1. Salwa Hassan Teama

2. Increased Plasma DNA Integrity Index in Breast Cancer Patients
Salwa Hassan Teama
Increased Plasma DNA Integrity Index in Breast Cancer Patients
Azza M Kamel 1; Salwa Teama 2; Amal Fawzy 1; Mervat El Deftar3
1 Clinical pathology department, NCI, Cairo University, Cairo, Egypt,
2 Molecular biology unit, Ain Shams University, Cairo, Egypt,
3 Pathology department, NCI, Cairo University, Cairo, Egypt

3. Presenter
Salwa Hassan Teama M.D. Clinical Pathology, National Cancer Institute Medical Research Center, Ain Shams University Cairo/Egypt

4. Salwa Hassan Teama
Introduction
Breast cancer is the most common cancer and the leading cause of cancer death in women worldwide. Current screening methods fail to detect many cancers that present at a later date as a result of symptoms. Early detection of breast cancer is crucial for successful treatment.
Shedding of nucleic acids (DNA) into the blood stream has been documented in various malignancies. Tumor-released DNA in blood represents a promising biomarker for cancer detection. 1

6. Cell Free DNA (Circulating DNA)

7. Salwa Hassan Teama
DNA is the repository of genetic information in each living cell, its integrity and stability is essential to life.
The presence of circulating cell free DNA has been documented as early as the 1940's (Mandel and Metais, 1947).
A baseline level of circulating DNA is generally present, even in healthy individuals and increased concentrations of DNA are present in the plasma of cancer patients.
Long DNA molecules broken into shorter fragments and a portion of these fragments find their way into the blood stream. 3

8. Salwa Hassan Teama
Salwa Hassan Teama
A number of sources appear to contribute to circulating cell free DNA; it arise from dying cells break up within every organ of the body, cells of a developing fetus, or a tumor cells that may be growing somewhere, it contains genetic information from all cell types of the body.  Among the many DNA fragments are those that carry recognizable signatures that identify their origin such as an undiagnosed tumor. 4

9. Salwa Hassan Teama
However it was not until the 1970's that a correlation between elevated levels of circulating DNA and several human disorders, including cancer.
The elevated levels of circulating DNA observed in cancer patients by researchers may be related to neoplastic cells, however this was not verified until the 1990's, when several reports were published showing that circulating DNA exhibited various tumor-like alterations. 5

10. Salwa Hassan Teama
Characteristics of tumor DNA have been found in genetic material extracted from the plasma of cancer patients
Decreased strands stability
Presence of specific oncogene, tumor suppressor gene and microsatellite alterations.
Point mutations of the ras genes have been detected in the plasma DNA of patients suffering from haematopoetic malignancies, colorectal and pancreatic cancer.
Rearranged immunoglobulin heavy chain DNA has been found in the plasma of patients with nonHodgkins lymphoma and acute B cell leukaemia.
Microsatellite instability, expressed either as a new allele or a loss of one allele (LOH) occurs in the plasma and serum DNA of patients suffering from head and neck, lung and renal cell cancer.
Features: 6

11. Salwa Hassan Teama
DNA integrity index as represented by the ratio of longer fragments to shorter DNA, may be clinically useful used as surrogate for detection of tumors. The DNA integrity assay (DIA) was originally characterized in DNA isolated from stool samples and was shown to be indicative of colorectal tumor presence.
DNA Integrity Index 8

12. Salwa Hassan Teama
Apoptosis vs. Necrosis
Cell death can occur by either of two distinct mechanisms; programmed cell death (apoptosis) or necrosis .
Apoptosis is a physiological and controlled process by which unwanted or useless cells are eliminated during development and other normal biological processes.
Necrosis is a pathological process which occurs when cells are exposed to a serious physical or chemical insult. 9

13. Apoptosis DNA Fragmentation is a key feature of programmed cell death.
Salwa Hassan Teama
Apoptosis DNA Fragmentation is a key feature of programmed cell death.
Apoptosis is characterized by the activation of endogenous endonucleases.
DNA cleavage during apoptosis occurs at sites between nucleosomes, protein-containing structures that occur in chromatin at ~200-BP intervals (cleavage of chromatin DNA into internucleosomal fragments of 200 BP).
This DNA fragmentation is often analyzed using agarose gel electrophoresis to demonstrate a "ladder" pattern at ~200-BP intervals.
DNA laddering (left) visualised in an agarose gel by ethidium bromide staining.
1 kb marker(middle) and
Control DNA (right) 11

14. Salwa Hassan Teama
Tumor necrosis is a frequent event in solid malignant neoplasms, and it generates a spectrum of DNA fragments with different strand lengths because of random and incomplete digestion of genomic DNA by a variety of deoxyribonucleases and death in the malignant tumors results not only from apoptosis, but also necrosis, autophagy, or mitotic catastrophe. Therefore, elevated levels of long DNA fragments may be a good marker for detection of malignant tumor DNA in blood. 12

