Timothy Farren

Frequency, demographics and risk (according to tumour type or site) of cancer-associated thrombosis among patients seen at outpatient DVT clinics

Venous thromboembolism (VTE) is a clinically important complication for both hospitalised and ambulatory cancer patients. In the current study, the frequency, demographics and risk (according to tumour site) of VTE were examined among patients seen at outpatient DVT (deep-vein thrombosis) clinics. Of 10,015 VTE cases, 1,361 were diagnosed with cancer, for an overall rate of cancer-associated VTE of 13.6% in this outpatient population. Patients with cancer-associated VTE were significantly older than cancer-free VTE cases (66.4 +/- 12.7 vs. 58.8 +/- 18.5 years; p<0.0001). The frequency of cancer-associated VTE peaked earlier among females than males, occurring in the sixth (137/639, 21.4% vs. 98/851, 11.3%; p<0.001) and seventh decades (213/980, 21.7% vs. 197/1096, 18%; p=0.036). VTE was described most frequently in common cancers - breast, prostate, colorectal and lung (56.1% of cases). The risk of VTE varied widely across 17 cancer types. Calculating odds ratios (OR) to assess the effect size of cancer type on VTE risk, the highest odds were observed for patients with pancreatic cancer (OR 9.65, 95% confidence interval [CI] (5.51-16.91). Tumours of the head and neck had higher odds than previously reported (OR 8.24, 95% CI 5.06-13.42). Reduced risk estimates were observed for skin cancers (melanoma and non-melanoma: OR 0.89, 95% CI 0.42-1.87; OR 0.74, 95% CI, 0.32-1.69, respectively). We conclude that outpatients have a similar rate of cancer-associated VTE as VTE patient populations previously reported, that cancer-associated VTE occurs in an older age group and earlier in females and that outpatients exhibit distinct tumour site-specific risk from that described among hospitalised cancer patients.

CD160 signaling mediates PI3K-dependent survival and growth signals in chronic lymphocytic leukemia

B-cell chronic lymphocytic leukemia (CLL) expresses CD160, a glycosylphosphatidylinositol-linked receptor found on normal natural killer (NK) and T cells, but not B cells. CD160 is a multifunctional molecule in normal lymphocytes, but its role in CLL biology is unknown. In vitro, CLL cells undergo rapid spontaneous apoptosis, which CD160 activation protected against-mean cell viability increased from 67% to 79% (P < .001). This was associated with up-regulation of Bcl-2, Bcl-xL, and Mcl-1, but not Bax. As expected from these changes in Bcl-2/Bax and Bcl-xL/Bax ratios, CD160 triggering reduced mitochondrial membrane potential collapse and cytochrome c release. CD160 stimulation also induced DNA synthesis, cell cycle progression, and proliferation. B-cell antigen receptor (BCR)-induced CLL proliferation was generally greater than with CD160, but marked variation was seen. Both BCR and CD160 signaling led to CLL secretion of interleukin-6 (IL-6) and IL-8, although CD160 induced greater increases of IL-6 (51-fold) and IL-8 (15-fold). Survival and activation signals mediated by CD160 showed dose-dependent suppression by phosphoinositide-3 kinase (PI3K) inhibitors. Thus, in vitro, CLL cells can use the CD160 pathway for survival and activation, mimicking CD160 signaling in normal NK and CD8(+) T cells. Establishing the pathophysiologic relevance of these findings may reveal new therapeutic targets.

Differential and tumor-specific expression of CD160 in B-cell malignancies

CD160 is a human natural killer (NK)-cell-activating receptor that is also expressed on T-cell subsets. In the present study, we examined 811 consecutive cases of B-cell lymphoproliferative disorders (B-LPDs), and demonstrated CD160 expression in 98% (590 of 600) of chronic lymphocytic leukemia (CLL) cases, 100% (32 of 32) of hairy cell leukemia (HCL) cases, 15% (5 of 34) of mantle cell lymphoma (MCL) in the leukemic phase, and 16% (23 of 145) of other B-LPD cases. CD160 transcript and protein were absent in the normal B-cell hierarchy, from stem cells, B-cell precursors, maturing B cells in the germinal center, and circulating B cells, including CD5(+)CD19(+) B1 cells in umbilical cord. CD160 positivity was significantly higher in CLL and HCL in terms of percentage (65.9% and 67.8%, respectively, P < .0001) and median fluorescence intensity (552 and 857, respectively, P < .0001) compared with all other B-LPD cases. Lymph node CLL samples were also CD160(+). Using the disease-specific expression of CD5, CD23, and CD160, a score of 3 characterized CLL (diagnostic odds ratio, 1430); a score of 0 excluded CLL, MCL, and HCL; and the CD23/CD5 ratio differentiated CLL from leukemic CD23(+) MCL. In the B-cell lineage, CD160 is a tumor-specific antigen known to mediate cellular activation signals in CLL, and is a novel target for therapeutic manipulation and monitoring of minimal residual disease.

