Antibody αPEP13h reacts with lymphangioleiomyomatosis cells in lung nodules.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is characterized by the proliferation in the lung, axial lymphatics (eg, lymphangioleiomyomas), and kidney (eg, angiomyolipomas) of abnormal smooth muscle-like LAM cells, which express melanoma antigens such as Pmel17/gp100 and have dysfunctional tumor suppressor tuberous sclerosis complex (TSC) genes TSC2 or TSC1. Histopathologic diagnosis of LAM in lung specimens is based on identification of the Pmel17 protein with the monoclonal antibody HMB-45. METHODS: We compared the sensitivity of HMB-45 to that of antipeptide antibody αPEP13h, which reacts with a C-terminal peptide of Pmel17. LAM lung nodules were laser-capture microdissected to identify proteins by Western blotting. RESULTS: HMB-45 recognized approximately 25% of LAM cells within the LAM lung nodules, whereas αPEP13h identified > 82% of LAM cells within these structures in approximately 90% of patients. Whereas HMB-45 reacted with epithelioid but not with spindle-shaped LAM cells, αPEP13h identified both spindle-shaped and epithelioid LAM cells, providing greater sensitivity for detection of all types of LAM cells. HMB-45 recognized Pmel17 in premelanosomal organelles; αPEP13h recognized proteins in the cytoplasm as well as in premelanosomal organelles. Both antibodies recognized a Pmel17 variant of approximately 50 kDa. CONCLUSIONS: Based on its sensitivity and specificity, αPEP13h may be useful in the diagnosis of LAM and more sensitive than HMB-45.

Human melanoma metastases demonstrate nonstochastic site-specific antigen heterogeneity that correlates with T-cell infiltration.

PURPOSE: Metastasis heterogeneity presents a significant obstacle to the development of targeted cancer therapeutics. In this study, we sought to establish from a large series of human melanoma metastases whether there exists a determined pattern in tumor cellular heterogeneity that may guide the development of future targeted immunotherapies. EXPERIMENTAL DESIGN: From a cohort of 1,514 patients with metastatic melanoma, biopsies were procured over a 17-year period from 3,086 metastatic tumors involving various anatomic sites. To allow specific tumor cell profiling, we used established immunohistochemical methods to perform semiquantitative assessment for a panel of prototypic melanocyte differentiation antigens (MDA), including gp100, MART-1, and tyrosinase. To gain insight into the endogenous host immune response against these tumors, we further characterized tumor cell expression of MHC I and MHC II and, also, the concomitant CD4(+) and CD8(+) T-cell infiltrate. RESULTS: Tumor cell profiling for MDA expression demonstrated an anatomic site-specific pattern of antigen expression that was highest in brain, intermediate in soft tissues/lymph nodes, and lowest in visceral metastases. Hierarchical clustering analysis supported that melanoma metastases have a phylogenetically determined, rather than a stochastic, pattern of antigen expression that varies by anatomic site. Furthermore, tyrosinase expression was more frequently lost in metastatic sites outside of the brain and was uniquely correlated with both endogenous CD8(+) and CD4(+) T-cell infiltrates. CONCLUSION: Site-specific antigen heterogeneity represents a novel attribute for human melanoma metastases that should be considered in future therapy development and when assessing the responsiveness to antigen-specific immunotherapies.

Occult metastases in lymph nodes predict survival in resectable non-small-cell lung cancer: report of the ACOSOG Z0040 trial.

