Glandular lesion and Iatrogenic lesion 연세대학교 원주의과대학 병리학교실 최준정 junjeong@yonsei.ac.kr

2nd edition of the Bethesda System – Glandular cells Atypical endocervical cells (NOS or specify in comments) endometrial cells (NOS or specify in comments) glandular cells (NOS or specify in comments) endocervical cells, favor neoplastic glandular cells, favor neoplastic Endocervical carcinoma in situ Adenocarcinoma endocervical, endometrial, extrauterine, NOS

Glandular Lesions of the Female Genital Tract Sampling techniques; Routine smears: The vaginal pool may contain exfoliated cells from the endocervix, endometrium, and endosalpinx, and even from the ovaries 2. Endometrial aspiration 3. Endometrial lavage

Normal endocervical cells Columnar cells with basal nuclei, in flat variable sized sheets or loosely associated Cyanophilic translucent cytoplasm or vacuolated Sheets have a honeycomb appearance Cells in ‘picket-fence’ palisades with basal nuclei and columnar cytoplasm Nuclei rounded, from the side the nuclei are oval Fine chromatin pattern, one or more small nucleoli often near the nuclear membrane Bare nuclei may accompany groups of degenerate endocervical cells

Normal endocervical cells

Endocervical Adenocarcinoma Risk Factors; Estrogen High socio-economic status HPV 16,18 (18 more common in adenoCa.) No relation with smoking Relation with squamous lesion; Endocervical adenoCa. → squamous lesion (>50%) Squamous lesion → adenocarcinoma (4%)

Endocervical Adenocarcinoma Glandular epithelial dysplasia (atypical glandular cells) Adenocarcinoma in situ (AIS) Invasive adenocarcinoma Well differentiated adenocarcinoma without mucin formation Well differentiated mucus-secretory adenocarcinoma Poorly differentiated adenocarcinoma

Adenocarcinoma in Situ (AIS) 10~25% of adenocarcinoma of the cervix Age: usually 10~20 yrs younger than invasive Ca. Symptom; Usually asymptomatic Vaginal bleeding Coexists with SIL or SqCC in 2/3 of cases

Cytologic Findings of AIS -Architectural Features- Cohesive sheets and tissue fragments of varying size in clean background Loss of honeycomb pattern: crowding and overlap of nuclei Specific architectural features; Pseudo-stratified strips of columnar epithelium Feathering (nuclear protrusion at group margins) Epithelial rosettes Crypt opening within an epithelial sheet

Cytologic Findings of AIS -Architectural Features- Exfoliation pattern; Cohesive sheets and tissue fragments of varying size in clean background

Cytologic Findings of AIS -Architectural Features- Exfoliation pattern ; Loss of honeycomb pattern; crowding and overlap of nuclei

Cytologic Findings of AIS -Architectural Features- Exfoliation pattern; Crypt opening may be seen within a sheet

Cytologic Findings of AIS -Architectural Features- Feathering; Stratification Cytoplasm is usually totally or partially lost Nuclei are somewhat attenuated

Cytologic Findings of AIS -Architectural Features- Rosettes;

Cytologic Findings of AIS -Nuclear Features- Higher than normal N/C ratio Nuclear enlargement with pleomorphism: elongation of nuclei common Distinctive coarsely granular, evenly distributed chromatin pattern Nucleoli are not typically prominent Mitoses and apoptotic bodies may be often present

Invasive Adenocarcinoma 8~25% of primary carcinoma of the cervix Age: usually 45~53 yrs Symptom; Watery discharge or vaginal bleeding Coexists with SIL or SqCC in 2/3 of cases

Invasive Adenocarcinoma Well differentiated adenocarcinoma without mucin formation Well differentiated mucus-secretory adenocarcinoma Poorly differentiated adenocarcinoma

Well differentiated adenocarcinoma without mucin formation Pattern: - Flat cell groups with nuclear crowding forming rosette pattern - Nuclear palisading arrangement, common Mitosis in 1/3 of the cases Cells: columnar with basal nuclei Nuclei: oval and hyperchromatic with coarse chromatin Nucleoli: may be present with halo Well-defined group borders (scalloped edges)

