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Background PD-1 inhibitors have shown significant efficacy in triple negative breast cancer (BC), however, durable responses are less common in estrogen receptor-positive (ER+) BC. Better markers are therefore needed that will identify likely responders to PD-1 inhibitors among patients with luminal BC. While most efforts have focused on the immune checkpoint protein PD-L1, the alternative PD-1 ligand, PD-L2, has been largely overlooked. We aimed to determine if PD-L2 is associated with unfavorable prognosis in ER+ BC. Methods PD-L2 protein levels in cancer cells and stromal cells were measured retrospectively by quantitative immunofluorescence histocytometry in tissue microarrays of therapy-naïve, localized or locoregional ER+ BC and correlated with progression-free survival (PFS). Evaluable tumor PD-L2 data were derived from a main study cohort A diagnosed between 1988-2005 (n=684) with extensive clinical and outcome data and from an independent validation cohort B diagnosed between 1992-2012 (n=273). Patients received standard-of-care adjuvant therapy without immune checkpoint inhibitors after tumor resection. Results Univariate analysis of the main cohort A revealed that high PD-L2 expression in cancer cells was associated with shorter PFS (HR=1.8; 95%CI:1.3-2.6; p=0.001), an observation that was validated in an independent cohort B (HR=2.3, 95%CI:1.1-4.8; p=0.026). Approximately one third of ER+ BC cases were classified as high PD-L2. After multivariable adjustment for common clinicopathological variables, high cancer cell levels of PD-L2 remained independently predictive of early recurrence (HR=2.0; 95%CI:1.4-2.9; p< 0.001). Sub-analysis of ER+ BC cases treated with adjuvant chemotherapy (n=197) suggested that high PD-L2 levels in cancer cells was associated with particularly increased risk of progression (multivariable HR=3.4; 95%CI:1.9-6.2; p< 0.001). The observed frequent expression of PD-L2 protein in BC provided scientific rationale for the design of our ongoing phase II clinical trial (NCT04243616) of neoadjuvant combined PD-1 inhibitor (cemiplimab; Regeneron Pharmaceuticals Inc) and chemotherapy in patients diagnosed with PD-L1+ and/or PD-L2+ BC. The primary objective of this trial is to assess pathologic responses to neoadjuvant treatment with secondary objective of assessing the correlation between PD-L1/PD-L2 status and tumor responses. Pathologist review of PD-L1 and PD-L2 expression in an initial set of ER+ tumors (n=15) screened for trial eligibility revealed frequent discordance between cancer cell positivity for PD-L1 and PD-L2 protein (PD-L1: median=0%; range 0-2% vs. PD-L2: median=18%; range < 1-60%) as well as immune cell positivity (PD-L1: median=5%; range 0-50% vs. PD-L2: median=0; range 0-50%). Conclusions In treatment-naïve ER+ breast tumors, high cancer cell expression of PD-L2 protein was an independent predictor of poor clinical outcome, with evidence of further elevated risk of progression in patients who received adjuvant chemotherapy. Preliminary analyses of ER+ tumors from our ongoing clinical trial showed frequent discordance between baseline PD-L1 and PD-L2 protein expression in both cancer cells and immune cells. Collectively, our analyses indicate that PD-L2 has prognostic value for ER+ BC, and our progress justify further studies to determine whether PD-L2, alone or in combination with PD-L1, may serve as a predictive marker of response to PD-1 inhibitors. Citation Format: Lubna N. Chaudhary, Inna Chervoneva, Amy R. Peck, Yunguang Sun, Misung Yi, John F. Langenheim, Julie M. Jorns, Sailaja Kamaraju, Yee Chung Cheng, John Burfeind, Christopher R. Chitambar, Jeffrey A. Hooke, Albert J. Kovatich, Craig Shriver, Hai Hu, Juan P. Palazzo, Marluce Bibbo, Terry Hyslop, Richard Pestell, Edith P. Mitchell, Hallgeir Rui. High PD-L2 Protein Expression in Cancer Cells is an Independent Marker of Unfavorable Prognosis in Luminal Breast Tumors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-13.

Supplementary Figure 7: Schematic representation of structure of wildtype Stat5a and Stat5b, dominant-negative Stat5 (DN-Stat5) and constitutively-active Stat5 (CA-Stat5) proteins.

Clinical Characteristics of Outcome Cohorts 1 and 2.

Purpose:Parathyroid hormone-related protein (PTHrP) is required for normal mammary gland development and biology. A PTHLH gene polymorphism is associated with breast cancer risk, and PTHrP promotes growth of osteolytic breast cancer bone metastases. Accordingly, current dogma holds that PTHrP is upregulated in malignant primary breast tumors, but solid evidence for this assumption is missing.Experimental Design:We used quantitative IHC to measure PTHrP in normal and malignant breast epithelia, and correlated PTHrP levels in primary breast cancer with clinical outcome.Results:PTHrP levels were markedly downregulated in malignant compared with normal breast epithelia. Moreover, low levels of nuclear localized PTHrP in cancer cells correlated with unfavorable clinical outcome in a test and a validation cohort of breast cancer treated at different institutions totaling nearly 800 cases. PTHrP mRNA levels in tumors of a third cohort of 737 patients corroborated this association, also after multivariable adjustment for standard clinicopathologic parameters. Breast cancer PTHrP levels correlated strongly with transcription factors Stat5a/b, which are established markers of favorable prognosis and key mediators of prolactin signaling. Prolactin stimulated PTHrP transcript and protein in breast cancer cell lines in vitro and in vivo, effects mediated by Stat5 through the P2 gene promoter, producing transcript AT6 encoding the PTHrP 1-173 isoform. Low levels of AT6, but not two alternative transcripts, correlated with poor clinical outcome.Conclusions:This study overturns the prevailing view that PTHrP is upregulated in primary breast cancers and identifies a direct prolactin–Stat5–PTHrP axis that is progressively lost in more aggressive tumors.