15. Salwa Hassan Teama
Brief summary of the cancer types that are positively correlated with elevated levels of circulating DNA
Cancer Type
Results
Shapiro et al. (1983) [20]
Gastrointestinal (pancreatic, gastric)
Patients with benign GI disease had mean DNA levels of 118 ± 14 ng/mL, whereas patients with malignant GI disease had 412 ± 63 ng/mL.
Giacona et al. (1998) [21]
Pancreatic Colorectal
90% of pancreatic carcinoma patients had serum DNA levels >100 ng/mL. Only 40% of colorectal carcinoma patients had DNA levels in similar concentrations.
Silva et al. (1999) [22]
Breast
Plasma DNA in breast cancer patients ranged from ng/mL; in healthy controls DNA levels ranged from 0-45 ng/mL
Wu et al. (2002) [23]
Non-Hodgkin lymphoma, Leukemia, Multiple myeloma, Lung, Bladder, Hepatoma, Cervic, Gastric, Esophageal,
Healthy volunteers were found to have serum DNA concentrations of 57.1  30.6 ng/mL, while the serum cell-free DNA was elevated in almost all types of malignant diseases.
Sozzi et al. (2003) [24]
Lung
Median concentration of circulating plasma DNA in lung cancer patients was almost eight times the value detected in age-, sex-, and smoking-matched controls (24.3 vs 3.1 ng/mL)
Allen et al. (2004)[25]
Prostate
Patients with benign prostatic hypertrophy (BPH) has significantly lower DNA levels than patients with either high-grade prostatic intraepithelial neoplasia (PIN) or adenocarcinoma.
Jung et al. (2004) [26]
Patients with localized cancer had DNA plasma within the reference interval. Increased plasma DNA was found in patients with lymph node and distant metastases.
Papadopoulou et al. (2004) [27]
Free circulating DNA can be detected in patients with prostate cancer compared with disease- free individuals.
Taback et al. (2004) [28]
Melanoma
Serum DNA in healthy donors ranged from 9-79 ng/mL, while patients with metastatic melanoma had serum DNA concentrations of ng/mL.
Boddy et al. (2005) [29]
Patients with prostate cancer had significantly higher level of DNA than those in a low-risk benign group as well as healthy controls The low-risk benign group has significantly higher DNA levels than the healthy control group.
Flamini et al. (2006) [30]
Colorectal
Median DNA concentration was about 5-fold higher in colorectal cancer patients than in healthy donors.
Ren et al. (2006) [31]
Hepatocellular
Compared with the healthy volunteers, a significantly higher circulating plasma DNA level was found in the patients with hepatocellular carcinoma (mean 47.1±43.7 ng/ml), or liver cirrhosis (mean 30.0±13.3 ng/ml).
Authors (year)
Cancer Type
Results
Table accessed at: 14
Authors (year)

16. Increased Plasma DNA Integrity Index in Breast Cancer Patients
Salwa Hassan Teama
Increased Plasma DNA Integrity Index in Breast Cancer Patients
Subjects:
Methods:
Objective:
Results:
Conclusion:
Key word: Breast cancer/DNA integrity index/Real time PCR 15

17. Subjects:
The study was performed on 64 female subjects including 32 with breast cancer at different disease stages; 12 with benign breast conditions and 20 healthy controls.
The American Joint Committee on Cancer (AJCC) system was used for staging of breast cancer patients. The DNA integrity index in plasma was determined using real-time PCR assay involving detection of 400 versus 100 bp PCR products of a housekeeping gene, the  -actin.
The study was conducted at the National Cancer Institute, Cairo University in the period form December 2008 till June 2010.

18. Group A: comprised 32 patients with breast cancer at different stages
Group A: comprised 32 patients with breast cancer at different stages. They had an age range of with a mean of 57.29.4 and a median of 56 years.
Group B: Included 20 apparently healthy female of matched age and sex as the malignant group without history of diseases. They had an age range of 30 – 69 with a mean of 54.29.5 and a median of 55 years.
Group C: comprised 12 patients diagnosed to have benign breast tumors by clinical evaluation and the fine needle aspiration cytology. Malignancy was ruled out by histopathological examination. They had age range of 39 – 66 with a mean of 55.88.4 and a median of 56 years.

