T-cell chronic lymphocytic leukaemia: anomalous cell markers, variable morphology, and marked responsiveness to pentostatin (2'-deoxycoformycin)

4 men with an unusual variant of T-cell chronic lymphocytic leukaemia are reported. The clinical features differed from the virus-associated T-cell disease reported from Japan, the Caribbean, and the southeastern United States. Cytology was pleiomorphic: cells with cerebriform nuclei resembling Sézary cells and lymphocytes with cytoplasmic granules resembling T-suppressor cells occurred in the same patients. Immunofluorescence studies with monoclonal antibodies suggested that the leukaemia cells expressed determinants of both helper- (OKT4+) and suppressor-(OKT8+) related antigens. The cells were TdT-negative. In all patients the disease was very refractory to conventional cytotoxic agents, but there was prompt and extensive response to the adenosine deaminase inhibitor pentostatin (2'-deoxycoformycin). This agent merits further study in the treatment of T-cell chronic lymphocytic leukaemia.     

B-cell chronic lymphocytic leukaemia with CD8 expression: report of 10 cases and immunochemical analysis of the CD8 antigen

We report 10 cases of B-cell chronic lymphocytic leukaemia (B-CLL) with expression of the T-cell antigen CD8. The majority of patients had typical B-cell CLL with stable and non-progressive stage A(O) disease except for more common expression of lambda light chain and CD25. Two patients had progressive disease and required therapy, one with atypical morphological and phenotypic features. The incidence of CD8 expression was approximately 0.5% of B-CLL patients from our institutions. Immunoprecipitation of the CD8 antigen from four of these B-CLLs showed identity to the CD8 antigen expressed on T cells with precipitation of CD8α bands of molecular weight ~34 kD. In view of the known intracellular signalling mechanism of CD8 using the tyrosine kinase p56-lck, we studied p56-lck expression by Western blot and found lack of consistent expression of the CD8 surface antigen, with most lacking p56-lck. Our report indicates that CD8 expression in B-CLL is probably under-recognized but is not a marker of disease progression. The CD8 on the B-CLL surface is immunochemically identical to the antigen on T cells, but is not accompanied by its usual signalling mechanism of p56-lck tyrosine kinase and therefore is unlikely to be a functionally active receptor.     

Second malignancies in B‐cell chronic lymphocytic leukaemia: possible association with human papilloma virus

Second primary malignancies have long been associated with chronic lymphocytic leukaemia (CLL). We assessed secondary tumour samples from CLL and control patients for the presence of human papilloma virus (HPV). 132 CLL patients with 44 second malignancies were compared to a matched randomly‐identified control population of 264 non‐CLL patients with 54 solid malignancies. Polymerase chain reaction was performed with the highly conserved MY09/MY11 HPV primer. None of control samples were HPV‐positive, while 53% of samples from the CLL group were positive. This report describes preliminary evidence for the presence of HPV in secondary malignancies, in patients with CLL.     

Immunoglobulin gene segment usage, location and immunogenicity in mutated and unmutated chronic lymphocytic leukaemia

The mutational status of the variable region of the immunoglobulin heavy chain gene (IgV(H)) is an important prognostic marker in B-cell chronic lymphocytic leukaemia (B-CLL), with mutated patients having improved outcome. To examine the impact of mutational status on V(H), D(H), and J(H) gene segment location and immunogenicity, we analysed 375 IgH sequences from 356 patients with B-CLL. Although V(H) and D(H) gene usage was different in mutated compared to unmutated patients, there was no impact of gene location on frequency of use or clinical outcome. Surprisingly, somatic mutations did not increase the immunogenicity of the Ig, as assessed by predicted binding affinity of Ig-derived peptides to major histocompatibility Class I and Class II molecules. Even excluding patients using V(H)1-69, cases using the V(H)1 gene family had a poor outcome. Both mutated and unmutated CLL patients demonstrated evidence of antigen selection. The worst outcome was seen in the subset of 14 unmutated patients with similar HCDR3 amino acid sequence using V(H)1-69, D(H)3-3 and J(H)6, suggesting an antigen-driven process modulating the clinical course.     