PURPOSE T-cell–mediated cytotoxicity is suppressed when programmed cell death-1 (PD-1) is bound by PD-1 ligand-1 (PD-L1) or PD-L2. Although PD-1 inhibitors have been approved for triple-negative breast cancer, the lower response rates of 25%-30% in estrogen receptor–positive (ER+) breast cancer will require markers to identify likely responders. The focus of this study was to evaluate whether PD-L2, which has higher affinity than PD-L1 for PD-1, is a predictor of early recurrence in ER+ breast cancer. METHODS PD-L2 protein levels in cancer cells and stromal cells of therapy-naive, localized or locoregional ER+ breast cancers were measured retrospectively by quantitative immunofluorescence histocytometry and correlated with progression-free survival (PFS) in the main study cohort (n = 684) and in an independent validation cohort (n = 273). All patients subsequently received standard-of-care adjuvant therapy without immune checkpoint inhibitors. RESULTS Univariate analysis of the main cohort revealed that high PD-L2 expression in cancer cells was associated with shorter PFS (hazard ratio [HR], 1.8; 95% CI, 1.3 to 2.6; P = .001), which was validated in an independent cohort (HR, 2.3; 95% CI, 1.1 to 4.8; P = .026) and remained independently predictive after multivariable adjustment for common clinicopathological variables (HR, 2.0; 95% CI, 1.4 to 2.9; P < .001). Subanalysis of the ER+ breast cancer patients treated with adjuvant chemotherapy (n = 197) revealed that high PD-L2 levels in cancer cells associated with short PFS in univariate (HR, 2.5; 95% CI, 1.4 to 4.4; P = .003) and multivariable analyses (HR, 3.4; 95% CI, 1.9 to 6.2; P < .001). CONCLUSION Up to one third of treatment-naive ER+ breast tumors expressed high PD-L2 levels, which independently predicted poor clinical outcome, with evidence of further elevated risk of progression in patients who received adjuvant chemotherapy. Collectively, these data warrant studies to gain a deeper understanding of PD-L2 in the progression of ER+ breast cancer and may provide rationale for immune checkpoint blockade for this patient group.

Purpose: Parathyroid hormone-related protein (PTHrP) is required for normal mammary gland development and biology. A PTHLH gene polymorphism is associated with breast cancer risk, and PTHrP promotes growth of osteolytic breast cancer bone metastases. Accordingly, current dogma holds that PTHrP is upregulated in malignant primary breast tumors, but solid evidence for this assumption is missing. Experimental Design: We used quantitative IHC to measure PTHrP in normal and malignant breast epithelia, and correlated PTHrP levels in primary breast cancer with clinical outcome. Results: PTHrP levels were markedly downregulated in malignant compared with normal breast epithelia. Moreover, low levels of nuclear localized PTHrP in cancer cells correlated with unfavorable clinical outcome in a test and a validation cohort of breast cancer treated at different institutions totaling nearly 800 cases. PTHrP mRNA levels in tumors of a third cohort of 737 patients corroborated this association, also after multivariable adjustment for standard clinicopathologic parameters. Breast cancer PTHrP levels correlated strongly with transcription factors Stat5a/b, which are established markers of favorable prognosis and key mediators of prolactin signaling. Prolactin stimulated PTHrP transcript and protein in breast cancer cell lines in vitro and in vivo, effects mediated by Stat5 through the P2 gene promoter, producing transcript AT6 encoding the PTHrP 1-173 isoform. Low levels of AT6, but not two alternative transcripts, correlated with poor clinical outcome. Conclusions: This study overturns the prevailing view that PTHrP is upregulated in primary breast cancers and identifies a direct prolactin–Stat5–PTHrP axis that is progressively lost in more aggressive tumors.

Objective: Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) has recently been used for the evaluation of various lesions arising in the porta hepatis. The purpose of this study is to evaluate the diagnostic yield of this increasingly utilized approach to porta hepatis lesions. Study Design: A retrospective study of 72 consecutive samples of porta hepatis lesions obtained via EUS-FNA between 2004 and 2015 was conducted. Clinical histories and endoscopic findings were available prior to the diagnostic interpretation. The diagnosis of each lesion was based on its cytologic features on smears, its histologic features on cell block, a comparison with any relevant prior specimens, immunohistochemistry and flow cytometric studies when applicable. Results: A total of 72 lesions (59 lymph nodes, 2 cysts and 11 masses) were biopsied in 70 patients. Adequate specimens were obtained in 65/72 cases (90%). Most of the lymph nodes were benign (n = 40, 67%) and most of the masses were malignant or suspicious (n = 8, 73%). A variety of diagnoses, primary and metastatic, were made, including hepatocellular carcinoma, cholangiocarcinoma and lymphoma. In addition, we have noted a significant increase in the number of EUS-FNAs in recent years. Conclusion: EUS-FNA is an effective and increasingly utilized diagnostic approach for the evaluation of multiple types of lesions in the porta hepatis.

Introduction Arginase-1 is a novel immunohistochemical (IHC) marker for hepatocellular differentiation. The purpose of this study was to evaluate the expression of Arginase-1 and HepPar-1 in lung adenocarcinoma to assess the potential value of these markers for diagnosing metastatic lung tumors, especially to the liver in fine-needle aspiration specimens. Materials and methods Forty-four cytology specimens of lung adenocarcinoma, obtained by endobronchial ultrasound-guided fine-needle aspiration were retrospectively reviewed. IHC stains for Arginase-1 and HepPar-1 were performed on formalin-fixed paraffin-embedded cell blocks. Tissue from confirmed hepatocellular carcinoma was used as the positive control. TTF-1 IHC stain was performed in all cases. Results All 44 lung adenocarcinoma cases (100%) were negative for Arginase-1, whereas HepPar-1 expression was detected in 3 (7%) of lung adenocarcinomas and negative in 41 (93%). The 3 HepPar-1–positive lung adenocarcinoma cases demonstrated positive TTF-1 IHC stain performed on the same cell block. Although both Arginase-1 and HepPar-1 are useful diagnostic IHC markers to differentiate metastatic lung adenocarcinoma from hepatocellular carcinoma, Arginase-1 IHC stain shows better specificity than HepPar-1 does (Arginase-1 specificity 100% and HepPar-1 specificity 93%). Conclusions Arginase-1 IHC can be used in combination with other markers in the workup of metastatic lung adenocarcinoma, especially to the liver.