MIMICERS Repair Non-keratinizing squamous cell carcinoma Predominantly syncytia Scattered single cells Vacuolated cyonoph

Well differentiated mucus-secretory adenocarcinoma Pattern: papillary groups / disorganized honeycomb pattern with unequal vacuoles Nuclei: - hyperchromatic, anisokaryotic - flattened by large mucous vacuoles

Poorly differentiated adenoCa. Pattern: Three-dimensional Cells: rounded Nuclei: round, hyperchromatic, several nucleoli, abnormal-shaped Cytoplasm: absent vacuoles Tumor diathesis: always present

Endocervical AIS vs. Invasive adenocarcinoma In situ Adenocarcinoma Invasive Adenocarcinoma Architecture Crowded gland/sheet, strips, rosettes Irregular and crowded glands Pseudostratification Stratification with supercrowding Polarity preserved Loss of polarity Peripheral feathering Peripheral feathering may be present Cells: normal or small in size Cells: large N/C: high Single malignant cells: uncommon Single malignant cells: common Nucleus Large Large, pleomorphic Hyperchromasia with evenly distributed chromatin Hyperchromasia, clearing and irregularly distributed chromatin Inconspicuous nucleoli Prominent nucleoli Cytoplsam Focal mucin loss Secretion varies Background: Clear or bloody with intact RBCs Necroinflammatory of bloody, with broken-down RBCs

Distinguishing Points Between SqCC and Adenocarcinoma Cell shape Polygonal Cuboidal, columnar Nuclei Central Eccentric Chromatin Coarse, compact Vesicular Nucleoli Not prominent Prominent Cytoplasm Thick , eosinophlic Thin, vacuoles Cell group Irregular Round, oval Lumen No Gland, acini, rosettes Row Yes

Atypical Endocervical Cells: NOS Cells occurs in sheets and strips with some cell crowding and nuclear overlap Nuclear enlargement, up to 3-5 times the area of normal endocervical nuclei, may be seen. Some variation in nuclear size and shape is present. Mild hyperchromasia is frequently evident. Nucleoli may be present. Mitotic figures are rare. Cytoplasm may be fairly abundant but the N/C ratio is increased. Distinct cell borders often are discernible.

Atypical Endocervical Cells, favor Neoplastic Cell morphology, either quantitatively or qualitatively, falls just short of an interpretation of endocervical adenocarcinoma in situ or invasive adenocarcinoma. Criteria Abrnomal cells occur in sheets and strips with nuclear crowding and overlap. Rare cell groups may show rosetting or feathering Nuclei are enlarged with some hyperchromasia. Occasional mitoses may be seen. N/C ratios are increased, quantity of cytoplasm is diminished, and cell borders may be ill defined.

Normal endometrial cells Epithelial cell aggregates in tubular or sheet-like arrangements Small cells with scanty cytoplasm  Uniform small round nuclei Fine granular chromatin with chromocentres Single or overlapping stromal cell groups or single spindle-shaped cells Epithelial and stromal cells are influenced by hormonal changes.

Endometrial cells may be seen normally in cervical samples up to 12 days from the onset of menstruation. Factors influencing their presence beyond this may reflect underlying endometrial pathology in a woman over 40 years of age or could be due to exogenous hormonal manipulation such as hormone replacement therapy or oral contraceptive use. Tamoxifen use, intrauterine device carriage and dysfunctional bleeding

During and shortly after menstruation they are grouped in well-formed tight three-dimensional clusters with a peripheral rim of epithelial cells and a central core of stromal cells

Degenerative changes quickly supervene, with crumpling of the nuclei and disorganisation of the cells. These then stand out as small clusters of densely hyperchromatic crowded cells Neutrophil polymorphs are often seen within endometrial clusters.

