Comprehensive Guide to Understanding & Interpreting OncoTrace Test Results

Purpose:

• Detects the presence of ALL types of malignant cells (CTCs) in circulation (whole blood) at a very low level of 1.3 CTC in 10 million WBCs or 1 CTC in 25 mL of whole blood.
• The OncoTrace test is used as a baseline for future comparison to assess therapy effectiveness or response.
• OncoTrace may be used as a screening tool, however the Onco-D-clare test is more appropriate. For cancer screening of a healthy patient with no history of cancer, order Onco-D-Clare. It’s important to note the following differences in sensitivity and specificity:
  • OncoTrace:
    • True positive 86.2%
    • True negative 83.9%
  • Onco-D-clare:
    • True positive 92.40%
    • True negative 94.78%
• Detects CD45 positive and CD45 negative cells
• The CD45 positive cells are used for hematological origin
• Determines the CTC Number & Immunophenotype
• The Circulating Tumor Cell (CTC) number is compared against a statistical reference number for a cancer type to assess extent of the disease (early or advanced). The CTC number or count is obtained using flow cytometry.
• The Immunophenotype identifies the protein markers, antigens, or receptors on the surface of the CTCs to provide information on:
  • Primary source or tissue origin, where feasible
  • Analysis of both hematological origin and solid tumor origin cell
  • Differentiation of solid tumor origin CTCs, that are of epithelial origin or EpCam+ (expressed as a percentage of all CTCs)
  • Risk behaviors as to aggressiveness, resistance to therapies, recurrence, and metastasis.
• In summary, the CTC and immunophenotype provide the identity and behavior of the dominant cancer cell subpopulation that drives the disease.

Method of Detection:

• Flow cytometry with antibody panel
• Sensitivity 86.2%
• Specificity 83.9%

Important: Since the sensitivity of this test is 86.2%, there are a few cases where CTCs may not be detected or identified and will be reported as 0 CTCs. The reasons are stated below:

1. The sample was not properly preserved during transportation and the CTCs were destroyed as a result but passed QC checks.
2. If the sample passed QC checks but testing showed endothelial cells were detected (CD31 positive), it is possible that the CD31 cells counteracted the number of CTCs, and this could be caused by failure to discard the first 2-3 ml of blood during blood draw.
3. The patient received systemic therapy (chemo agents, steroids, anti-hormone therapies, immunotherapy, or targeted therapies, including
   natural substances like IV therapies or oral natural extracts, within the 14-day pre-blood draw period as outlined in the Pre & Post Blood Draw Guidelines. These therapies will eliminate the CTCs from circulation and will also affect the solid tumor at a later stage. This is why it is essential to adhere to the 14-day, pre-blood draw, “no therapy” waiting period prior to the RGCC blood draw.
4. There is a chance that CTCs will merge or fuse with monocytes and together generate hybrid cells that are very difficult to isolate (only by size
   and content of nucleus). In this rare case, the isolation process is different than the routine approach and is very time-consuming. If this is the case, then the testing analysis has to be repeated but with different steps of isolation.

Interpretation of Results:

• CTC is compared to a reference or cut-off number (this statistical reference is only useful if it is a baseline test, and the patient has not yet received any cancer treatment)
  • Breast cancer < 5 cells/7.5 mL
  • Prostate cancer < 20 cells/mL
  • Sarcoma < 15 cells/6.5 mL
  • Colon cancer < 5 cells/mL
  • Lung cancer < 10 cells/mL
  • All other cancer types other than the ones listed above < 5 cells/mL
• If CTC is > cut-off, this indicates advanced or progression of disease.
• If CTC is < cut-off, this indicates early disease or disease is responding to a treatment plan.
• Note: if baseline CTC is < cut-off, following treatments (conventional or holistic), this might indicate minimal residual disease. If baseline CTC is >
  cut-off, during or following treatments (conventional or holistic), this indicates advanced or progression of disease post-treatment or with ongoing
  treatment. There are occasions where the CTC is low, but the disease has progressed. One of the plausible reasons for this is the active and dominant
  CTCs known as CSCs (CTCs with the tumor stem cell markers) are the last ones to be destroyed by therapy. So even when the CTCs are decreasing, the
  CSCs can remain the same in their percentages until the CTC number has reached low levels (in our more experienced doctor’s observation - CTC
  approaching 1). And if the CSCs are present, there is still risk for disease progression despite a low CTC number.
• The CTC baseline of the patient is always used as a point of reference for future comparison and relevance rather than the statistical cut-o reference
  point.
• The CTC number is the concentration number per unit volume that represents the TOTAL number of the various subclones or subpopulations of CTCs per unit volume.
• There is another CTC number that is associated with only the EpCAM+ve (EpCAM+) CTCs. This is shown at the bottom of page 2 of the OncoTrace in the Markers table. This number is also shown in the Markers table on page 12 of the Onconomics Plus and on page 8 of the Onconomics Extracts+. To show favorable therapy response, this EpCAM+ CTC number must also decrease.
  
