CAM 154

DNA Ploidy Cell Cycle Analysis

Category:Laboratory   Last Reviewed:July 2019
Department(s):Medical Affairs   Next Review:July 2020
Original Date:October 2016    

Description
DNA ploidy is an indication of the number of chromosomes in a cell. Ploidy analysis is a test performed on cells or tissues using either image analysis microscopes or flow cytometers.  DNA ploidy basically measures the DNA content within tumor cells. Due to anomalies in DNA replication, some cell populations such as cancer cells can have abnormal DNA content, and therefore, a different ploidy. 

The DNA content is generally expressed as a DNA index, which is the quantity of DNA in the test cell population in relation to that in normal diploid cells. A DNA index of 1.0 indicates normal diploid cells in the G0/G1 phase (BD Biosciences)

Policy
Application of medical necessity criteria is dependent upon an individual’s benefit coverage at the time of the request.

  1. Measurement of flow cytometry-derived DNA content (DNA Index), or cell proliferative activity (S-phase fraction or % S-phase) is NOT MEDICALLY NECESSARY for prognostic or therapeutic purposes in the routine clinical management of cancers.  

Policy Guidelines 
The American Society of Clinical Oncology (2001) prepared evidence-based guidelines on the use of tumor markers in breast cancer and colorectal cancer, and reached the following conclusions.  Regarding DNA ploidy or flow cytometric proliferation analysis as a marker for colorectal cancer, ASCO concluded that "[p]resent data are insufficient to recommend DNA flow cytometrically-derived ploidy (DNA index) for the management of colorectal cancer."  

Baldettorp et al., 2003, in a study among labs, have concluded that using "generalized guidelines" increased the inter-laboratory agreement for flow cytometric DNA ploidy and SPF analysis in breast cancer.   

Most labs in the United States  follow the guidelines from the National Institutes of Health consensus development conference. Australia follows the “international consensus,” which is same as the NIH consensus conference outcome. 

Rationale 
DNA ploidy is a tool to predict the behavior of abnormal DNA content in individual cells within mixed populations of cells.  It is measured in the form of histogram. The results are interpreted as:

      • Tumors, with a diploid pattern of DNA amounts, tend to be benign.
      • Tumors with highly variable DNA patterns tend be more aggressive.

Benign cells and well-behaved tumor cells grow and divide in an orderly fashion. In the resting state, they contain one complete set of chromosomes (this is the diploid condition). This complete set of chromosomes consists of 23 chromosomes (or N)  from Ma and 23 (N again)  chromosomes from Pa (equaling a total of 2N). Before a cell can divide it must double the number of its chromosomes, creating two complete sets of chromosomes (this is 4N, or the tetraploid state). After division is completed, each new cell gets half the booty of genetic material and so is diploid (2N) once again. 

Chromosome Count Nomenclature
Per Harvard Edu glossary, 2n if diploid, n if haploid. In humans, 2n = 46 in diploid somatic cells, and n = 23 in haploid germ cells.  Cells are described according to the number of sets present (the ploidy level): monoploid (1 set), diploid (2 sets), triploid (3 sets), tetraploid (4 sets), pentaploid (5 sets), hexaploid (6 sets), heptaploid or septaploid (7 sets), etc. The generic term polyploid is frequently used to describe cells with three or more sets of chromosomes (triploid or higher ploidy). 

DNA Ploidy Analysis
If DNA ploidy analysis were to be performed on a group of these cells, one would see that most of the cells would be diploid and a small fraction of them (those getting ready to divide) would be tetraploid. If we were to measure the amount of genetic material in each cell and create a graph, we would see a dominant diploid peak and a minor tetraploid peak. We can measure the amount of DNA in a cell by staining it with a dye that binds to the genetic material. The concentration and distribution of this dye can be measured by image analysis microscopy. (Gerald et al., 2012) (Stephan B et al., 2011) 

Now, when tumors go from bad to worse, they tend to not divide as orderly as they once did. Instead of the resting state having a complete set of chromosomes, they might have a set and a half. Such cells would have a DNA content that was neither diploid nor tetraploid but mid-way between. Plotting these cells on a graph would yield an aneuploid peak midway between the other two.

Some studies have shown that tumors that have a significant aneuploid peak do not behave as well as those that do not. Since there is a strong correlation between ploidy status and nuclear grade, this is not surprising. A nuclear grade can be assessed by any pathologist with enough experience with prostate cancer.

Many, but not all, doctors consider the value that DNA ploidy analysis adds is that it is an objective measurement that can be compared between labs using standardized techniques and that can be used to perform a quick check on the “ball-park” accuracy of Gleason scoring. For instance, any Gleason score 2+2=4 or 2+3=5 tumor that has an aneuploid peak, you should consider discussing if a re-evaluation for possible score adjustment is desirable. 

