paris system of urine (cytology)Dr.Ankita Singh

Dr.Ankita

1. Urine Cytology

2. Where it started 2013, May 18 international congress of cytology, Paris 1 st international attempt to standardize urine cytology . Joint initiative of the American Society of cytopathology (ASC) & the international academy of cytology.( IAC)

3. Introduction Standardize reporting of urine cytology is necessary t o provide a clinically relevant & synaptic report. Main purpose of urine cytology is t o detect urothelial carcinoma. D r G eorge P apanicolaou (1945)- 1 st to hypothesize microscopic evaluation of exfoliated cells in urine.

4. Type of urine specimens- Voided urine- it has low cellularity with presence of umbrella cells, few intermediate cells and squamous cells(women). Cell clusters are rare. Instrumented specimens- forced exfoliative specimens that includes urinary bladder washing specimens. Other specimens are washings and brushings from urethra, ureters and renal pelvis. Cellularity is high due to contamination with non-urothelial cells. Numerous cell clusters seen.

5. Ileal conduit Urinary diversion specimens are urines obtained from patients who underwent cystectomy by one of the surgical procedures designed to reroute the urine flow (ileal conduit, neobladder ). Very cellular and composed mainly of degenerated glandular cells, either single or in clusters, resembling histiocytes in a dirty background with mucus and bacteria.

6. Pathogenesis Schematic representation of the two major pathways of urothelial carcinogenesis: while recurrences are common in both pathways, invasive disease is seen only in the dysplasia pathway (HGUC); the dotted line represents a questionable transition pathway from LGUC to HGUC. Image courtesy- Paris system of reporting urine cytology

7. Diagnostic categories for Paris system Non diagnostic/ unsatisfactory. Negative for high grade urothelial carcinoma (NHGUC) Atypical urothelial cells (AUC) Suspicious for high grade urothelial carcinoma (SHGUC) High grade urothelial carcinoma (HGUC) Low grade urothelial neoplasm (LGUN) Other: primary & secondary malignancies and miscellaneous lesions.

8. Adequacy Determined by 4 factors Collection type Cellularity Volume Cytomorphologic findings .

9. Adequacy Cytomorphological findings should be considered first since presence of any atypical, suspicious or malignant findings make a specimen adequate regardless of collection time, cellularity and volume. Fresh voided urine should be >30 ml volume. In instrumented sample- at least 20 well preserved, well visualized urothelial cells per 10 high power field.

10. 1. Non diagnostic/ unsatisfactory : S ample quality compromised due to- Degenerative changes Overgrowth of contaminant, microbes or Cell obscured by blood, inflammatory cells or artefacts

11. 2.Neg a tive for high grade urothelial carcinoma (NHGUC) Benign urothelial , squamous and glandular cells. Benign urothelial tissue fragments/ sheets. Changes associated with urolithiasis, viral cytopathic effect, polyoma virus. Post treatment/ radiation induced changes. A bsence of atypical, suspicious or malignant cells in an adequate sample. Inflammation may be referred as reactive changes but reported as negative for HGUC .

12. B enign sBuperficial (umbrella) urothelial cells Superficial cells are large, shaped like the canopy of an umbrella, with rounded (convex) luminal surfaces and scalloped (concaved) borders onto which the underlying intermediate cells are sometimes attached. Cytoplasm is abundant and vacuolated or foamy. Often bi- or multinucleated with single large nucleus. The nuclei are centrally located, round to oval, with smooth nuclear membranes. The chromatin is fine and an occasionally prominent nucleolus is present. Characteristically, the N/C ratio is low.

13. Benign squamous cells. Two benign squamous cells line up below an umbrella cell with three nuclei. The presence of squamous cells in voided urine may come from external genitalia, including the vagina. In a catheterized patient, their origin is usually in an area of metaplasia in the lining of the bladder between the ureteral orifices and the urethra, the trigone. Image courtesy- Paris system of urine vytology .