19. Histopathologic grade G1 G2 G3 Histopathologic type Invasive ductal
Salwa Hassan Teama
Histopathological characteristics of 32 breast cancer patients
Parameter
Histopathologic grade G1 G2 G3
Histopathologic type
Invasive ductal
Invasive lobular
Other No 23 9 18 8 6 %
71.8
28.2
56.3
25.0
18.7

20. Salwa Hassan Teama
Objectives:
Assess of plasma DNA integrity index ; the ratio of longer fragments to shorter DNA, as a marker for detection of malignant tumor DNA in blood and whether it could discriminate breast cancer from healthy and benign breast disease and its role in prediction of tumor progression. 18

21. DNA Strand Integrity Analysis:
According to method described by Wang et al., DNA strand integrity was measured by quantitative PCR (QPCR) using the Stratagene MX3000 real-time system.
Plasma DNA was tested by amplifying  -actin as a house keeping gene. Both 400 and 100 bp products were amplified and the integrity index calculated as the ratio between them.
The threshold (Ct) value for each reaction was calculated by the Real Time software. The Ct value for the 400-bp product for a sample was subtracted from that for the reference control to obtain the normalized Ct value for the 400-bp product (ΔCt-400). Likewise, the Ct value for the 100-bp product for a sample was subtracted from that for the reference control to obtain the normalized Ct value for the 100-bp product (ΔCt-100). The normalized Ct value for the 400-bp product was then subtracted from that for the 100-bp product to obtain a final ΔΔ Ct value. The integrity index was calculated as exponential of (- ΔΔCt x LN 2) by comparative Ct method.

22. Salwa Hassan Teama
Results:
Plasma DNA Integrity Index is significantly higher in breast cancer as compared to both benign breast lesion patients and healthy controls. The mean Plasma DNA integrity index was 0.67 ( ) in the neoplasic group which was significantly higher than benign and healthy control groups (0.34 and 0.23 respectively, (p=<0.001). Although patients with benign breast condition exhibited detectable DNA integrity index higher than those with healthy control group patients, yet the difference is statistically insignificant (p=0.057).
Plasma DNA integrity directly correlated with advanced (AJCC) breast cancer stage. The mean plasma DNA integrity in patients with stage I, II, III, and IV was 0.45, 0.57, 0.81 and 0.91 respectively. DNA integrity index is significantly higher in breast cancer stage III &IV than stage I &II (p<0.05).
This rapid and minimally invasive approach may be a promising marker for detection of malignant tumor DNA in blood and may have a significant prognostic utility for detection of breast cancer progression. 22

23. DNA Integrity index in breast cancer and Controls
Salwa Hassan Teama
DNA Integrity index in breast cancer and Controls
Parameter No
DNA integrity index
Mean  SD
Median
Range
Cancer Breast 32
0.710.24
0.67
Benign Breast lesions 12
0.300.17
0.34
Healthy Control 20
0.180.11
0.23

24. Salwa Hassan Teama 21

25. Salwa Hassan Teama
The highest sensitivity and specificity were obtained at a threshold level for integrity index of When the index cutoff of 0.48 was applied, none of the 32 patients with non-neoplastic diseases was positive, whereas 27 of 32 neoplastic patients were positive sensitivity = 84.4%.
A scatter plot of the DNA integrity index in plasma samples from 32 patients with cancer breast (Lane 1), 20 healthy controls (Lane 2) and 12 with benign breast lesions (Lane 3) 26

26. Salwa Hassan Teama
ROC curve for analysis of DNA integrity index in plasma. The area under the ROC curve for DNA integrity index was in distinguishing breast cancer from benign breast lesions 27

27. Salwa Hassan Teama
ROC curve for analysis of DNA integrity index in plasma. The area under the ROC curve for DNA integrity index was in distinguishing breast cancer from healthy controls. 28

28. Receiver operating characteristic curves (ROC Curve)
Salwa Hassan Teama
Receiver operating characteristic curves (ROC Curve)
ROC Curves were used to assess the feasibility of using plasma DNA concentration for detecting breast cancer and other cancers.
ROC Curves: graphic representation of sensitivity plotted against the false positive rate (1- specificity) and the ROC curve is used to evaluate the performance of a test at different thresholds of diagnostic measure.
The area under the curve is a measure of test accuracy (measure overall ability of a diagnostic test with multiple cutoffs to distinguish between diseased and control individuals.

29. Brief summary of the cancer types that are positively correlated with increased DNA integrity index.
Authors (year)
Cancer Type
Results
Boynton 2003 [43]
Colon Cancer
DNA fragments isolated from CRC patients were of higher molecular weight (>18 bands detected of a total of 24 possible bands) than fragments isolated from fecal DNA of the colonoscopy-negative control group.
Wang et al. (2003) [1]
Gyn.& breast
The findings suggest that increased DNA integrity in plasma DNA is associated with cancer
Jiang et al. (2006) [44]
Head &Neck
This study shows that DNA integrity index in the plasma of the patients with HNSCC is increased in comparison with that in the plasma from non-HNSCC control subjects
Robert Hanley[38]
Prostate Cancer
Patients with prostate cancer (86 of 123; 69.9%) were determined to have a positive DIA score of ≥7.
Chan et al. (2008) [45]
Nasopharyngeal carcinoma
The plasma DNA integrity index of the NPC patients was significantly higher than that of the healthy controls (median, versus 0.238; P < 0.001).
Gao et al. (2010) [46].
Acute Leukaemia
Plasma DNA integrity is increased in acute leukemia
El-Shazly SF(2010)
Hepatocellular carcinoma
DNA integrity is a promising molecular biomarker for detecting HCC in patients with chronic HCV infection; it reflects the progression and metastatic potential of the tumor, and high DNA integrity is associated with short overall survival in HCV-related HCC.

