Monitoring of circulating tumour DNA in advanced pancreatic ductal adenocarcinoma predicts clinical outcome and reveals disease progression earlier than radiological imaging.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with a need for better tools to guide treatment selection and follow-up. The aim of this prospective study was to investigate the prognostic value and treatment monitoring potential of longitudinal circulating tumour DNA (ctDNA) measurements in patients with advanced PDAC undergoing palliative chemotherapy. Using KRAS peptide nucleic acid clamp-PCR, we measured ctDNA levels in plasma samples obtained at baseline and every 4 weeks during chemotherapy from 81 patients with locally advanced and metastatic PDAC. Cox proportional hazard regression showed that ctDNA detection at baseline was an independent predictor of progression-free and overall survival. Joint modelling demonstrated that the dynamic ctDNA level was a strong predictor of time to first disease progression. Longitudinal ctDNA measurements during chemotherapy successfully revealed disease progression in 20 (67%) of 30 patients with ctDNA detected at baseline, with a median lead time of 23 days (P = 0.01) over radiological imaging. Here, we confirmed the clinical relevance of ctDNA in advanced PDAC with regard to both the prediction of clinical outcome and disease monitoring during treatment.

Prognostic value of disseminated tumor cells in unresectable pancreatic ductal adenocarcinoma: a prospective observational study.

BACKGROUND: Although pancreatic ductal adenocarcinoma (PDAC) rarely metastasizes to the skeleton, disseminated tumor cells have been detected in bone marrow samples from patients with this disease. The prognostic value of such findings is currently unclear. Thus, the current study aimed to clarify the prognostic information associated with disseminated tumor cell detection in samples from patients with PDAC. METHODS: Bone marrow aspirates were obtained from 48 patients with locally advanced (n = 11) or metastatic (n = 37) PDAC, before and after 2 months of chemotherapy. Disseminated tumor cells were detected with an mRNA panel and quantitative reverse transcription PCR. We used the highest levels measured in healthy bone marrow (n = 30) as a threshold to define the positive detection of disseminated tumor cells. Progression-free and overall survival were analyzed with Kaplan-Meier and Cox proportional hazards regression analyses. RESULTS: Disseminated tumor cells were detected in 15/48 (31%) bone marrow samples obtained before starting chemotherapy and in 8/25 (32%) samples obtained during chemotherapy. Patients with disseminated tumor cells detected before therapy had significantly shorter progression-free (p = 0.03; HR = 2.0) and overall survival (p = 0.03; HR = 2.0), compared to those without disseminated tumor cells in the bone marrow. When restricting disseminated tumor cell detection to keratins KRT7 and KRT8, the prognostic information was substantially stronger (p = 1 × 10-6; HR = 22, and p = 2 × 10-5; HR = 7.7, respectively). The multivariable Cox regression analysis demonstrated that disseminated tumor cell detection prior to treatment had independent prognostic value. In contrast, disseminated tumor cells detected during treatment did not have prognostic value. CONCLUSIONS: Disseminated tumor cells detected before commencing chemotherapy had prognostic value in patients with inoperable PDAC.

Fragment size and level of cell-free DNA provide prognostic information in patients with advanced pancreatic cancer.

BACKGROUND: It was recently demonstrated that the size of cell-free DNA (cfDNA) fragments that originates from tumor cells are shorter than cfDNA fragments that originates from non-malignant cells. We investigated whether cfDNA fragment size and cfDNA levels might have prognostic value in patients with advanced pancreatic cancer. METHODS: Blood samples were obtained from patients with advanced pancreatic cancer, before (n = 61) initiation of chemotherapy and after the first cycle of chemotherapy (n = 39). Samples were separated with density centrifugation and plasma DNA was isolated. Mode cfDNA fragment size and cfDNA levels were then determined using a 2100 Bioanalyzer. A cohort of partially age-matched healthy volunteers (n = 28) constituted the control group. RESULTS: Both a pre-treatment cfDNA fragment size of ≤ 167 bp (mode) and high pre-treatment cfDNA levels were associated with shorter progression-free survival (PFS) (p = 0.002 and p < 0.001, respectively) and overall survival (OS) (p = 0.001 and p = 0.001, respectively). Furthermore, multivariable Cox regression analyses demonstrated that pre-treatment cfDNA levels could independently predict prognosis for both PFS (HR = 3.049, p = 0.005) and OS (HR = 2.236, p = 0.028). CONCLUSION: This study demonstrates that cfDNA fragment size and cfDNA levels can be used to predict disease outcome in patients with advanced pancreatic cancer. The described approach, using a rapid, economic and simple test to reveal prognostic information, has potential for future treatment stratification and monitoring.

