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Use & Interpretation of Laboratory Tests Books
Use & Interpretation of Laboratory Tests Books

Bladder Cancer: DNA Ploidy and S-Phase Fraction

DNA ploidy by flow cytometry can be performed on biopsied fresh, frozen or paraffin-embedded tumor tissue and bladder irrigation samples. DNA ploidy should be used in patients with tumor, a past history of tumor or where there is a strong suspicion of tumor. Bladder irrigation is preferred to voided urine in monitoring patients for recurrence. Less than 50% of voided urine specimens have adequate cellularity for flow cytometry and up to one-third can have uninterpretable histograms due to cell degeneration in unfixed specimens. Irrigated bladder specimens should be processed fresh but may be stored at refrigerated temperatures up to 12 hours or fixed in 25% ethanol to prevent cell degeneration. Samples that contain too few cells to be analyzed by flow cytometry can be reflexed to image analysis.1

The majority of grade 1 transitional cell carcinoma of the bladder (TCC) are DNA diploid; whereas, most grade 3 are DNA aneuploid. Grade 2 TCC are a heterogeneous group with equal number of DNA diploid and aneuploid cases; DNA ploidy adds more prognostic information in this group than grade.1 In superficial grade 2 TCC, the 5-year recurrence free rates are 75% for DNA diploid compared to 29% for DNA aneuploid, respectively.2 The percentage of hyperdiploid and hypertetraploid cells are important factors that help sub-classify grade 2 TCC into prognostic groups.3

In patients with superficial bladder cancer (Ta, T1 and tumor in situ [TIS]), DNA ploidy is most helpful in providing prognostic information for progression of tumor into muscle. About 10%-20% of Ta/T1 TCC will eventually develop muscle invasion. Progression is found in 2%, 10% and 50% of low grade (1 or 2) Ta or T1 tumors containing DNA diploid, DNA tetraploid and DNA aneuploid cell populations, respectively. The presence of multiple DNA aneuploid populations is associated with a very poor prognosis. Grade 3 Ta or T1 TCC are nearly all DNA aneuploid, and DNA ploidy is not of value in their prognosis.4-6 DNA diploid Ta/T1 TCC with low S-phase fraction (SPF) (<11%) have a longer recurrence-free survival than those with high SPF.7 In patients with TCC, survival can be stratified depending on the DNA ploidy status and the stage. The mean survival time for DNA diploid T3a or less is 91 months versus 26 months for DNA aneuploid, T3b or T4.8,9

TIS should be suspected when the irrigation specimen is DNA aneuploid and the tumor biopsy is DNA diploid suggesting residual tumor. In TIS the 5-year progression-free survival in patients with one and multiple DNA aneuploid populations is 67% and 20%, respectively. A change from a single DNA aneuploid cell population to multiple aneuploid populations precedes tumor progression by 2 years.10

DNA ploidy can be used with cytology to improve the sensitivity for detection of recurrent TCC. The presence of a clear non-tetraploid DNA histogram is diagnostic of a recurrent tumor. The majority of patients (97%) with cytological diagnosis of cancer have abnormal DNA ploidy; whereas, only 5% of patients with normal cytology have abnormal DNA ploidy.11 Cytology is negative in 98% of DNA diploid, 8% of DNA aneuploid and 69% of DNA tetraploid.12 In patients with atypical cytological findings, 20% with abnormal DNA ploidy have a recurrence compared to only 5% with normal DNA ploidy.11 Comparison of cytology and DNA ploidy in urine and bladder washes for the detection of recurrent TCC shows that cytology has a sensitivity of 57% to74% and a specificity of 96%; DNA ploidy has a sensitivity of 81% to 85% and specificity of 57%.13,14 Combining cytology and DNA ploidy a 95% sensitivity can be achieved with no loss of specificity in detecting recurrent TCC in bladder irrigation specimens.15-17 After a diagnosis of DNA tetraploidy, 45% of patients have no cystoscopic or pathologic evidence of neoplasia, and a diagnosis of DNA tetraploidy with negative cytology should not be equated with recurrent cancer.12

Prediction of recurrence in grade 1 Ta/T1 tumors, which by conventional methods is difficult, can be enhanced by quantitative digital image analysis of chromatin patterns. Combining DNA ploidy and the morphonuclear score (an image analysis-based nuclear grading system) recurrence versus nonrecurrence is predicted in 91% of patients with grade 1 Ta/T1 TCC.18

Patients treated with external beam radiation for muscle-invasive TCC have a clinical response in 100%, 54% and 30% of cases for tumors that are DNA diploid, aneuploid and multiploid, respectively.19,20 About 70% of patients with an SPF <10% survive 10 years in contrast to only 30% of patients with an SPF >10%.19,21

In patients monitored during intravesicular therapy (chemotherapy or Bacillus Calmette-Guerin [BCG]), DNA diploid cells in the bladder wash indicate a good response to treatment; whereas, an aneuploid DNA histogram indicates treatment failure. After radiation treatment, a DNA tetraploid population can be expected up to 2 years post-therapy and should not be interpreted as treatment failure.5
REFERENCES