The Normal Cell Cycle and DNA Ploidy
The amount of DNA (deoxyribonucleic acid) in each (normal) cell is a constant value within an individual species. A normal amount of DNA is originally derived from combination of the egg and sperm at fertilization. Egg and sperm contain one half (haploid) amount of DNA, fertilized cell is diploid with respect to the DNA content.

Ploidy analysis is based on the paradigm that the G0/G1DNA content of a cell nucleus is correlated with its chromosomal number, since the DNA within the cell is contained within the chromosomes. Chromosomes are only seen in cells during cell division; however, DNA can be measured at any point in cell cycle. Ploidy analysis is a useful tool to measure a cell cycle as the cell duplicates its DNA content (2C). When the cell is ready to divide, the DNA content becomes double or tetraploid (4C). In normal tissues, number of cells active in cell division are relatively low as the rate of new cell formation is matched to the rate of cell loss from normal “wear and tear.” If the rate of new cell formation goes up, it is an indication of either increased cell loss or of tumor formation. DNA Ploidy measurements can easily detect cells that have elevated amount of DNA and are therefore engaged in proliferation. 

Specific pathologic conditions involve loss or the addition of a chromosome number, referred to as Aneuploidy.  Aneuploidy is mostly associated with malignancies. Disease states also result from alterations in chromosome structures – small losses of chromosomes (deletions), shuffling of portions of chromosomes (translocations) or duplication of portions of chromosomes (amplifications). Such changes may not produce significant changes in total cell DNA content, and may not be detected by Ploidy analysis, but can be seen by alterations in the proliferation index

Human neoplasms actively synthesizing DNA replicate through a process similar to that of normal cells known as the cell cycle. Cells in the resting diploid state (G0) phase contain 7.14 picograms of DNA and enter the cell cycle as gap 1 (G1) cells. During the synthesis phase (S phase), cells increase their DNA content continuously from 7.14 to 14.28 pg/cell until they reach the tetraploid state with twice the diploid DNA content. The second gap (G2 phase) refers to the tetraploid, pre-mitotic fraction of cells that undergo mitosis in the M phase to generate two diploid G0 cells, which may re-enter the cell cycle or persist in the resting state.

A DNA index of 1.0 corresponds to a 2N or 46 chromosome number characteristic of G0 and G1 cells. The G2 and M cells feature a 2.0 DNA index that corresponds to a 4N chromosome count of 92. The distribution of a population of cells within the cell cycle generates a pattern known as a histogram and represents DNA ploidy.

A DNA histogram is defined as diploid when the predominant or G0/G1 peak is equal to the G0/G1 peak of a known diploid reference cell population and the S and G2M phase contents are relatively low.

In normal tissues and most low-grade or slowly proliferating neoplasms, approximately 85% of the cell population forms the G0/G1 peak and 15% of the cells are in the S phase and G2 and M phases. DNA aneuploidy is defined as a DNA content of the G0/G1 peak of a cell population that varies significantly from the mean peak of the known diploid reference cell population. The DNA index of an aneuploid cell population may rarely be < 1.0 (hypodiploid) or > 1.0 (hyperdiploid). Aneuploid cell populations with a DNA index near 2.0 must be differentiated from diploid cell populations with significant G2M phases. Table 1 summarizes the terminology used for DNA ploidy definitions. 

Technique
The basic technique involves passage of a monocellular st flow cytometry is an emerging technique, which involves the separation, classification and quantitation of cell types by:

(i)    cell surface antigens (phenotype);

(ii)   DNA content (ploidy) (DNA index); and

(iii)  DNA flow cytometric proliferation analysis (S-phase fraction or % S-phase). 

The basic technique involves passage of a monocellular stream of cells through a beam of laser light after cell surface antigens have been tagged with fluorescent monoclonal antibodies; complex computerized instruments are then used to sort normal from abnormal cells and also subgroups of the same cell type.  Data are most often collected as a bar-graph histogram, which is then displayed visually as a densitometer tracing of the bar graph; the concentration of cells in each bar appears as a separate peak for each cell category, with a peak height proportional to the number of cells in each bar of the bar graph.  For example, lymphocytes can be separated into B- and T-cell categories; the T-cells can be further phenotyped as helper/inducer, suppressor/cytotoxic or natural killer cell types.

Using fluorescent dye that stains nucleic acids, flow cytometry methods have also been applied to measure nuclear deoxyribonucleic acid (DNA) content (ploidy) as a prognostic indicator of solid tumors based on the fact that malignant cells sometimes show abnormalities in total chromosome number and the frequency of these abnormalities generally increases with progression to higher-grade tumors.  In such testing, DNA diploid tumors are those in which a single peak containing an amount of DNA similar to normal control cells is generated by flow cytometry.  DNA aneuploid tumors have additional peaks on DNA histogram, presumably representing cells containing more or less nucleic acid than is found in 46 normal chromosomes.  A more quantitative method of expression is the DNA index (DI), which is the ratio of the mean tumor sample G0 /G1 DNA content of normal diploid reference cells.  The greater the deviation of the DI from 1, the more "aneuploid" the tumor.