PURPOSE: The survival of patients with non-small-cell lung cancer (NSCLC), even when resectable, remains poor. Several small studies suggest that occult metastases (OMs) in pleura, bone marrow (BM), or lymph nodes (LNs) are present in early-stage NSCLC and are associated with a poor outcome. We investigated the prevalence of OMs in resectable NSCLC and their relationship with survival. PATIENTS AND METHODS: Eligible patients had previously untreated, potentially resectable NSCLC. Saline lavage of the pleural space, performed before and after pulmonary resection, was examined cytologically. Rib BM and all histologically negative LNs (N0) were examined for OM, diagnosed by cytokeratin immunohistochemistry (IHC). Survival probabilities were estimated using the Kaplan-Meier method. The log-rank test and Cox proportional hazards regression model were used to compare survival of groups of patients. P < .05 was considered significant. RESULTS: From July 1999 to March 2004, 1,047 eligible patients (538 men and 509 women; median age, 67.2 years) were entered onto the study, of whom 50% had adenocarcinoma and 66% had stage I NSCLC. Pleural lavage was cytologically positive in only 29 patients. OMs were identified in 66 (8.0%) of 821 BM specimens and 130 (22.4%) of 580 LN specimens. In univariate and multivariable analyses OMs in LN but not BM were associated with significantly worse disease-free survival (hazard ratio [HR], 1.50; P = .031) and overall survival (HR, 1.58; P = .009). CONCLUSION: In early-stage NSCLC, LN OMs detected by IHC identify patients with a worse prognosis. Future clinical trials should test the role of IHC in identifying patients for adjuvant therapy.

Association of occult metastases in sentinel lymph nodes and bone marrow with survival among women with early-stage invasive breast cancer.

CONTEXT: Immunochemical staining of sentinel lymph nodes (SLNs) and bone marrow identifies breast cancer metastases not seen with routine pathological or clinical examination. OBJECTIVE: To determine the association between survival and metastases detected by immunochemical staining of SLNs and bone marrow specimens from patients with early-stage breast cancer. DESIGN, SETTING, AND PATIENTS: From May 1999 to May 2003, 126 sites in the American College of Surgeons Oncology Group Z0010 trial enrolled women with clinical T1 to T2N0M0 invasive breast carcinoma in a prospective observational study. INTERVENTIONS: All 5210 patients underwent breast-conserving surgery and SLN dissection. Bone marrow aspiration at the time of operation was initially optional and subsequently mandatory (March 2001). Sentinel lymph node specimens (hematoxylin-eosin negative) and bone marrow specimens were sent to a central laboratory for immunochemical staining; treating clinicians were blinded to results. MAIN OUTCOME MEASURES: Overall survival (primary end point) and disease-free survival (a secondary end point). RESULTS: Of 5119 SLN specimens (98.3%), 3904 (76.3%) were tumor-negative by hematoxylin-eosin staining. Of 3326 SLN specimens examined by immunohistochemistry, 349 (10.5%) were positive for tumor. Of 3413 bone marrow specimens examined by immunocytochemistry, 104 (3.0%) were positive for tumors. At a median follow-up of 6.3 years (through April 2010), 435 patients had died and 376 had disease recurrence. Immunohistochemical evidence of SLN metastases was not significantly associated with overall survival (5-year rates: 95.7%; 95% confidence interval [CI], 95.0%-96.5% for immunohistochemical negative and 95.1%; 95% CI, 92.7%-97.5% for immunohistochemical positive disease; P = .64; unadjusted hazard ratio [HR], 0.90; 95% CI, 0.59-1.39; P = .64). Bone marrow metastases were associated with decreased overall survival (unadjusted HR for mortality, 1.94; 95% CI, 1.02-3.67; P = .04), but neither immunohistochemical evidence of tumor in SLNs (adjusted HR, 0.88; 95% CI, 0.45-1.71; P = .70) nor immunocytochemical evidence of tumor in bone marrow (adjusted HR, 1.83; 95% CI, 0.79-4.26; P = .15) was statistically significant on multivariable analysis. CONCLUSION: Among women receiving breast-conserving therapy and SLN dissection, immunohistochemical evidence of SLN metastasis was not associated with overall survival over a median of 6.3 years, whereas occult bone marrow metastasis, although rare, was associated with decreased survival. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00003854.

The clinical immunohistochemistry laboratory: regulations and troubleshooting guidelines.

The Clinical Laboratory Improvement Amendments (CLIA) set standards designed to improve the quality of all laboratory testing. In the first portion of this chapter, we discuss the CLIA requirements that apply to most Immunohistochemistry laboratories, and explain topics such as certification, test complexity, patient test management, proficiency testing, personnel, quality control, quality assurance, and compliance. The second portion of this chapter addresses the most common problems encountered in immunohistochemical procedures and the appropriate solutions to correct them.

