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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 TSC2or TSC1. Histopathologic diagnosis of LAM in lung specimens is based on identification of the Pmel17 protein with the monoclonal antibody HMB-45.

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.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.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 μg/μL to 7.8 ng/μL total protein/array spot (R2 = 0.9887) for a membrane receptor protein (epidermal growth factor receptor; R2 = 0.9935).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. Cancer (Cancer Cytopathol) 2007. Published 2007 by the American Cancer Society.

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.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.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.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. Cancer (Cancer Cytopathol) 2006. Published 2006 by the American Cancer Society.

We assessed the cerebrospinal fluid (CSF) by flow cytometry and cytology in 51 newly diagnosed and 9 treated aggressive B-cell lymphomas at risk for central nervous system (CNS) involvement to examine the utility of flow cytometry, incidence of CSF disease, and clinical surrogates of CNS spread. Multicolor flow cytometry using multiple antibody panels for light chains and B- and T-cell antigens identified neoplastic clones that constituted as little as 0.2% of total CSF lymphocytes. Among 51 newly diagnosed patients, 11 (22%) had occult CSF involvement. All 11 were detected by flow cytometry but only 1 by cytology (P = .002). Among 9 treated patients, CSF involvement was detected by flow cytometry alone in 2 and also by cytology in 1 case. CSF chemistry and cell counts were similar in patients with and without CSF lymphoma. Only the number of extranodal sites was associated with occult CSF lymphoma in newly diagnosed patients by univariate (P = .006) or logistic regression analysis (P = .012). We hypothesize that the biologic phenotype associated with colonization of extranodal sites leads to CNS spread, possibly related to the microenvironment. Patients at risk for CNS spread should undergo staging CSF evaluation by flow cytometry.

We assessed the cerebrospinal fluid (CSF) by flow cytometry and cytology in 51 newly diagnosed and 9 treated aggressive B-cell lymphomas at risk for central nervous system (CNS) involvement to examine the utility of flow cytometry, incidence of CSF disease, and clinical surrogates of CNS spread. Multicolor flow cytometry using multiple antibody panels for light chains and B- and T-cell antigens identified neoplastic clones that constituted as little as 0.2% of total CSF lymphocytes. Among 51 newly diagnosed patients, 11 (22%) had occult CSF involvement. All 11 were detected by flow cytometry but only 1 by cytology (P= .002). Among 9 treated patients, CSF involvement was detected by flow cytometry alone in 2 and also by cytology in 1 case. CSF chemistry and cell counts were similar in patients with and without CSF lymphoma. Only the number of extranodal sites was associated with occult CSF lymphoma in newly diagnosed patients by univariate (P= .006) or logistic regression analysis (P= .012). We hypothesize that the biologic phenotype associated with colonization of extranodal sites leads to CNS spread, possibly related to the microenvironment. Patients at risk for CNS spread should undergo staging CSF evaluation by flow cytometry.

Proteomic studies of cells via surface-enhanced laser desorption/ionization spectrometry (SELDI) analysis have enabled rapid, reproducible protein profiling directly from crude samples. We applied this technique to archival cytology material to determine whether distinct, reproducible protein fingerprints could be identifiedfor potential diagnostic purposes in blinded specimens. Rapid Romanowsky-stained cytocentrifuged specimens from fine-needle aspirates of metastatic malignant melanoma (with both known cutaneous primary and unknown primary sites), clear cell sarcoma, and renal cell carcinoma and reactive effusions were examined using the SELDI technology. A unique characteristic fingerprint was identified for each disease entity. Fifteen "blinded" unknown samples then were analyzed. When the protein profilefingerprints were plotted against the known fingerprints for the aforementioned diagnoses, the appropriate match or diagnosis was obtained in 13 (87%) of 15 cases. These preliminary findings suggest a substantial potential for SELDI applications to specific pathologic diagnoses.

We retrospectively analyzed 155 urine cytology samples (78 from patients treated with indinavir; 77, no indinavir) from 90 HIV+ patients to evaluate possible association between human polyomavirus and hematuria and to describe indinavir-associated urinary cytologic findings. The CD4 count also was recorded. Variables studied included the presence of cellular viral changes consistent with polyomavirus infection (PVCs), microscopic hematuria, multinucleated cells, indinavir crystals, neutrophils, and eosinophils. Twenty-two samples (15.8%) from patients with CD4 counts of more than 200/microL (>200 x 10(6)/L) showed PVCs. Multinucleated cells, of presumed histiocytic origin based on morphologic features and selective immunocytochemical findings, were present in a higher percentage of samples from indinavir-treated patients. Neutrophils were present in a higher percentage of indinavir-treated patients. Indinavir crystals were identified in 9 samples (12%) from patients receiving indinavir The lower percentage of PVCs in HIV+ patients with high CD4 counts likely represents an indirect antipolyomavirus indinavir effect by boosting immunity. Multinucleated cells (presumably histiocytic) and acute inflammation are associated with indinavir therapy. Indinavir crystals have a characteristic fan or circular lamellate appearance. Because indinavir crystals may be associated with genitourinary disease, recognizing and reporting them is clinically relevant in HIV+ patients.

