Dietary Intervention With High Phenolic EVOO in CLL
Condition: Chronic Lymphocytic Leukemia (CLL)
MeSH terms: Leukemia; Leukemia, Lymphoid; Leukemia, Lymphocytic, Chronic, B-Cell
Study type: Interventional

Summary: 
      Daily intake of extra virgin olive oil (EVOO), which is the major component of the
      Mediterranean diet and also a source of monounsaturated fat, may be partly responsible for
      the increased life expectancy of the Mediterranean people. A high dietary intake of EVOO is
      correlated with lower incidence of cancer, cardiovascular disease, metabolic diseases,
      Alzheimer's disease and osteoporosis Oleocanthal, a phenolic derivative of extra virgin olive
      oil, has important health promoting anti-cancerous properties, since it can inhibit the
      growth and promote the apoptosis of several cancer cells.

      The purpose of the present study was to investigate the effect of dietary intake of olive oil
      rich in oleocanthal on hematological, metabolical, cell progression markers and disease
      progression in patients with Chronic Lymphocytic Leukemia. The aim is also to study the
      possible association of apoptosis in the mechanism of action of virgin olive oil phenols in a
      patient with CLL in order to find the possible mechanism of the cellular action of
      oleocanthal in neoplasia.

      After the screening of >300 EVOO samples the investigators selected an EVOO with high
      oleocanthal and oleacin concentration of 416 and 284 mg/Kg respectively (EVOO OC/OL). Pilot
      dietary intervention was made in a group of 21 patients with chronic lymphocytic leukemia
      (CLL) who did not follow any treatment. EVOO was administered 40 ml/day for six months.
      Biochemical, hematological and molecular markers were studied six month before the
      intervention and six month during the intervention
    

Detailed description: 
      Extra virgin olive oil (EVOO) is the major component of the Mediterranean diet. More than 200
      different chemical compounds have been detected in olive oil, including fatty acids, sterols,
      carotenoids, terpenoids, flavonoids, tocopherols and olive polyphenols. Virgin olive oil
      contains a variety of important phenolic components, mainly corresponding to the compounds
      tyrosol, hydroxytyrosol, oleocanthal (OC) ,oleacin (OL) ,ligstroside and oleuropein aglycones
      .Among these olive phenols, oleocanthal , a dialdehydic form of dicarboxymethyl ligstroside
      aglycone which has been isolated from EVOO, presents a wide range of biological effects
      including antioxidant, anti-inflammatory, anticancer, neuroprotective and antidiabetic
      activities and for this reason has attracted more scientific attention.

      Chronic Lymphocytic Leukemia (CLL), the most commonly diagnosed adult leukemia in Western
      countries, is responsible for about 1 in 4 cases of all leukemias. It is characterized by the
      accumulation of monoclonal B-cells in bone marrow, peripheral blood, lymphatic tissues and
      spleen, while it is often asymptomatic and slow in its development. Patients with CLL do not
      always require immediate therapy. However, patients who appear symptoms have an approximate
      median survival range from 18 months to 6 years depending on the clinical stage. Criteria for
      the diagnosis of CLL are monoclonal B lymphocytes ≥5000 lymphocytes/ml in the peripheral
      blood for at least 3 months, prolymphocytes ≤55%, co-expression of claster of diferentiation
      (CD)5 and B-cell surface antigens CD 19, CD 20, and CD 23, low levels of CD 20, CD 79b and
      low expression of surface immunoglobulins (sIg), and kappa or lambda light chain restriction.
      The standard clinical procedures to estimate prognosis are the clinical staging systems
      developed by Rai et al and Binet et al.

      The level of apoptosis in hematopoiesis may provide information about the mechanism which is
      responsible for the regulation of progenitor's and stem cell's proliferation. The dysfunction
      of apoptosis has found to be associated with leukemia. An increase in the expression of
      Apo-1/fas (CD 95) apoptotic marker in myeloid progenitor cells in CLL patients and a
      corresponding decrease in the apoptotic B-cell lymphoma 2 (BCL-2) inhibitor has been
      described. Recent studies showed also that the increase in CLL patients' lymphocytes may be
      due to the disturbed balance between the proliferation and apoptosis of CLL cells while the
      changes in apoptosis-related protein expression are recognized during all the stages of Binet
      in CLL. The p38 levels are reduced (resistance to apoptosis) and Induced myeloid leukemia
      cell differentiation protein (Mcl-1) and Survivin levels are elevated, so as to enhance the
      survival of B lymphocytes.

      Survivin is a protein inhibitor of apoptosis which inhibits caspases and blocks cell death.
      It is highly expressed in most cancers, including hematological, and is associated with a
      poor clinical outcome. The apoptotic marker CD 95 (Apo-1/fas) and its ligand, CD 95 is a
      death receptor ligand that mediates apoptosis induction in order to maintain immune
      homeostasis and has the capacity to mediate apoptosis induction in cancer cells, generating
      an 18-kilodalton fragment termed caspase-cleaved cytokeratin-18 (cCK-18).

      Oleocanthal has been shown to induce cytotoxicity and apoptosis in vitro in human acute
      promyelocytic leukemia and in myeloma cells. It may also cause primary necrosis and cellular
      apoptosis by inducing lysosomal membrane permeability in different cancer cell lines.

      The aim of this study taking into account the strong apoptotic properties of oleocanthal in
      vitro, was to investigate its ability, under real clinical settings, to affect the
      hematological and biochemical profile; also the cellular apoptotic markers Survivin, cCK18-
      (caspase activity marker) and Apo1fas/CD 95 in CLL patients after taking into consideration
      the progression of their disease. For this reason was conducted a dietary intervention study
      comparing the antineoplastic effects of an oleocanthal-rich EVOO in CLL patients.