Persistent non-neoplastic gammadelta-T cells in cerebrospinal fluid of a patient with hepatosplenic (gammadelta) T cell lymphoma: a case report with 6 years of flow cytometry follow-up.

Hepatosplenic (gammadelta) T-cell lymphoma (HSTCL) is an uncommon T-cell lymphoma with an aggressive clinical course and poor prognosis. Bone marrow and peripheral blood are frequently involved, with central nervous system involvement less common. We describe a case of a 31-year old man diagnosed with a gammadelta HSTCL in 2003, successfully treated with chemotherapy and allogeneic stem cell transplantation, and followed from 2003 to present. Four-color flow cytometry (FC) was performed on a BD FACSCalibur and data analyzed with CellQuest Pro and FCS Express software. For cerebrospinal fluid (CSF), all cells were acquired due to limited material. Cytological correlation was available on all specimens. Molecular studies for T-cell gene rearrangement were non-contributory. By FC, the diagnostic HSTCL immunophenotype was CD3 (+), CD7 (+), CD2 (+), CD5 (-), CD4 (-), CD8 (-), TCR gammadelta (+). Subsequent CSF FC analysis revealed a distinct population of gammadelta T-cells in all specimens, ranging from <1% to 13% of lymphocytes. Consistently, the gammadelta T-cells exhibited a different immunophenotypic profile from the reported diagnostic immunophenotype; they expressed CD5, and exhibited a heterogeneous pattern of CD8 expression. Comparison to in-house cases from patients with hairy cell leukemia and concomitant increases in non-neoplastic gammadelta T-cells was performed. The persistent gammadelta T-cells from the CSF of the patient with HSTCL were immunophenotypically consistent with non-neoplastic gammadelta T-cells. We describe an unusual case of persistent gammadelta T-cells in the CSF of a patient during 6 years of flow cytometric follow-up after treatment for gammadelta HSTCL. By cytology, non-neoplastic and malignant gammadelta T-cells are often difficult to distinguish. FC analysis helps to make this distinction, even with a limited panel. By FC, the gammadelta-T cells in the CSF of this patient are immunophenotypically consistent with non-neoplastic gammadelta T-cells. Remarkably, this finding is underscored by the patient's unusual clinical picture; he remains well and disease free.

Ductal lavage in women from BRCA1/2 families: is there a future for ductal lavage in women at increased genetic risk of breast cancer?

PURPOSE: Ductal lavage has been used for risk stratification and biomarker development and to identify intermediate endpoints for risk-reducing intervention trials. Little is known about patient characteristics associated with obtaining nipple aspirate fluid (NAF) and adequate cell counts (> or =10 cells) in ductal lavage specimens from BRCA mutation carriers. METHODS: We evaluated patient characteristics associated with obtaining NAF and adequate cell counts in ductal lavage specimens from the largest cohort of women from BRCA families yet studied (BRCA1/2 = 146, mutation-negative = 23, untested = 2). Fisher's exact test was used to evaluate categorical variables; Wilcoxon nonparametric test was used to evaluate continuous variables associated with NAF or ductal lavage cell count adequacy. Logistic regression was used to identify independent correlates of NAF and ductal lavage cell count adequacy. RESULTS: From 171 women, 45 (26%) women had NAF and 70 (41%) women had ductal lavage samples with > or =10 cells. Postmenopausal women with intact ovaries compared with premenopausal women [odds ratio (OR), 4.8; P = 0.03] and women without a prior breast cancer history (OR, 5.2; P = 0.04) had an increased likelihood of yielding NAF. Having breast-fed (OR, 3.4; P = 0.001), the presence of NAF before ductal lavage (OR, 3.2; P = 0.003), and being premenopausal (OR, 3.0; P = 0.003) increased the likelihood of ductal lavage cell count adequacy. In known BRCA1/2 mutation carriers, only breast-feeding (OR, 2.5; P = 0.01) and the presence of NAF (OR, 3.0; P = 0.01) were independent correlates of ductal lavage cell count adequacy. CONCLUSIONS: Ductal lavage is unlikely to be useful in breast cancer screening among BRCA1/2 mutation carriers because the procedure fails to yield adequate specimens sufficient for reliable cytologic diagnosis or to support translational research activities.