Objective: Liquid-based cytology of nongynecological specimens is commonly used in cytology laboratories throughout the world and various processing methods, such as ThinPrep and SurePath, have been reported. The cytological features and performance of liquid-based cytology for various cytology specimens, including body cavity fluids, urine, brushing specimens and fine-needle aspiration of various lesions, were reviewed and compared with the experience of our laboratory and the literature published in PubMed. Study Design: The parameters for the evaluation of liquid-based cytology and conventional smears were described in the various types of specimens. Criteria for the interpretation of nongynecological liquid-based cytology were highlighted to show differences in cell morphology, background and artifacts. Results: The interpretation requires familiarity with the appearance of liquid-based cytology in the various types of preparations to avoid misdiagnosis. Conclusions: Cell blocks can be prepared with specimens preserved in a liquid-based cytology medium and immunocytochemical stains and molecular testing can be successfully performed. These are important adjuncts in order to reach a definitive diagnosis.

Objectives To report our experience with ureteroscopic laser ablation of upper tract urothelial carcinoma (UTUC) in patients with Lynch Syndrome (LS), as defined by a documented germline mutation in the MSH-2 gene. To increase awareness among urologists about UTUC in this unique patient population and refer to genetic counselling when appropriate. Patients and Methods Demographic, clinical and pathological data on 13 consecutive patients with UTUC and documented MSH-2 mutation comprising 15 involved renal units were retrospectively collected. Ureteroscopic evaluations involved biopsy and laser treatment with combination holmium/neodymium yttrium aluminum garnet (YAG) lasers. Tumours were graded from 1 to 3 according to the 1973 World Health Organisation classification by a single pathologist evaluating cell block preparations. Results The mean patient age at initial presentation was 56.5 years, with six of 13 patients having metachronous bilateral UT disease. The mean follow-up was 59 months with a mean number of surveillances of 12. Of 15 affected renal units, 10/15 (67%) of initial tumours involved the ureter with mean lesion size of 17.5 mm, while five of 15 (33%) involved the intrarenal collecting system with mean lesion size of 25 mm. Ureteroscopy cleared 13/15 (87%) lesions and four of those 13 (31%) needed staged procedures. Renal preservation rate was 14/15 (93%) with one nephroureterectomy and one segmental ureterectomy performed. One patient developed metastatic UTUC after 40 months surveillance. No patient presented with bladder tumours but seven of the 13 (54%) developed them within 10 months of the initial ureteroscopy. Conclusions Patients with LS who develop UTUC present at younger ages and appear to be more likely to have bilateral UT disease over their lifetimes vs sporadic UTUC patients. Ureteroscopic laser ablation offers a good renal preservation rate with reasonable cancer control in patients willing to undergo endoscopic surveillance. Development of new bladder tumours is common.

Objective: We performed a retrospective review of Papanicolaou (Pap) testing to assess whether the cytology practice in our institution was affected by the introduction of high-risk (HR) human papillomavirus (HPV) assays over time. Study Design: Cytology, HPV and histopathology records were retrieved from our laboratory information system from 2003 to 2015. Records for Digene Hybrid Capture 2®, Hologic Cervista® and Roche Cobas® HPV assays were obtained. A 3-month follow-up for HPV detected cases was performed, and results were correlated with cytology and biopsies. A 1-year follow-up of HPV 16/18 and other HR HPV detected cases was also performed. Results: From 2008 to 2015, a noticeable decrease in Pap testing volume occurred, from 11,792 to 4,664, while the percentage of HPV testing increased from 19 to 59%. Similar HPV detection rates and follow-up results for both reflex and cotesting were observed in the 3 HPV assays. Conclusions: The decrease in Pap testing was due to the lengthening of the test interval when cotesting results were negative. Practitioners adhering to guidelines accounts for increased molecular testing volume. A trend towards higher-grade cervical intraepithelial neoplasia in the follow-up of detected HPV 16/18 was noted. So far there has been no demand for HPV as a stand-alone test.

Characterization of pancreatic cysts by using EUS-FNA includes chemical and cytologic analysis.To evaluate whether material obtained from FNA of the cyst wall increases diagnostic yield.Prospective series.Tertiary referral center.Consecutive patients with pancreatic cysts referred for EUS-FNA between March 2010 and March 2011.FNA was performed with aspiration of cyst fluid for carcinoembryonic antigen (CEA) and cytology, followed by cyst wall puncture (CWP). CWP is defined as puncturing the far wall of the cyst and moving the needle back and forth through the wall to sample the wall epithelium.The diagnostic yield for mucinous cystic pancreatic neoplasms by CEA and cytology obtained from cyst fluid compared with cytology obtained from CWP. CEA ≥192 ng/mL was considered mucinous.A total of 69 pancreatic cysts from 66 patients were included. Adequate amounts of fluid were aspirated for CEA, amylase, and cytology in 60 cysts (81%). Cellular material adequate for cytologic assessment from CWP was obtained in 56 cysts (81%). Ten (30%) of 33 cysts with CEA192 ng/mL and negative results of cyst fluid cytology had a mucinous diagnosis from CWP; 6 of 9 (67%) cysts with an insufficient amount of fluid for CEA analysis and cyst fluid cytology had a mucinous diagnosis from CWP. Furthermore, 4 malignant cysts were independently diagnosed by CWP cytology. The incremental diagnostic yield of CWP for mucinous or malignant cysts was therefore 29% (20 of 69 cysts, P = .0001). An episode of pancreatitis (1.45%) occurred.Lack of surgical criterion standard.CWP during EUS-FNA is a safe and effective technique for improving the diagnostic yield for premalignant and malignant pancreatic cysts.