In vitro crosstalk between fibroblasts and native human acute myelogenous leukemia (AML) blasts via local cytokine networks results in increased proliferation and decreased apoptosis of AML cells as well as increased levels of proangiogenic Interleukin 8.

Interactions between native human acute myelogenous leukemia (AML) blasts and nonleukemic cells in the bone marrow microenvironment seem important both for disease development chemosensitivity. Native human AML blasts from consecutive patients were cultured with normal human bone marrow stromal cells and two fibroblast lines (HFL1 and Hs27) separated by a semipermeable membrane. This bidirectional crosstalk via the cytokine network between AML blasts and fibroblasts caused (i) increased proliferation, (ii) mediated antiapoptotic signalling and (iii) increased local levels of proangiogenic IL8.

Coculture of native human acute myelogenous leukemia blasts with fibroblasts and osteoblasts results in an increase of vascular endothelial growth factor levels.

OBJECTIVES: Angiogenesis seems important in the development of acute myelogenous leukemia (AML). Proangiogenic vascular endothelial growth factor (VEGF) is constitutively secreted by the AML blasts for a subset of patients, but it can also be released by non-leukemic bone marrow cells. METHODS: VEGF levels were determined after coculture of native human AML blasts with fibroblast lines, osteoblastic sarcoma cell lines, normal bone marrow stromal cells and normal osteoblasts. Cultures were prepared with leukemic and non-leukemic cells separated by a semipermeable membrane or in direct contact. RESULTS: The non-leukemic cells usually showed higher spontaneous VEGF release than AML cell populations. Coculture of AML blasts with HFL1 fibroblasts caused a supra-additive increase of VEGF levels when the cell populations were cultured separately, and the increase was also observed when cells were cultured in direct contact. An increase was also observed when AML blasts were cultured with osteoblastic sarcoma cells, normal bone marrow stromal cells and normal osteoblasts. Coculture had divergent effects on VEGF mRNA levels both for leukemic and non-leukemic cells, but increased mRNA levels were commonly observed especially for the non-leukemic cells. Cytokine inhibition experiments suggest that IL1 is important for the VEGF-increasing crosstalk, whereas the mechanisms are probably heterogeneous for coculture with osteoblasts. CONCLUSION: The bi-directional crosstalk via local cytokine networks between AML blasts and non-leukemic cells results in increased local VEGF levels, an observation suggesting that VEGF-targeting antiangiogenic therapy should be considered as a general therapeutic strategy in AML.

In vitro effects of native human acute myelogenous leukemia blasts on fibroblasts and osteoblasts.

Bone marrow stromal cells constitute a heterogeneous population, and in the present study we investigated intercellular crosstalk via release of soluble mediators between native human AML blasts and fibroblasts/osteoblasts. Coculture of nonleukemic stromal cells and AML blasts separated by a semipermeable membrane decreased proliferation of the fibroblast line HFL1, and the inhibition was maintained when HFL1 and AML cells were cultured in direct contact. A similar inhibitory effect was observed for osteoblastic sarcoma cell lines (Cal72, SJSA-1) and normal osteoblasts. GM-CSF was released by both nonleukemic cells and a subset of AML blast populations, and increased levels of GM-CSF were detected in AML cocultures with fibroblasts and osteoblastic sarcoma cells when testing AML cell populations with constitutive GM-CSF release. Furthermore, constitutive IL-1beta secretion by AML blasts was detected only for a subset of patients, whereas relatively high levels of IL-1RA were observed for all patients; coculture of AML blasts with HFL1 fibroblasts and osteoblastic sarcoma cells increased IL-1beta levels for patients with constitutive IL-1beta secretion, whereas IL-1RA levels were slightly decreased but still generally higher than IL-1beta levels (tested only for HFL1 fibroblasts). The bidirectional crosstalk between AML blasts and stromal cells with increased release of AML growth factors may be important in leukemogenesis, whereas the decreased stromal cell proliferation combined with the persistent release of IL-1RA may in addition inhibit remaining normal hematopoiesis and thereby contribute to bone marrow failure in AML.

Osteoblasts increase proliferation and release of pro-angiogenic interleukin 8 by native human acute myelogenous leukemia blasts.