Adult T‐cell leukemia/lymphoma (ATLL) is a postthymic lymphoproliferative neoplasm of T cells caused by human T‐cell lymphotropic virus (HTLV‐1). Most cases are found in Japan, the Caribbean basin, and West Africa.To identify diagnostic parameters for cytology in this neoplasm, the authors undertook a retrospective review of all ATLL samples from 1990 to 2000.One hundred fourteen samples from 34 patients with the diagnosis of ATLL were reviewed: 80 cerebrospinal fluids, 7 pleural effusions, 4 bronchoalveolar lavages, 2 peritoneal effusions as well as fine‐needle aspirations of 15 lymph nodes, 4 subcutaneous lesions, and 2 breast nodules. Twenty‐one patients were women and 13 were men, with an age range of 30 to 71 years. Morphologically, all specimens were characterized by the presence of a polymorphous population of lymphocytes ranging from small bland‐appearing lymphocytes to large atypical ones with bizarre, multilobulated nuclei (flower‐like or clover leaf cells) with coarse chromatin and prominent nucleoli. The cytoplasm was deeply basophilic with occasional vacuoles. Immunocytochemistry was performed on 17 specimens from 14 patients. In all cases tested, tumor cells were immunoreactive for CD3, CD4, CD5, and CD25 and were nonimmunoreactive for CD7 and CD8. Flow cytometry was performed on 12 specimens from 9 patients. The tumor cells in all cases tested were positive for CD2, CD3, CD4, CD5, and CD25 and were negative for CD7.Despite the polymorphous nature of ATLL, diagnosis can be established by close attention to nuclear cytologic features in conjunction with ancillary studies such as immunocytochemistry and/or flow cytometry. Cancer (Cancer Cytopathol) 2002. © 2002 American Cancer Society. Cancer (Cancer Cytopathol) 2002. © 2002 American Cancer Society.

Recent studies have characterized the ABC half-transporter associated with mitoxantrone resistance in human cancer cell lines. Encoded by the ABCG2 gene, overexpression confers resistance to camptothecins, as well as to mitoxantrone. We developed four polyclonal antibodies against peptides corresponding to four different epitopes on the mitoxantrone resistance-associated protein, ABCG2. Three epitopes localized on the cytoplasmic region of ABCG2 gave rise to high-affinity antibodies, which were demonstrated to be specific for ABCG2. Western blot analysis of cells with high levels of ABCG2 showed a single major band of the expected 72-kDa molecular size of ABCG2 under denaturing conditions. Immunoblot analysis performed under non-reducing conditions and after treatment with cross-linking reagents demonstrated a molecular weight shift from 72 kDa to several bands of 180 kDa and higher molecular weight, suggesting detection of dimerization products of ABCG2. Evidence of N -linked glycosylation was also obtained using tunicamycin and N -glycosidase F. Finally, both by light, fluorescence and electron microscopic immunohistochemical staining, we demonstrate cytoplasmic and predominantly plasma membrane localization of ABCG2 in cell lines with high levels of expression. Plasma membrane staining was observed on the surface of the chorionic villi in placenta. These results support the hypothesis that ABCG2 is an ABC half-transporter that forms dimers in the plasma membrane, functioning as an ATP-dependent outward pump for substrate transport.

MART‐1 and gp100 currently are utilized as targets in immunotherapy protocols for metastatic malignant melanoma (MMM). Enrollment of patients into ongoing peptide vaccination trials at the National Cancer Institute includes immunophenotyping of samples of metastatic lesions obtained by fine‐needle aspiration (FNA). As therapy progresses, immunocytochemistry is performed on serial FNAs of metastatic lesions to monitor changes in antigen expression during treatment. It is theorized that antigen expression of melanoma cells may be diminished because of selective immunodestruction of tumor cells, or perhaps intentionally, to escape immunosurveillance.Thirty‐eight lesions from 33 patients were serially monitored for the expression of gp100 (clone HMB‐45) and MART‐1 (clone M2‐7C10), using an avidin‐biotin peroxidase technique. The staining intensity of tumor cells was scored on a scale of 0 to 3+, with the proportion of positive cells categorized as less than 25%, 25–50%, 50–75%, and greater than 75%. All lesions were examined within approximately 2 months after the start of peptide vaccination, providing a consistent timepoint for analysis.Using the Wilcoxon signed rank test, the authors found that there were no significant changes from baseline compared with 2 months later for quantitative antigen expression of HMB‐45 or MART‐1. However, there was a trend toward a decline in staining intensity of tumor cells for HMB‐45.Preliminary results evaluating antigen expression during selective immunotherapy indicate a trend in the decline of staining intensity of tumor cells to HMB‐45. Thus, although other studies have shown that peptide‐based immunotherapy results in immune selection, this does not hinder the diagnostic utility of antibodies to HMB‐45 and MART‐1 in FNA samples of MMM. Cancer (Cancer Cytopathol) 2001;93:409–14. © 2001 American Cancer Society.