Minimal residual disease detection with tumor-specific CD160 correlates with event-free survival in chronic lymphocytic leukemia

In chronic lymphocytic leukemia (CLL), the detection of minimal residual disease (MRD) correlates with outcome in the trial setting. However, MRD assessment does not guide routine clinical management and its assessment remains complex. We incorporated detection of the B cell, tumor-specific antigen CD160 to develop a single-tube, flow cytometry assay (CD160FCA) for CLL MRD to a threshold of 10−4 to 10−5. One hundred and eighty-seven patients treated for CLL were enrolled. Utilizing the CD160FCA methodology, there was a high level of comparison between blood and bone marrow (R=0.87, P<0.001). In a validation cohort, CD160FCA and the international standardised approach of the European Research Initiative on CLL group demonstrated high concordance (R=0.91, P<0.01). Patients in complete remission (CR) and CD160FCA negative had longer event-free survival (EFS) (63 vs 16 months, P<0.01) and prolonged time to next treatment (60 vs 15 months, P<0.001) vs MRD positive patients; with a median time to MRD positivity of 36 months. In multivariate analysis, CD160FCA MRD detection was independently predictive of EFS in patients in CR and even predicted EFS in the good-risk cytogenetic subgroup. CD160FCA offers a simple assay for MRD detection in CLL and gives prognostic information across different CLL risk groups.

Impact of venous thromboembolism on survival in patients with malignancy

An association between venous thromboembolism (VTE) and cancer is well recognised (Lee, 2004). Recent populationbased studies have provided some insights into the incidence and timing of VTE events in patients with common cancers (White et al, 2007), and we have recently described increased relative risks of VTE in common and rare cancer types (Rose et al, 2007). There is evidence that patients with cancer who develop VTE have shortened survival (Sorensen et al, 2000; Alcalay et al, 2006; Chew et al, 2006, 2007) but the impact of VTE on survival in patients with specific cancers remains to be fully defined. This study aimed to compare outcome in VTE patients with specific cancer types with VTE-free patients and assess if VTE affected overall survival. Patients were enrolled at two University teaching hospitals (Walsgrave Hospital, Warwickshire and Derriford Hospital, Plymouth) into prospectively maintained databases of patients. Patients with VTE participated in the VEnous thromboembolism RegIsTY (VERITY), which prospectively enrols patients attending deep vein thrombosis (DVT) outpatient clinics. Details of the VERITY registry have been published previously (O’Shaughnessy et al, 2004). Data are collected by trained hospital staff in a standardised electronic case report form and submitted to the electronic database. Patient data are anonymised and identified in the database only by a VERITY number. Demographic characteristics, medical history, presenting symptoms, diagnosis, treatment practices including location of treatment, and follow-up data are collected. All patients underwent a Doppler ultrasound examination to confirm the diagnosis and determine the extent of DVT. Additional investigations were undertaken if appropriate according to local clinical algorithms. All patients with VTE received standard treatment with low molecular weight heparin and warfarin. Patients without VTE were enrolled in a separate, prospective database of consecutive patients maintained at Walsgrave Hospital. This database included all patients suspected of having a DVT but for whom the diagnosis was excluded after Doppler ultrasound examination. The presence of known malignancy (defined by ongoing treatment for cancer or metastatic disease) and type of malignancy detected at the time of thrombosis or subsequently during follow-up was documented in the database. The outcome parameter used in this analysis was death. Statistical analysis was carried out using the Statistical Package for the Social Sciences (spss) version 13.0 (SPSS Inc., Chicago, Ill, USA) for Windows and GraphPad InStat version 3.06 (GraphPad Software, San Diego, CA, USA) for Windows software. Overall survival was estimated by the Kaplan–Meier method. The log-rank test was used to compare survival between groups across the whole follow-up period. The alpha level for statistical significance was set at 0Æ05. Between February 2001 and December 2006, 902 patients with confirmed VTE were included in this analysis, 699 consecutive patients at Walsgrave Hospital (described previously, Paneesha et al, 2006) and 203 patients at Derriford Hospital. Between February 2001 and December 2005, 2263 consecutive patient episodes from 2016 patients without VTE were recorded. For patients with VTE, median age at presentation was 66 years (range 16–96 years) and 51Æ3% were male; for VTE-free patients, median age was 69 years (18– 105 years) and 67Æ1% were male. Cancer was present in 42% of patients with confirmed VTE (379/902) and in 11% of nonVTE cases (222/2016). Cancer type was specified in 73Æ1% of cancer cases; 19Æ7% had bowel cancer, 19Æ6% had breast, 16Æ9% had prostate, 10Æ5% had lung and 7Æ6% had gynaecological cancer. Median follow-up was 21 months (range 0–74 months) in patients with VTE and 22Æ2 months (0–65 months) in those negative for VTE. Mean overall survival in non-VTE patients without malignancy was 56 months compared with 54 months in VTE patients with malignancy. Survival was analysed between cancer patients with and without VTE according to cancer type. Median survival in patients with and without VTE was 30 vs. 31 months for prostate cancer, 17 vs. 50 for gynaecological cancer and 8 vs. 3Æ5 for lung cancer. For patients with unspecified cancer type, median survival was 9 vs. 30 months. For breast and bowel cancer, the numbers were large enough to allow calculation of Kaplan–Meier estimates of survival, which are shown in Fig 1. For patients with breast or bowel cancer, the log-rank test revealed a significant difference between the survival rates over time with VTE patients having shorter survival (P = 0Æ016 and P = 0Æ05, respectively). For bowel cancer patients, for whom the statistical significance was marginal, median survival time was 8 months vs. 36 months in those with and without VTE. This analysis suggests that common cancers, specifically breast and bowel, are associated with adverse outcome in the presence of objectively confirmed VTE. A previous study of cancer survival based on the California Cancer Registry and the California Patient Discharge dataset reported reduced survival at 2 years for breast cancer (Chew et al, 2007) and colorectal cancer (Alcalay et al, 2006) patients. In those analyses, the authors were able to assess the impact of Correspondence

