High PD-L2 Predicts Early Recurrence of ER-Positive Breast Cancer.

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.

Loss of Nuclear Localized Parathyroid Hormone-Related Protein in Primary Breast Cancer Predicts Poor Clinical Outcome and Correlates with Suppressed Stat5 Signaling.

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.

Review of Cytology Practice at Thomas Jefferson University Hospital before and after High-Risk Human Papillomavirus Testing.

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.

Diagnostic Yield of Endoscopic Ultrasound-Guided Fine-Needle Aspiration Cytology of Porta Hepatis Lesions: A Retrospective Study.

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.

Arginase-1 and HepPar-1 expression in fine-needle aspiration specimens of primary lung adenocarcinoma.

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.

Overview of nongynecological samples prepared with liquid-based cytology medium.

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.

Mutational analysis of cytocentrifugation supernatant fluid from pancreatic solid mass lesions.

Diagnosis of fine-needle aspirations of pancreatic solid masses is complicated by many factors that keep its false-negative rate high. Our novel approach analyzes cell-free cytocentrifugation supernatant, currently a discarded portion of the specimen. Supernatant and cytology slides were collected from 25 patients: 11 cases with confirmed outcome [five positive (adenocarcinoma) and six negative (inflammatory states)], plus 14 without confirmed outcomes. Slides were microdissected, DNA was extracted from microdissections and corresponding supernatants, and all were analyzed for KRAS point mutation and loss of heterozygosity. Notably, higher levels of free DNA were found in supernatants than in corresponding microdissected cells. Supernatants contained sufficient DNA for mutational profiling even when samples contained few to no cells. Mutations were present in 5/5 malignancies and no mutations were present in inflammatory states. In conclusion, these findings support using supernatant for mutational genotyping when diagnostic confirmation is required for pancreatic solid masses.

Ureteroscopic biopsy of upper tract urothelial carcinoma: comparison of basket and forceps.

PURPOSE: To compare two different biopsy devices for upper tract urothelial carcinoma (UTUC) and evaluate the pathologic result obtained by these devices. PATIENTS AND METHODS: From January 2008 to December 2010, 414 ureteroscopies were performed and 504 biopsies were taken for evaluation of UTUC. Two biopsy devices were compared: 2.4F stainless steel flat wire basket and 3F cup biopsy forceps. The effect of the biopsy device on obtaining an adequate pathologic specimen was evaluated using univariate and multivariate binary logistic regression analysis. We also investigated whether tumor grade determination was affected by the biopsy device among patients with a diagnostic biopsy. RESULTS: Diagnosis was successful in 63% and 94% in the forceps and basket groups, respectively (P < 0.0001). Among biopsies with a definite diagnosis of UTUC, specific grade was determined in 80% and 93% in the forceps and basket groups, respectively (P = 0.033). In subgroup analysis of tumors larger than 10 mm in diameter, diagnosis was obtained in 80% and 94% in the forceps and basket groups, respectively (P = 0.037). Cytologic evaluation was found to increase diagnostic rates. CONCLUSIONS: The stainless steel flat wire basket was shown to be superior to the 3F cup biopsy forceps in terms of obtaining tissue diagnosis and providing specific grade.

Thyroglobulin wash testing in the surveillance of patients with thyroid carcinoma: proposal for a reflex test.

OBJECTIVE: Fine needle aspiration (FNA) cytology with thyroglobulin wash (TG-W) testing is recommended for follow-up of patients with differentiated thyroid carcinoma (DTC). The goal of this retrospective study was to determine if TG-W results contributed to the management of cases with positive FNA cytology. STUDY DESIGN: We reviewed data on patients with positive and suspicious cytology results, undergoing lymph node or thyroid bed FNA with TG-W testing as part of the preoperative or follow-up investigation of histologically proven DTC in our institution and from the literature. RESULTS: Of 30 positive/suspicious lymph node and thyroid bed FNAs in our institution, 22 (73%) had an elevated (>1 ng/ml) TG-W level. Seven of 8 TG-W-negative cases had DTC on follow-up. Of 577 cytology-positive/suspicious FNAs in the literature, 557 (97%) showed TG-W-positive results. Fourteen of 20 TG-W-negative cases had DTC on follow-up. All patients in retrospective and literature review groups with positive and suspicious FNA cytology and available follow-up were treated for recurrent or metastatic disease regardless of TG-W results. CONCLUSION: Observations of both our and other institutions support a recommendation of reflex FNA TG-W testing only for cases with negative or indeterminate cytology results.

The potential value of phosphohistone-h3 mitotic index determined by digital image analysis in the assessment of pancreatic endocrine tumors in fine-needle aspiration cytology specimens.

OBJECTIVE: According to the World Health Organization, pancreatic endocrine tumors are graded by assessment of the Ki67 proliferation index and/or mitotic count. The objective was to find comparable grading on the basis of the novel mitotic marker phosphohistone-H3 (PHH3). STUDY DESIGN: A computer-assisted system was used to assess 23 cell blocks stained with PHH3 and Ki67 antibodies. We investigated possible cut-points for PHH3 and computed percent agreement between the PHH3- and Ki67-based grading. RESULTS: The Spearman correlation between percent Ki67 positive and percent PHH3 positive was 0.76 (p = 0.001). A value of 0.3% for the lower cut-point ('cut-point 1', differentiating between grades 1 and 2) and values of about 1.8-1.9% for the higher cut-point ('cut-point 2', differentiating between grades 2 and 3) shows optimal agreement between PHH3 and Ki67 grading. The percentage of positive cells was much higher for Ki67 than for PHH3 (mean 10.6 vs. 3.0%). CONCLUSIONS: PHH3 has good correlation with Ki67, but the range of PHH3 positivity is much narrower than that of Ki67 (range 0-4% for PHH3 vs. 0-50% for Ki67). Therefore, to be as accurate, grading on the basis of PHH3 requires evaluation of a larger number of tumor cells for a precise determination of percent PHH3-positive nuclei.