Cytology plays a pivotal role in the diagnosis of pleural effusions. In many cases, immunocytochemistry (ICC) is required to elucidate the etiology of the atypical cells. Effusions are samples that present unique problems for ICC. To date there is no standardization of ICC methods for effusions and cytology in general.The authors review the most commonly used cytologic preparations, fixatives, and antibodies used in effusion ICC.Through the utilization of cell block preparations and a panel of antibodies appropriate for the differential diagnosis in question, ICC conditions utilized in surgical pathology can be most closely replicated.ICC may provide reliable insights into various diagnostic dilemmas in effusion cytology, provided that laboratory standardization practices are followed. Cancer (Cancer Cytopathol) 2001;93:293–308. © 2001 American Cancer Society.

Tyrosinase, the rate-limiting enzyme in melanin synthesis, is a melanoma associated antigen that is recognized by both CD4+ and CD8+ T-cells in an HLA-restricted fashion. Peptides derived from the tyrosinase antigen currently are being utilized as a target for T-cells in several immunotherapy protocols for metastatic malignant melanoma (MMM) at the National Institutes of Health/National Cancer Institute. Serial fine-needle aspirations of metastatic lesions are performed to monitor the antigen expression of tyrosinase during treatment by immunostaining cytologic preparations with the monoclonal antibody T311. In the current study, 62 samples of MMM were evaluated for tyrosinase immunoreactivity on air-dried, acetone fixed cytospins and the corresponding formalin fixed, paraffin embedded cell block using an avidin-biotin immunoperoxidase method. Positive immunoreactivity revealed a granular cytoplasmic staining in melanocytic cells. The current study results showed that 92% of samples (57 of 62) were T311 immunoreactive on cell block preparations, whereas only 61% (38 of 62) were immunoreactive on cytospin preparations. In 66% of samples (41 of 62) immunoreactivity for T311 was greater in the cell block sample than in the corresponding cytospin, whereas in only 3% of samples (2 of 62) was it greater in the cytospins. In 31% of samples (19 of 62) there was no significant difference in immunoreactivity between the 2 sample types. The results of the current study show that tyrosinase is a sensitive marker for the detection of MMM; however, the optimal method of sample preparation for immunoperoxidase staining appears to be formalin fixation and paraffin embedding as tyrosinase immunoreactivity is diminished significantly in air-dried cytospin samples despite subsequent acetone fixation. Cancer (Cancer Cytopathol) 2000;90:252–7. © 2000 American Cancer Society.

Mechanisms of drug resistance other than P-glycoprotein are of increasing interest as the list of newly identified members of the ABC transport family has grown. We sought to characterize the phenotype of the newly discovered ABC transporter encoded by the mitoxantrone resistance gene, MXR, also known as ABCP1 or BCRP. The pharmacodynamics of mitoxantrone and 12 other fluorescent drugs were evaluated by confocal microscopy in four multidrug-resistant human colon (S1) and breast (MCF-7) cancer cell lines. We utilized two sublines, MCF-7 AdVp3000 and S1-M1-80, and detected overexpression of MXR by PCR, immunoblot assay and immunohistochemistry. These MXR overexpressing sublines were compared to cell lines with P-glycoprotein- and MRP-mediated resistance. High levels of cross-resistance were observed for mitoxantrone, the anthracyclines, bisantrene and topotecan. Reduced levels of mitoxantrone, daunorubicin, bisantrene, topotecan, rhodamine 123 and prazosin were observed in the two sublines with high MXR expression. Neither the P-glycoprotein substrates vinblastine, paclitaxel, verapamil and calcein-AM, nor the MRP substrate calcein, were extruded from MCF-7 AdVp3000 and S1-M1-80 cells. Thus, the multidrug-resistant phenotype due to MXR expression is overlapping with, but distinct from, that due to P-glycoprotein. Further, cells that overexpress the MXR protein seem to be more resistant to mitoxantrone and topotecan than cells with P-glycoprotein-mediated multidrug resistance. Our studies suggest that the ABC half-transporter, MXR, is a potent, new mechanism for conferring multiple drug resistance. Definition of its mechanism of transport and its role in clinical oncology is required.