BACKGROUND AND OBJECTIVES: Interactions between acute myelogenous leukemia (AML) blasts and non-leukemic cells in the bone marrow seem to be important for both disease development and susceptibility to chemotherapy. Recent studies have focused on the endothelial cells, but other non-leukemic cells may also be involved. In the present study we investigated how osteoblasts affect native human AML blasts. DESIGN AND METHODS: AML cells were derived from a large group of consecutive patients. The AML blasts and osteoblastic sarcoma cell lines (Cal72, SJSA-1) were incubated together in different chambers separated by a semipermeable membrane. We investigated effects of co-culture on proliferation, apoptosis and cytokine release. RESULTS: The cross-talk between these two cell populations, achieved via release of soluble mediators, resulted in increased AML blast proliferation, including increased proliferation of clonogenic progenitors, but did not affect spontaneous in vitro apoptosis. Both interleukin (IL) 1-b and granulocyte-macrophage colony-stimulating factor were involved in this growth-enhancing cross-talk, and normal osteoblasts could also increase the AML blast proliferation. Furthermore, co-culture of AML blasts with osteoblastic sarcoma cells as well as normal osteoblasts increased the levels of the pro-angiogenic mediator IL8. INTERPRETATION AND CONCLUSIONS: Our in vitro results suggest that the release of soluble mediators by osteoblasts supports leukemic hematopoiesis through two major mechanisms: (i) direct enhancement of AML blast proliferation; and (ii) enhanced angiogenesis caused by increased IL8 levels.

The angioregulatory phenotype of native human acute myelogenous leukemia cells: influence of karyotype, Flt3 abnormalities and differentiation status.

INTRODUCTION: The cytogenetic abnormalities and the response to induction therapy have been regarded as the most important prognostic parameters in acute myelogenous leukemia (AML) patients. Recent studies have demonstrated that internal tandem duplications and specific D-835 point mutations of the Flt3 gene, as well as the angioregulatory phenotype represent additional adverse prognostic factors. The aim of the study was to investigate possible associations between genetic abnormalities, differentiation status and angioregulatory phenotype in native human AML blasts. METHOD: Native AML blasts derived from consecutive patients were cultured in vitro and concentrations of angioregulatory molecules determined in the supernatants. RESULTS: Most patients released at least two different angioregulatory mediators. Pro-angiogenic interleukin 8 (IL8) was released at relatively high levels for most patients, many of these patients showed additional release of pro-angiogenic vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). High release of anti-angiogenic IL12 was associated with high release of pro-angiogenic IL8 and VEGF. Furthermore, patients with D-835 mutations showed increased IL12 release, whereas patients with normal karyotype had decreased HGF release. Myelomonocytic differentiation was associated with IL18 release and CD34 expression with low IL12 release. CONCLUSION: Our results suggest that native human AML blasts have a pro-angiogenic phenotype. Although the investigated genetic abnormalities are associated with variation in the in vitro release of angioregulators, these differences are relatively small and do not quantitatively involve the most important IL8 release. It therefore seems unlikely that this phenotypic variation can explain the prognostic impact of the genetic abnormalities.

Effects of angiogenic regulators on in vitro proliferation and cytokine secretion by native human acute myelogenous leukemia blasts.

Angiogenesis seems to be important in the pathogenesis of acute myelogenous leukemia (AML). The endothelial cell proliferation and microvessel formation are regulated by a wide range of soluble mediators, including angiogenin, angiopoietin-2, basic fibroblast growth factors, vascular endothelial growth factor (VEGF), VEGF-D, angiostatin and endostatin. In the present study, it has been investigated whether these mediators have an additional direct effect on the proliferation and cytokine release by native human AML blasts. AML cells derived from a large group of consecutive patients were investigated. All these mediators could alter the proliferation and cytokine release [interleukin (IL) 1beta, IL6, IL8, tumor necrosis factor alpha] for a minority of patients. Alteration of spontaneous proliferation by at least one mediator was detected in five of 38 patients; whereas, altered cytokine (Flt3-ligand, granulocyte-macrophage colony-stimulating factor, stem cell factor)-dependant proliferation was observed for 10 patients. Growth enhancement was most frequently observed, whereas growth inhibition was uncommon. The effects on AML blast proliferation were often dependant on or were modulated by the presence of the three hematopoietic growth factors. Based on the present results, it is concluded that angioregulatory mediators have additional growth-enhancing effects directly on the AML blasts for certain patients. However, based on the results from this investigation and previous studies it is suggested that their major contribution to the pathogenesis of AML is through their effects on regulation of bone marrow angiogenesis, and future studies of these mediators in AML should probably focus on these effects.

