Experiences of DNA and cell proliferation analysis, and its clinical importance

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Experiences ofDNA and cell proliferation
analysis, and its clinical importance

• Bo Baldetorp
• Department of Oncology
• Lund University
• SWEDEN

Email bo.baldetorp_at_med.lu.se
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Cell cycle
G2M
DNA Histogram
S
G0
G0-G1
G1
G2-M
of Events
S
Fluorescence Intensity
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Diploid/low SPF
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Multiple DNA stemlines
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Non-diploid/high SPF
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Non-diploid/low SPF
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Prognostic value of DNA FCM in different
malignant diseases

• The way is long
• Importance of relevant clinical studies and
  materials
• Lack of Guidelines
• Lack of Accreditation
• Standardized instrumentation, protocols and
  softwares

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Whats the biologic meaning of

• DNA diploidy vs DNA non-dipoidy
• One vs two or more DNA stemlines
• Low vs high DNA index
• Low vs high proliferation status (S-phase)
• The length of DNA synthesis time

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Correlations to..

• Clinical parameters
• Histopathologic data
• Traditional as well as new tumor cell biological
  factors

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DNA FCM- applikationer

• 1. Huvudsakligen maligna tumörsjukdomar
  DNA-ploidi och S-fas bestämning som
  tilläggsinformation för diagnostik prognostik,
  behandlings prediktion och -val
• 2. Cellbiologi t ex cell- respektive växtkinetik
• 3. Övrigt

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DNA FCM

• Blåscancer
• Lungcancer
• Bröstcancer
• Gyn-cancer
• Huvud-halscancer
• Prostatacancer
• Colo-rectalcancer
• Lymfom
• Leukemi
• Skelett- och mjukdelstumörer
• Övriga tumörsjukdomar

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Features of DNA FCM

• Stright forward!
• .and we should continue with DNA FCM!
• Because.
• DNA ploidy can be determined to 100
• S-phase in 90
• Highly reproducible
• Prognostic strength
• Inexpensive

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Estimation of the S-phase planimetrically

• Expand the ordinate in order to more easily find
  a representative S-phase region. 

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DNA FCM S-phase reproducibility. 1st round 12
lab and 67 samples
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Reproducibility in DNA flow cytometric analysis
of breast cancer comparison of 12 laboratories'
results for 67 sample homogenates.Baldetorp B,
Bendahl PO, Ferno M, Alanen K, Delle U, Falkmer
U, Hansson-Aggesjo B, Hockenstrom T, Lindgren A,
Mossberg L, et al.Department of Oncology,
University Hospital, Lund, Sweden.Flow
cytometric (FCM) DNA analysis yields information
on ploidy status and the S-phase fraction (SPF),
variables of prognostic importance in breast
cancer. The clinical value of the SPF is
currently being evaluated in prospective
randomized trials. The widespread use of FCM DNA
analysis emphasizes the importance of
reproducibility (both intra- and
interlaboratory). In this study, 67 nuclear
suspensions of breast cancer samples were
analyzed by 12 laboratories routinely performing
FCM DNA analysis in breast cancer. No general
guidelines were imposed each laboratory used its
own standard protocols. For DNA ploidy status
(diploid vs. non-diploid), agreement was complete
for 79 (53/67) of the samples, compared with 64
(43/67) of samples when tetraploidy was
considered i.e., euploid (diploidtetraploid)
vs. aneuploid (the remaining non-diploid). For
the SPF, pairwise comparison of the results of
all 12 laboratories yielded a mean Spearman's
rank correlation of 0.78 (range 0.54-0.93). For
those 39 samples being categorized in low or high
SPF by all laboratories, all agreed in 14 samples
(36). Similar patterns were obtained with kappa
measures, agreement being good for ploidy status
(diploid vs. non-diploid overall kappa 0.87
and 0.74 for euploid vs. aneuploid), but moderate
for the SPF overall kappa 0.47 (for low SPF
vs. high SPF vs. "no SPF reported").
Discrepancies were chiefly attributable to
differences in the categorization of the S-phase
values, rather than in FCM procedures, other
critical differences being in the detection and
interpretation of near-diploid and small
non-diploid cell populations, the definition of
tetraploidy, and the choice and execution of the
method used for S-phase estimation. Based on the
observations of this study, detailed guidelines
for FCM analysis and interpretation of data are
proposed in the Appendix. Some issues remain,
however, e.g., to standardize a method for
S-phase calculation and tetraploid definition.
Cytometry 22(2) 1995
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DNA FCM 1st Round

• All participating labs attended a workshop where
  the results were discussed.
• Generalized Guidelines were constructed.