• The therapy outcome goal is to see the CTC reduced below 2 (because statistically this lowers the risk of active disease progression), and then as
  close to 0 as possible, and the tumor stem cell markers (CD34, CD133, CD44, Nanog, OKT-4, and Sox-2) to be close to negative or negative.
• The CTC number of a patient with advanced disease cannot be compared with the CTC number of another patient with advanced disease, even with
  similar diagnosis and stage because the detection of CTC is unique to each patient in terms of the degree of disease burden, the potential formation of anaplastic CTCs (seen in advanced disease), along with the fact that no test is 100% accurate.
• CTC number below the reference cut-off point can still be considered advanced disease if the patient’s cancer is becoming anaplastic. That is why assessing the phenotype biomarkers takes precedence over the CTC number (this information is a summary from a verbal explanation by Dr. Papasotiriou).
• Risk of cancer progression is not reduced until the CTC number is near 1 or 0.
• When CTC is near 1 or zero and the tumor stem cell markers have reduced to DIM or become negative, usually this state is similar to “remission status”. (Add the explanation of what DIM means)
• When conventional oncology reports NED (no evidence of disease) even though there is still presence of CTCs, this means the patient is tumor-free but NOT cancer-free.
• If subsequent follow-up tests show the CTC decreased by 0.3 but the subpopulation EpCAM+ CTC increased by 0.3 (as an example), this is not a favorable outcome since this clinically significant subgroup is higher than previous. On the other hand, if the EpCAM+ve CTC is decreasing more in proportion than the total CTC, it usually is a favorable outcome.
  
• CTCs with tumor stem cell markers are known as CSCs (cancer stem cell-like) or TICs (tumor initiating cells). Please note that all CSCs are CTCs, but CTCs are NOT all CSCs.
• CSCs are the dominant CTCs that drive the disease and exhibit cell-renewal, tumorigenesis, and resistance to chemotherapy. The CSCs with the OKT-4 and Sox-2 positive or Dim are the most dominant CTCs that drive the disease.
• CSCs are shown as percentages of total CTC shown in the phenotype report.
• CTCs that are NOT CSCs, are followers or “enslaved cells”. These cells are easily destroyed by chemotherapy.
• Some CSCs express only 1 type of tumor stem cell marker on their surface, while others have more than 1. CSCs that have all the tumor stem cell markers
  detected are the worst and most aggressive and resistant to treatment, these CSCs are likely the last ones to be destroyed.
• If the CSCs (CTCs having the tumor stem cell markers) are decreasing towards negative but the CTC number have increased or remained the same, this constitutes a favorable therapeutic response. Decreasing CTCs but increasing CSCs constitute poor therapy response due to likelihood of resistant clones forming. Example, if CTC decreased from 3.5 down to 2.8 but CSCs’ expression increased from OKT-4 DIM to Positive (25% of all CTC), this constitutes an unfavorable therapeutic response.
• There is a hierarchy in the CSC subpopulation. CSCs with Sox-2 and/or OKT-4 are at the highest rank. Below that is Nanog. The lowest rank CSCs are the ones with only the CD133 and/or CD44 on them.
• CSCs with the presence of both OKT-4 and Sox-2 constitute “high resistance to treatment and high recurrence risk”.
• CSCs with the presence of only OKT-4 or Sox-2 but not both constitute “less resistance to treatment and less recurrence risk”.
• CSCs with the highest tumor stem cell marker percentage at 50% constitute a moderate to high aggressive risk. If the resistance and recurrence risk is high, the aggressive risk is interpreted as moderate to high aggressive risk; If the percentage is 75% or higher, it constitutes a high aggressive risk.
• Just the presence of CSCs carries potential risk for metastasis.
  
• If the biomarker c-MET is positive the risk for metastasis is greatly increased; the interpretation for the test will need to state, “high metastatic risk” because the CSCs are actively trying to metastasize.
• Even if the CTC number has reduced to near 1 but not 0, and c-MET has become Dim or Positive from Negative, this constitutes a high metastatic risk and therapy is not yet over. When c-MET is positive, cancer is actively trying to metastasize and that is about a 3-month window.
• Risk of cancer metastasis is low when CTC number is 1.2 or less, OKT-4 and Sox 2 are negative, and the rest of the tumor stem cell markers show mostly DIM or Negative, and c-MET is negative.
• If EpCAM+ CTC decreases in percentage, disease prognosis has worsened even if EpCAM+ CTC number has decreased because they are becoming less differentiated or are becoming anaplastic. Prognosis is much better if the percentage of all EpCAM+ CTC has remained the same or has increased, and
  the CTC number has decreased.
  