In an attempt to understand the mechanisms of cell injury in the inflammatory myopathies, we analyzed the expression of costimulatory molecules, CTLA4, CD28, CD86, CD40, and CD154 as well as HLA class I, HLA class II, and ICAM-I in normal muscle and in muscle biopsies from patients with polymyositis (PM) or dermatomyositis (DM). By immunohistochemical staining, DM and PM biopsies showed the presence of CTLA4, CD28, CD86, and CD40 on inflammatory cells. More strikingly, however, low levels of CTLA4 and CD28 were observed on muscle cells. The expression of CTLA4 and CD28 on nonlymphoid cells has not been previously reported. These unexpected findings were confirmed in cultured normal human myoblasts: various proinflammatory cytokines induced the expression of CTLA4 and CD28 on normal human muscle cells. The sequences of the cDNAs were found to be identical to the sequences for these molecules in T cells. The data suggest a novel complexity in the network of cellular interactions between the infiltrated immune cells and the muscle cells in which the normal relationship between infiltrating inflammatory cells and target tissue is under a previously unrecognized set of controls.

To determine whether the multidrug resistance gene MDR1could act as a selectable marker in human subjects, we studied engraftment of peripheral blood progenitor cells (PBPCs) transduced with either MDR1 or the bacterial NeoR gene in six breast cancer patients. This study differed from previous MDR1 gene therapy studies in that patients received only PBPCs incubated in retroviral supernatants (no nonmanipulated PBPCs were infused), transduction of PBPCs was supported with autologous bone marrow stroma without additional cytokines, and a control gene (NeoR) was used for comparison with MDR1. Transduced PBPCs were infused after high-dose alkylating agent therapy and before chemotherapy with MDR-substrate drugs. We found that hematopoietic reconstitution can occur using only PBPCs incubated ex vivo, that theMDR1 gene product may play a role in engraftment, and that chemotherapy may selectively expand MDR1 gene-transduced hematopoietic cells relative to NeoR transduced cells in some patients.

The Tall cell variant of papillary carcinoma of the thyroid (TCV) is characterized by the proliferation of oxyphilic, tall, columnar cells with a height-to-width ratio of at least 2:1. TCV exhibits more aggressive clinical behavior than conventional thyroid papillary carcinoma (CPC). Cytologic features suggestive of TCV have been described in fine-needle aspiration material from primary tumors. Similarly, loss of heterozygosity (LOH) for chromosome 1 (D1S243) and the p53 gene (TP53) have been reported in TCV but not in CPC, thus making exploitation of this genetic feature a potential tool for molecular discrimination between these two neoplasms. Cytology samples of metastatic and/or recurrent neoplasms (M/R) (12 cases) and 7 cases of primary TCV obtained from 12 patients were evaluated. The cytologic findings of these cases were compared with previously published findings. Microdissection and polymerase chain reaction for LOH for chromosome 1 and p53 (D1S243 and TP53 markers) were performed on cytologic smears from 6 cases of M/R tumors and 3 cases of primary tumors. More then 50% of M/R showed atypical follicular cells with enlarged nuclei, granular chromatin, nuclear grooves, pseudoinclusions, and abundant finely granular cytoplasm. Cells were disposed in monolayers (58%) and papillary clusters (50%). Similar findings were present in cases of primary TCV. LOH studies showed that 4 of 6 M/R were noninformative and 2 of 3 cases of primary TCV were informative for the D1S243 marker; however, in contrast with previously published reports, no LOH was detected for the markers evaluated. M/R and primary TCV have similar cytologic features. Additional studies of larger series of M/R and primary TCV should be performed to delineate further any potential application of LOH for chromosome 1 and the p53 gene as a tool for diagnosing TCV with cytologic preparations. Cancer (Cancer Cytopathol) 1999;87:238–42. © 1999 American Cancer Society.

Loss of heterozygosity (LOH) for several tumor suppressor genes (including loci on 3p, 1p, and 17p,) has been documented in surgical specimens of adrenal cortical carcinomas (ACCA) without accompanying losses in benign hyperplastic and adenomatous adrenal cortical lesions (ACL). This disparate pattern of LOH raises the possibility of exploitation of these differences for diagnostic utilization. Cytologic differentiation of benign versus malignant ACL may be impossible based solely on fine-needle aspiration (FNA) material. The authors attempted to extrapolate the genetic findings on surgical specimens to FNA specimens of ACL to determine whether LOH studies could be utilized as a definitive diagnostic tool. Microdissection of archival material was performed on FNAs of ten ACCAs (stained with the Papanicolaou and Diff-Quik stains) with corresponding histologic material (stained with hematoxylin and eosin), one FNA of a benign ACL, and three touch preparations of benign adrenal cortex. LOH analysis was performed by polymerase chain reaction (PCR) with flanking markers for the following putative tumor suppressor genes: p53 (17p13; TP53), 1p (1p36; D1S165), and the von Hippel-Lindau gene at 3p25 (D3S1038 and D3S1110). Similar results were obtained with cytologic and histologic material. As expected, benign ACL showed no LOH for the markers examined. Of the informative ACCA cases, 70% showed LOH for at least 1 of the 3 markers tested on both FNA and histologic samples. For all cases with amplifiable DNA, there was a 100% concordance rate for LOH between cytologic and histologic material, with at least 7 of the 10 cytologic samples originating from metastatic lesions and all of the surgical material originating from the primary adrenal neoplasm. The results of this study suggest that the combination of microdissection and PCR for LOH of p53, 1p, and 3p25 from FNA material has the potential to be utilized to distinguish ACCA from benign ACL in informative cases. It also shows a 100% concordance rate between metastatic and primary ACCAs for the losses observed, a finding that can be extremely useful for the definitive identification of metastatic lesions. Archival cytologic preparations of ACCA are a reliable source of DNA for LOH studies. [See editorial counterpoint on pages 173–5 and reply to counterpoint on pages 176–7, this issue.] Cancer (Cancer Cytopathol) 1999;87:231–7. © 1999 American Cancer Society.