Objective: We aimed to supplement microscopic examination of biliary cytobrush specimens to improve sensitivity by mutational profiling of: (1) selected cells microdissected from cytology slides and (2) corresponding cell-free DNA in residual supernatant fluid. Study Design: From 43 patients with brushings of bile or pancreatic duct strictures, DNA was extracted from microdissected cells and 1–2 ml of cytocentrifugation supernatant fluid. Mutational analysis targeted 17 genomic sites associated with pancreaticobiliary cancer, including sequencing for KRAS point mutation and loss of heterozygosity (LOH) analysis of microsatellites located at 1p, 3p, 5q, 9p, 10q, 17p, 17q, 21q, and 22q. Results: Mutations were found in 25/28 patients with malignancy, and no mutations were found in 5/5 patients with benign surgical results. The cell-free supernatant fluid generally contained higher levels and quality of DNA, resulting in increased detection of mutations in most patients. KRAS mutations only occurred in patients with pancreatic cancer. Mutational profiling of supernatant fluid specimens resulted in high sensitivity and specificity for malignancy, improving the detection of malignancy over cytology alone. Conclusion: Brush cytology specimens yielded supernatant fluid enriched with DNA, probably from actively proliferating cells. Mutational profiling can enhance the cytologic evaluation and characterization of specimens suspected to contain pancreatic or bile duct cancer.

Fluorescence in situ hybridization is gaining popularity for transitional cell carcinoma screening. We determined the accuracy of fluorescence in situ hybridization for identifying upper tract transitional cell carcinoma.A retrospective review of our upper tract transitional cell carcinoma database from 2005 to 2008 identified 35 patients with upper tract transitional cell carcinoma who submitted voided urine specimens for fluorescence in situ hybridization at commercial laboratory during a routine office visit. Each patient was evaluated endoscopically in the operating room within 3 months of sampling. Suspicious lesions were biopsied and treated. Transitional cell carcinoma in the lower or upper tract was proved by direct visualization, positive biopsy or upper tract cytology read as positive or highly suspicious for malignancy.Of the patients 35 satisfied study inclusion criteria. A total of 67 fluorescence in situ hybridization specimens were submitted. Upper tract transitional cell carcinoma was identified on 51 operative evaluations, of which 23 showed concurrent bladder tumor. For all encounters the sensitivity of fluorescence in situ hybridization was 56% and specificity was 80%. Sensitivity for low and high grade lesions was 68% and 67%, respectively. Only upper tract tumors were noted in 28 patients, in whom there were 2 false-positive and 13 false-negative voided fluorescence in situ hybridization results. In these cases sensitivity was 54% and specificity was 78% compared to the 18% sensitivity and 100% specificity of bladder cytology. Sensitivity for low and high grade upper tract transitional cell carcinoma was 60% and 50%, respectively.Voided fluorescence in situ hybridization has become an adjunct for bladder transitional cell carcinoma surveillance. However, it has limited value for upper tract tumor surveillance.

Objective To evaluate whether B72.3 and CEA could identify duodenal and gastric contamination in cell blocks of clinically proven cases of pancreatic ductal carcinoma, intraductal papillary mucinous neoplasm (IPMN) and mucinous cystic neoplasm (MCN). Study design Cell blocks of pancreatic fine needle aspirates from 19 ductal adenocarcinomas, 9 IPMNs, 5 MCNs, and 22 cases containing gastrointestinal epithelial contamination (GIC) (7 gastric, 15 duodenal) were stained with antibody to carcinoembryonic antigen (CEA) and B72.3. Results CEA was positive in 89% of adenocarcinomas and 92% of mucinous lesions. It was never expressed in gastric contamination and was positive in 2/15 (13%) duodenal contaminants. B72.3 was positive in 95% of adenocarcinomas and 85% of mucinous lesions. It was positive in 2/7 (28%) gastric and 7/15 (47%) duodenal contaminants. Conclusion In contrast to previous work, our preliminary results indicate that B72.3 expression cannot be reliably used to identify GIC. A lack of CEA expression, however, can be used to identify both gastric and duodenal contamination. This represents an important diagnostic aid in the evaluation of suspected low grade mucinous lesions.

OBJECTIVE To evaluate the diagnostic yield and cytologic accuracy of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in cases of clinically suspected epithelial malignancy, sarcoidosis and lymphoma. STUDY DESIGN Over a 9-month period from inception at Thomas Jefferson University Hospital, a retrospective analysis of the cytologic diagnoses of all EBUS-TBNA procedures performed in 48 patients was undertaken. The patients were divided into 2 groups, those with clinical suspicion of an epithelial malignancy and those with clinical suspicion of sarcoidosis or lymphoma. RESULTS Of the 48 patients who underwent EBUS-TBNA, 39 had adequate fine needle aspiration biopsy samples (60 of 78) with a diagnostic yield of 77%; the pre-EBUS yield was 58%. For the group with malignant disease the calculated sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were all 100%. For the group with benign disease the calculated sensitivity, specificity, PPV and NPV were also 100%. CONCLUSION Preliminary results show that cytologic samples obtained via BUS-TBNA are accurate and specific in making a diagnosis of an epithelial malignancy or benign disease.