Check Sample Abstracts.

The following abstracts are compiled from Check Sample exercises published in 2008. These peer-reviewed case studies assist laboratory professionals with continuing medical education and are developed in the areas of clinical chemistry, cytopathology, forensic pathology, hematology, microbiology, surgical pathology, and transfusion medicine. Abstracts for all exercises published in the program will appear annually in AJCP.

The needle in the haystack: application of breast fine-needle aspirate samples to quantitative protein microarray technology.

BACKGROUND: There is an unmet clinical need for economic, minimally invasive procedures that use a limited number of cells for the molecular profiling of tumors in individual patients. Reverse-phase protein microarray (RPPM) technology has been applied successfully to the quantitative analysis of breast, ovarian, prostate, and colorectal cancers using frozen surgical specimens. METHODS: For this report, the authors investigated the novel use of RPPM technology for the analysis of both archival cytology aspirate smears and frozen fine-needle aspiration (FNA) samples. RPPMs were printed with 63 breast FNA samples that were obtained before, during, and after treatment from 21 patients who were enrolled in a Phase II trial of neoadjuvant capecitabine and docetaxel therapy for breast cancer. RESULTS: Based on an MCF7 cell line model of breast adenocarcinoma, the sensitivity of the RPPM detection method was in the femtomolar range with a coefficient of variance <13.5% for the most dilute sample. Assay linearity was noted from 1.0 microg/microL to 7.8 ng/microL total protein/array spot (R(2) = 0.9887) for a membrane receptor protein (epidermal growth factor receptor; R(2) = 0.9935). CONCLUSIONS: The results from this study indicated that low-abundance analytes and phosphorylated and nonphosphorylated proteins in specimens that consist of a few thousand cells obtained through FNA can be quantified with RPPM technology. The ability to monitor the in vivo state of cell-signaling proteins before and after treatment potentially will augment the ability to design individualized therapy regimens through the mapping of aberrant cell-signaling phenotypes. The mapping of these protein pathways will further the development of rational drug targets.

The effects of antibody clone and pretreatment method on the results of HER2 immunostaining in cytologic samples of metastatic breast cancer: A query and a review of the literature.

The standardization and use of heat-induced epitope retrieval (HIER) is particularly important with immunohistochemical markers that direct the course of cancer treatment, such as Herceptin therapy. Increasingly, many laboratories are performing immunohistochemical analysis using various antibodies and methodologies for HER2/neu. We attempted to determine the effects of antibody clone and pretreatment methods on the interpretation of HER-2/neu staining in cytologic samples. Cell block sections from 54 cases of metastatic breast cancer (24 FNAs, 30 effusions) were analyzed for HER2 expression using antibodies to CB-11, TAB250, and A0485. Antibodies were analyzed with and without HIER. One pathologist using the FDA-approved scoring system for the HercepTest reviewed all slides in a blinded fashion. Five of fifty-four cases (9%) using CB-11 showed a significant increase in HER2 immunoreactivity using HIER (i.e. from 0/1+ to 2-3+). However, in twenty-nine of fifty-four cases (54%), the cytoplasmic background was significantly higher after HIER. With the A0485 antibody, two of fifty four cases (4%) showed a significant increase in immunoreactivity using HIER, while seventeen of fifty-four cases (31%) exhibited only more pronounced cytoplasmic staining. HIER pretreatment did not increase HER2 staining in any TAB250 stained sample, rather four of fifty-four cases (7%) showed a significant decrease in staining with HIER. We conclude that HIER may enhance membrane staining with the CB-11 and A0485 antibodies, but also increases cytoplasmic background. Loss of antigenicity is seen when HIER is used with TAB250.

Sequential gene profiling of basal cell carcinomas treated with imiquimod in a placebo-controlled study defines the requirements for tissue rejection.

