USING BIOMARKERS TO MONITOR THE DYNAMICS OF

TUMOR RESPONSE TO THERAPY

BY SUMMAR ELMORSHIDY

ASSISTANT LECTURER OF CLINICAL ONCOLOGY CLINICAL ONCOLOGY DEPARTMENT

ASSIUT UNIVERSITY

TUMOR MARKER is produced by a tumor or by the host in response to a tumor which is used to differentiate a tumor from normal tissue or to detect the presence of a tumor based on measurements in blood or secretions.

They are found in cells, tissues or body fluids.

Measured qualitatively or quantitatively by chemical immunological or molecular biological methods.

They are biochemical or immunologic counterparts of differentiation states of tumor.

Cancer a multigene disease (cluster of diseases)which arises as a result of mutational & epigenetic changes coupled with activation of complex signaling networks.

It involves alteration of three main classes of genes – 1)ProtoOncogenes 2)Tumor suppressor genes 3) DNA repair genes.

This contribute to development of cancer genotype & phenotype.

This alterations resist the natural & inherent death mechanisms embedded in cells(apoptosis) coupled with dysregulation of cell proliferation events.

These genetic alterations include gene rearrangements, point mutations & gene amplifications leading to disturbances in molecular pathways regulating cell growth, survival & metastasis.

When these changes manifest in majority of patients with specific type of tumour this can be used as tumour markers ( Biomarkers ) .

This new tumor markers include a broad range of biochemical entities such as:

1. nucleic acids 2. proteins 3. sugars 4. lipids 5. small metabolites 6. cytogenetic & cytokinetic parameters 7. whole tumor cells 8. cancer stem cells

Ideal Tumour Marker should be….

Highly specific i.e. detectable in only one tumor, not detectable in benign disease and healthy subjects

Highly sensitive i.e. detectable when only a few cancer cells are present

specific to a particular organ Correlate with the tumour stage or tumour mass correlate with the prognosis have a reliable prediction value But ideal tumour marker doesn’t exists

Clinical applications of tumor markers

screening Diagno

sis progno

sis

Monitor of

treatment

Diagnosis

Detection of

recurrence

Types of tumor markers

Cell surface markers: essential for typing and diagnosis of certain

malignancies An important example is the cluster of differentiation(CD) antigens on cells which are useful in diagnosis of haematological malignancies

Hormones : HUMAN CHOROINIC GONADOTROPIN : CHORIOCARCINOMA,

EMBRYONAL CALCITONIN :MEDULLARY CA THYROID VASOACTIVE INTESTINAL PEPTIDE :PHEOCHROMOCYTOMA,

NEUROBLASTOMA ACTH :CUSHING’S SYNDROME,LUNG CANCER

cancer Antigens: CA 125, CA15-3, CA 19-9

Enzymes: ALKALINE PHOSPHATASE :BONE, LIVER LEUKEMIA PROSTATIC ACID PHOSOHATASE :PROSTATE NEURON SPECIFIC ENOLASE : SMALL CELL LUNG CANCER,

NEUROBLASTOMA,MELANOMA LACTATE DEHYDROGENES : LYMPHOMA , LEUKEMIA

Oncofetal Proteins: AFP : HEPATOCELLULAR ,GERM CELL TUMOR, CEA : COLORECTAL, GASTROINTESTINAL, LUNG, BREAST ,PANCREATIC

Receptors: ER ,, PR: indicators of hormonal treatment in breast cancer

ANDROGEN RECEPTORS : PROSTATE CANCER EPIDERMAL GROWTH FACTOR RECEPTORS

Genetic : Sister chromatid exchanges & translocations give

rise to structural aberrations are scored using various banding techniques. Philadelphia chromosome is associated with CML due to translocation between chromosomes 9 & 22.

Mutations & loss of heterozygosity within several proto-oncogenes can lead to microsatellite instability . Detection of this MSI in pathological tissue samples & comparison with normal tissue represents a valuable tool for early detection , at pre neoplastic stage.

Classification according to Potential uses for cancer biomarkers

Estimate risk of developing cancer :BRCA1germline mutation (breast and ovarian cancer)

Screening : Prostate specific antigen (prostate cancer)

Differential diagnosis :Immunohistochemistry to determine tissue of origin

Determine prognosis of disease 21 gene recurrence score (breast cancer),,,,KRAS mutation and anti-EGFR antibody (colorectal cancer)

Predict response to therapy HER2 expression and anti-Her2 therapy (breast and gastric) Predict response to therapy,,,,,Estrogen receptor expression (breast cancer)

Monitor for disease recurrence:CEA (colorectal cancer) AFP, LDH, βHCG (germ cell tumor)

An important distinction should be made between biomarkers and targets, since in many cases these are not equivalent. For example, as mentioned above, KRAS is an excellent biomarker in colorectal cancer, even though it is not the actual target of therapy. Instead, mutations in KRAS render tumors less responsive to anti-EGFR therapies

Cells as Biomarkers

Circulating Tumor Cells [CTCs] : Distant metastasis is the main cause of tumour-related death, but the occult spread of isolated tumour cells (ITCs) in the earliest stage of breast cancer remains undetected by conventional imaging technologies.