Routine use of intraoperative pathologic examination (IOPE), including frozen section (FS) and scrape preparation cytology (SPC), during diagnostic thyroid lobectomy continues to be a source of controversy. We sought to better delineate the usefulness and cost-benefit ratio of IOPE in the context of cytologically diagnosed cellular follicular lesion (CFL) or follicular neoplasm (FN).Records of 205 patients who underwent thyroidectomy for cytologically diagnosed FN or CFL between 1997 and 2005 were retrospectively reviewed. IOPE results, patient demographics, and tumor characteristics were correlated to final histopathologic diagnoses. Sensitivity, specificity, predictive values, accuracy, and costs of IOPE were calculated.IOPE correctly identified 3 of 16 follicular carcinomas and 9 of 36 papillary carcinomas. Sensitivity, specificity, and accuracy were 23%, 99%, and 78%, respectively. On univariate analysis, malignancy risk among follicular nodules did not correlate with age, gender, or nodule size. On multivariate analysis, nodule size was predictive of malignancy (p0.05). Over the entire patient series, routine IOPE resulted in a net cost savings of $74,304.33.IOPE reduced costs and limited the number of completion thyroidectomies necessary. IOPE is specific, cost effective, and of minimal additional risk when performed routinely for patients with CFL or FN.

To pay tribute to the Founders of Acta Cytologica, this Golden Anniversary symposium on nongynecologic cytology revives the written symposium style of the 1950s. Participants from countries throughout the world were asked how new technologies are currently applied in their laboratories and whether future advances and challenges can be predicted. The specific questions and the participants' answers follow.

In this chapter we describe the special procedure to handle ureteroscopic samples in the cytopathology laboratory at Thomas Jefferson University Hospital to achieve the best diagnostic results. Urinary specimens, such as voided urine, catheterized bladder urine, and aspiration of ureter and pelvis are collected in saline and rapidly delivered to the cytopathology laboratory to keep the cellular preservation. Biopsies samples of the tumors are also delivered to the cytopathology laboratory and prepared as cell blocks for histologic assessment. The cytology slides and the cell blocks are then evaluated by experienced pathologists taking into account the cytomorphologic and histopathologic features. Our ability to diagnose and grade UTUC has markedly improved since adopting the described sample processing, providing better guidance for patient management. Immunohistochemical stains, FISH or other molecular studies can be used for tumor diagnosis and grading.

The category “negative for intraepithelial lesion or malignancy” is used for specimens that show a spectrum of nonneoplastic changes, including those associated with protective and reactive responses to inflammation, hormonal alterations, and colonizing or infectious organisms. Recognition of the wide variety of normal cellular presentations is key for the accurate discrimination from neoplastic conditions. Many characteristic patterns of reaction in normal cells can be important clues to the underlying causative process. Some of these patterns have features which are also seen in neoplasia, hence recognition of the key reactive changes is essential for overall accuracy. An expanded variety of “normal” findings as well as non-neoplastic mimics of classic epithelial abnormalities are included in this chapter, providing a more complete representation of the morphologic variations that can be encountered in cervical cytology preparations.

Objective To validate proteins identified by proteomics as potentially usable markers in thyroid pathology. Study design Frozen sections of thyroid tumors were manually micro-dissected and proteins extracted. Two-dimensional (2D) gel electrophoresis and subsequent liquid chromatography/mass spectroscopy were performed, and differentially expressed proteins were identified. Validation of candidates for tumor markers (galectin-1, galectin-3, S100C and voltage-dependent anion channel 1 [VDAC1]) was done by immunohistochemistry in 21 cell blocks from fine needle aspiration biopsies (FNAB) and corresponding histology specimens (13 cases). Results Galectin-3 was negative in benign lesions and positive in FNAB from papillary carcinoma (5 of 5), follicular variant of papillary carcinoma (1 of 4) and follicular carcinoma (1 of 2). S100C was positive in some benign lesions: hyperplasia (2 of 4), goiter (1 of 3) and follicular adenoma (1 of 3), with predominantly nuclear pattern of staining. S100C was positive in malignant lesions, showing cytoplasmic location. Galectin-1 was negative in benign lesions and positive in follicular carcinoma (1 of 2), papillary carcinoma (2 of 5) and follicular variant of papillary carcinoma (1 of 4). VDAC1 was detected in benign and malignant lesions, showing a strong positivity in follicular carcinomas. Conclusion Immunohistochemical validation of potential markers is a crucial step before clinical application in diagnosis. Galectin-3, galectin-1 and S100C can be used to help in discriminating benign and malignant thyroid lesions.

Residual material from liquid-based pap (LBP) test has been used for molecular testing of Human Papilloma Virus (HR HPV) DNA. LBP test specimens (1,025) were evaluated to determine how often residual material was insufficient to perform microbiological testing. The specimens were tested for Chlamydia trachomatis and Neisseria gonorrhea (CCGT) alone and in combination with HR HPV(CCGT + HR HPV), using PCR method and HYBRID CAPTURE 2, respectively. A minimum volume of 5 ml (CCGT: 0.5-1 ml, HPV: 4 ml) of residual material is required for both the tests. The residual material was measured at >5 ml, < or =5 ml, and 0.5 ml volumes and documented. Results were tabulated. The insufficient rate for cases submitted for CCGT was 0.2% and 7.6% for HR HPV, with a detection rate of 40.9% for HR HPV. Utilizing residual material from LBP test for microbiological testing, as well as DNA testing for HR HPV is a convenient, cost-effective means to enhance patient care.

