Ricardo S. Cajulis

Expression of the double-stranded RNA-dependent protein kinase (p68) in human breast tissues

P68 is a potent inhibitor of protein synthesis in virally infected cells and has been suggested to function in noninfected cells as a tumor suppressor gene. We have previously demonstrated that p68 expression correlates directly with cellular differentiation and inversely with proliferative activity in normal epithelium and in several human tumor systems. In order to determine the role of p68 in human breast cancer, we utilized immunohistochemistry and mapped the expression of p68 in tissue from 200 breast biopsy specimens. A total of 434 foci, ranging from normal breast tissue to infiltrating carcinoma were examined. We found that p68 was present at basal levels in normal lobular and luminal ductal epithelial cells, with higher levels present in myoepithelial cells. Nonproliferative fibrocystic lesions showed variable expression of p68, with high levels seen within foci of apocrine metaplasia and low levels in cystically dilated terminal duct units. Low levels of p68 were seen in typical ductal proliferations, lobular neoplasia (atypical lobular hyperplasia and lobular carcinoma in situ), and in fibroadenomas. Foci of atypical ductal hyperplasia in situ and invasive ductal carcinoma generally showed higher levels of p68 expression. Among the infiltrating carcinomas, p68 expression correlated with nuclear grade. This suggests that the ability of p68 to inhibit cellular proliferation may be impaired in breast cancer and that its expression, although modestly paralleling cellular differentiation, is not a predictive indicator of improved survival.

Detection of numerical chromosomal abnormalities by fluorescence in situ hybridization of interphase cell nuclei with chromosome-specific probes on archival cytologic samples

Fluorescence in situ hybridization (FISH) is rapidly emerging as a tool for analyzing numerical and structural chromosomal abnormalities in both liquid and solid tumors. Most studies make use of fresh samples. To determine the feasibility of detecting numerical chromosomal abnormalities (NCA) by FISH using chromosome‐specific probes 8, 12, 17, and X (Vysis, Inc., Downers Grove, IL) on archival cytologic preparations, we studied 23 patient samples, one Papanicolaou‐ and one Diff‐Quik‐stained slide per case (46 slides), and two additional unstained slides (fresh ascitic fluids) as controls. Included in this study were nine ascitic fluids (four benign and five malignant), four malignant pleural fluids, three benign bladder washes, and seven malignant fine‐needle aspirates (FNA) from various sites. The slides ranged from 1–94 days old. After removal of coverslips using xylene, all slides were destained in a series of alcohol and water washes. Pretreatment of slides with pepsin was followed by the in situ hybridization procedure. Two hundred cells per slide were evaluated for distinct separate signals. Results showed the following: 1) all slides were evaluable except for eight (8/46) which had either too few cells or enough cells but with faint signals, 2) the oldest sample showed distinct signals, 3) previously Diff‐Quik‐stained slides showed relatively better signals than Papanicolaou‐stained slides, 4) samples less than a month old showed relatively better signals, and 5) malignant samples showed various NCA, but not the benign samples. We conclude that FISH on archival cytologic preparation 1) is feasible, although age of the slide is a factor since better signals were seen in those less than a month old, 2) shows better results in previously Diff‐Quik‐stained slides, and 3) is a tool that can be used in the retrospective study of various liquid and solid neoplasms. Diagn Cytopathol 1996;14:178–181.

Diagnostic significance of signet ring cells in fine-needle aspirates of the breast.

Fine‐needle aspiration (FNA) is a reliable and cost‐effective procedure in the evaluation and management of breast lesions. One diagnostic dilemma that may sometimes arise is the finding of signet ring cells. The isolated finding of such cells in aspirate smears may be particularly problematic in cases of low cellularity or those with otherwise benign features. Although it is generally held that such cells are almost exclusively associated with carcinoma (particularly the lobular subtype), their significance in FNA smears has never been systematically evaluated. To establish their diagnostic utility, we evaluated aspirate smears from 150 cases of histologically proven benign (77) and malignant (73) breast lesions for the presence of signet ring cells, defined as those containing a prominent intracytoplasmic vacuole with nuclear displacement. Signet ring cells were identified in 71% of malignant cases (75% of ductal carcinomas and 71% of lobular carcinomas), mostly as single cells or within small, loosely cohesive tissue fragments. Such cells were also present in 6% of histologically proven benign lesions, most commonly within large tissue fragments. Many of these cells were proven to be vacuolated myoepithelial cells, based on histologic correlation and immunostaining results using anti‐muscle‐specific actin. On the basis of these findings, we conclude that (1) the presence of signet ring cells within small loose tissue fragments or as single cells in FNA smears should prompt close clinical follow‐up (including repeat FNA and perhaps surgical biopsy), regardless of smear cellularity, (2) the presence of signet ring cells in cases of adenocarcinoma does not predict a particular tumor subtype, and (3) rare benign breast lesions may contain signet ring cells, particularly within large tissue fragments, and do not, in isolation, warrant surgical biopsy to exclude malignancy. Diagn. Cytopathol. 16:117–121, 1997.