Ureteroscopic management of upper tract urothelial carcinoma (UTUC) in patients with Lynch Syndrome (hereditary nonpolyposis colorectal cancer syndrome).

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.

Molecular analysis of centrifugation supernatant fluid from pancreaticobiliary duct samples can improve cancer detection.

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.

Classification of the four main types of lung cancer using a microRNA-based diagnostic assay.

For patients with primary lung cancer, accurate determination of the tumor type significantly influences treatment decisions. However, techniques and methods for lung cancer typing lack standardization. In particular, owing to limited tumor sample amounts and the poor quality of some samples, the classification of primary lung cancers using small preoperative biopsy specimens presents a diagnostic challenge using current tools. We previously described a microRNA-based assay (miRview squamous; Rosetta Genomics Ltd., Rehovot, Israel) that accurately differentiates between squamous and nonsquamous non-small cell lung cancer. Herein, we describe the development and validation of an assay that differentiates between the four main types of lung cancer: squamous cell carcinoma, nonsquamous non-small cell lung cancer, carcinoid, and small cell carcinoma. The assay, miRview lung (Rosetta Genomics Ltd.), is based on the expression levels of eight microRNAs, measured using a sensitive quantitative RT-PCR platform. It was validated on an independent set of 451 samples, more than half of which were preoperative cytologic samples (fine-needle aspiration and bronchial brushing and washing). The assay returned a result for more than 90% of the samples with overall accuracy of 94% (95% CI, 91% to 96%), with similar performance observed in pathologic and cytologic samples. Thus, miRview lung is a simple and reliable diagnostic assay that offers an accurate and standardized classification tool for primary lung cancer using pathologic and cytologic samples.

Does mitosis-specific marker phosphohistone H3 help the grading of upper tract urothelial carcinomas in cell blocks?

BACKGROUND: Grading upper tract urothelial carcinomas (UTUC) in cell blocks with small distorted tissue fragments can be challenging; interobserver agreement is poor among pathologists. Mitotic figure (MF) counting along with nuclear features is important in grading these tumors. We evaluated the use of the mitotic-specific marker phosphohistone H3 (PHH3) as an adjunct to hematoxylin and eosin (H&E) stain for grading UTUC in cell blocks. METHODS: Formalin-fixed, paraffin-embedded tissues from the cell blocks of 61 UTUC were stained with H&E and PHH3 antibody. The grading of tumors was performed independently by 3 pathologists, on both H&E-stained and PHH3 plus H&E-stained slides. The grading system used was the 1973 WHO 3-point grading system. Gradings were compared by all the pathologists for H&E staining versus PHH3 plus H&E staining with the Stuart-Maxwell test of marginal homogeneity that accounts for the matched data. RESULTS: The average pairwise agreement by H&E alone was 55%, and 80% by PHH3 plus H&E. CONCLUSION: By adding PHH3 immunostain to the H&E, the agreement in grading the carcinomas among the 3 pathologists improved dramatically. PHH3 immunostain may play an important role in grading UTUC in small cell block samples.

Assessment of fine needle aspiration specimen adequacy for high-risk HPV detection and genotyping in oropharyngeal squamous cell carcinoma.

OBJECTIVE: The aim of this study was to determine the adequacy of archived and fresh fine needle aspiration (FNA) specimens from metastatic head and neck squamous cell carcinoma (SCC) for the molecular detection and genotyping of high-risk (HR) HPV. STUDY DESIGN: Thirty-seven specimens from 26 patients diagnosed by FNA with metastatic SCC were included as retrospective specimens [19 slides stained with Papanicolaou (Pap) and 18 with Diff-Quik® (DQ)]. Twenty fresh FNA specimens from 18 patients were included as prospective specimens. These specimens were analyzed using the standard protocol for ThinPrep® cervical specimens, with a Cervista HR HPV detection kit. The positive specimens were tested for the HPV 16 and 18 genotypes. RESULTS: Forty-four of 57 specimens (77%) had sufficient cells to yield a valid HPV result. The adequacy rate for Pap-stained slides was 15/19 (79%), for DQ-stained slides it was 13/18 (72%), and for fresh needle aspirates it was 16/20 (80%). HR HPV was detected in 23/44 (52%) specimens. Among the 23 HPV-positive specimens, 19 were genotyped as HPV 16 and 1 as HPV 18. CONCLUSIONS: HR HPV detection and genotyping can be performed on FNA specimens of head and neck SCC prospectively collected in PreservCyt as well as on archival slides with either Pap or DQ stain.

Targeted cyst wall puncture and aspiration during EUS-FNA increases the diagnostic yield of premalignant and malignant pancreatic cysts.

BACKGROUND: Characterization of pancreatic cysts by using EUS-FNA includes chemical and cytologic analysis. OBJECTIVE: To evaluate whether material obtained from FNA of the cyst wall increases diagnostic yield. DESIGN: Prospective series. SETTING: Tertiary referral center. PATIENTS: Consecutive patients with pancreatic cysts referred for EUS-FNA between March 2010 and March 2011. INTERVENTION: 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. MAIN OUTCOME MEASUREMENTS: 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. RESULTS: 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 CEA <192 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. LIMITATION: Lack of surgical criterion standard. CONCLUSIONS: CWP during EUS-FNA is a safe and effective technique for improving the diagnostic yield for premalignant and malignant pancreatic cysts.