Anti-α-inhibin, an antibody directed against a peptide hormone, has been shown to be a useful diagnostic aid in surgical pathology material for the identification of sex cord–stromal neoplasms and recently has been described in adrenocortical carcinoma (ACC). The diagnosis of ACC versus renal cell carcinoma (RCC) may be difficult morphologically, particularly in fine-needle aspiration (FNA) material. To date, the immunohistochemical distinction of ACC from RCC is based on a panel of antibodies that include vimentin, cytokeratins, and epithelial membrane antigen. However, the reliability of this panel is weakened by inconsistent staining patterns. Archival formalin fixed, paraffin embedded cell block sections from 45 FNAs of known primary and metastatic ACC and RCC as well as benign adrenocortical nodules were stained with anti-α-inhibin using an avidin-biotin procedure. All samples were microwave pretreated and a biotin block was performed to reduce the background stain due to the high endogenous biotin often present in these types of samples. All cases of ACC (n = 7; 100%) and benign adrenocortical cells (n = 15; 100%) were immunoreactive with the α-inhibin antibody, showing a diffuse cytoplasmic and granular staining pattern. The staining intensity and number of immunoreactive cells varied within each sample, with the cases of ACC having the greatest proportion of immunoreactive cells and the strongest intensity. None of the cases of RCC (n = 23; 0%) were immunoreactive with anti-α-inhibin. The morphologic distinction of ACC versus RCC in FNA material from renal, adrenal, and metastatic neoplasms is not always feasible based on cytology alone. However, due to the advent of the α-inhibin antibody, the reliable distinction of these entities now may be possible. The intense and specific immunostaining pattern for cells of adrenal origin, even in paucicellular samples, suggests potential for the widespread clinical utility of this marker by cytopathologists. Cancer (Cancer Cytopathol) 1999;87:168–72. © 1999 American Cancer Society.

HMB-45, an antibody directed against a premelanosome glycoprotein, has thus far been considered the most specific antibody for the immunocytochemical substantiation of the diagnosis of malignant melanoma (MM). A recently described antigen, MART-1, is a transmembrane protein that is present in normal melanocytes and widely expressed in MM. Antibodies to MART-1 have recently become commercially available. Both HMB-45 and MART-1 form the basis of ongoing immunotherapy protocols at the National Institutes of Health/National Cancer Institute. The authors evaluated 207 lesions from 160 patients with metastatic MM procured via fine-needle aspiration (FNA) for expression of MART-1 (clone M2-7C10) and HMB-45 prior to commencement of immunotherapy. FNAs were performed on subcutaneous soft tissue masses (190 lesions), lung (8 lesions), liver (5 lesions), pancreas (3 lesions), and brain (1 lesion). To test the specificity of the monoclonal antibody directed against MART-1, the authors evaluated its reactivity in normal tissues as well as in various nonpigmented neoplasms that are often included in the differential diagnosis of MM. Of all lesions tested, 13 (6%) were negative for both MART-1 and HMB-45. Of all patients tested, 20% had 1 or more lesions that were non-immunoreactive with HMB-45, whereas only 10% had 1 or more lesions that were nonimmunoreactive for MART-1. Eight percent of the lesions tested were negative for MART-1 only, whereas 16% of lesions tested were negative for HMB-45 only. In 35% of the lesions, MART-1 stained more cells than HMB-45. In 13%, MART-1 stained fewer cells than HMB-45, and in 52% both antibodies stained an equivalent number of cells. All samples of normal tissue were negative for staining with MART-1, as were the nonpigmented lesions tested. Melanocytes in normal skin samples stained positively for MART-1. The MART-1 antibody is a superior immunohistochemical marker for the diagnosis of MM. It has the potential to become the preferred antibody over HMB-45 for the diagnosis of metastatic MM in FNA material, as MART-1 stains a higher percentage of lesions in a higher percentage of patients than does HMB-45. Cancer (Cancer Cytopathol) 1999;87:37–42. © 1999 American Cancer Society.

