Proteomic markers with prognostic impact on outcome of chronic lymphocytic leukemia patients under chemo-immunotherapy: results from the HOVON 109 study.

Despite recent identification of several prognostic markers, there is still a need for new prognostic parameters able to predict clinical outcome in chronic lymphocytic leukemia (CLL) patients. Here, we aimed to validate the prognostic ability of known (proteomic) markers measured pretreatment and to search for new proteomic markers that might be related to treatment response in CLL. To this end, baseline serum samples of 51 CLL patients treated with chemo-immunotherapy were analyzed for 360 proteomic markers, using Olink technology. Median event-free survival (EFS) was 23 months (range: 1.25-60.9). Patients with high levels of sCD23 (>11.27, p = 0.026), sCD27 (>11.03, p = 0.04), SPINT1 (>1.6, p = 0.001), and LY9 (>8.22, p = 0.0003) had a shorter EFS than those with marker levels below the median. The effect of sCD23 on EFS differed between immunoglobulin heavy chain variable gene-mutated and unmutated patients, with the shortest EFS for unmutated CLL patients with sCD23 levels above the median. Taken together, our results validate the prognostic impact of sCD23 and highlight SPINT1 and LY9 as possible promising markers for treatment response in CLL patients.

CMV-specific CD8+ T-cell function is not impaired in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), CD8(+) T cells exhibit features of exhaustion and impaired functionality. Yet, reactivations of latent viruses such as cytomegalovirus (CMV) are uncommon in untreated CLL, suggesting that antiviral responses are uncompromised. We analyzed phenotypical and functional characteristics of CMV-specific CD8(+) T cells in CLL patients in comparison with age-matched healthy controls (HCs). Despite increased expression of the inhibitory receptors PD1, CD160, and CD244 on total CD8(+) T cells in CLL, expression levels of these markers were decreased on CMV-tetramer(+)CD8(+) T cells. Second, cytokine production upon stimulation with both phorbol 12-myristate 13-acetate/ionomycin and CMV-peptide-loaded antigen-presenting cells was intact in CMV-tetramer(+)CD8(+) T cells. Third, CMV-tetramer(+)CD8(+) T cells of CLL patients and HCs were equally effective in killing CMV-peptide-loaded target cells. Finally, quantitative imaging flow cytometry revealed that the proportion of CD8(+) T cells forming immunologic synapses with CMV-peptide-loaded B cells was intact. In conclusion, despite evidence for global T-cell dysfunction in CLL, we show here that CLL-derived CMV-specific CD8(+) T cells display lower expression of exhaustion markers and are functionally intact. These data indicate that the changes in the T-cell compartment in CLL may be more heterogeneous than presently assumed.

Targeted resequencing for analysis of clonal composition of recurrent gene mutations in chronic lymphocytic leukaemia.

Recurrent gene mutations contribute to the pathogenesis of chronic lymphocytic leukaemia (CLL). We developed a next-generation sequencing (NGS) platform to determine the genetic profile, intratumoural heterogeneity, and clonal structure of two independent CLL cohorts. TP53, SF3B1, and NOTCH1 were most frequently mutated (16.3%, 16.9%, 10.7%). We found evidence for subclonal mutations in 67.5% of CLL cases with mutations of cancer consensus genes. We observed selection of subclones and found initial evidence for convergent mutations in CLL. Our data suggest that assessment of (sub)clonal structure may need to be integrated into analysis of the mutational profile in CLL.

Overview of available p53 function tests in relation to TP53 and ATM gene alterations and chemoresistance in chronic lymphocytic leukemia.

The ATM-p53 DNA damage response pathway plays a crucial role in chemoresistance in chronic lymphocytic leukemia, as indicated by the adverse prognostic impact of deletions of 17p (locus of TP53) and 11q (locus of ATM) detected by fluorescence in situ hybridization (FISH) analysis. In addition to deletions, mutations in these respective genes are also associated with chemoresistance, and add to the prognostic information provided by FISH. In order to explore the possibility that dysfunction of the ATM-p53 pathway might also result from mechanisms other than ATM/TP53 deletion/mutation, assays have been developed that probe the functional integrity of the ATM-p53 pathway. Currently, four different p53 function assays have been developed that are based on the measurement of p53 and p53-dependent genes at the RNA (real-time polymerase chain reaction [RT-PCR]p21; RT-PCRmiR34a; reverse transcription-multiplex ligation-dependent probe amplification assay [RT-MLPA]p21, bax, puma and CD95) or protein (fluorescence activated cell sorting [FACS]p53-p21) level in untreated cells or following irradiation or drug treatment. Here we provide an overview of these assays based on the available literature.

Chronic lymphocytic leukemia specific T-cell subset alterations are clone-size dependent and not present in monoclonal B lymphocytosis.