BACKGROUND: Imiquimod is a Toll-like receptor-7 agonist capable of inducing complete clearance of basal cell carcinoma (BCC) and other cutaneous malignancies. We hypothesized that the characterization of the early transcriptional events induced by imiquimod may provide insights about immunological events preceding acute tissue and/or tumor rejection. RESULTS: We report a paired analysis of adjacent punch biopsies obtained pre- and post-treatment from 36 patients with BCC subjected to local application of imiquimod (n = 22) or vehicle cream (n = 14) in a blinded, randomized protocol. Four treatments were assessed (q12 applications for 2 or 4 days, or q24 hours for 4 or 8 days). RNA was amplified and hybridized to 17.5 K cDNA arrays. All treatment schedules similarly affected the transcriptional profile of BCC; however, the q12 x 4 days regimen, associated with highest effectiveness, induced the most changes, with 637 genes unequivocally stimulated by imiquimod. A minority of transcripts (98 genes) confirmed previous reports of interferon-alpha involvement. The remaining 539 genes portrayed additional immunological functions predominantly involving the activation of cellular innate and adaptive immune-effector mechanisms. Importantly, these effector signatures recapitulate previous observations of tissue rejection in the context of cancer immunotherapy, acute allograft rejection and autoimmunity. CONCLUSION: This study, based on a powerful and reproducible model of cancer eradication by innate immune mechanisms, provides the first insights in humans into the early transcriptional events associated with immune rejection. This model is likely representative of constant immunological pathways through which innate and adaptive immune responses combine to induce tissue destruction.

Diagnostic effects of prolonged storage on fresh effusion samples.

The effects on morphology and diagnostic interpretation of delayed processing of refrigerated effusion samples have not been well documented. The potential for cellular degeneration has led many laboratories to reflexively fix samples rather than submit fresh/refrigerated samples for cytologic examination. We sought to determine if effusion specimens are suitable for morphologic, immunocytochemical, and DNA-based molecular studies after prolonged periods of refrigerated storage time. Ten fresh effusion specimens were refrigerated at 4 degrees C; aliquots were processed at specific points in time (days 0, 3, 5, 7, 10, 14). Specimens evaluated included four pleural (3 benign, 1 breast adenocarcinoma) and six peritoneal (2 ovarian adenocarcinomas, 1 malignant melanoma, 2 mesotheliomas, 1 atypical mesothelial) effusions. The morphology of the cytologic preparations from the 10 effusions was preserved and interpretable after 14 days of storage at 4 degrees C. The immunocytochemical profile of the samples (AE1/AE3, EMA, calretinin, and LCA) was consistent from day 0 to day 14. Amplifiable DNA was present in all samples tested on day 14. We conclude that cytopathologic interpretation of effusion samples remains reliable with refrigeration at 4 degrees C even if processing is delayed.

Fine-needle aspiration of a Xp11.2 translocation/TFE3 fusion renal cell carcinoma metastatic to the lung: report of a case and review of the literature.

A 57-yr-old woman presented to the National Cancer Institute (NCI) with a history of nephrectomy for a clear cell renal cell carcinoma (RCC), Fuhrman grade 3 of 4 diagnosed 1 yr prior to admission to the NCI. A CT scan done upon admission revealed multiple bilateral lung masses. A CT-guided fine-needle aspiration (FNA) of one of the lung masses revealed a cellular specimen composed primarily of follicular structures surrounding dense hyalinized central cores. The cells in the follicular structures displayed bland nuclei and had granular to vacuolated cytoplasm. Papillary structures were also appreciated. Immunocytochemical studies showed tumor cells that were strongly vimentin and TFE3 positive. Focal staining for AE1/AE3 and CD10 was observed, as was negative staining for EMA. A surgical biopsy specimen reflected the FNA findings and demonstrated a similar immunoprofile. These findings correspond to the recently described Xp11.2 translocation/TFE3 fusion renal cell carcinoma. To our knowledge, this is the first report describing the cytologic features of an Xp11.2 translocation/TFE3 fusion RCC.

Combined breast ductal lavage and ductal endoscopy for the evaluation of the high-risk breast: a feasibility study.