OBJECTIVE: To evaluate the morphologic characteristics of gynecologic samples prepared by 3 different methods of liquid-based cytology. STUDY DESIGN: Cytologic samples from representative cases of each diagnostic category of squamous epithelial lesion, prepared by automated and manual liquid-based systems, were analyzed by 3 laboratories in the United States, Portugal and Brazil. The systems included: ThinPrep® (automated, U.S. Food and Drug Administration approved; Cytyc Corp., Boxborough, Massachusetts, U.S.A.), Autocyte® PREP (South American system, manual; TriPath Imaging, Inc., Burlington, North Carolina, U.S.A.) and DNACITOLIQ® (manual; Digene Brazil, Sao Paulo, Brazil). A panel of 16 morphologic parameters was evaluated: cellularity, clean background, uniform distribution, artifacts, cellular overlapping, architectural and cytoplasmic distortion, cytoplasmic vacuolization, cellular elongation, imprecise cytoplasmic borders, fold-ed cytoplasmic borders, nuclear hyperchromasia, coarse chromatin, prominent nucleolus, irregular nuclear borders, atypical mitosis and inflammatory infiltrate. Negative, atypical squamous cells of undetermined significance, low grade squamous intraepithelial lesion (LSIL) and high grade squamous intraepithelial lesion (HSIL) cases were included. Cases without biopsies were confirmed by consensus. RESULTS: Cellularity was adequate in all samples. Clean background was observed in the vast majority of samples with all liquid-based systems. Uniform distribution was frequently found in ThinPrep® and Autocyte® PREP samples but not in DNACITOLIQ®. Artifacts were not present in DNACITOLIQ® samples, rare in ThinPrep® and observed in 8 (34.7%) Autocyte® PREP. Cellular overlapping was observed in all 3 system samples: 11 (31.42%) cases in ThinPrep®, 16 (69.56%) in Autocyte® PREP and 17 (68%) in DNACITOLIQ® System. Architectural and cytoplasmic distortion were present in 3 cases of HSIL (13%) and cytoplasmic vacuolization in 2 cases of LSIL and 1 HSIL of Autocyte® PREP. Cellular elongation was found in 13 (56.5%) Autocyte® PREP and in 5 (20%) DNACITOLIQ® samples. Inflammatory infiltrate was found in all 3 system samples but with more frequency in the Autocyte® PREP (69.56%) and DNACITOLIQ® System (72%). CONCLUSION: This study clearly indicated that in spite of the different methodologies, the 3 methods adequately preserved cellular structure for morphologic evaluation. The choice of method will depend on price, availability and procedures involved.

BACKGROUND The cytologic diagnosis of adenocarcinoma in a liver mass usually is straightforward. Identifying where the adenocarcinoma arose from is much more problematic. The Das-1 immunostain is directed against a colon specific antigen and has shown excellent sensitivity and specificity for adenocarcinoma of the colon in surgical pathology studies. In the current study, the authors examined the clinical utility of the Das-1 immunostain in the setting of fine-needle aspiration cell block material from the liver. METHODS The cell block material from 77 fine-needle aspiration biopsy specimens from the liver were studied. These included 17 hepatocellular carcinomas, 20 colon adenocarcinomas that were metastatic to the liver, and 40 other malignancies, predominantly adenocarcinomas, that were metastatic to the liver from a variety of primary tumor sites. Each case was stained with the Das-1 immunostain using the avidin-biotin complex method and evaluated in a blinded fashion for membranous and/or cytoplasmic staining. The diagnoses were unblinded and correlated with staining and clinical history. RESULTS Thirteen of 20 metastatic colon carcinoma samples exhibited immunostaining whereas only 2 of the remaining 57 samples of malignancy exhibited immunostaining. CONCLUSIONS The results of the current study suggest that the Das-1 immunostain may prove to be helpful in identifying adenocarcinomas in the liver as arising from the colon. Cancer (Cancer Cytopathol) 2002. © 2002 American Cancer Society.

BACKGROUND Diagnosing liver tumors can be difficult in the setting of a poorly differentiated tumor or tumors with no known prior malignancy. Frequently, α-fetoprotein, carcinoembryonic antigen, factor VIII, and mucicarmine have been employed to distinguish hepatocellular carcinoma (HCC) from adenocarcinoma. However, these stains have their limitations. CA 15-3 and Glut-1 are expressed in a variety of carcinomas. To the authors' knowledge, their expression in HCC has not been studied extensively. The authors examined the clinical utility of CA 15-3 and Glut-1 in the setting of fine-needle aspiration biopsy samples from the liver. METHODS Thirty-five cases of HCC and 59 cases of tumors metastatic to the liver were studied. These cases previously were studied with the hepatocyte paraffin-1 antibody. Each case was stained with CA 15-3 and Glut-1 using the avidin-biotin complex method. Each case was evaluated in a blinded fashion for membranous staining that was stronger than cytoplasmic or background staining. The diagnoses were unblinded and staining patterns were compared. RESULTS CA 15-3 stained 43 of 59 metastatic carcinoma samples and 3 of 35 HCC samples. Glut-1 stained 34 of 59 metastases and 2 of 35 HCCs. Together, the 2 immunostains stained 51 of 59 metastases and 5 of 35 HCCs. Diagnostic accuracy was improved by adding hepatocyte paraffin-1 to the staining panel. CONCLUSIONS CA 15-3 and Glut-1, especially in conjunction with hepatocyte paraffin-1, appear to be helpful in discriminating HCC from other carcinomas. Cancer (Cancer Cytopathol) 2002;96:53–7. © 2002 American Cancer Society.