In patients with chronic lymphocytic leukemia (CLL), effector and memory CD4 + and CD8 + T cells are expanded. We investigated whether these CLL specific T-cell expansions also occur in monoclonal B lymphocytosis (MBL), the pre-leukemic state of CLL, which is currently distinguished from CLL by an arbitrarily chosen cut-off value of CD19 of 5.0 × 10(9)/L. Whereas an increase in effector and memory CD4 + and CD8 + T cells was found in CLL, these expansions could not be found in MBL. Although a significant correlation was found between absolute B cell count (CD19) and T cell numbers, correlation coefficients were rather low. Therefore, we analyzed whether an optimal threshold for CD19 number could be defined which best related to an expansion of T cells. The B-cell threshold that best predicted expansion of CD3 +, CD4 + and CD8 + T cells, respectively, was 10 × 10(9)/L. Our study indicates that a higher cut-off value than the current 5.0 × 10(9)/L relates better to the biological impact of CLL.

Use of the CD19 count in a primary care laboratory as a screening method for B-cell chronic lymphoproliferative disorders in asymptomatic patients with lymphocytosis.

BACKGROUND: Detection of absolute and relative lymphocytosis in otherwise asymptomatic elderly patients is very common in the primary care setting and frequently results in referral for screening of lymphoproliferative disorders. Since many B-cell chronic lymphoproliferative disorders (B-CLPD) are indeed asymptomatic at diagnosis in most patients with lymphocytosis, no sign of such a disorder is usually detected. Currently, specific guidelines for screening of patients with lymphocytosis are lacking. We investigated the practicability and clinical value of a single colour CD19 count performed by a primary care laboratory in order to improve the diagnostic follow-up of patients with lymphocytosis in a primary care laboratory. METHODS: The capability of detecting monoclonal B-cell lymphocytosis and B-CLPD by CD19, was first confirmed in patient samples with known B-CLPD. Next, in a previously defined geographic area, a CD19 count was performed on all samples for patients aged ≥40 years with relative or absolute lymphocytosis but without neutropenia. Clinical follow-up, with a median of 4 years, was performed using both a survey among the requesting general practitioners and by analysis of the records of the referral hospitals within the borders of the defined area. RESULTS: A total of 520 cases with asymptomatic lymphocytosis were identified. In all cases, the CD19 count was performed; 207 (40%) showed increased values and 313 (60%) showed normal values. An increase in CD19 proved highly sensitive for detection of B-CLPD (98%, 95% CI; 94%-100%) with a high positive predictive value (57%, 95% CI; 50%-63%). The area under curve, the receiver-operating characteristic curve of the CD19 count (0.93, 95% CI; 0.91-0.96), was significantly higher compared to the absolute lymphocyte count (0.86, 95% CI; 0.83-0.89), especially in patients with moderate lymphocytosis. CONCLUSIONS: This study indicates that the CD19 count, performed by a primary care laboratory, is feasible and a promising tool for initial screening of lymphocytosis to discriminate B-CLPD from benign causes of lymphocytosis.

Treatment options in massive pulmonary embolism during pregnancy; a case-report and review of literature.

UNLABELLED: Systemic thrombolysis with recombinant tissue plasminogen activator (rt-PA), streptokinase or urokinase is considered as high-risk treatment in pregnancy. However, several reports have described the successful use of systemic thrombolysis in pregnant patients with massive pulmonary embolism and haemodynamic instability. CASE: We describe a 34-year old, pregnant female, who presented at 25 weeks of gestation with an acute collapse with reduced consciousness and shortness of breath caused by massive pulmonary embolism. Because of significant haemodynamic instability, increased right ventricular pressure and no improvement after intravenous heparin, thrombolytic therapy was administered. The response to thrombolytic therapy was excellent. No severe haemorrhagic complications were observed. Anticoagulant therapy with LMWH was continued until delivery. A healthy child was born at term. REVIEW: In English literature, 13 patients received thrombolysis during pregnancy because of pulmonary embolism. No maternal deaths, four non-fatal maternal major bleeding complications, 30.8%;95%CI(9.1-61.4), two fetal deaths, 15.4%;95%CI(1.9-45.5), and five preterm deliveries, 38.5%;95%CI(13.9-68.4), were observed. Surgical embolectomy and catheter embolectomy or catheter thrombolysis has only been performed in 12 patients. CONCLUSION: The number of reports on thrombolytic therapy, surgical embolectomy and catheter embolectomy or thrombolysis for massive pulmonary embolism during pregnancy are limited. We suggest an international registry for pregnant patients undergoing thrombolysis or embolectomy to gain more information about these treatment options. Nevertheless, complication rates of thrombolytic therapy are acceptable in the light of the underlying disease, and in the meantime, current data do not justify withholding pregnant women from thrombolytic therapy in case of life-threatening PE.