• Prognosis is better if the EpCAM+ or PanCK+ percentages remain or increase over time throughout therapy. 75-100% is “well-differentiated”, 50% is
  “moderately differentiated”, 25% or less is “poorly dierentiated”, and if it decreases to “Negative” it has become anaplastic (poor prognosis). (explain what anaplastic means)
• The primary sources that can be detected (but are not always detected) that are CD45- cells are: CD34 (epithelioid), CD99 (sarcoma), VHL mut (renal carcinoma), PSMA (prostate), CD19 (lung neuroendocrine), MUC-1 (breast), and CD63 (melanoma), and for CD45+ cells (lymphoma/leukemia)
• If the primary sources for CD45- cells (CD34, CD99, VHL mut, PSMA, CD19, MUC-1, and CD63) are decreasing in percentages, prognosis is not good because they are becoming anaplastic. Good prognosis is if percentages remain the same or increase over time)
  
• If CD31 (endothelial cell membrane antigen) and panCK (epithelial origin cell marker) are present (detected) in a test sample, whether CTC is detected or not, this means the blood sample is suboptimal due to contamination by endothelial cells from the blood draw. Even after passing QC check, there is still a possibility of endothelial cell contamination, which can hide the presence of CTC and may present as false negative on a “0” CTC report or if CTC is detected, it may be falsely low.

Practical Examples of Interpretation and Reporting:

Example 1:
6-months post chemo treatment of Hodgkin’s Lymphoma 

Results:
Baseline CTC Number = 3.5 cells/mL (reference cut-off is < 5 cells/mL)

Phenotype BioMarkers:
CD45 positive cells (Hematologic origin):
Results:

• CD15 negative
• CD30 positive
• CD34 positive
• CD19 diminished positive (Dim)

Index of Markers:
CD30+ (Hematologic malignancy marker, Hodgkin’s lymphoma)
CD34+ (Hematological stem cell and blast cell marker, tumor initiating cell)
CD19 Dim (Hematologic malignancy marker, B cell origin)

Final Interpretation Report:
Malignant cells detected in circulation.
CTC at 3.5 cells/mL baseline, ongoing residual disease, 6 months post-chemo
Positive for Hodgkin’s Lymphoma, B cell origin
Positive for blast cell marker (tumor stem cell marker; risk for metastases)

Example 2:
No pre-treatment of a biopsy-proven breast cancer

Results:
Baseline CTC Number = 4.5 cells/7.5 mL (reference cut-off is < 5 cells/7.5 mL)

Phenotype BioMarkers
CD45 negative cells (non-Hematologic origin)

Results:

• EpCam positive (50% of all CTC), EpCAM+ CTC 3.9 cells/7.5 mL
• CD133 positive (50% of all CTC)
• CD44 positive (25% of all CTC)
• Nanog Dim
• OKT-4 positive (25% of all CTC)
• Sox-2 Dim
• C-MET Negative
• MUC-1 positive (25% of all CTC)
• PanCK positive (50% of all CTC

Index of Markers:
EpCam (epithelial origin marker)
CD133, CD44, Nanog, OKT-4, Sox-2 (tumor stem cell markers)
c-MET (mesenchymal to epithelial transition)
MUC-1 (breast cancer antigen)
PanCK (epithelial origin cell marker)

Final Interpretation Report:
Malignant cells detected in circulation.
CTC at 4.5 cells/7.5 mL, baseline, no-pretreatment, early disease (because CTC is <5 cells/7.5 mL; this CTC population includes both EpCAM+ and EpCAM- CTCs) EpCAM+ CTC at 3.9 cells/7.5 mL (this is the subpopulation that carries clinical
significance as it carries most of the CSC subpopulation)
Breast origin (because MUC-1 is positive)
Epithelial origin (because it is EpCAM positive)
Moderately differentiated (because EpCAM is positive in 50% of all CTC)
Moderate to high aggressive risk (because one of the tumor stem cell markers,
CD133, shows 50% of all CTC, which is moderate, and since both OKT-4 and Sox-2 are present, it raises the risk from moderate to high)
High resistance to treatment risk (because of the presence of both OKT-4 and Sox-2)
High recurrence risk (because of the presence of both OKT-4 and Sox-2)

Less metastatic risk (because c-MET is negative)

Example 3:
No oncology pre-treatment for a biopsy-proven breast cancer (Stage 0 DCIS) but received holistic treatment several months ago

Results:
No malignant cells or 0 cells/7.5 mL +/- 0.3 cells

Phenotype BioMarkers:
CD45 negative cells (non-hematologic origin)

Results:

• CD31 Positive (0% of all CTC)
• panCK Positive (0% of all CTC)
• The rest of the markers are negative.

Index of Markers:
CD31 (endothelial cell membrane antigen)
panCK (epithelial origin cell marker)

Final Interpretation Report:
No malignant cells detected in circulation.
CTC at 0 cells/7.5 mL

Supplemental explanation to provider:
This does not necessarily mean there is no CTC because the blood sample has been contaminated by endothelial cells, which can hide CTCs. The fact the test was run means it passed the QC check however the results are not reliable to a certain degree. Since CTCs were not detected, the actual CTC number may be very low to begin with. This does not mean a re-sampling or recollection of blood needs to be done now but it is strongly recommended that the test be repeated within a few months (maybe 3 to 6 months) especially if this established cancer case carries a certain degree of risk. In this real patient example, the OncoTrace test was repeated in 6 months and this time still showed no malignant cells in circulation and both CD31 and panCK markers were negative. So, the confidence now is high that the results are accurate.