14. Intermediate urothelial cells:-N uclei with basically the same size and character as superficial cells with less cytoplasm, imparting a higher N/C ratio. Nuclear chromatin may be a bit coarser than the superficial cells, but thin nuclear membranes and uniform chromatin distribution will support their totally benign condition. Cytoplasm will not be as vacuolated as superficial cells, but is not completely opaque (homogeneous), the latter feature being cited as a characteristic of low-grade urothelial neoplasms.

15. Superficial (umbrella) cells and clusters of smaller cells are seen ( arrows ). The nuclei are darker and slightly smaller than superficial cells but the nuclear shapes are round, nuclear membranes are smooth, and architecture is uniform. N/C ratios are high due to the small amount of cytoplasm of each cell. Image courtesy- Paris system of urine cytology.

16. Benign glandular cells—Glandular cells in a urinary tract specimen may be native to the urinary collecting system, or external to it. The larger cells are urothelial or superficial squamous . The Benign cluster of small dark cells originated in an endometriosis of the ureter. The patient was presenting with hematuria and pain coincident with the patient’s menstrual periods

17. Benign urothelial tissue fragments Causes of BUTF in voided urines are multifold and include prostate/rectal manipulation prior to collection of the sample, jogging, abdominal palpation, etc. Most often BUTF are of no clinical importance. Instrumented urine specimens are often cellular, consisting of numerous benign appearing cells arranged in groups that resemble papillary clusters with smooth borders that lack fibrovascular cores. These are defined as “true tissue fragments”. If all the criteria of benign urothelial cells are present then the call is BUTF and the sample is considered NHGUC, unless other criteria of atypia, suspicious or HGUC are present in the specimen, or if evidence of another significant lesion, e.g. LGUN is seen.

18. Benign urothelial tissue fragment (BUTF). ( a ) Voided. BUTF can be seen in voided urines, and should not mandate a diagnosis of “atypical”. In this fragment, nuclei are uniform in size and shape, evenly spaced, and with finely granular chromatin ( b ) Instrumented from renal pelvis. Cell fragments from the renal pelvis should be cautiously considered. In this case, the diagnosis rendered was “suspicious for low-grade neoplasm”. The excision of the kidney revealed only urothelial hyperplasia overlying a subepithelial hemangioma. Retrospective review recognized the uniform nuclear size and round shape. Image courtesy- Paris system for reporting urine cytology.

19. Urothelium with Nephrolithiasis Voided urine specimens from patients who may present with hematuria or filling defect . cellular smear consist of three-dimensional urothelial fragments composed of cells that may exhibit significant pleomorphism .If a cause of the atypia is found, such as a stone, then the diagnostic category should be NHGUC. .The cells in the fragments or clusters may also exhibit nuclear enlargement and atypia, slightly increased N/C ratios, nuclear and cytoplasmic degeneration, and/or squamous metaplasia. With a confident diagnosis of lithiasis, and without single HGUC cells in the sample, the findings may be placed in the NHGUC category

20. Kidney and bladder stones can cause serious changes in the urothelium, sometimes resembling neoplasms. Careful examination of the cells in a three-dimensional TTF is critical to an accurate diagnosis. These cells have round nuclei which are evenly spaced. Chromatin is finely granular and nucleoli are inconspicuous. A renal calculus was discovered on imaging studies and from the clinical history.

21. Urothelium changes characteristic of inflammation Acute bacterial infection- cellular specimen comprising of reactive urothelial cells with slight enlarged nuclei and prominent nucleoli but with fine granular chromatin and thin nuclear membrane. Presence of infiltrating neutrophils admixed with reactive urothelial cells supports a reactive benign process.

22. Reactive umbrella cells. ( a ) Most inflamed epithelial cells demonstrate changes, especially in the nuclei. Nucleoli may become prominent, but nuclear chromatin will remain finely granular and shapes will remain round. The cytoplasm retains its transparency. Neutrophils are infl ammatory cells, but lymphocytes may be present if a chronic process is ongoing ( b ) Changes consistent with epithelial repair are identical with those in all other body sites. The epithelium appears stretched but all cells stay connected, retaining their intercellular connections. Image courtesy- Paris system of urine cytology.