CD126 and Targeted Therapy with Tocilizumab in Chronic Lymphocytic Leukemia

Purpose: IL6 promotes tumor growth and signal transduction via both its membrane-bound (CD126) and soluble receptors (sCD126). We aimed to study whether the levels of CD126 expression in chronic lymphocytic leukemic (CLL) cells can predict in vitro and in vivo treatment response. Experimental Design: The levels of membrane-bound CD126 expression were determined on freshly isolated CLL B cells (n = 58) using flow cytometry. These CLL cells were treated with chlorambucil or fludarabine with or without anti-CD126 antibody tocilizumab for 24 hours and IL6-mediated STAT3 transcriptional activity and cell-cycle alteration were evaluated. Results: CD126 surface expression was found in all cases and positively correlated with the levels of in vivo constitutive STAT3 activity. The levels of CD126 expression were significantly and positively correlated with the resistance of CLL cells to in vitro treatment with chlorambucil or fludarabine and poor in vivo treatment response of CLL patients. Blocking IL6 signaling with the anti-CD126 antibody, tocilizumab, had profound effects on STAT3-mediated survival and growth signals: decreased Mcl-1 and Bcl-xL, favoring an apoptotic profile; and decreased p27 with increased cyclin E and CDK2 expression, leading to cell-cycle shift from G0–G1. These tocilizumab-mediated changes induced chemosensitization in resistant CLL cells, with the greatest effect seen in cells with higher CD126 expression (P < 0.001). Conclusions: CLL cells with higher CD126 expression are more resistant to treatment in vivo and in vitro via IL6–CD126–STAT3 axis. Blocking CD126 using tocilizumab sensitizes CLL cells to chemotherapy. Clin Cancer Res; 22(10); 2462–9. ©2015 AACR.

STAT3 and NF-κB cooperatively control in vitro spontaneous apoptosis and poor chemo-responsiveness in patients with chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is an adult disease characterized by in vivo accumulation of mature CD5/CD19/CD23 triple positive B cells and is currently incurable. CLL cells undergo spontaneous apoptosis in response to in vitro cell culture condition but the underlying mechanism is unclear. We hypothesize that the sensitivity of CLL cells to spontaneous apoptosis may be associated with the constitutive activities of transcription factors STAT3 and/or NF-κB. We now show that the sensitivity of fresh CLL cells to spontaneous apoptosis is highly variable among different patients during 48 hours’ cell culture and inversely correlated with in vivo constitutively activated STAT3 and NF-κB (p < 0.001). Both activated STAT3 and NF-κB maintain the levels of anti-apoptotic protein Mcl-1/Bcl-xL and autocrine IL-6 production. CLL cells with higher susceptibility to in vitro spontaneous apoptosis show the greatest chemosensitivity (p < 0.001), which is reflected clinically as achieving a complete response (CR) (p < 0.001), longer lymphocyte doubling times (p < 0.01), time to first treatment (p < 0.01), and progression free survival (p < 0.05). Our data suggest that the sensitivity of CLL cells to in vitro spontaneous apoptosis is co-regulated by constitutively activated STAT3 and NF-κB and reflects the in vivo chemo-responsiveness and clinical outcomes.