Much remains to be learned about drug resistance in the biology of RCC and its metastases.We measured MDR-1/P-glycoprotein expression in 19 tumor samples from patients with metastatic RCC by RNase protection and quantitative PCR assays. The median level of the 16 tumor metastases was 4.9 (range: 0.10 to 156.2) relative to the level of 10 assigned to a reference cell line, SW620, which has been characterized as expressing a minimum level of MDR-1. Since these levels were lower than expected for RCC, we asked whether the metastases possessed a phenotype different from primary RCC and examined MDR-1 expression in 5 paired cell lines derived from primary and metastatic RCC. In 8/10 lines, MDR-1 expression was >10. Relative to the level in the primary line, MDR-1 expression was decreased (3 to 50-fold) in 3 metastatic lines, was increased in 1, and unchanged in 1. MRP mRNA expression was lower in the metastatic lines while EGFR expression was variable. IC 50values for 6 compounds (including 4 standard agents and one new Phase 1 agent) were determined for the paired lines. Rhodamine and calcein efflux assays were performed as measures of P-glycoprotein and MRP function. Rhodamine efflux correlated with MDR-1 mRNA expression (r = 0.87) and with the IC 50s (r = 0.60) for paclitaxel in the paired cell lines. In contrast, calcein efflux did not correlate with MRP expression. Lastly, MDR-1 expression correlated with cytokeratin 8 (CK8) protein levels, a measure of cellular differentiation. In sum, these data suggest renal cell carcinoma (RCC) metastases have altered MDR-1 expression potentially due to altered differentiation relative to the primary tumor. Thus, the drug resistance phenotype of primary RCC tumors may not reflect that of their metastases.

To determine whether the multidrug resistance gene MDR1could act as a selectable marker in human subjects, we studied engraftment of peripheral blood progenitor cells (PBPCs) transduced with either MDR1or the bacterial NeoR gene in six breast cancer patients. This study differed from previous MDR1gene therapy studies in that patients received only PBPCs incubated in retroviral supernatants (no nonmanipulated PBPCs were infused), transduction of PBPCs was supported with autologous bone marrow stroma without additional cytokines, and a control gene (NeoR) was used for comparison with MDR1. Transduced PBPCs were infused after high-dose alkylating agent therapy and before chemotherapy with MDR-substrate drugs. We found that hematopoietic reconstitution can occur using only PBPCs incubated ex vivo, that theMDR1gene product may play a role in engraftment, and that chemotherapy may selectively expand MDR1gene-transduced hematopoietic cells relative to NeoR transduced cells in some patients.

Cytopathology is no longer simply a screening modality limited to the “Pap mills” of yore. The news in cervicovaginal cytology is automation. The news in FNA cytology is the application of molecular techniques. Whether it is the detection of specific proteins/antigens for definitive diagnoses/treatment guidance in immunotherapy, or it is “reading nucleic acids,” the cytopathologist of the future will be called upon to gather and report more detailed and precise information. As we develop methods for extrapolating the secrets previously locked within the individual cells, it becomes evident that the cells were talking all along, we just did not know how to listen.

This report characterizes nine new cell lines derived from patients with malignant pleural mesothelioma. The lines were initiated between July 1990 and July 1992 from solid tumors (5 lines) or effusions (4 lines) and had proliferated for a period of at least 2 months without senescence. They were characterized by cell size, doubling time, immunohistochemical analyses, electron microscopy, and chromosomal karyotyping. Growth factor/cytokine elaboration was determined using enzyme-linked immunoassays. The established lines were similar in morphology to their parent tumor (ie, epithelial or sarcomatoid). Cell sizes ranged from 59 to 81 μm, and the doubling times varied from 31 to 65 hours. The lines stained with cytokeratin and showed expected negative staining for adenomarkers including B72.3 and carcinoembryonic antigen. All cell lines exhibited aneuploidy, with modal chromosome numbers between 40 and 81 and had multiple chromosomal aberrations. Significant production of granulocyte—monocyte colony-stimulating factor, leukemia inhibitory factor, platelet-derived growth factor, and interleukin-6 was seen. These new cell lines derived from human mesotheliomas can now be used to aid in the design of innovative treatment strategies.

The prognosis of metastatic thyroid carcinoma is dependent on the age of the patient, the histologic characteristics of the neoplasm, and the site of metastasis. A more favorable prognosis is found in patients less than 40 years old with follicular carcinoma and without any bony metastases. Metastatic thyroid carcinoma presenting as distal spinal cord compression is extremely rare. We report one such case and review the literature. As reported in the literature, the combination of decompressive laminectomy followed by total thyroidectomy and radioactive iodine therapy has proved to be effective in the treatment of patients with thyroid carcinoma metastatic to the distal vertebral bodies.