ITCs in secondary sites, such as blood and bone marrow (BM), are assumed to be precursors of (micro)metastatic disease.

The phenomenon of haematogenous dissemination in the metastatic cascade was recognised by several researchers in 19th century .

Therefore, detection and characterisation of these cells have become a major focus of translational cancer research. Sensitive assays enable reproducible evaluation of disseminated tumour cells (DTCs) and circulating tumour cells (CTCs) at the single-cell stage.

As demonstrated by a large pooled analysis, the presence of DTCs in BM at the time of diagnosis is associated with reduced survival . In recent years, numerous research groups have endeavoured to replace the invasive and painful BM biopsy with a simple blood test which is the CTC .( Liquid biopsy )

The low frequency of ITCs, estimated at one tumour cell/107–108 blood cells in patients with advanced cancer, explains the need for extremely sensitive detection assays and tumour cell enrichment . Currently, antibody-based and molecular methods are the main techniques for CTC detection.

Antibody-based CTC detection The majority of translational research trials use

antibodies against markers absent from other blood cells; due to the lack of breast cancer-specific antigens, commonly used markers are of epithelial origin (e.g., EpCAM and CKs) . CTCs are then identified by the staining pattern and morphological criteria.

positive selection leads to the enrichment of CTCs through the use of an antibody targeted against, e.g., cytokeratins (CKs) or epithelial cell adhesion molecule (EpCAM); or (c) negative selection, where the antibody is targeted against a leucocyte antigen (e.g., CD45).

Circulating tumor cells, disease recurrence and survival in newly diagnosed breast cancer

Bas Franken1, Marco R de Groot1, Walter JB Mastboom2, Istvan Vermes3, Job van der Palen4,5, Arjan GJ Tibbe6 and

Leon WMM Terstappen7* Abstract Introduction: The presence of circulating tumor cells (CTC) is an independent prognostic factor

for progression free survival and breast cancer-related death (BRD) for patients with metastatic breast cancer beginning a new line of systemic therapy. The current study was undertaken to explore whether the presence of CTC at the time of diagnosis was associated with recurrence-free survival (RFS) and BRD.

Methods: In a prospective single center study, CTC were enumerated with the CellSearch system in 30 ml of

peripheral blood of 602 patients before undergoing surgery for breast cancer. There were 97 patients with a

benign tumor, 101 did not meet the inclusion criteria of which there were 48 patients with DCIS, leaving 404 stage I to III patients. Patients were stratified into unfavorable (CTC ≥1) and favorable (CTC = 0) prognostic groups.

Results: ≥1 CTC in 30 ml blood was detected in 15 (15%) benign tumors, in 9 DCIS (19%), in 28 (16%) stage I, 32

(18%) stage II and in 16 (31%) patients with stage III. In stage I to III patients 76 (19%) had ≥1 CTC of whom 16(21.1%) developed a recurrence. In 328 patients with 0 CTC 38 (11.6%) developed a recurrence. Four-year RFS was 88.4% for favorable CTC and 78.9% for unfavorable CTC (P = 0.038). A total of 25 patients died of breast cancerrelated causes and 11 (44%) had ≥1 CTC. BRD was 4.3% for favorable and 14.5% for unfavorable CTC (P = 0.001).

In multivariate analysis ≥1 CTC was associated with distant disease-free survival, but not for overall recurrence-free survival. CTC, progesterone receptor and N-stage were independent predictors of BRD in multivariate analysis.

Conclusions: Presence of CTC in breast cancer patients before undergoing surgery with curative intent is associated with an increased risk for breast cancer-related death.

ONCOTYPE DX

Although patients diagnosed with axillary node–negative estrogen receptor–positive breast cancer have an excellent prognosis, about 15% of them fail after 5 years of tamoxifen treatment.

Clinical trials have provided evidence that there is a significant benefit from chemotherapy for these patients, but it would be significant overtreatment if all of them were treated with chemotherapy. Therefore, context-specific prognostic assays that can identify those who need chemotherapy in addition to tamoxifen, or those who are essentially cured by tamoxifen alone, and can be performed using routinely processed tumor biopsy tissue would be clinically useful.

a 21-gene recurrence score (RS), based on monitoring of mRNA expression levels of 16 cancer-related genes in relation to five reference genes, has been developed. The RS identified approximately 50% of the patients who had excellent prognosis after tamoxifen alone. Subsequent study suggested that high-risk patients identified with the RS preferentially benefit from chemotherapy.