OBJECTIVE: To develop a procedure for the immunocytochemical detection of P 16INK4A in ThinPrep specimens. STUDY DESIGN: Archived ThinPrep, liquid-based cervical/endocervical cytology specimens (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) diagnosed as LSIL, HSIL and WNL were resampled and fixed in 95% ethanol for at least three days. Rehydration and endogenous peroxidase blocking of both ThinPreps and formalin-fixed, paraffin-embedded tissues were accomplished on a Leica Autostainer (Leica, Deerfield, Illinois, U.S.A.). Microwave antigen retrieval with CitraPlus (Biogenex, San Ramon, California, U.S.A.) was performed using a Panasonic microwave oven (Matsushita Cooking Appliances, Franklin Park, Illinois, U.S.A.) on the high setting twice for five minutes each. After cooling for 20 minutes and undergoing a buffer rinse, the slides were placed in a Dako autostainer (Dako-USA, Carpinteria, California, U.S.A.). The P 16INK4A primary antibody, clone E6H4 (MTM Laboratories, Heidelberg, Germany) was diluted 1:200 in antibody diluent buffer. Detection was accomplished with a mouse nonavidin-biotin EnVision+ polymer (Dako). The expression of P 16INK4A in ThinPreps and corresponding biopsies were scored by two pathologists. A ThinPrep case was scored as positive if it contained >10 abnormal cells with nuclear and cytoplasmic immunocytochemical staining. Corresponding biopsies were scored as exhibiting negative, sporadic, focal or diffuse staining, as described by Klaes et al, Overexpression of P 16INK4A as specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri (Int J Cancer 2001;92:276-284). RESULTS: The P 16INK4A antibody assay was positive in 14 of 19 (73.68%) LSIL ThinPrep cases and in 25 of 26 (96.15%) HSIL ThinPrep cases. Thirty-eight of the 39 (97.44%) biopsies corresponding to the positively stained ThinPreps also were positive, with a staining score of at least focal positivity in the dysplastic regions. The P 16INK4A antibody assay was negative in 5 of 19 (26.32%) LSIL ThinPrep cases and negative in 1 of 26 (3.85%) HSIL ThinPrep cases. The six biopsies corresponding to the negative ThinPreps were similarly negative. The two cytologic specimens diagnosed as WNL were negative for P 16INK4A , as were two tissue control cases with benign diagnoses. Nondysplastic squamous epithelium, identified in 17 biopsy cases, did not stain, nor did nondysplastic squamous cells identified in ThinPrep cases. Sporadic staining of bacteria, inflammatory cells and occasional endocervical glandular cells was identified. CONCLUSION: P 16INK4A expression in ThinPrep specimens correlates with tissue expression of P 16INK4A , as implemented in the above protocol. P 16INK4A may thus serve as a surrogate marker in gynecologic cytology for high-risk HPV infection and for the development of cervical neoplasia.

BACKGROUND Diagnosing liver tumors by fine-needle aspiration biopsy is safe and accurate. However, there are cases that prove diagnostically difficult. Traditionally, immunostains for α-fetoprotein and polyclonal carcinoembryonic antigen have been used to distinguish adenocarcinomas from hepatocellular carcinomas (HCCs). In poorly differentiated tumors, these immunostains have limitations in both sensitivity and specificity. An hepatocyte-specific immunostain has been described in the surgical pathology literature. To the authors' knowledge, this hepatocyte antibody has not been studied in liver fine-needle aspiration biopsies. The authors examined the Hepatocyte Paraffin 1 (HP1) antibody for its diagnostic utility in this cytologic setting. METHODS Cell-block material from 40 cases of HCC and 53 cases of metastatic adenocarcinoma were studied. Slides were stained for HP1 by the avidin-biotin complex method following antigen retrieval. The percentage of malignant cells that exhibited coarse granular staining in the cytoplasm was estimated for all cases of HCC, poorly differentiated HCC, and metastatic adenocarcinoma. RESULTS HP1 was expressed in 83% of all HCCs but in only 56% of poorly differentiated HCCs. Only 2 of 53 (4%) of metastatic tumors expressed HP1. The overall sensitivity of HP1 was 79% and its specificity was 96%. CONCLUSION HP1 was found to be a specific immunostain that may prove helpful in diagnosing all but the most undifferentiated liver tumors biopsied by fine-needle aspiration. Cancer (Cancer Cytopathol) 2001;93:288–291. © 2001 American Cancer Society.

OBJECTIVE: To develop a cytopreparation protocol suitable for satisfactory processing by the AutoCyte® PREP System with the gynecologic specimens collected in PreservCyt® fluid for the ThinPrep® machine. STUDY DESIGN: The residue of a number of gynecologic specimens collected in PreservCyt® and processed by ThinPrep® were processed by AutoCyte® PREP. Some modifications were made in the cytopreparatory protocol in order to obtain satisfactory specimens. RESULTS: The ThinPrep® and AutoCyte® PREP specimens were examined independently. The results were comparable, with a high degree of concordance between the two techniques. CONCLUSION: Gynecologic specimens collected in PreservCyt® and following the ThinPrep® specimen collection protocol can be processed using the AutoCyte® PREP System. Minor protocol modifications provided comparable diagnostic material. Additional studies are needed to explore the feasibility of this approach and fulfill the various U.S. regulatory agency requirements for the liquid-based Pap test.

OBJECTIVE: To determine the utility of immunohistochemical staining for p53 in cell block material for distinguishing reactive mesothelium from borderline or low grade ovarian carcinoma. STUDY DESIGN: Paraffin-embedded cell blocks from paracentesis and pelvic wash fluid of 44 cases of ovarian carcinoma and 20 cases containing only reactive mesothelium were immunostained for p53 using monoclonal antibody DO-7. Tumor grades ranged from borderline to high grade and were serous papillary (33), clear cell (3), mucinous (2), endometrioid (2), mixed serous papillary/clear cell (3) and undifferentiated (1). The three authors independently evaluated the staining, including estimation of the percentage and intensity of positive nuclear staining. RESULTS: A separation of positive from negative cases was seen when staining intensity was considered the critical parameter ; moderate to strong staining was considered truly positive. Seventy-three percent (8/11) of borderline tumors, 80% (8/10) of low grade tumors and 65% (15/23) of intermediate to high grade tumors showed moderate to strong positivity. Percentage of staining was a less-reliable parameter as 25% of negative cases were positive by this assessment. CONCLUSION : p53 Immunohistochemistry, using monoclonal antibody DO-7 combined with standard morphologic evaluation, may be useful in distinguishing benign reactive mesothelium from borderline or low grade ovarian carcinoma.