BACKGROUND AND OBJECTIVES: Evaluation of the ductal epithelium of the breast at increased risk for breast cancer is needed to define the carcinogenic pathway, for risk assessment, and to improve selection of women for chemoprevention therapy. We studied the feasibility of combining breast ductal endoscopy with ductal lavage in the high-risk contralateral breast of women with ipsilateral breast cancer for the evaluation of high-risk ducts and acquisition of ductal epithelial cells for analysis. METHODS: Breast ducts were studied by ductal lavage and ductal endoscopy, and epithelial cell content studied cytologically and quantitatively. RESULTS: Twenty-five subjects and 44 ducts, including 22 (50.0%) which did not produce nipple aspirate fluid (NAF), were studied. Cellular atypia was present in five subjects. Ductal endoscopy was performed on 1 or more ducts in 24 subjects. Structural changes were noted in 63.6% of the ducts, most commonly fibrous stranding or bridging. Ductal sampling with endoscopic brush and coil sampling devices provided additional cellular samples of relatively pure ductal epithelial content (> or = 91% purity) in 8/11 subjects. CONCLUSIONS: Breast ductal endoscopy combined with ductal lavage represents a feasible approach for characterizing the ducts and ductal epithelium of the high-risk breast, especially in a research setting.

Cytological identification of metastatic epithelial nephroblastoma in pleural fluid: report of a case and review of literature.

Nephroblastoma (Wilms' tumor) is the most common childhood renal tumor and usually presents with a histology and cytology consisting of blastemal, epithelial, and stromal cells. Effusions are not uncommon and may suggest an unfavorable prognosis when containing anaplastic tumor cells. In the present case, we report the cytological appearance of a Wilms' tumor metastatic to the pleura. The effusion consisted primarily of tumor cells demonstrating epithelial differentiation. The tumor cells mostly presented as three-dimensional aggregates in an inflammatory background. Many cystic and tubular structures were identified. The tumor cells demonstrated strong CD56 and WT1 immunoreactivity. The histology of a subsequent surgical specimen reflected the features seen in cytology.

Granulocytic invasion of the central nervous system after hematopoietic stem cell transplantation for systemic lupus erythematosus.

We report on the likely mechanism of an exacerbation of neurological symptoms developed during immune reconstitution after autologous non-myeloablative hematopoietic stem cell transplantation in a 33-year-old man with systemic lupus erythematosus- associated recurrent transverse myelitis. Cerebrospinal fluid examination revealed prominent neutrophilic pleocytosis and no evidence of infection or of reactivation of lupus. Following a course of corticosteroid treatment the exacerbation resolved completely and the patient's neurological function continued to improve, resulting in net gain above pre-treatment for over 1 year follow-up without maintenance immunosuppression. Granulocytic invasion of the central nervous system represents a novel and possibly preventable cause of neurological complications during haematologic reconstitution.

Detection of malignant hematopoietic cells in cerebral spinal fluid previously diagnosed as atypical or suspicious.

BACKGROUND: Involvement of the cerebrospinal fluid (CSF) by hematopoietic malignancies may be difficult to document by morphology alone. In cases with low numbers of cells or ambiguous morphology, the diagnoses of "atypical" or "suspicious" may be used. The significance of these diagnostic terms in this scenario has not been well established. METHODS: Between January 2000 and July 2004, 32 patients with known lymphoma or leukemia and an initial diagnosis of "atypical" or "suspicious" using morphologic criteria were identified. Subsequent flow cytometry (FC) and cytologic data from these patients were evaluated. RESULTS: Of the 32 patients with an initial diagnosis of "atypical" or "suspicious," 40.6% (n = 13) had negative first and subsequent FC and morphologic evaluation of their CSF samples with follow-up up to 1 year. Nineteen patients (59.4%) had malignant hematopoietic cells identified in subsequent CSF samples by cytology and/or FC. CONCLUSIONS: In patients with a previous history of lymphoma or a hematopoietic malignancy, a majority of the patients (59.4%) with an "atypical" or "suspicious" diagnosis on CSF will ultimately have malignant cells identified in the CSF by cytology and/or FC. Many of these patients can be identified more expediently with the concurrent utilization of flow cytometry.