      Material and Methods

      In the current study, a screening of 300 commercial samples (selected among a database of
      >3000 samples), obtained from olives (Olea europaea L.) harvested in 2017-2018 season, was
      performed by the Quantitative NMR spectroscopy (qNMR) method in order to identify oils which
      could fulfill the following criteria: The extravirgin oil would have high oleocanthal and
      oleacin content (D1 index) and low or even no content in other secoiridoid conjugated
      phenolics (3-8) or free tyrosol and hydroxytyrosol Specifically Oil rich in Oleocanthal was
      obtained from Monovarietal oil of Lianolia Kerkyras variety provided by "The Governor"
      Company from Agios Matthaios, Corfu, Greece. This oil contained: Oleocanthal 416±7 mg/Kg,
      oleacin 284±10 mg/Kg, Tyrosol < 10 mg/Kg, Hydroxytyrosol < 10 mg/Kg (D1 = 700 mg/Kg). It
      should be mentioned that apart the qNMR analysis, the hydroxytyrosol and tyrosol content was
      also measured by the standard IOC method for measurement of olive oil biophenols. Selected
      EVOO immediately after their analysis and until the beginning of the study period were stored
      at 4 celsious degree in order to minimize possible alterations in chemical composition.

      Subjects Twenty one patients from the hematologic clinic of the General hospital of Lakonia
      (Greece), were enrolled to participate in the dietary intervention. CLL diagnosis was
      confirmed by standard criteria. Patients were untreated, in Rai stage 0 to II.

      Dietary Intervention At the begin of the pilot study medical exams of each patient's history
      were recorded for third and sixth month before dietary the intervention. The clinical record
      before the dietary intervention concerns the hematological profile, biochemical exams
      including, lipidemic profile, fasting glucose, Liver and kidney function markers, as well as
      data concerning blood marrow biopsy, neoplastic markers and immunophenotype. Drug treatment
      or comorbidities were also recorded.

      At the begin of the pilot dietary intervention a group of 21 patients with CLL consumed
      before their meals, 40 ml/day of EVOO rich in olecanthal and oleacin (OC/OL) for 6 months, in
      order to explore the effect of the dietary consumption of the A-EVOO on the studied
      hematological, biochemical and molecular markers. The amount of the consumed EVOO OC/OL was
      chosen according to a previous study. (15) Patients were advised to continue their regular
      diet; their adherence to the study protocol was assessed by two 24-h telephone recalls every
      two weeks.

      Peripheral blood samples from the 21 patients with CLL were gathered at the begin, after 3
      months and after 6 months from the begin of the dietary intervention. Whole blood count and
      complete biochemical tests were performed for fasting glucose, blood lipid profile, and
      markers of liver and kidney function. Serum analyses were conducted using the same procedure
      and at the same lab with the use of a biochemical analyzer (Olympus AU-600, Olympus, Tokyo,
      Japan).

      A commercially available enzyme-linked immunosorbent assay (ELISA) was used according to the
      instructions of the supplier to assay the apoptotic markers M30 Apoptosense-cCK18 (VLVbio
      Hästholmsvägen, Sweden), Apo-1/Fas/CD 95 (Novus Biologicals, Colorado USA) and the
      antiapoptotic marker Survivin (OriGene Technologies, Inc USA) into plasma samples of all the
      patients at all the studied time points.

      Statistical analysis was performed using SPSS v.24 (SPSS Inc., Chicago, IL. USA) and the
      significance level was set at 0.05. Initially a descriptive analysis of the sample was
      performed in terms of age, sex, clinical, biochemical and molecular markers. The markers were
      found to be skewed at p < 0.05 significance level when checked by using the
      Kolmogorov-Smirnov test. Therefore non-parametric Mann-Whitney U test were used to find out
      the differences between the intervention and the control group.

      The differences between the time intervals of the intervention was assessed with Friedman
      non-parametric test followed by Dunn's test for multiple comparison Finally, the correlations
      of the molecular markers with the clinical parameters included in the study and were checked
      with Spearman Rho.
    

Intervention name(s): High phenolic EVOO intake; No High phenolic EVOO intake
Intervention type(s): Dietary Supplement; Dietary Supplement
Intervention description(s): During the pilot dietary intervention patients with CLL consumed before their meals, 40 ml/day high phenolic EVOO for 6 months; Medical record of each patient's was followed during 6 month before dietary the intervention
Allocation: Randomized
Intervention model: Crossover Assignment
Intervention model description: Twenty one patients with Chronic lymphocytic leukemia (CLL) from the hematologic clinic of the General hospital of LaKonia (Greece), were enrolled to participate in the dietary intervention. CLL diagnosis was confirmed by standard criteria. Patients were untreated, in Rai stage 0 to II, did not met criteria to initiate chemotherapy and had no neoplasmatic comorbidity. Subjects were randomly selected from the Hematology Department of the General Hospital of Lakonia. Exclusion criteria were neoplastic comorbidities or severe metabolic diseases. Only one group participates in this study, which performed dietary intervention consuming EVOO rich in oleocanthal and oleacin. Clinical and molecular markers were registered 6 months before the intervention and 6 months during the intervention.
Masking: None (Open Label)
Primary purpose: Treatment
Keywords: CLL; oleocanthal; Extra Virgin Olive Oil (EVOO); phenols; apoptosis