Controversy exists concerning the use of DNA content (aneuploid versus diploid status) as an independent prognostic indicator.  Basic and clinical studies have reached different conclusions concerning its value.  While many of the earlier studies reported that diploid carcinomas had significantly or considerably better prognosis than aneuploid ones, some more recent studies do not confirm this or do not find that ploidy is a significant independent risk factor.  The results of studies on DNA content in different types of tumors have yielded varying results.

Aneuploidy, which is thought to be most often associated with malignancy, has been shown to occur in some non-neoplastic cell populations as part of the reaction to or regeneration after inflammation or tissue destruction and has also been reported in some benign tumors.  While groups of diploid patients have better prognoses than groups of non-diploid patients, ploidy status may have uncertain prognostic value in individual patients.  A small biopsy demonstrating diploid tumor may be missing a significant underlying non-diploid component.  Controversy remains as to the nature of the relationship between histologic grade and tumor stage with the degree of aneuploidy.  And, lastly, standards for tissue preparation and analysis to insure reproducibility are not yet established from lab to lab. 

In conclusion, DNA flow cytometry-derived estimates of DNA ploidy and S-phase proliferation have been correlated with cancer patient outcome in many studies.  However, the evidence is almost entirely from retrospective studies with multiple cut-off points for defining high- and low-risk populations.

INTERPHASE
After M phase, the daughter cells each begin a new cycle by proceeding to interphase. Each stage of interphase has a distinct set of specialized biochemical processes that prepares the cell for initiation of cell division.

G1 phase
Interphase begins with G1 (G stands for gap) phase. During this phase, the cell makes a variety of proteins that are needed for DNA replication.

S phase
During S phase, which follows G1 phase, all of the chromosomes are replicated. Following replication, each chromosome now consists of two sister chromatids (figure below). Thus, the amount of DNA in the cell has effectively doubled, even though the ploidy, or chromosome count, of the cell remains at 2n

It is important to note that chromosomes double their number of chromatids post replication but the nuclei remains diploid as the number of centromeres and chromosomes remains unchanged. Hence, the number of chromosomes in the nucleus, which determines the ploidy, remains unchanged from the beginning to the end of the S phase.

In a study of single-Cell Analysis of Ploidy and Centrosomes in hepatic cells, Francesca Faggioli et al. (2011) summarizes the percentage of mono and bi-nucleated hepatocytes at different mouse ages as analyzed by classical eosin/hematoxilin staining.  

The results are measured against a known normal control cell or control value:

    • Rat hepatocytes for epithelial
    • Normal lymphocytes (lymphoproliferative disorders).

Controls are stained with each patient batch as external control to ensure proper staining techniques and correct instrument setup. The specimen results are interpreted by the degree of hyperdiploidy and proliferation index. The histogram provides a quick determination of the presence of abnormal cells in a mixed population. 

The proliferation index provides rapid verification of the cellular proliferation in the sample without search of visible mitotic figures. Many tumor populations exhibit abnormal cell cycle transit patterns, and a significant population may be in various stages. Such a population would be difficult to recognize without either histograms or proliferation indices.

Ploidy analysis can be performed either by flow cytometry or image analysis:

FLOW CYTOMETRY IMAGE ANALYSIS
Specimen should be in the form of individual cells or isolated nuclei Microscopic slides as cell smears, cytospins, cell imprints or tissue sections
Requires significant amount of sample, multi parameter analysis Small sample size, and study more parameters per cell in greater detail.
A rapid assay Retains morphological information.
A small tumor cell population mixed with large number of normal tissue cells, abnormal cells may not be recognized Requires longer time for analyzing
  Tumor cells can be selectively imaged.

Application of DNA Ploidy (ASC)

  • An improved marker to predict outcome
  • As a modifier of established prognostic parameter
  • To predict velocity of tumor growth
  • As a reference benign vs. malignant
  • To design of future prospective treatment protocol

References: 