30. Circulating Free DNA :Uses and Limitation
Salwa Hassan Teama
Circulating Free DNA :Uses and Limitation
The use of circulating DNA would be to monitor for disease recurrence or to establish disease free status of an individual and as a prognostic tool to detect early stage cancers that may be amenable to treatment or surgical resection and cure. Variations in the sample type and processing techniques were shown to affect the quantity of cell free DNA rescued. While no correlation has yet been found between circulating DNA levels and the size or location of the primary tumor.
DNA Concentration; many results suggested that plasma DNA concentration may not be sensitive or specific enough for cancer screening or diagnosis because it is difficult to eliminate the influence of circulating DNA concentration variance among different kinds of cancers which restrict plasma DNA concentration as a general tool for cancer diagnosis, but it may have different diagnostic or prognostic value in different cancers. Also other conditions such as pregnancy, systemic lupus erythematosus, rheumatoid arthritis, pulmonary embolism and myocardial infarction have been linked to elevated DNA levels.
Plasma DNA Alteration; the low sensitivities reported for detection of DNA alterations in plasma, however, make these approaches challenging for immediate clinical applications. 30

31. Salwa Hassan Teama
DNA integrity index may be a promising tumor marker for diagnosis and prognosis but assessment of the integrity of free circulating DNA is not yet practical for clinical use because the sensitivity and specificity of these methods have not been validated
A potential limitation may be the purification of DNA from serum or plasma, which decreases DNA yield. DNA loss may be inversely dependent on fragment size, which would affect DNA integrity values.
Other important variables have included time between collection and processing of samples in addition to storage prior to performing the assay, i.e., freeze-thawing of samples and the effects on DNA stability. 31

32. Summary and Conclusion
Salwa Hassan Teama
Summary and Conclusion
The circulating DNA values significantly differ in different cancer which restricts concentration of plasma DNA as a general tool for cancer diagnosis, but it may have different diagnostic or prognostic value in different cancers.
The low sensitivities reported for detection of DNA alterations in plasma, however, make these approaches challenging for immediate clinical applications.
Existing Data on the relationship between DNA integrity index and cancer are limited.
Due to lack of current research and discrepancies of various results; further validation studies are needed to assess the feasibility of using plasma DNA integrity index as a screening tool for detection of malignancy and/or tumor progression.
DNA integrity index, simple; non-invasive approach is important for the future of cancer. Using DNA integrity index may be a promising tumor marker for diagnosis and prognosis and follow-up tracking and could complement to established markers and newly diagnostic markers with especial emphasis on stringent control over the variables associated with sample collection and processing. 32

33. References 33 Salwa Hassan Teama
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35. Salwa Hassan Teama
Salwa Hassan Teama
Glossary
Loci = an arbitrary region of the genome that can have mutations/polymorphisms
Allele = a particular configuration of a loci with particular DNA sequence (can be many alleles for a particular loci, depending on its size)
Microsatellite: A microsatellite is a simple sequence repeat (SSR). It might be a homopolymer ('...TTTTTTT...'), a dinucleotide repeat ('....CACACACACACACA.....'), trinucleotide repeat ('....AGTAGTAGTAGTAGT...') etc. Due to polymerase slip (a.k.a. polymerase chatter), during DNA replication there is a slight chance these repeat sequences may become altered; copies of the repeat unit can be created or removed. Consequently, the exact number of repeat units may differ between unrelated individuals. Considering all the known microsatellite markers, no two individuals are identical. This is the basis for forensic DNA identification and for testing of familial relationships (e.g. paternity testing).
Loss of heterozygosity (LOH) in a cell is the loss of normal function of one allele of a gene in which the other allele was already inactivated.
Autophagy, or autophagocytosis, is a catabolic process involving the degradation of a cell's own components through the lysosomal machinery. It is a tightly-regulated process that plays a normal part in cell growth, development, and homeostasis, helping to maintain a balance between the synthesis, degradation, and subsequent recycling of cellular products. It is a major mechanism by which a starving cell reallocates nutrients from unnecessary processes to more-essential processes. . 35

36. Salwa Hassan Teama

37. Further comments or Question Number or title of slide
Number or title of slide

38. References
Thank You