Endometrial Glandular Lesions Endometrial hyperplasia Endometrial atypical hyperplasia and in situ adenocarcinoma Endometrial adenocarcinoma

“TBS” – Glandular cells Atypical endocervical cells (NOS or specify in comments) endometrial cells (NOS or specify in comments) glandular cells (NOS or specify in comments) endocervical cells, favor neoplastic glandular cells, favor neoplastic Endocervical carcinoma in situ Adenocarcinoma endocervical, endometrial, extrauterine, NOS

Endometrial cells, day 4 Exodus, day 7

Endometrial hyperplasia Should be suspected when benign endometrial cells are found 10 days after the end of the last menstrual period Abundance of thick clusters of reactive endometrial cells In postmenopausal women, any crowded clusters of reactive endometrial cells

Cystic hyperplasia, day22 Adenomatous hyperplasia, day22

Endometrial atypical hyperplasia and in situ adenocarcinoma (AIS) Variable numbers of polymorphic endometrial cells Cytoplasm: abundant and eosinophilic Nuclei: enlarged with granular chromatin, irregularly distributed Nucleoli: often prominent

Normal Atypical hyperplasia Atypical hyperplasia, day 25

Malignant glandular lesions Endometrial adenocarcinoma Detection rate; Cervical scraping only: 15% Vaginal pool smear and cervical scraping: 45% Endocervical aspiration & vaginal pool smears: 65% Intrauterine & endocervical aspiration and lavage: 90%

Endometrial adenocarcinoma Risk factors; Estrogen (granulosa cell tumor) Obesity Hypertension Diabetes mellitus High socio-economic status

Helpful hints for diagnosis of endometrial adenocarcinoma (1) Presence of degenerate or well-preserved normal endometrial cells in a post-menopausal woman In an asymptomatic premenopausal woman, the presence of normal-appearing endometrial cells at mid-cycle or post-ovulatory time Presence of an increased number of small or large histiocytes, often with phagocytized necrotic cellular debris, in the non-atrophic smear of any post-menopausal woman (small: 4%, large: 17%)

Helpful hints for diagnosis of endometrial adenocarcinoma (2) Increased number of foreign body giant cells in the non-atrophic smear of any post-menopausal woman Presence of unexplained fresh and old blood or an increase in necrotic debris or fibrin (tumor diathesis) in the smear of a post-menopausal woman A history of DM, obesity, hypertension, breast cancer, family history of breast and ovarian cancer, late menopause, or post-menopausal bleeding

General diagnostic cellular criteria in endometrial adenocarcinoma Number of diagnostic cells: very scanty or abundant Imitate the appearance of the benign columnar cells (arranged in row) Shed in tight clumps, in loose clusters, or singly Background: usually dirty (watery diathesis)

Architectural Loss of polarity Papillary cell clusters Discohesive cells Cellular High nucleo/cytoplasmatic ratio Anisonucleosis and poikilonucleosis Coarse and/or marginated chromatin Nucleolar prominence Nuclear membrane indentations Cell cannibalism Background Scarcity of stromal cells Necrosis.

Differentiation Well Moderate Poor

DDx. between endometrial and endocervical adenocarcinoma Number of cells Less abundant Abundant Degeneration Marked Mild Size 160±40 u2 190±60 u2 Ingestion of leukocytes Rare Nuclear size 60±10 u2 80±20 u2 Chromatin Moderately coarse Finely granular Nucleoli Multiple 20%, Macronucleoli 20% Multiple 60%, Macronucleoli 40% Background Very dirty Relatively clean Squamous dyskaryosis Very rare Occasional

Atypical endometrial cells Cells occur in small groups, usually 5-10 cells/group Nuclei are slightly enlarged compared to normal endometrial cells. Mil hyperchromasia may be seen. Small nucleoli may be present. Scant cytoplasm is occasionally vacuolated. Cell borders are ill defined.