Risk factors for cancer-associated venous thromboembolism in outpatient deep venous thrombosis clinics

Venous thromboembolism (VTE) in cancer patients is increasingly frequent (Khorana et al, 2007), is associated with worse survival (Paneesha et al, 2009) and its treatment poses particular challenges (Noble, 2007), including increased bleeding and recurrence risks. A number of important factors influence thrombotic risk, including site of cancer, presence of metastatic disease and certain chemotherapy and hormonal treatment regimens (Wun & White, 2009), but, for most types of cancer, there are few data describing the occurrence or relevance of common VTE risk factors and it is not clear to what extent major risk factors such as surgery or previous history of VTE impact the risk already associated with the presence of cancer. This is particularly the case for ambulant cancer patients. While there have been attempts to identify cancer outpatients at high-risk of chemotherapy-associated VTE (Khorana et al, 2008), there is a paucity of data on risk factors that might allow identification of those cancer patients most at risk of VTE. In this analysis of patients seen at outpatient deep venous thrombosis (DVT) clinics, we wished to determine if the incidence of established VTE risk factors differed in patients with VTE according to cancer status (cancer diagnosis vs. no cancer diagnosis). The presence of seven established risk factors for VTE (Arya et al, 2009) was compared in cancer and non-cancer patients with confirmed VTE using the chi-square test with Yates correction. All analyses were conducted using a commercial software package (spss version 16; SPSS Inc., Chicago, IL, USA) and a P-value <0Æ05 was considered statistically significant. The risk factors were medical inpatient history or immobilization for more than 3 d within the last 4 weeks, major surgery in the last 4 weeks, hormonal risk (use of hormone-replacement therapy or oral contraceptives; pregnant or post-partum), personal history of VTE, known thrombophilia, intravenous drug abuse and current smoking, as previously described (Arya et al, 2009). The data set has been described previously (Paneesha et al, 2010). The VEnous thromboembolism RegIsTrY (VERITY) is an ongoing prospective registry of patients attending outpatient DVT clinics. A variety of algorithms, mainly based on D-dimer measurement and pre-test probability, are used to exclude VTE. Confirmatory, objective testing is undertaken according to local protocols, usually including ultrasonography or venography for suspected DVT and pulmonary angiography, lung scintigraphy or helical computerized tomography for suspected pulmonary embolism. Cancer (current or having received treatment for cancer in the last 6 months) is recorded as one of 17 specific cancer types [bone/ sarcoma, breast, central nervous system (CNS), colorectal, endocrine, gynaecological, head/neck, leukaemia, lung, lymphoma, melanoma, myeloma, non-melanoma, pancreas, prostate, urological, upper gastrointestinal (GI)]. No record is made of metastatic disease, but patients with multiple cancer sites are recorded; no histological description, tumour grade or cancer stage are recorded. In addition, a detailed history of the cancer patients is not recorded, some of whom may have been hospitalized during their care and only briefly used the outpatient service. Of 39 618 patients enrolled in VERITY by 43 UK hospitals, 10 015 (25Æ3%) had confirmed VTE and 1361 (13Æ6%) were recorded as having cancer. 49% of the non-cancer VTE cases were female, compared with 52% of the cancer VTE patients. Patients with cancer were significantly older than cancer-free VTE cases (66Æ4 ± 12Æ7 vs. 58Æ8 ± 18Æ5 years; P < 0Æ0001) (Paneesha et al, 2010). Breast, prostate, colorectal and lung cancer were the most common cancers accounting for 56Æ1% of the cancer-associated VTE cases (Paneesha et al, 2010). Data on the seven established VTE risk factors were available in 78–94% of cases (numbers are shown in Table I). Cancerassociated thrombosis cases were more likely to have experienced a medical inpatient stay/immobilization ( 14Æ8% vs. 10Æ6%; v = 17Æ7; P < 0Æ001), but less likely to have a history of VTE (16Æ0% vs. 25Æ6%; v = 51Æ3; P < 0Æ001), thrombophilia (0Æ7% vs. 2Æ2%; v = 10Æ8; P = 0Æ001), intravenous drug abuse (0Æ5% vs. 7Æ7%; v = 80Æ3; P < 0Æ001) and to smoke (14Æ5% vs. 26Æ2%; v = 61Æ9; P < 0Æ001) than cancer-free cases; there was no difference in hormonal risk and recent surgery (see Table I). This analysis of an observational registry that enrolled more than 10 000 patients with VTE at outpatient DVT clinics showed that established VTE risk factors – previous history of VTE, thrombophilia, intravenous drug abuse and smoking – were less common in patients with cancerassociated VTE. Of the seven risk factors compared, the only risk factor more common in patients with cancer was a medical in-patient stay/immobilization, despite the finding that patients with cancer were significantly older than cancerfree VTE cases. Identification of those cancer patients most at risk of VTE is an important clinical goal. Previously, we reported extreme differences in VTE risk associated with different cancer sites in this patient cohort (Paneesha et al, 2010). Taken together with our current findings, we suggest that established risk factors for VTE, such as previous thrombosis history, are of less Correspondence