  1. Baldetorp B, Bendahl PO, Fernö M, Stål O.; Improved DNA flow cytometric, DNA ploidy, and S-phase reproducibility between 15 laboratories in analysis of breast cancer using generalized guidelines; Cytometry A 2003 Nov; 56 (1): 1-7  
  2. Cell Cycle and DNA Content Analysis, BD Bio Sciences  
  3. Francesca Faggioli, Paolo Vezzoni, Cristina Montagna; Single-Cell Analysis of Ploidy and Centrosomes Underscores the Peculiarity of Normal Hepatocytes; Plos, October 12, 2011 
  4. Gerald Li, B.Sc. (Hons),* Martial Guillaud, Ph.D., Michele Follen, M.D., Ph.D., and Calum MacAulay, Ph.D.; Double Staining Cytologic Samples with Quantitative Feulgen-Thionin and Anti–Ki-67 Immunocytochemistry as a Method of Distinguishing Cells with Abnormal DNA Content from Normal Cycling Cells; Anal Quant Cytopathol Histpathol. 2012 Oct; 34(5): 273–284. 
  5. Harvard Edu glossary  
  6. Interphase, Harvard. edu
  7. NIH Consensus Development Program  
  8. Stefan Biesterfeld, Sascha Beckers, Maria del Carmen and Martin Schramm; Feulgen Staining Remains the Gold Standard for Precise DNA Image Cytometry; International journal of cancer research and treatment; Jan 2011, Vol 31, No 1 53-58

Coding Section 

Code Number Description
CPT  88182 Flow cytometry, cell cycle or DNA analysis 
ICD-10-CM  C16.0-C16.9 Malignant neoplasm of stomach [gastric, localized without metastatic disease
  C38.1-C38.2 Malignant neoplasm of anterior and posterior mediastinum (neuroblastoma, localized without metastatic disease)
  C54.1 Malignant neoplasm of endometrium (localized without metastatic disease)
  C56.1-C56.9 Malignant neoplasm of ovary (localized without metastatic disease)
  C57.4 Malignant neoplasm of uterine adnexa, unspecified (localized without metastatic disease)
  C61 Malignant neoplasm of prostate
  C64.1-C64.9 Malignant neoplasm of unspecified kidney, except renal pelvis [localized without metastatic disease
  C67.0-C67.9 Malignant neoplasm of bladder (localized without metastatic disease)
  C71.0-C71.9 Malignant neoplasm of brain (medullo-blastoma in adults only)
  C81.00-C86.6, C88.4, C88.8-C88.9, C90.00-C94.42, C94.80-C95.91, C96.0-C96.4, C96.A-C96.9 Malignant neoplasm of lymphoid, hematopoietic and related tissue
  D00.2 Carcinoma in situ of stomach [gastric]
  D07.0 Carcinoma in situ of endometrium
  D07.5 Carcinoma in situ of prostate
  D09.0 Carcinoma in situ of bladder
  D43.0-D43.3 Neoplasm of uncertain behavior of brain and cranial nerves (intracranial in adults only)
  D45 Polycythemia vera
  D46.0-D46.9 Myelodysplastic syndromes
  D47.0-D47.1, D47.3-D47.9 Other neoplasms of uncertain behavior of lymphoid, hematopoietic and related tissue
  D49.6 Neoplasm of unspecified behavior of brain (adults only)
  D56.4 Hereditary persistence of fetal hemoglobin (HPFH)
  D57.00 - D57.3
D57.80 - D57.819
Sickle-cell disorders
  D58.0 Hereditary spherocytosis
  D58.2 Other hemoglobinopathies
  D59.5 - D59.8 Acquired hemolytic anemia
  D75.81 Myelofibrosis
  D80.0 - D81.2, D81.4
D81.89 - D82.1
D83.0 - D84.9
D89.810 - D89.9
Certain disorders involving the immune mechanism (B-cell monitoring for immunosuppressive disorders and primary immunodeficiencies)
  D86.0 - D86.9 Sarcoidosis
  O01.0 - O01.9 Hydatidiform mole
  R89.7 Abnormal histological findings in specimens from other organs, systems and tissues
  T86.00 - T86.99 Complications of transplanted organs and tissue (postoperative monitoring)
  Z21 Asymptomatic human immunodeficiency virus [HIV] infection status (T cell monitoring)
  Z94.0 - Z94.9, Z95.3 Transplanted organ and tissue status [postoperative monitoring]

Procedure and diagnosis codes on Medical Policy documents are included only as a general reference tool for each Policy. They may not be all-inclusive. 

This medical policy was developed through consideration of peer-reviewed medical literature generally recognized by the relevant medical community, U.S. FDA approval status, nationally accepted standards of medical practice and accepted standards of medical practice in this community, Blue Cross and Blue Shield Association technology assessment program (TEC) and other non-affiliated technology evaluation centers, reference to federal regulations, other plan medical policies and accredited national guidelines.

"Current Procedural Terminology© American Medical Association.  All Rights Reserved" 

History From 2016 Forward     

07/12/2019 

Annual review, no change to policy intent. One clarification made regarding DNA index. Also updating coding. 

06/03/2019 

Updated category from medicine to laboratory. No other changes made. 

07/18/2018 

Annual review, no change to policy intent. 

07/19/2017 

Annual review, updating policy criteria for clarity. 

10/05/2016

New Policy


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