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2nd Round 15 labs and 46 samples

• 10 labs from the 1st round participated in the
  2nd round
• The 46 samples in this round were also used in
  the 1st round
• All labs had to follow the generalized guidelines

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DNA FCM
All labs agreed concerning DNA ploidy status
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DNA FCM
S-phase concordance
DNA ploidy concordance All labs agreed in all
samples
Moreover The aquired DNA ploidy and S-phase data
from the five new labs, participating in the
2nd round, were in concordance with those
reported from the ten experienced labs.
Using guidelines, DNA FCM is highly reproducible
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Scandinavian Guidelines for DNA FCM ploidy and
planimetric S-phase estimation

• Fresh/frozen biopsy samples
• - Breast Cancer
• - Lymphoma
• - Bladder cancer
• - Gynaecologic cancer
• - Soft tissue tumors
• Bone marrow samples
• - Acute lymphoblastic leukemia

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Could we be even more reproducible?

• The answer is YES

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Guidelines and tutorials for ModFitLT

• Tutorials
• Video demonstration
• Rules

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Most laboratories in Sweden have now adopted
ModFitLT

• Combination of the Scandinavian Guidelines and
  the ModFitLT-Rules

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Which program?

• ModFitLT
• MultiCycle
• Other

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Indicators of prognosis in node-negative breast
cancer.Sigurdsson H, Baldetorp B, Borg A,
Dalberg M, Ferno M, Killander D, Olsson
H.Department of Oncology, University Hospital,
Lund, Sweden.Measures of the proliferative
activity of tumor cells have prognostic value in
patients with node-negative breast cancer. We
studied 367 women in southern Sweden who had
undergone surgical resection for such cancer.
Tumor specimens were analyzed with DNA flow
cytometry in order to estimate both the DNA
content (ploidy) and the fraction of cells in the
synthetic phase of the cell cycle (S phase). The
median duration of follow-up was four years 28
percent of the patients received adjuvant
therapy, usually with tamoxifen (n 83). A
multivariate analysis based on complete data on
250 patients included the following covariates
age (greater than or equal to 75, 50 to 74, and
less than or equal to 49 years), tumor size (less
than or equal to 20 vs. greater than 20 mm),
concentration of estrogen and progesterone
receptors (less than 10 vs. greater than or equal
to 10 fmol per milligram of protein), ploidy
(diploid vs. nondiploid), and S-phase category
(fraction of cells in S phase less than 7.0
percent, 7.0 to 11.9 percent, and greater than or
equal to 12 percent). The S-phase fraction
yielded the most prognostic information, followed
by progesterone-receptor status and tumor size. A
prognostic model based on these three variables
identified 37 percent of the patients as
constituting a high-risk group with a fourfold
increased risk of distant recurrence. In the
remaining 63 percent of the patients, the
five-year overall survival rate (92 /- 4 /-
SE percent) did not differ from the expected
age-adjusted rate for Swedish women. We conclude
that a prognostic index that includes indicators
of the proliferative activity of tumor cells may
be able to identify women with node-negative
breast cancer in whom the risk of recurrence is
sufficiently low that adjuvant chemotherapy can
be avoided.
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DNA FCM
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Validation of the Baylor model in a prospective
study

• Node-negative cases of invasive breast cancer
  from 237 consecutive premnopausal patients.
• Median follow-up time is 8 years
• Endpoint Distant recurrence free survival

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Fanconi diagnostics
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I. Vid diagnos, behandlingsdag 0. Fraktion
non-diploida lymfatiska celler(gul markering)
97, DNA- Index 1.15, S-fas 5.9. Fraktion
normala diploida celler (röd markering) 3,
DNA-index 1.00
III. Behandlingsdag 15. Fraktion nondiploida
celler (gul markering) 17, S-fas 1.8. Till
följd av pågående behandling har den sjuka
cellpopulationen reducerats men är dock inte
borta. Normala celler (röd markering) 83.

S- fas
IV. Behandlingsdag 43. Den sjuka cellpopulationen
tycks nu vara helt borta. Det kan dock
ej uteslutas en mycket liten kvarvarande
fraktion av sjuka lymfatiska celler som inte kan
detekteras med DNA flödes- cytometri.
II. Behandlingsdag 29. Fraktion non-diploida
celler (gul markering) 5, S-fas 2. Fraktion
normala diploida celler 95. Den sjuka
cellpopulationen har reducerats ytterligare.
Fig 1. Flödescytometriska DNA histogram från en
2-årig patients benmärgsceller, diagnos ALL.
Provtagning och analys vid fyra olika
tillfällen. Med tekniken mäts relativa mängden
DNA i de individuella cellerna från provet.
Histogrammens x-axel anger cellernas DNA mängd,
y-axeln antalet analyserade celler. Vid analysen
tillsätts två olika typer av kontrollceller, vars
respektive DNA mängder (i histogrammen markerade
som gråfärgade toppar) används som interna
kontroller i provet vid beräkningen av DNA index
hos testprovets cellpopulationer.
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