Bendamustine + rituximab chemoimmunotherapy and maintenance lenalidomide in relapsed,refractory chronic lymphocytic leukaemia and small lymphocytic lymphoma: A Wisconsin Oncology Network Study

Bendamustine + rituximab (BR) has demonstrated high response rates in relapsed/refractory (R/R) chronic lymphocytic leukaemia (CLL) and small lymphocytic lymphoma (SLL). However, progression‐free survival (PFS) after BR is <18 months. This study was designed to determine if maintenance lenalidomide after BR induction could improve PFS in R/R CLL/SLL. Thirty‐four patients with R/R CLL/SLL who had received 1–5 prior chemotherapy regimens were treated with 6 cycles of BR induction. Patients achieving at least a minor response received twelve 28‐d cycles of lenalidomide 5–10 mg/d. The primary endpoint was PFS. The median age was 67 years, with a median of 2 prior therapies. Eleven patients had confirmed presence of 17p and/or 11q deletions. Twenty‐five (74%) completed 6 cycles of induction BR (response rate 56%). Nineteen (56%) patients received maintenance lenalidomide; only 6 patients completed the intended 12 cycles, highlighting the limited feasibility of lenalidomide in this setting, primarily due to haematological and infectious toxicities. The observed median PFS of 18·3 months is not significantly different from that of BR induction in R/R CLL/SLL without maintenance therapy (15·2 months). It is possible that lenalidomide maintenance may be more feasible and effective in the front‐line setting, which is being tested in an ongoing trial (NCT01754857).     

Migration of leukaemic lymphocytes (chronic lymphocytic leukaemia) through a 3-D collagen gel: a possible prognostic factor

Migratory properties of malignant lymphocytes in 27 patients with chronic lymphocytic leukaemia (CLL) were investigated using 3-D collagen gel. There was significant impairment of migration in malignant lymphocytes as compared with normal lymphocytes. Moreover there was an inverse correlation between Rai's staging and the migration index. Patients with progression of disease also showed a decrease in migration index. There was no correlation between kappa and lambda type in terms of migration index. The prognostic significance of the migratory properties of malignant lymphocytes is also discussed.     

The Dohner fluorescence in situ hybridization prognostic classification of chronic lymphocytic leukaemia (CLL): the CLL Research Consortium experience

This study revisited the Dohner prognostic hierarchy in a cohort of 1585 well‐documented patients with chronic lymphocytic leukaemia. The duration of both time to first treatment (TTFT) and overall survival (OS) were significantly longer than observed previously, and this is at least partly due to improved therapeutic options. Deletion 13q remains the most favourable prognostic group with median TTFT and OS from fluorescence in situ hybridization (FISH) testing of 72 months and >12 years, respectively. Deletion 11q had the poorest median TTFT (22 months) and 17p deletion the poorest median OS (5 years). The percentages of abnormal nuclei were significantly associated with differential TTFT for the trisomy 12, 13q and 17p deletion cohorts but not for the 11q deletion cohort. From the date of the first FISH study, patients with >85% 13q deletion nuclei had a notably shorter TTFT (24 months). Patients with ≤20% 17p deletion nuclei had longer median TTFT and OS from the date of the first FISH study (44 months and 11 years), and were more likely to be IGHV mutated.     

Ribosome‐associated nucleophosmin 1: increased expression and shuttling activity distinguishes prognostic subtypes in chronic lymphocytic leukaemia

