We collected 557 patients from 18 centers, among which 513 were evaluable for the study purpose and characterized in Table 2. Forty-four patients (7.9%) were excluded because of suspected diagnosis of HCL variant (17 cases), which generally have a reduced rate of response compared with classic HCL, concomitant treatment with Rituximab (15 cases), or incomplete data (12 cases).

Male/female ratio was 4.5 with a median age of 54 years (range 24–88); more than half of patients (298, 58%) were diagnosed in the 2010–2019 years. BRAF-V600E mutation result was only available for 136 (26.4%) patients and positive in all cases. Splenomegaly was detected in 241 (46.9%) patients, with a median diameter of 18 cm (range 15–30) in the 230 patients with this radiological information available. At the onset, 436 patients (84.9%) were in good clinical condition with ECOG 0, while 71 (13.9%) and 6 (1.2%) had an ECOG 1 and 2, respectively. We recorded a low comorbidity rate in this population. Thirty-seven (7.2%) patients presented with an infection at the onset of disease, that was pneumonia in 24 cases (64.9%), fever of unknown origin in 7 (20%), skin and abdominal abscess in 2 (5%), and 4 (10%) cases respectively.

All patients received 2CDA, after a median time from diagnosis of 1.2 months (range 0.1–261.8) (Table 3), administered intravenously in 330 (64.3%) patients and subcutaneously in 183 (35.7%) patients. Among patients with intravenous 2CDA, 250 (75.8%) received a daily infusion for 5–7 consecutive days and 80 (24.2%) a weekly infusion for 5–7 consecutive weeks.

Overall, adverse events were reported in 175 patients (34.1%) (Table 4). Allergic reactions were reported in 48 (9.2%) patients, only 1 of which was recorded as grade 3. Hemorrhagic events were reported in 3 (0.6%) patients: 2 cases of low-grade skin bleeding and 1 case of grade 3 gastrointestinal bleeding; 3 (0.6%) cases of thrombosis, deep vein of lower limbs (2), and peri-catheter upper limb (1), were also reported. Other adverse events (5 hepatic, 1 gastrointestinal, and 1 cardiovascular; 1.4%) were reported: 4 were grade 3 (3 hepatic and 1 gastrointestinal) and 2 were grade 4 (1 hepatic and 1 cardiovascular). Infection was reported in 141 (27.7%) cases: 97 (19.2%) were treated as inpatients due to grade 3 or higher severity of the event, whereas 44 were treated as outpatients due to grade 2 or lower severity. Most infections were recorded as fever of unknown origin (98, 69.5%): these events are more likely the result of cytokine release rather than true infection. Pneumonia was detected in 23 (15.6%) patients: in 5 cases it was clinically documented only, while in 18 cases it was attributable to bacteria (8, 44.5%), fungi (6, 33.4%), or viruses (4, 22.1%). Other sites of infections were bloodstream (9, 6.8%), upper respiratory tract (4, 2.7%), skin (4, 2.7%), and lower urinary tract (3, 2.7%). Overall, 6 patients died of infectious complications: none of them presented an uncontrolled infection before treatment. The 6 fatal infective events included invasive aspergillosis, bacterial pneumonia, and bacterial sepsis (2 cases each).

Hematological toxicities are detailed in Table 5. Briefly, among 111 patients with ANC > 1.5 × 109/l, grade 2 and above neutropenia occurred in 29 patients (26%), while among the 295 patients with grade 2 or 3 neutropenia worsening to grade 3 and 4 occurred in 128 cases (43%). In 49 patients with normal platelet count, grade 1–2 thrombocytopenia occurred in 14 cases (29%), while among the 464 thrombocytopenic patients, worsening to grade 2 and above occurred in 108 cases (23%). In 357 patients with hemoglobin level above 11 g/dl, grade 1 to 3 anemia occurred in 102 cases (29%; grade 2 in 55 cases, 15%), while among 157 patients with anemia grade 1 or above worsening to grade 2 or above occurred in 48 cases (31%).

Transfusions of red blood cells (median 3 units; range: 1–27) and platelets (median 2 units; range: 1–9) were required in 98 (19%) and 17 (3%) of all 513 treated patients, respectively. The timing of cytopenia recovery was as follows (Table 2). In 284 neutropenic patients, recovery above 1.5 × 109/l of ANC was reported in 169 (59.5%) patients after a median of 30 days (range 4–302). Recovery above 100 × 109/l of platelet count was reported in 214 (59%) of 363 thrombocytopenic patients after a median of 22 days (range 6–300), while recovery above 11 g/dl of hemoglobin value was reported in 81 (68.1%) of 119 patients with anemia after a median of 50 days (range 7–182).