The differential diagnosis of metastatic clear cell carcinoma is broad. To date, there are no specific immunohistochemical markers for renal cell carcinoma (RCC) in general use. Loss of heterozygosity (LOH) at 3p25.5, the von Hippel-Lindau (VHL) gene locus, is frequent in sporadic clear cell RCC. The authors compared LOH in primary and metastatic RCC through microdissection and the polymerase chain reaction (PCR) to evaluate these techniques as potential diagnostic tools. The authors identified 14 patients with known clear cell RCC who underwent fine-needle aspiration (FNA) evaluation of presumed metastatic lesion. Direct-visualization microdissection was performed from archival histologic glass slides of the primary neoplasm and the adjacent normal kidney parenchyma. Malignant cell clusters were microdissected from archival FNA slides of metastatic lesions. The cytology slides were previously stained with either Diff-Quik or Papanicolaou stain. This was followed by a single-step DNA extraction and PCR amplification for evaluation of LOH using polymorphic markers, D3S1038 and D3S1110, flanking the VHL gene. Thirteen of the 14 cases contained DNA suitable for PCR in both the paraffin embedded and the FNA material. Eight of the 13 cases were heterozygous (informative) for the above markers, and 6 of these showed identical allelic loss in the primary and metastatic tumor for either one or both of the markers used. The remaining two cases did not show LOH at the VHL locus with the two polymorphic markers used. DNA from archival cytologic material stained with Papanicolaou stain or Diff-Quik is reliable for PCR amplification. Visually directed microdissection in combination with PCR has the potential to be a useful technique for confirmatory identification and diagnosis of metastatic clear cell RCC in cytologic material, because a specific genetic abnormality is present in the primary tumor. As characteristic genetic abnormalities are identified in various neoplasms, the use of this technique has the potential for conclusive evaluation of metastatic disease with FNA material, when used in comparison with surgical or cytologic material from the primary tumor. The utility of this combination of techniques has the potential for the molecular diagnosis of morphologically ambiguous cell populations. Cancer (Cancer Cytopathol) 1997; 81:180-6. © 1997 American Cancer Society.

Malignant effusions are complications of metastatic malignant melanoma (MM). Differential diagnosis often involves distinguishing MM from adenocarcinoma and reactive mesothelial cells. Descriptions in the literature of the morphologic and immunocytochemical (IM) staining characteristics of MM in effusions are sparse. A combination of morphology and immunocytochemistry should yield the most accurate diagnostic results. The MART-1 antigen, a transmembrane protein, is specifically expressed in melanocytes and MM. A recently developed monoclonal antibody to the MART-1 antigen may represent a useful marker for the identification of MM in effusions. The authors conducted a retrospective review of 32 effusion samples diagnosed as MM. The review consisted of morphologic and IM analyses of the effusion samples with antibodies to MART 1, HMB45, S-100, and cytokeratins (AE1/AE3).IM stains were performed on cell block or cytospin material, depending on availability. In the morphologic review, emphasis was placed on Diff Quik-stained material, due to its enhanced cytoplasmic volume and detail. Predominant cytologic features noted were lack of cellular cohesion (in 100% of cases), large eccentric nuclei with prominent nucleoli (in 100%), multinucleation (in 84%), variable cytoplasmic vacuolization (in 75%), pigment (in 72%), and cell-in-cell engulfment (in 47%). All immunoreactive cases with sufficient material stained with at least one of the markers used. Tumor cells were positive with IM stains to MART-1 in 78% of cases, HMB45 in 81%, and S-100 in 81%. Coexpression of MART-1, HMB45, and S-100 was noted in 63% of cases. Of cases that showed expression for only 1 of the 3 antigens, the MART-1 was positive in 1 case, and HMB45 and S-100 were positive in 2 cases each. Three cases showed immunoreactivity for cytokeratins in the melanoma cells. The diagnosis of MM in effusions can be made reliably through a combination of morphologic and IM features. Differential diagnosis often involves distinguishing MM from adenocarcinoma or reactive mesothelial cells. Cytoplasmic vacuolization, multinucleation, prominent nucleoli, and cell-in-cell engulfment are cytologic features common to all three. The lack of IM staining for cytokeratins alone cannot reliably distinguish MM; 11% of cases showed positive staining with this antibody in the melanoma cells. The use of a panel of antibodies increases the accuracy of diagnosing MM. In this study, MART-1 proved a useful adjunct to the HMB45/S-100/cytokeratin panel for the diagnosis of MM in effusions, staining 78% of the immunoreactive cases, with positivity in 1 case that was negative for HMB45 and S-100. Cancer 1997; 81:57-63. © 1997 American Cancer Society.