Two distinct groups of chronic lymphocytic leukaemia (CLL) are distinguished by the presence or absence of somatic hypermutation of the immunoglobulin heavy‐chain gene. CLL without somatic hypermuataion has an adverse outcome, but the precise biological differences that underlie this more aggressive clinical‐course are unclear. Using a proteomic approach, we found that the two prognostic forms of CLL were consistently distinguished according to their protein expression pattern. The most important difference observed related to the different expression of nucleophosmin 1 between the two forms of CLL. This different expression was not related to apoptosis, proliferation or gene mutation. However, co‐immunoprecipitation experiments identified an association between nucleophosmin 1 and ribosomal proteins. Using immunocytofluorescence, nucleophosmin 1 expression was identified in the nucleoli and nucleoplasm of all cells, but in a proportion of cells, nucleophosmin had been transferred from the nucleoplasm to the cytoplasm. Both the fluorescent intensity, and the frequency of cytoplasmic nucleophosmin 1 expression, was higher in CLL without somatic hypermutation. We propose therefore, that nucleophosmin 1, in association with ribosomal proteins, undergoes nucleo‐cytoplasmic shuttling in CLL. This process is most prominent in un‐mutated CLL and may signify altered protein biosynthesis.     

Angiogenic factors in chronic lymphocytic leukaemia (CLL): Where do we stand?

The role of angiogenesis in haematological malignancies such as chronic lymphocytic leukaemia (CLL) is difficult to envision, because leukaemia cells are not dependent on a network of blood vessels to support basic physiological requirements. Regardless, CLL cells secrete high levels of major angiogenic factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet derived growth factor (PDGF). Nonetheless, it remains unclear how most angiogenic factors regulate accumulation and delayed apoptosis of CLL cells. Angiogenic factors such as leptin, granulocyte colony-stimulating factor (G-CSF), follistatin, angiopoietin-1 (Ang1), angiogenin (ANG), midkine (MK), pleiotrophin (PTN), progranulin (PGRN), proliferin (PLF), placental growth factor (PIGF), and endothelial locus-1 (Del-1), represent novel therapeutic targets of future CLL research but have remained widely overlooked. This review aims to outline our current understanding of angiogenic growth factors and their relationship with CLL, a still uncured haematopoietic malignancy.     

Reconstitution of the T-cell repertoire following treatment with alemtuzumab (anti-CD52 monoclonal antibody) in patients with B-cell chronic lymphocytic leukaemia

In this pilot study, T-cell receptor B-variable (TCR-BV) gene usage in CD4 and CD8 T cells was assessed, by real-time polymerase chain reaction, as well as complementarity-determining region 3 (CDR3)-length polymorphism, before and after therapy in five patients with B-cell chronic lymphocytic leukaemia who received alemtuzumab (anti-CD52 monoclonal antibody) as first-line therapy. A decline in expression of most BV family genes in both CD4 and CD8 T cells was observed after alemtuzumab treatment, which was followed by a gradual increase in most BV families during long-term follow-up. After treatment, CDR3-length polymorphism showed an even more restricted pattern in CD4 T cells compared with pretreatment, with a shift towards a monoclonal/oligoclonal pattern. The clonally restricted pattern was significantly reduced in CD4 (P < 0.01) but not in CD8 T cells. This was followed by a gradual increase in the number of peaks within the CDR3 region of the different TCR-BV families, i.e. a polyclonal repertoire, during long-term follow-up. A restricted CDR3 pattern became even more restricted after treatment, but normalised during unmaintained follow-up. These results indicate that perturbations in the T-cell alterations following alemtuzumab are complex and include not only changes in CD4/CD8 T-cell numbers but also a highly restricted T-cell repertoire especially in CD4 T cells.     

Relationship between co‐morbidities at diagnosis,survival and ultimate cause of death in patients with chronic lymphocytic leukaemia (CLL): a prospective cohort study

The ultimate cause of death for most patients with newly diagnosed chronic lymphocytic leukaemia (CLL) and its relationship to co‐morbid health conditions is poorly defined. We conducted a prospective cohort study that systematically followed 1143 patients diagnosed with CLL between June 2002 and November 2014. Comorbid health conditions at the time of CLL diagnosis and their relationship to survival and cause of death were evaluated. Collectively, 1061 (93%) patients had at least one co‐morbid health condition at the time of CLL diagnosis (median number 3). Despite this, 89% of patients had a low‐intermediate Charlson Comorbidity Index score (CCI) at diagnosis. After a median follow‐up of 6 years, 225 patients have died. Death was due to CLL progression in 85 (46%) patients, infection in 14 (8%) patients, other cancer in 35 (19%) patients and comorbid health conditions in 50 (27%) patients. Higher CCI score and a greater number of major comorbid health conditions at the time of CLL diagnosis was associated with shorter non‐CLL specific survival, but not with shorter CLL‐specific survival on multivariate analysis. In conclusion, CLL and CLL‐related complications (infections and second cancers) are the overwhelming cause of death in patients with CLL, regardless of CCI score and number of comorbid health conditions at diagnosis.     