A prospective study—the Trial Assigning Individualized Options for Treatment (Rx) (TAILORx)—to examine whether chemotherapy is required for the intermediate-risk group defined by the RS is accruing in North America.

TPS (a circulating tumor marker in breast cancer.) Tissue polypeptide specific antigen (TPS) measures an

antigenic determinant associated with human cytokeratin 18. TPS is the only test that specifically measures cytokeratin 18. TPS is a marker of tumor cell activity in contrast to markers

related to tumor burden. The value of detecting circulating TPS lies in the early detection

of recurrence by serial determinations and in the rapid assessment of the efficacy of the treatment. Pretreatment levels of TPS in patients with metastatic breast cancer are related with prognosis. Decreasing TPS levels during therapy monitoring indicate response and a fast response is correlated to favourable prognosis.

TPS for monitoring and follow up According to studies published during the last

15 years TPS have been shown to have the strongest association with clinical response .

In total ,studies of more than 3000 patients have been reported.

TPS has been found to be a better indicator of disease progression than CA 15-3.

In a study by Van Dalen et al sensitivity of TPS to detect progressive disease was found to be 83% compared to 30% for CA 15-3.

TPS has also been shown to be the marker that exhibits the most frequent and rapid decrease when the applied therapy is effective .

Low baseline levels of TPS indicates a better prognosis .

Summary of tumor markers

Beta-tubulin NSCLC IHC High expression of beta-tubulin confers worse

prognosis BRCA1 Breast IHCHigh expression of BRCA1 confers worse prognosis

in untreated patients CA19-9 Pancreatic IHC Higher preoperative CA19-9 levels are

associated with lower resectability,  more advanced stage and inferior survival

CAIX RCC IHC High expression of CAIX is associated with a better

prognosis CD44 Bladder RT-PCRExpression of CD44 is associated with poor prognosis CEA CRC IHCpreoperative CEA levels in resectable colorectal

cancer is  associated with poor prognosisIHC c-KIT GIST patients have a better

prognosis if they harbor a mutation in exon 11 of the c-KIT gene

ColoPrint CRC Prognosis for colorectal cancer patients

CTC Melanoma Increased number of circulating melanoma cells is associated with poor  prognosis CRC Colorectal patients with ≥3 CTC/7.5 ml of peripheral blood were  associated with shorter

PFS and OS, i.e. poor prognosis  tumor

Breast cancer patients with ≥5 CTC/7.5 ml of peripheral blood are associated  with shorter PFS

and OS, i.e. poor prognosis Prostate≥5 CTC/7.5 ml of peripheral blood is associated with poor prognosis

EGFR BladderOverexpression of EGFR is associated with

high grade and high stage NSCLC High gene copy number of EGFR in NSCLC

patients is associated with  poor prognosis NSCLC EGFR mutation in NSCLC patients is

associated with better prognosis  in untreated patients

RectalOverexpression of EGFR in rectal cancers is also associated with  poor prognosis

ER BreastPatients with ER-positive breast tumors have better survival than  patients with hormonal negative tumors

MSI status CRC High frequency MSI colorectal tumors are associated with better  

prognosis and show improved relapse-free survival K-ras NSCLC K-ras mutation is associated with poor prognosis in NSCLC patients

Oncotype DX BreastA 21-gene multiplex test used for prognosis to determine 10-year disease  recurrence for ER-positive, lymph node negative breast cancers using a  continuous variable algorithm and assigning a tripartite recurrence score

VEGF RCC Overexpression of VEGF is associated

with poor prognosis in clear cell renal carcinoma patients

Her2/neuBreast Patients with Her2/neu-positive breast tumors are more aggressive and  have a worse prognosis compared to Her2/neu-negative tumors

Metabolic Biomarkers

Bio-energetic index of cell has been suggested for classification and prognosis of cancer, besides predicting the response to therapy.

Positron emission tomography allows non invasive and quantitative analysis of various biologic process.

It uses a glucose analogue [2-deoxy-D-glucose] labelled with positron emitter Fluorine 18.

FDG that is partially metabolized and trapped as its phosphate [2-DG-6-P] in the tumor tissue, thus, localizing the tumor

Extent of increase in glucose utilization measured by FDG-PET has been co-related with degree of malignancy in some tumors.

Studying cancer through metabolomics could reveal new biomarkers for cancer that could be useful for its future prognosis ,diagnosis & therapy.