  1. Walther PJ. The role of flow cytometry in the management of bladder cancer. Hematol Oncol Clin North Am 1992;6:81-98.
  2. Tachibana M, Miyakawa A, Nakamura K, Baba S, et al. Role of proliferative activity estimated by bromodeoxyuridine labeling index in determining predictive factors of recurrence in superficial intermediately malignant bladder tumors. J Urol 1996;156:63-9.
  3. Decaestecker C, van Velthoven R, Petein M, Janseen T, et al. The use of decision tree technique and image cytometry to characterize aggressiveness in World Health Organization (WHO) grade II superficial transitional cell carcinomas of the bladder. J Pathol 1996;178:274-83.
  4. Al-Abadi H, Nagel R. Deoxyribonucleic acid content and survival rates of patients with transitional cell carcinoma of the bladder. J Urol 1994;151:37-42.
  5. Wheeless LL, Badalament RA, deVere White RW, Fradet Y, Tribukait B. Consensus review of the clinical utility of DNA cytometry in bladder cancer. Cytometry 1993;14:478-81.
  6. Norming U. DNA flow cytometry: an update of its use in assessing prognosis for transitional cell cancer of the bladder. Semin Urol 1993;XI:154-63.
  7. Tetu B, Allard P, Fradet Y, Roberge N, Bernard P. Prognostic significance of nuclear DNA content and S-phase fraction by flow cytometry in primary papillary superficial bladder cancer. Hum Pathol 1996;27:922-6.
  8. Lee SE, Park MS. Prognostic factors for survival in patients with transitional cell carcinoma of the bladder: evaluation by histopathologic grade, pathologic stage and flow cytometic analysis. Eur J Urol 1996;29:193-8.
  9. Bittard H, Lamy B, Billery C. Clinical evaluation of cell deoxyribonucleic acid content measured by flow cytometry in bladder cancer. J Urol 1996;155:1887-91.
  10. Norming U, Tribukait B, Gustafson H, Nyman CR, Wang N, Wijkstrom H. Deoxyribonucleic acid profile and tumor progression in primary carcinoma in situ of the bladder: a study of 63 patients with grade 3 lesions. J Urol 1992;147:11-5.
  11. Slaton JW, Dinney CP, Veltri RW, Miller CM, et al. Deoxyribonucleic acid ploidy enhances the cytological prediction of recurrent transitional cell carcinoma of the bladder. J Urol 1997;158:806-11.
  12. Kline MJ, Wilkinson EJ, Askeland R, Given RW, et al. DNA tetraploidy in Feulgen-stained bladder washings assessed by image cytometry. Anal Quant Cytol Histol 1995;17:129-34.
  13. Mora LB, Nicosia SV, Pow-Sang JM, Ku NK, et al. Ancillary techniques in the followup of transitional cell carcinoma: a comparison of cytology, histology and deoxyribonucleic acid image analysis cytometry in 91 patients. J Urol 1996;156:49-5.
  14. Cajulis RS, Haines GK 3rd, Frias-Hidvegi D, McVary K, Bacus JW. Cytology, flow cytometry, image analysis, and interphase cytogenetics by fluorescence in situ hybridization in the diagnosis of transitional cell carcinoma in bladder washes: a comparative study. Diagn Cytopathol 1995;13:214-23.
  15. Giella JG, Ring K, Olsson CA, Karp FS, Benson MC. The predictive value of flow cytometry and urinary cytology in the follow-up of patients with transitional cell carcinoma of the bladder. J Urol 1992;148:293-6.
  16. Murphy WM, Emerson LD, Chandler RW, Moinuddin SM, Soloway MS. Flow cytometry versus urinary cytology in the evaluation of patients with bladder cancer. J Urol 1986;136:815-9.
  17. Amberson JB, Laino JP. Image cytometric deoxyribonucleic acid analysis of urine specimens as an adjunct to visual cytology in the detection of urothelial cell carcinoma. J Urol 1993;149:42-5.
  18. van Velthoven R, Tetein M, Oosterlinck WJ, Roels H, et al. The use of digital image analysis of chromatin texture in Feulgen-stained nuclei to predict recurrence of low grade superficial transitional cell carcinoma of the bladder. Cancer 1995;75:560-8.
  19. Wijkstrom H, Tribukait B. Deoxyribonucleic acid flow cytometry in predicting response to radical radiotherapy of bladder cancer. J Urol 1990:144:646-50.
  20. Lipponen PK, Eskelinen MJ, Nordling S. Progression and survival in transitional cell bladder cancer: a comparison of established prognostic factors, S-phase fraction and DNA ploidy. Eur J Cancer 1991;27:877-81.
  21. Lipponen PK. Review of cytometric methods in the assessment of prognosis in transitional cell bladder cancer. Eur Urol 1992;21:177-83.





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