Effects of Irradiation and Other Techniques Cellular changes from radiotherapy; Inhibit the formation of DNA or RNA or cause cross-links and breaks in the DNA double strands of benign & malignant cells (G2-M) Cytoplasm: more abundant, may have peri-nuclear halo Nuclei: - often enlarged and hyperchromatic with prominent Nu. - differ from dysplasia or cancer cells by (1) finely granular or amorphous chromatin (2) smooth regularity of the nuclear membrane

Cellular changes from radiation (radiation response) Mechanism of action; 1. Prevent the synthesis of DNA proteins → rapid death of cells 2. Inhibit cellular mitosis 3. Change the chromosomal or genetic information of cells Radiation responses (RR) = cellular changes resulting from irradiation appear first in the nuclei, then in the cytoplasm

Cellular changes from radiation (radiation response) Some irradiation cellular changes may persist for the whole life of patients Acute reaction: - appear about 1 day after the start of irradiation and continue for 6 weeks after the end of the treatment - cellular necrosis and increase in the exfoliation of degenerated, nonviable, benign or malignant cells Healing stage: mixed with repair & inflammatory cells

Cellular changes from radiation (radiation response) Cytoplasmic changes; Similar changes in chemotherapy, UV irradiation, or cautery 1) Edema and hypertrophy (3~4 times) 2) Biphasic stain and loss of normal fine granularity → amorphous, homogeneous, amphophilic, or eosinophilic 3) Cytoplasmic fibrils, often seen in concentric or irregular 4) Vacuolization 5) Pseudo-cannibalism 6) Cytoplasmic fragments and naked nuclei: -mainly in the late stage of irradiation -indicate the death of cells

Cellular changes from radiation (radiation response) Changes in the nuclei; 1) Nuclear hypertrophy (2-10 times): - most consistent and common changes - may be extremely irregular, with nuclear wrinkling 2) N/C ratio remains within the normal limits 3) Peri-nuclear halo, sometimes with pyknotic nuclei 4) Vacuolization: indicates death of the cell 5) Chromatin (the amount of DNA): -increases but finely granular & uniformly distributed -decreases: pale and empty-appearing nuclei 6) Multi-nucleation

Other irradiation changes; 7) Nucleoli: enlarged, remain round and regular in shape 8) Changes indicating the death of the cells (benign or malignant) (1) Peripheral condensation of the chromatin (2) Vacuolization (3) Fragmentation (4) Nuclear pyknosis (5) Karyorrhexis Other irradiation changes; 1) Soon after irradiation: large No. of inflammatory cells 2) In the healing stage: an increase in repair cells 3) Increase in mucus secretion during radiation

Cytology of irradiated malignant lesions During RTx. & in the 2 wks following it; (1) Cancer cells are shed in large numbers (2) Absence of cancer cells may indicate a poor prognosis (3) Most of malignant cells show irradiation changes similar to those described for benign cells (4) The diagnosis of persistent carcinoma should not be made at this time, because the exfoliated cells with these changes are dead or are still alive (5) The death of a malignant cell should be suspected when the nucleus show pyknosis, karyorrhexis, and karyolysis or vacuolization.

Cytology of irradiated malignant lesions B. 4 wks after irradiation; (1) The presence of malignant cells showing minimal irradiation changes suggest the possibility of persisting cancer (2) These cells usually have the same features as the original non-irradiated malignant cells

Cytology of irradiated malignant lesions C. Recurrence of carcinoma; (1) Should be suspected when malignant cells appear after an interval of smears free of tumor cells (2) Recurrent cancer cells tend to be smaller and less differentiated D. All patients after radiotherapy; Need to be closely followed by periodic repeated smears, taken every 3 ~ 6 months

SqCC, radiotherapy 2 wks

Endometrial adenoCa, radiotherapy 3 wks

Cellular changes from chemotheraphy Similar to cellular changes of irradiation Systemic effect Cyclophosphamide (urinary bladder) & busulfan (lung, pancreas, spleen, urinary tract, uterine cervix) More prominent in nuclei Atrophic smear Need close long-term follow-up: 3 ~ 6 months