Saliva from Patients with cancer: searching for novel prognostic markers in Acute Myeloid Leukemia

State of the art: Acute Myeloid Leukemia (AML) results from the malignant expansion of myeloid progenitors. Treatment response rates are around of 80% but the relapse remains significantly high, rendering this patients with a dismal prognosis without any curative options (1-3).
The saliva is a biofluid that reflect the status of the oral cavity. Recently, the saliva began to sample systemic diseases and biomarkers were related to several cancers (4, 5), but no studies were reported in the context of AML.
Research plan: The Clinical Hematology service (Hospital da Luz – Lisbon) will provide the samples upon informed consent and we aim to unravel AML prognostic markers using saliva, achieving a better stratification to improve prognosis. We propose the following tasks:
1. Sampling AML patients: The samples will be collected from primary diagnosed AML patients and during the treatment or disease progression in the course of clinical procedures. Healthy subjects will be also sampled. Samples will be processed as follows:
i) From the peripheral blood and bone marrow aspirates, the plasma will be collected, the mononuclear cells isolated and lysed for RNA and protein extraction (6);
ii) Saliva samples will be processed by direct spiting to the test tube containing stabilizing agents and the samples will be shipped to SalivaTec for processing (7).
2. Transcriptomics in AML biopsies: Upon collection of at least 8 patient samples we will analyze their transcriptional profile (RNA-seq):
i) 19 saliva samples (3 healthy donors + 8 diagnosis + 8 disease progressed);
ii) 16 bone marrow samples (8 diagnosis + 8 disease progressed) and;
iii) 3 peripheral blood samples (3 healthy donors).
3. Novel prognostic markers in AML: Bioinformatic analysis will occur at two levels: a) to identify the biomarkers (differences between the diagnosis and progression) and b) to integrate with saliva data. This will allow the identification of AML progression markers and verify whether such markers are detectable in saliva. These findings will be validated by quantitative PCR (qPCR), western-blot and Multiplex xMap assays. A preliminary analysis of the oral microbiome by qPCR will be done to determine their usefulness in prognosis.