In vitro culture of human acute lymphoblastic leukemia (ALL) cells in serum-free media; a comparison of native ALL blasts, ALL cell lines and virus-transformed B cell lines.

The aim of this study was to standardize in vitro culture conditions for human acute lymphoblastic leukemia (ALL) cells. The cells were cultured in medium containing 10% fetal calf serum (FCS) and in the four serum-free media X-vivo 10, X-vivo 15, X-vivo 20 and Stem Span. Native ALL blasts could proliferate in all four serum-free media, but the strongest responses were usually observed with Stem Span. Native leukemia blasts were also cultured in the presence of various single cytokines or cytokine combinations. The highest proliferation was usually observed in the presence of Flt3-Ligand (Flt3-L) when single cytokines were examined, and these responses could be further increased especially by combining Flt3-L with interleukin 3 (IL3), IL7 or stem cell factor (SCF). Proliferation could also be increased when ALL blasts were cultured in the presence of two commercially available fibroblast cell lines (Hs27 and HFL1). Based on these results we suggest that in vitro culture conditions for native human ALL blasts can be standardized by using serum-free culture media supplemented with exogenous Flt3-L+IL3+SCF, and the use of accessory cells can also be standardized by using well-characterized fibroblast cell lines. Detectable ALL blast proliferation can then be observed for most patients. Our experimental model can thereby be used for in vitro evaluation of possible antileukemic treatment strategies, and it will then allow comparison of experimental results between different studies.

Interleukin-7 (IL-7) in patients receiving intensive chemotherapy for acute myelogenous leukemia: studies of systemic IL-7 Levels and IL-7 responsiveness of circulating T lymphocytes.

Early immune reconstitution after intensive chemotherapy for acute myelogenous leukemia (AML) occurs after 2-4 weeks of cytopenia, but T cell reconstitute is usually completed after several months. Interleukin-7 (IL-7) is a T cell growth factor involved in the late immune reconstitution, but its function during the early period of cytopenia has not been investigated. In the present study, we found that patients with untreated AML had decreased IL-7 serum levels, and induction chemotherapy had divergent effects on these levels. In contrast, patients in complete remission (CR) had intermediate levels immediately before consolidation therapy, and these levels decreased significantly when the patients developed therapy-induced cytopenia. Systemic IL-7 levels showed only minor increases during febrile neutropenia. Furthermore, IL-7 enhanced in vitro proliferative responses of polyclonal T cells derived from cytopenic patients, and the majority of circulating clonogenic CD4(+) and CD8(+) T cells from cytopenic patients could respond to both IL-2 and IL-7. To conclude, patients with untreated AML and severe chemotherapy-induced leukopenia (1) differ from other patients with CD4(+) T lymphopenia in that they show decreased IL-7 serum levels, and (2) the detection of circulating IL7-responsive T cells indicates that variations in systemic IL-7 levels are functionally important and contribute to an additional qualitative T cell defect in these severely T lymphopenic patients.

A critical review of T cell targeting immunotherapy in pancreatic cancer: only a hypothesis or a real hope for the patients?

T cell targeting immunotherapy is now being investigated in patients with pancreatic carcinomas. Until recently this therapeutic strategy has been used as a single specific treatment and clinical studies have demonstrated that tumour-specific T cell responses can be induced in a subset of patients and even in patients with advanced disease. However, it remains to be demonstrated whether these T cell responses can mediate clinically relevant antitumour reactivity. Furthermore, in vitro studies of cancer cell susceptibility to antitumour T cell reactivity are often limited to cancer cell lines because native tumour cells are frequently not available from patients. It is therefore still a hypothesis that T cell targeting immunotherapy can be developed into an efficient therapeutic strategy in patients with inoperable pancreatic cancer. Future investigations of this therapeutic strategy should probably focus on its incorporation into treatment regimens that also include other newer treatment strategies, for example tumour-reactive antibodies coupled with toxins or cytotoxic drugs, improved vaccination procedures probably including the use of dendritic cells and enhancement of antigenic presentation in the tumour microenvironment. T cell targeting immunotherapy will contribute to the hope of curative treatment in patients with inoperable tumours if such combinatory approaches can be developed.