Although fine-needle aspiration biopsy of salivary gland masses has been reported in the otolaryngology literature, the use of sonography to guide the biopsy of nonpalpable masses and masses seen on other cross-sectional imaging studies has not been described. Our goal was to evaluate sonographically guided biopsy of masses and lymph nodes related to the salivary glands. We analyzed the records of 18 patients who had undergone fine-needle aspiration biopsy of a salivary gland mass or lymph node with a 25-, 22-, or 20-gauge needle. A definitive cytologic diagnosis was made for 13 of the 18 patients (72%); cytology was suggestive but not definitive in three patients (17%) and insufficient in two (11%). Definitive diagnoses were made in three cases of reactive lymph node, in two cases each of lymph node metastasis and Warthin's tumor, and in one case each of pleomorphic adenoma, adenoid-cystic carcinoma, schwannoma-neurofibroma, parotid metastasis, parotid lymphoma, and Sjögren ‘s-related lymphoid-epithelial lesion. Sonographically guided biopsy allows for confident needle placement in masses seen on computed tomography and magnetic resonance imaging. Sonography can usually distinguish a perisalivary lymph node from true intrasalivary masses, and it can help the surgeon avoid the pitfall of a nondiagnostic aspiration of the cystic component of masses. We conclude that sonographically guided biopsy of salivary gland masses can provide a tissue diagnosis that can have a direct impact on clinical decision making.

Recent work has shown CytoRich Red fixative system is effective in lysing red blood cells and reducing background in bloody fluid specimens. The scope of this study was to see whether CytoRich Red can lyse red blood cells in freshly prepared Pap and fine-needle aspiration smears. Paired smears from 20 bloody fine-needle aspirations were prepared. One slide was initially placed in CytoRich Red for up to 30 sec, removed, and then fixed in 95% alcohol. The other slide was placed directly into 95% alcohol. Ten paired Pap smears, one fixed with a commercial fixative and another immersed in a solution of CytoRich Red, were evaluated. All slides were stained with the Papanicolaou stain and analyzed for the amount of red blood cells, background material, and nuclear and cytoplasmic staining. In 100% of all smears utilizing CytoRich Red, red blood cells were significantly reduced without hindering staining. Significant loss of cells from the slides sent in CytoRich Red solution was not observed. CytoRich Red fixative can be effective in reducing red blood cells and background on freshly made smears. In both gynecological and nongynecological cases, diagnostic cells were well preserved and not compromised by blood.

OBJECTIVE: To correlate the ranked review slides by the AutoPap Assisted Primary Screener with the final cytologic diagnosis and to assess whether the ranking of slides improves diagnostic accuracy. STUDY DESIGN: A total of 5,865 consecutive conventional and suitable cervical/ vaginal smears, including high-risk (HR) cases, were processed by the AutoPap System. All slides designated Review were manually screened by two cytotechnologists using the Ranked Review Report. All abnormal findings and reactive/ reparative changes were referred to two attending cytopathologists for a final diagnosis. After screening, the Review slides called Within Normal Limits (WNL) were selected according to rank for a further Quality Control (QC) review by the supervisor. All HR cases not selected by AutoPap for QC review were also rescreened. The final diagnoses of the possibly abnormal/reactive/reparative referred slides were recorded and distributed in five ranks according to the Ranked Review Report assignment. RESULTS: Of 5,865 slides, 5,120 (87%) qualified for scanning. The AutoPap System designated 3,840 (75%) slides for manual review. One thousand three hundred forty-five slides were assigned for QC review. After elimination of nonqualified slides, 763 remained. Rescreening detected 1 high grade squamous intraepithelial lesion (HSIL), 2 low grade squamous intraepithelial lesions (LSIL), 5 atypical squamous cells of undetermined significance (ASCUS) and 5 Benign Reactive Changes (BRC). QC of 364 HR cases revealed 1 LSIL, 2 ASCUS and 3 BRC. Ultimately, 313 (8.1%) smears were diagnosed as ASCUS or a more severe abnormality, 262 (6.8%) as reactive/reparative changes, 3,259 (84.8%) as WNL and 6 (0.1%) as unsatisfactory. Of 313 abnormal slides, 181 were ranked by the system in the 1st rank, 61 in the 2nd, 38 in the 3rd, 19 in the 4th and 14 in the 5th. No HSIL or more severe lesions occurred in the fourth and fifth ranks. CONCLUSION: Our study validated the claim by the manufacturer that a significant epithelial abnormality is more likely to be present if a high score is assigned to a slide. The preliminary results support use of the AutoPap Assisted Primary Screener to improve cytologic diagnosis.

OBJECTIVE: To assess whether a light microscopic, semiquantitative approach could reliably distinguish between benign nonneoplastic, benign neoplastic and malignant oncocytic lesions of the breast and thyroid. STUDY DESIGN: Alcohol-fixed, Papanicolaou-stained fine needle aspiration smears of histologically proven goiter and chronic thyroiditis (18 cases), Hurthle cell adenomas (7 cases), Hurthle cell carcinomas (6 cases), fibrocystic disease (17 cases), papillomas and papillomatosis (7 cases) and apocrine carcinomas (6 cases) were rated by three independent observers using the following 10 cytologic criteria: cellularity, nuclear-cytoplasmic ratio, multinucleation, nuclear size, nuclear shape, anisonucleosis, multinucleolation, nucleolar-nuclear ratio, nucleolar size and nucleolar shape. Each of these 10 cytologic criteria was rated using a 1-3 scale. The scores for all 10 features were summed to give a total score for each case. The total scores were statistically analyzed to determine the validity and reproducibility of the summed criteria. RESULTS: The summed criteria of the total scores were reproducible between the three observers, with standard deviations ranging from 1.36 to 2.88 for thyroid and 1.72 to 2.00 for breast oncocytic lesions. The ability of the total scores to differentiate benign from malignant oncocytic lesions of the breast and thyroid was confirmed by a positive predictive value for malignancy of 67% for thyroid and 72% for breast and a negative predictive value for malignancy of 100% for nonneoplastic oncocytic lesions and >90% for benign oncocytic neoplasms in both. The Kruskal-Wallis test revealed that the total scores were able to distinguish three diagnostic categories of nonneoplastic, benign neoplastic and malignant oncocytic breast and thyroid lesions, with P