23. Viral cytopathic effects The most important and most common virus identified in urine specimens is polyoma virus. Other viruses may be seen are HPV, CMV. Polyoma virus infected cells are enlarged with single, homogeneous basophilic inclusions occupying most of the enlarged nuclear area. Nuclear membranes of those cells are smooth and regular in shape as compared to the irregular nuclear membranes in high-grade malignant cells, which they often mimic (decoy cells).

24. ( a ) Polyoma cytopathic effect. The classic changes include enlargement of the nucleus and nuclear chromatin homogenization, caused by the viral infection. The shape of the nucleus is always round or oval with a very smooth outline. Cytoplasm is almost gone ( b ) dissolution of the nuclear chromatin is also a characteristic of polyoma (BK) virus infection. The size and shape of the nucleus are the same as the classic features ( c ) If the focal plane is changed, then a spider web of degenerated chromatin comes into view

25. Urothelial Changes Associated with Treatment Effects Radiation-induced cytomorphologic changes will display significant cytomegaly and nucleomegaly and preserved N/C ratio. Multinucleation may be seen and nuclear and cytoplasmic vacuoles larger than the native cytoplasmic vacuoles, are often demonstrated. Intravesical BCG immunotherapy can cause granulomatous inflammation in urine specimens. On the other hand, intravesical mitomycin and thiotepa usually affect superficial cells and cause nuclear enlargement, multinucleation and hyperchromasia of those cells, all of which are non-specific but may be worrisome.

26. Seminal vesicle cells Seminal vesicle cells in urine specimens often have a bizarre appearance with greatly enlarged nuclei and foamy fragmented minimal cytoplasm. Chromatin is hyperchromatic, degenerated, and smudgy. In contrast, the chromatin of malignant cells is coarse. As in prostatic specimens, seminal vesicle cells may be distinguished from cancer cells by the presence of golden brown lipofuscin pigment.

27. Seminal vesicle cells are unusual and may provide confusion with HGUC cells because of their large size and nuclear hyperchromasia . Two clues to their identity include intracytoplasmic yellow lipofuscin pigment ( arrow ), and accompanying sperm.

28. 3. Atypical urothelial cells Major + 1minor MAJOR( required ) Non-superficial & nondegenerated urothelial cells with high N/C >0.5 MINOR ( only one required ) Mild nuclear hyperchromasia Irregular nuclear membrane, Irregular, coarse, clumped chromatin.

29. Atypical urothelial cells (AUC). Two groups of urothelial cells are shown. The group at the top is composed of intermediate type urothelial cells with smooth nuclear contours, and no features of atypia. The urothelial cells in the group on the bottom have high N/C ratios, and nuclear contour irregularity. Nuclear chromasia is similar in both groups. Due to the cytologic atypia seen in the group on the bottom this case should be categorized as AUC

30. 4.Suspicious for high grade urothelial carcinoma : Nonsuperficial & nondegenerated urothelial cells with increased N/C >0. 5-0.7 Moderate to severe nuclear hyperchromasia and Any 1 of the following : Irregular clumpy chromatin Marked Irregular nuclear membrane.

31. Suspicious for high-grade urothelial carcinoma (SHGUC). Rare but abnormal well preserved intermediate urothelial cells showing increased N/C ratios, hyperchromasia , and irregular nuclear membranes

32. Suspicious for high-grade urothelial carcinoma (SHGUC). A few abnormal well preserved intermediate urothelial cells display increased N/C ratios, hyperchromasia , prominent nucleoli, and irregular nuclear membranes in the absence of evaluable chromatin details

33. 5.High grade urothelial carcinoma(HGUC) Criteria of malignancy Cellularity: At least 5–10 abnormal cells N/C ratio: 0.7 or greater Nucleus: Moderate to severe hyperchromasia Nuclear membrane: Markedly irregular Chromatin: Coarse/clumped

34. Other notable features Cellular pleomorphism Marked variation in cellular size and shapes, i.e., oval, rounded, elongated, or plasmacytoid (Comet cells) Scant, pale, or dense cytoplasm Prominent nucleoli Mitoses Necrotic debris Inflammation