MART-1/MelanA and Pmel17/gp100 are melanoma-associated antigens (MAAs) that can be recognized by tumor-infiltrating lymphocytes (TILs) capable of mediating successful adoptive therapy in vivo. Analysis of melanoma cell lines in vitro has demonstrated that heterogeneous antigen expression in the context of class I MHC is a significant co-factor in determining the recognition of melanoma targets by cytotoxic lymphocytes (CTLs). In this study, 217 specimens from 103 patients with metastatic melanoma were examined for the expression of MART-1/MelanA (monoclonal antibody \[MAb\] M27C10) and Pmel17/gp100 (HMB45 MAb) by immuno-histochemistry. Marked heterogeneity in the expression of both MAAs was confirmed by analysis of the percentage of positively staining tumor cells or the average intensity of tumor staining. We also noted heterogeneity of expression among multiple lesions taken from different anatomic sites within a patient. A dissociation was noted in the detection of MART-1 and gp100 in some lesions, with gp100 being undetectable in 24% of the lesions and MART-1 being undetectable in 11%. In several cases, loss of one MAA was not associated with loss of the other MAA, suggesting that MART-1 can represent a useful additional marker for the diagnosis of melanoma in gp100 (HMB45)-negative lesions. Of the 217 specimens, 155 were obtained from HLA-A*0201 patients, of which 6% were negative for HLA-A2, 8% were negative for MART-1/MelanA and 21% were negative for Pmel17/gp100. The potential significance of our findings is illustrated by a case study in which a patient with melanoma experienced rapid tumor progression in association with loss of either MAA or HLA expression in several lesions. Int. J. Cancer 75:517-524, 1998. Published 1998 Wiley-Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America.

Squamous atypia in postmenopausal (PM) cervical vaginal smears (CVS) only rarely is associated with a biopsy-proven squamous intraepithelial lesion (SIL), and thus most commonly represents an atrophy-associated benign reactive change. To distinguish atypical squamous cells of undetermined significance (ASCUS) and SIL from atrophy-associated benign reactive changes, a review of atypical atrophic PM CVS was performed. Ninety CVS exhibiting an atrophic smear pattern were considered appropriate for study. Repeat smears and/or biopsy after local estrogen therapy were requested to distinguish atrophic/reactive from dysplastic changes. Generally, atrophic CVS exhibit uniform nuclear enlargement in the squamous cell population, which, using the criterion of nuclear enlargement alone, would qualify the majority of these cases to be classified as ASCUS. The nuclear enlargement associated with atrophy resolves with the local application of estrogen. Follow-up after local estrogen treatment was available for 84 of 90 patients and revealed 10 cases of SIL (12%) and 9 cases of ASCUS (11%), 6 of which were favored to be of a reactive etiology. Nuclear features most commonly noted in the cases considered to be ASCUS (nonreactive) and SIL were nuclear hyperchromasia and nuclear contour irregularities. Nuclear enlargement alone is not sufficient for diagnosing ASCUS or SIL in PM CVS. Nuclear enlargement in squamous cells is an expected normal reactive change present in PM CVS that resolves with the application of local estrogen. Nuclear hyperchromasia and irregular nuclear contours remain the most reliable cellular characteristics for diagnosing SIL in atrophic CVS. [See editorial on pages 200-1, this issue.] Cancer (Cancer Cytopathol) 1998;84:218-225. © 1998 American Cancer Society.

The distinction between malignant mesothelioma (MM) and adenocarcinoma (ACA) in cytologic specimens frequently is difficult, often requiring immunocytochemistry to support the diagnosis. Recent reports have proposed the utilization of antibodies to mesothelial cell clone HBME-1 and thrombomodulin (TM), because they are immunoreactive in MM and less commonly reactive in ACA. Immunoreactivity for the monoclonal antibody CA 19-9 has been observed in many ACAs and reportedly is absent in MM. In this study, immunostaining was performed on formalin fixed, paraffin embedded cell blocks from effusions or fine-needle aspirations using the avidin-biotin-peroxidase method. Thirty-eight MMs and 49 ACAs were tested using antibodies to CA 19-9, HBME-1, and TM. Anti-CA 19-9 stained only 1 of the 37 cases of MM tested (3%), but stained 24 of the 49 cases of ACA (49%). Anti-HBME-1 stained 34 of 38 cases of MM (89%), and 28 of 43 cases of ACA tested (65%). Anti-TM stained 24 of 36 cases of MM (67%), and 21 of 40 cases of ACA tested (53%). CA 19-9 has utility as part of an immunocytochemical panel for distinguishing ACA from MM, because a positive staining reaction would make the diagnosis of MM unlikely. Although HBME-1 and TM can identify MM positively, each frequently is detected in ACA, thus limiting the utility of these antibodies in cytologic specimens.Cancer (Cancer Cytopathol) 1998;84:101-8. © 1998 American Cancer Society.

No abstract.

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 × 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-α 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.