and high IPI. Interestingly, six patients presented with complex cytogenetics, which did not appear to impact on the response to induction or duration of remission. As expected in patients with advanced phase of NHL, performance status was poor at presentation. This may have contributed, in addition to the high tumour burden and despite appropriate tumour lysis syndrome prophylaxis and cytoreduction prior to start of therapy, to significant morbidity and an early mortality of 14%. This early mortality is higher than what was observed in patients with DLBCL treated with RCHOP (6%), (Coiffier et al, 2002) and in patients with stage IV aggressive NHL treated with RHCVAD (0%) (Muringampurath-John et al, 2012).
Using predominantly rituximab-based chemotherapy regimens, the overall response was 88%, with 54% achieving CR. These regimens included RCHOP (or variants of this regimen with different anthracycline), RHCVAD, and CODOXM/IVAC. The number of patients was too small to define an optimal regimen for this patient population, but none of the two patients receiving CODOXM/IVAC responded. With a median follow-up of 4 years, OS was 47%, and appeared comparable to the survival observed in high IPI score patients with DLBCL without circulating lymphomatous cells receiving RCHOP (48% 5-year survival) (Feugier et al, 2005).
This study is limited by the retrospective nature of the analysis, the small number of patients, and the lack of data on gene expression (activated B cell-like versus germinal centre B cell-like subtypes) and other molecular markers. Additionally, this study, through its design, could not determine the prevalence of this unusual presentation of DLBCL, as some patients may not have had blood flow cytometry done as part of their work-up.
This study is, to our knowledge, the first report to describe clinical characteristics, response to modern therapy and outcomes in patients with DLBCL in leukaemic phase. These patients present with extra nodal disease and have a high rate of induction-associated early complications and death. However, treatment with anthracycline and rituximab-based regimens (RCHOP or RHCVAD) is effective and is associated with an approximately 50% 4-year survival.
DMJ, DLJ, ZC, DS, MB and HJK performed the research. DMJ and HJK designed the research study. DLJ, CRF, MJL, DDW, MLA, LBM, LTH, MM, JLK, EFW, SL, JOA, and HJK contributed essential reagents or tools. DMJ, DLJ, ZC, DS, and HJK analysed the data. DMJ, DLJ, CRF, DS, MJL, MLA, LBM, LTH, MM, JLK, EFW, SL, JOA, and HJK wrote the paper.
Conflict of interest disclosure
The authors have no conflict of interest to declare.