35. High-grade urothelial carcinoma (HGUC) displays coarse chromatin and nuclear membrane irregularity High-grade urothelial carcinoma (HGUC) with cytoplasmic vacuolization reflects glandular differentiation. Nuclear membrane irregularity, hyperchromasia , and coarse chromatin typify HGUC

36. High-grade urothelial carcinoma. ( a ) The sample is hypercellular showing numerous tumor cells that demonstrate pleomorphism and necrosis in the background . ( b ) The sample was full of these abnormal cells with high N/C ratios and prominent nuclear profiles

37. However, percentage of urinary cytology cases reported as “positive for malignancy” is relatively low.

38. 6. Low grade urothelial neoplasm ( LGUN ): LGUN – it is a combined cytologic term for low grade papillary urothelial neoplasm( includes- urothelial papilloma, PUNLMP, & LGPUC ) Criteria for LGUN is three- dimensional cellular papillary clusters- with fibrovascular cores including capillaries. Cellular papillary cluster are defined as cluster of cells with nuclear overlapping forming papillae.

39. Positive for LGUN. Three-dimensional papillary structures have central cores. Notice mild cytologic atypia and disorganization of cells forming papillae.

40. Other Malignancies Primary malignancy of bladder other than urothelial origin- SCC, adeno ca, small cell ca. SCC is the second most common malignancy accounting for 2-5% of all malignancies. However, in countries endemic for Schistosoma hematobium infection (North Africa and the Middle East), it is responsible for about 25–30 % of all bladder malignancies.

41. SqCC of urinary bladder with coarse dysplastic nuclear features. Compare the size and staining of the neoplastic cell nuclei to the umbrella cell in the upper right hand corner of the image Non-keratinizing SqCC of urinary bladder shows metaplastic type of cells with rigid basophilic cytoplasm and hyperchromatic angulated malignant nuclei

42. Primary AdCa of the urinary bladder is a malignant neoplasm derived from metaplastic urothelium showing histologically pure glandular differentiation. Urachal AdCa develops within urachal remnants . Extrapulmonary small cell carcinoma ( SmCC ) is rare and can occur at diverse sites including the bladder and accounts for less than 1 % of all urinary bladder carcinomas.

43. Adenocarcinoma, not otherwise specified ( AdCa , NOS) displays a cluster of cells with eccentrically placed irregular nuclei, prominent nucleoli, and finely vacuolated cytoplasm. In an individual fragment it is difficult to determine if the apparent vacuolization is due to glandular secretion or degeneration, but the observation of many such fragments results in a conclusion of glandular differentiation

44. Metastatic tumors are adenocarcinoma prostate, colorectal adenoca and squamous cell carcinoma of uterine cervix. RCC, breast carcinoma and gastric carcinoma can also metastasize to bladder.

45. Ancillary studies There have been many ancillary studies used for urine cytology, but only a few are currently US Food and Drug Administration (FDA) approved to be used in the laboratory setting; namely: UroVysion FISH (Abbott Molecular Inc, Des Plaines, Ill., USA), ImmunoCyt ( Scimedx , Denville, N.J., USA), BTA stat ( Polymedco , Cortlandt Manor, N.Y., USA), and NMP 22 ( Allere , Waltham, Mass., USA). The FDA approval for these tests are for voided urine specimens only. Of these, one of the most commonly used to clarify inconclusive cytological findings is the UroVysion FISH test, likely because of its morphologic applicability to the cytopathology laboratory.

46. Relative Risk of the diagnostic categories outlined in The Paris System, based on few studies

47. Take Home message Algorithmic approach to diagnosis of urinary cytology in The Paris System 1 .

48. THANK YOU

49. References Barkan , G. A., Wojcik, E. M., Nayar , R., Savic -Prince, S., Quek, M. L., Kurtycz , D. F., & Rosenthal, D. L. (2016). The Paris System for Reporting Urinary Cytology: The Quest to Develop a Standardized Terminology.  Acta cytologica ,  60 (3), 185–197. Diagnostic cytopathology:- winifred Gray .