Acute lymphocytic leukaemia in the elderly: characteristics and outsome with the vincristine-adriamycin-dexamethasone (VAD) regimen

To analyse the pretreatment characteristics, responseto therapy, and overall prognosis of elederly patients with acute lymphocytc leukaemia (ALL). 268 consecutive adults with newly-diagnosed ALL who received the vincristine-adria-mycin-dexamethasone (VAD) regimens were reviewed; Pretreatment fharacteristics, response to VAD therapy, and overall outcomewere analysed in patients 60 years or older, and compared to younger patients. Fifty-two patients (19%) were 60 years or older. Compared with younger patients, elderly patients with ALL had a significantly worse performance status at presentation (P < 0.001); higher incidences of FAB L2 morphology (P=0.03). thrombocytopenia (P < 0.01), hypoalbuminaemia (P < 0.001) and renal dysfunction (P=0.03); and trends for worse anaemia (P = 0.06) and a higher rate of myeloid markeers positivity (P= 0.06). With VAD induction therapy, elderly patients had a lower CR rate than younger patients following two induction courses (CR rate 58% rate 58% v 82%; P < 0.01), and overall (CR rates 65% v 90%; P <).01). The response rate to VAD chemotherapy, however, appeared superior to the 30–35% CR rates reported from other series in elderly ALL patients. The lower CR rate was due to both higher induction mortality (P = 0.02) and more resistant disease (P < 0.01). The longterm CR rate was 20% in elderly patients achieving CR, but the overall survival was poor (< 10% at 3 years). There were strong associations between older age and other poor prognostic features. However, multivariate analyses identified older age to be an independent poot prognostic factor for response to chemotherapy. In summary, elderly patients with ALL have an appreciable CR rate with the VAD regimens. Patients achieving CR may have durable remissions. The myelosuppression-associated mortality in both remission induction and maintenance may be reduced with the addition of growth factors in both treatment phases, and by using standard maintenance therapy without consolidation-intensification phases.     

Dasatinib inhibits B cell receptor signalling in chronic lymphocytic leukaemia but novel combination approaches are required to overcome additional pro-survival microenvironmental signals

As antigenic stimulation of the B cell antigen receptor (BCR) is key to chronic lymphocytic leukaemia (CLL) pathogenesis, targeting dysregulated kinases involved in BCR signalling is an attractive therapeutic approach. We studied the effects of the Src/c‐Abl tyrosine kinase inhibitor dasatinib on BCR signal transduction in CLL cells. Treatment of CLL cells with 100 nmol/l dasatinib induced apoptosis by an average reduction in viability of 33·7% at 48 h, with dasatinib sensitivity correlating with inhibition of Syk^Y348 phosphorylation. Dasatinib inhibited calcium flux, phosphatidylinositol‐3‐kinase and mitogen‐activated protein kinase activation following BCR crosslinking, and blocked the Mcl‐1‐dependent increase in CLL cell survival on prolonged BCR stimulation. However, the pro‐apoptotic effect of dasatinib was abrogated by stromal cell contact alone or in the presence of CD154 and interleukin (IL)‐4 (CD154L/IL‐4 system). Whilst dasatinib retained the ability to sensitize CLL cells in stromal co‐culture to both fludarabine and chlorambucil, the addition of CD154 and IL‐4 rendered cells resistant to these drug combinations. We demonstrate that the HSP90 inhibitor 17‐DMAG exhibited synergy with dasatinib in vitro, and moreover, induced apoptosis of CLL cells in the CD154L/IL‐4 system. Our data provide evidence that dasatinib would be most clinically effective in combination with agents able to target antigen‐independent microenvironmental signals.