Cytomorphology of primary CNS lymphoma: review of 23 cases and evidence for the role of EBV.

Primary non‐Hodgkins lymphoma of the central nervous system (PCNSL) has recently increased in incidence, due primarily to an enlarging immunosuppressed patient population. The pathogenetic role of Epstein‐Barr virus (EBV) is of interest due to its established role in other lymphoproliferative disorders in immunosuppressed patients. Twenty‐three cases of histologically confirmed PCNSL with corresponding cytology were identified, all obtained under stereotactic guidance. Twenty patients were human immunodeficiency virus (HIV) positive, two were HIV negative, and one was of unknown status. Papanicolaou‐stained slides were selected from each case and evaluated for the presence of EBV RNA via in situ hybridization (ISH) utilizing a biotinylated probe specific for EBER 1 RNA, and detected by a conventional streptavidin‐peroxidase system. The cases included immunoblastic (12), large cell (10), and mixed small and large cell lymphoma (1). The predominant immunophenotype was B‐cell (19), although T‐cell (2) and biphenotypic (1) cases were also identified. ISH showed nuclear positivity for EBV RNA in 19 of 23 cases (83%). This study confirms the presence of EBV in PCNSL in immunosuppressed patients and implies a potential etiologic role. The ability to demonstrate EBV RNA in cytologic preparations by ISH also raises the possibility of early identification of high‐risk patients through detection of EBV‐infected lymphocytes in CSF specimens. Diagn Cytopathol 1996; 14:114–120.

Intraoperative Diagnostic Techniques for Stereotactic Brain Biopsy: Cytology versus Frozen-Section Histopathology

Stereotactic brain biopsy has gained widespread acceptance as a primary diagnostic tool for the evaluation of intracranial lesions. Intraoperative evaluation of such specimens has included the use of both cytological and frozen section histologic techniques. The current study seeks to compare the diagnostic utility of frozen section histopathology and cytology in the intraoperative evaluation of stereotactic brain biopsies in HIV-seropositive patients. Seventy-five HIV-seropositive patients undergoing stereotactic brain biopsy for the evaluation of intracranial lesions were evaluated; intracranial diseases were predominantly infectious or hematologic malignancies. Comparison of frozen section and cytology as a means of intraoperative evaluation showed cytology to have a greater sensitivity (86 vs. 78%), positive (95 vs. 90%) and negative (50 vs. 39%) predictive values and a greater overall diagnostic efficiency (84 vs. 75%) than frozen section. Thus, cytology is a highly effective tool equaling and in some cases surpassing frozen section in terms of sensitivity, predictive value, and overall accuracy. Cytologic examination may often be used as the sole means of intraoperative diagnosis, obviating the need for the freezing and sectioning of fresh tissue and potentially reducing specimen turn around time as well. In other cases, cytology can be used in conjunction with other methodologies for arriving at both intraoperative and final diagnoses in these often difficult cases.

The immediate postconization endocervical smear: evaluation of its utility in the detection of residual dysplasia.

OBJECTIVE To determine the diagnostic value of obtaining an endocervical smear for cytologic examination immediately following cervical conization (by either the loop electrosurgical excision procedure or large loop excision of the transformation zone) in the detection of residual squamous dysplasia. STUDY DESIGN Thirty-eight cases were identified in which cervical conization was immediately followed by endocervical sampling (most commonly using a brush) and smear. RESULTS Twenty-one of the 38 postconization endocervical smears (55%) were either unsatisfactory or sub-optimal for evaluation due to cellular distortion (i.e., cautery artifact), degeneration or obscuring blood. Histologic in evaluation showed negative endocervical margins in 32 cases (84%) and positive endocervical margins in 6 cases (16%), including both low and high grade squamous intraepithelial lesions. The endocervical smears in the 32 cases with a negative surgical margin did not demonstrate evidence of dysplasia. However, in the six cases with histologically positive margins, postconization endocervical smears also failed to identify any evidence of dysplasia. CONCLUSION Immediate postconization endocervical smears do not appear to be useful for the detection of residual disease in patients undergoing conization for squamous dysplasia of the cervix.