Response was evaluated after a median time of 3.3 months (range 0.3–9.8). Overall response rate (ORR) was 91.8%: CR was obtained in 335 (65.3%) patients, PR in 96 (18.7%) and hematological response (HR) in 40 (7.8%); 42 (8.2%) patients did not show any response (no response) (Fig. 1). Characteristics associated in multivariate analysis with CR compared to PR were higher hemoglobin value (OR: 1.12, 95% CI: 1.02–1.22, p = 0.021) and lower number of circulating hairy cells (OR: 3.72, 95% CI: 1.35–13.2, p = 0.020 (Table 6). No other differences between complete versus partial responders were identified, according to sex, age, calendar decade of treatment, route of administration, baseline values of white blood cells, neutrophils, and platelets, comorbidities at onset, time from diagnosis to treatment start, percentage of leukemic infiltration and bone marrow Hairy Cell Index (total bone marrow cellularity multiplied by leukemic cellularity).

The diagram shows the deep of response and the outcome after first-line therapy, the number of patients who underwent a second line teatment, and the deep of response and outcome of second-line therapy. CR complete response, PR partial response, HI hematological improvement, NR no response.

Among 42 non-responder patients, 25 (4.9%) underwent a second course of treatment: 13 obtained a CR (7 with rituximab and 6 with 2CDA), 5 a PR (2 with 2CDA, 2 with pentostatin, 1 with interferon-α, and 1 with rituximab) and 2 an HR (2 cases with interferon-α). Five patients did not improve after receiving 2CDA (2 cases), rituximab (2 cases), or interferon-α (1 case). Of the remaining 17 non-responder patients, 10 were lost to follow-up a few weeks after bone marrow re-evaluation and 7 died soon after first treatment. Overall, 11 and 4 patients deceased of natural causes and infections, respectively, 7 before and 8 after second-line therapy (Fig. 1).

At a median follow-up of 6.83 years (range (0.04–28.52) in the 431 patients achieving CR or PR, the median time to relapse was 12.2 years: particularly, 75%, 53.6%, and 45.4% of patients were expected to be free from relapse at 5, 10, and 15 years, respectively (Fig. 2A). A statistically significant difference in duration of response was identified between patients that obtained a CR compared to patients in PR (19.4 years versus 4.8 years, p < 0.0001) (Fig. 2B). No other differences in RFS were identified, according to the route of administration, detection of BRAF-V600E mutation, calendar decade of treatment, age at treatment start, sex, baseline values of white blood cells, neutrophils, platelets and hemoglobin, and infection at onset, percentage of leukemic infiltration and bone marrow Hairy Cell Index. No differences in depth and duration of response, RFS, infections, bone marrow recovery, and survival were reported among 183 patients receiving subcutaneous administration of 2CDA, compared to intravenous administration.

A Global RFS of the study population. B RFS of the study population according to response. C OS of the study population.

Of all 431 responders, 118 (27.3%) patients relapsed, including 70 of the 335 with a previous CR (20.9%) and 48 of the 96 with a previous PR (50%). Among these 118 relapses, 4 patients were lost to follow-up whereas 114 patients underwent a second-line therapy (Fig. 1) after a median of 53.2 months from response (range 7.5–211.5): 85 (74.5%) patients were retreated with 2CDA, alone (64 cases) or associated with rituximab (21 cases); 12 patients (10.5%) with pentostatin, alone (8 cases) or associated with rituximab (4 cases); 8 patients (7%) with interferon α, 7 patients (6.1%) with rituximab, 1 patient (0.9%) with vemurafenib and zanubrutinib each. ORR2 was 89.4%: 58 patients (50.9%) obtained a CR (42 after 2CDA), 37 patients (32.4%) a PR (32 after 2CDA), 7 patients (6.1%) an HR (4 after 2CDA) and 12 patients (10.5%) had no response (6 after 2CDA) (Fig. 2). Regarding the 40 patients obtaining only HR after first-line cladribine (Fig. 1), 11 patients underwent a second-line treatment after a median time of 4.3 years (range 0.9–17.7 years): 9 (81.8%) were retreated with cladribine and 2 (18.2%) were treated with pentostatin. ORR was 81.8%: 3 and 6 patients obtained CR and PR, respectively. Moreover, 22 patients did not receive any other treatment and 7 were lost to follow-up.

Median OS was not reached: particularly, 95.3%, 92.4%, and 81.8% of patients were estimated to be alive at 5, 10, and 15 years, respectively (Fig. 2C). Overall, 49 patients (9.5%) deceased: causes of death were infection in 14 cases (2.7%), natural causes in 14 (2.7%), cardiovascular events not related to HCL or treatment in 13 (2.5%), a second neoplasm in 6 (1.2%) and progression of HCL in 2 cases only (0.4%).