Serum levels of angiogenin, basic fibroblast growth factor and endostatin in patients receiving intensive chemotherapy for acute myelogenous leukemia.

Angiogenesis seems to be important both in the pathogenesis of acute myelogenous leukemia (AML) and for the susceptibility of AML blasts to chemotherapy. Recent clinical studies even suggest that antiangiogenic therapy can induce disease control in patients with AML relapse. In this context we have investigated the profile of the systemic component of angiogenic regulation in AML by characterizing the serum levels of (i) the angiogenic regulators angiogenin, basic fibroblast growth factor (bFGF) and endostatin; (ii) the endothelial cell marker soluble (s) E-selectin. Patients with untreated AML had increased levels of angiogenin, endostatin and sE-selectin, whereas the levels of bFGF were not significantly altered. The systemic levels of the proangiogenic bFGF, the antiangiogenic endostatin and the endothelial cell marker sE-selectin showed significant correlations, whereas angiogenin and sE-selectin levels were not correlated. Furthermore, intensive chemotherapy resulted in decreased systemic levels of the 2 proangiogenic mediators angiogenin and bFGF, whereas endostatin levels remained high after treatment. Although angiogenin normally is a part of the acute phase reaction, its systemic levels were not altered when patients with chemotherapy-induced cytopenia developed complicating bacterial infections. Our results suggest that intensive chemotherapy can modulate the systemic component of angiogenic regulation in AML patients.

Leptin in human acute myelogenous leukemia: studies of in vivo levels and in vitro effects on native functional leukemia blasts.

BACKGROUND AND OBJECTIVES: Leptin receptors can be expressed by acute myelogenous leukemia (AML) cells, but the functional effects of leptin on native AML blasts have not been characterized in detail. We investigated systemic leptin levels in AML patients and in vitro effects of leptin on cultured AML blasts. DESIGN AND METHODS: Serum leptin levels were compared for patients with untreated AML and healthy controls. Native AML blasts were derived from a large group of consecutive patients, and effects of leptin on proliferation (suspension cultures and colony formation), constitutive cytokine secretion, differentiation and apoptosis regulation were assayed in vitro. RESULTS: Systemic leptin levels were decreased in patients with untreated AML, and leptin levels in acute leukemia patients were not altered during severe chemotherapy-induced cytopenia and complicating febrile neutropenia. In vitro studies demonstrated that leptin increased AML blast release of interleukin (IL) 1beta, IL6, tumor necrosis factor (TNF) alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF). This enhancing effect showed no correlation with CD34 expression and was not dependent on the presence of serum, induction of differentiation or alteration of caspase 3 activity with decreased in vitro apoptosis. Leptin also increased spontaneous AML blast proliferation, whereas divergent effects on blast proliferation were observed in the presence of exogenous cytokines. The in vitro effects were usually observed at concentrations exceeding the systemic levels. INTERPRETATION AND CONCLUSIONS: Our results suggest that systemic leptin levels alone do not have a major influence on native AML blasts, but the systemic levels in combination with local leptin release in the bone marrow may affect the functional characteristics of these cells.

Use of marine toxins in combination with cytotoxic drugs for induction of apoptosis in acute myelogenous leukaemia cells.

Intensive chemotherapy for acute myelogenous leukaemia (AML) results in an overall long-term disease-free survival of < 50%. This percentage reflects an improved survival for certain subsets of patients with low-risk cytogenetic abnormalities after treatment with high-dose cytarabine, whereas lower long-term survival is seen for other patients and especially for the large group of elderly patients. New treatment strategies are therefore considered in AML and one approach is to target the regulation of apoptosis in AML cells with new pharmacological agents. Regulation of apoptosis seems to be clinically important in AML as intracellular levels of apoptosis-regulating mediators can be used as predictors of prognosis in AML. It is also well documented that cytotoxic drugs exert important antileukaemic effects through induction of apoptosis. Marine toxins represent new pharmacological agents with proapoptotic effects and should be considered for combination therapy with cytotoxic drugs. These agents are already useful laboratory tools for in vitro studies of AML cells but it is still too early to conclude whether they will become useful in clinical therapy. One of the major problems to be investigated is the toxicity of combination therapy, although this may be solved by the coupling of toxins to antibodies or growth factors with a preferential binding to AML cells. Other problems that have to be addressed are the possible effect of the toxins' tumour promoting effects on chemosensitivity in relapsed AML and the possibility of cross-resistance between cytotoxic drugs and toxins.