Cytologic Grading of Fine Needle Aspirates of Breast Carcinoma by Private Practice Pathologists

OBJECTIVE To determine the reproducibility of two clinically proven grading systems for breast carcinoma assessed by private practice pathologists. STUDY DESIGN Twenty fine needle aspiration (FNA) slides of histologically proven breast carcinoma were submitted to 15 private practice pathologists practicing in 11 separate groups who interpret cytology as part of their daily practice. The pathologists received the same set of slides. They graded the FNAs using the modified Black (MB) (grades 1-3) and simplified Black (SB) (low grade, high grade) grading systems. Specified criteria and guidelines as well as case samples were provided. RESULTS There was complete agreement among the 15 pathologists on only one case using the MB grading system as compared to five using the SB grading system. In MB, > or = 10 pathologists were in agreement on 14 cases as compared to 19 cases using SB. There were three cases in MB where the grades ranged from 1 to 3. Also noted in MB was the high number of cases graded 2 (intermediate grade). The predominant comment made by the pathologists was the easier, more objective and practical application of the SB. CONCLUSION High reproducibility in the cytologic grading of FNA of breast carcinoma can be more readily attained among private practice pathologists using the two-tier SB grading system.

Touch preparation cytological evaluation of radical prostatectomy specimens

The failure of current histological techniques to predict local failure and disease progression after radical prostatectomy is supported by substantial evidence. Moreover, the characterization of histological findings is hampered by the lack of uniform interpretation. We report a prospective study of 92 patients undergoing radical prostatectomy for clinical stages A and B prostate cancer in which the technique of touch preparation cytological analysis of surgical margins is compared to the standard histological approach. We evaluated 47 pathological stage B, 43 stage C and 2 stage D specimens. Specimens initially assigned to pathological stage B were upstaged to stage C on review by 1 blinded pathologist in 19 of 65 cases (29%). Overall, 15 of 47 histological stage B specimens (32%), 20 of 43 histological stage C specimens (47%) and 2 of 2 histological stage D specimens (100%) had malignant cells identified on the margins by touch preparation cytology. Postoperative mean followup of 7 months (range 0.4 to 26) revealed that 7 of 14 nonstage D cancer patients (50%) with elevated serum prostate specific antigen levels had positive cytology results, including 1 with histologically confirmed organ-confined disease. Among the stage C specimens cytology was more likely to be positive if there was concomitant seminal vesicle invasion. Correlation of this information with eventual patient course during the long term will be necessary to assess its role in patient management.

Modified interphase cytogenetics technique as an adjunct in the analysis of atypical cells in body fluids

Our objective was to determine the value of modified interphase cytogenetics technique (MICT) by fluorescence in situ hybridization (FISH) in the study of atypical cells in body fluids in previously stained slides, allowing a direct morphologic‐cytogenetic correlation. Thirty‐five cases (29 bladder washes, four pleural fluids, two ascitic fluids) initially diagnosed as “atypical” with subsequent histologic confirmation were included. Histologically, there were 25 malignant, four dysplastic, and six benign lesions. Previously Papanicolaou or Diff‐Quick‐stained slides were marked to determine the location of the cells of interest prior to FISH analysis. A pretreatment modification using pepsin digestion was utilized. Chromosome‐specific probe 8 (Vysis) was used to detect numerical chromosomal abnormalities (NCA) involving chromosome 8. Various NCA (aneuploid) were detected in the atypical cells of histologically proven malignant cases but not in the benign cases. Using histology as a “gold standard.” FISH has a sensitivity of 83% and a specificity of 100%. In conclusion this study shows that a MICT by FISH on previously stained slides can serve as an adjunct in the study of atypical cells in body fluids. This technique allows a direct morphologic‐cytogenetic correlation which in the future may aid in the better understanding of carcinogenesis. Diagn. Cytopathol. 16:331–335, 1997.

Cytomorphology of progressive multifocal leukoencephalopathy (PML): review of sixteen cases occurring in HIV-positive patients.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disorder of the central nervous system (CNS) resulting from infection of oligodendrocytes by JC virus. Although all patients immunocompromised by any congenital, acquired, or iatrogenic condition are at risk, the population which currently accounts for the majority of new cases is that infected with the human immunodeficiency virus (HIV). Though the clinical/radiologic presentation is characteristic, biopsy confirmation is necessary, as these patients are at risk for other primary CNS disorders which may produce similar clinical findings. Immediate assessment of tissue adequacy by cytologic smear is generally preferred in these specimens due to its relative reduced risk of disease transmission when compared to conventional frozen section. We report here the cytologic findings seen in touch imprints and squash preparations of 16 cases of PML, all occurring in HIV‐positive patients and obtained by stereotactic guided needle biopsy. Typical cytomorphologic findings are described and correlated with histologic sections. In addition, features useful in the exclusion of other differential diagnostic possibilities are discussed. Diagn Cytopathol 1996;14:4–9.