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BAT is an immune-modulatory monoclonal antibody that exhibits strong lymphocyte-mediated anti-tumor activity against a variety of murine and human tumors. Peptide A is a vaccine we have developed by screening a phage display peptide library on BAT monoclonal antibody. Anti-tumor activity was obtained in mice inoculated with B16 melanoma by either a single injection with BAT or immunization with peptide A. The aim of this study was to follow and compare histopathologically the process of prevention of melanoma metastases in lungs of treated and immunized mice. Mice were sacrificed on different days after tumor inoculation, their lungs were weighed and the number of metastases was counted. The lungs were then fixed in formalin, embedded in paraffin, and stained with hematoxylin and eosin. Histological examination of tumor inoculated mice on day 10 revealed the existence of microscopic melanoma lesions (0.01-0.012 mm) that increased gradually in number and size and on day 21, most of the metastases were large and spanned entire lobes, from the pleura to the hilum, measuring up to 3.5 mm and coexisting with scattered, small metastases showing the same morphology and pattern of lung involvement. On day 24, the lungs of untreated mice were massively infiltrated by coalescing metastases replacing up to 50% of the lung tissue and measuring up to 7.0 mm. The number of lung metastases and weight was dramatically decreased by a single injection of BAT monoclonal antibody ten days post tumor inoculation. The treated mice clearly had fewer and smaller metastases in different mice at the different days post tumor inoculation. On day 21, there were few small metastases measuring up to 1.6 mm and on day 24 no lung metastases were detected in this group that appeared with a completely normal lung structure. Immunization with peptide A started one day post tumor inoculation and was compared to immunization with control peptide N. Fourteen days post tumor inoculation, mice immunized with peptide A had only 1-2 metastases (0.012-0.076 mm) and on day 24 ranged up to 2 mm compared to control immunized mice where the tumor developed up to 5-7 mm. Foci of lung inflammation in both the untreated, treated or immunized mice were rare, small, and not preferentially associated with the lung metastases. They were composed mainly of small lymphocytes and a few macrophages. This study is the basis of histopathological understanding of metastases prevention in lungs of mice immunized or treated by BAT monoclonal antibody.

Background Mycosis fungoides (MF) is an epidermotropic cutaneous T-cell lymphoma in which the tumor cells express a mature T-helper memory phenotype, ie, CD3 + , CD4 + , CD8 − , CD45RO + , with a T-cell receptor (TCR) of the α/β heterodimer. A minority of patients have an unusual immunohistochemical profile consisting of a CD4 − , CD8 + mature T-cell phenotype. An aberrant CD4/CD8 double-negative (DN) immunophenotype in patients with early MF has rarely been reported. Objectives We sought to evaluate the frequency of CD4/CD8 DN immunophenotype in patients with early MF, and to study their clinical, histopathologic, and immunohistochemical features, and the course of their disease. Methods Our departmental archives were searched for patients with early-stage MF and CD4/CD8 DN immunophenotpye. Results Of the 140 patients with early MF immunophenotyped in our laboratory, 18 (12%) showed CD4 and CD8 expression in less than 10% of their intraepidermal T cells on fresh-frozen and paraffin-embedded samples. The group included 13 male and 5 female patients; 14 adults and 4 children; and 15 Jews and 3 Arabs. In all, 8 had classic MF and 10 had unusual clinical variants (5 hypopigmented, 3 localized, 1 ichthyosiform, 1 purpuric). All received skin-targeted therapies and all had an indolent course (mean follow-up 3.5 years). Histopathology revealed early MF. Results of immunohistochemical analysis of the intraepidermal lymphocytes were as follows: CD3 + , CD4 − , CD8 − in all patients; CD7 − in all of 17; CD45RO + in 15 of 16; T-cell–restricted intracellular antigen-1 + in 11 of 15; CD30 + in 2 of 16; and CD56 + in 2 of 16. A βF1 + /δ − phenotype, indicating a TCR of the α/β heterodimer, was found in 8 of 16; βF1 − /δ + phenotype, indicating a TCR of the γ/δ heterodimer, in 1 of 16; βF1 − / δ − in 5 of 16; and no determinable phenotype in 2 of 16. The TCR γ gene was clonally rearranged in 10 of 16 patients. Limitation This was a single-center case series. Conclusions There is a subgroup of patients with early MF that exhibit a CD4/CD8 DN immunophenotype. In our region, this aberrant immunophenotype is not as rare as reflected in the literature, is overrepresented in the unusual clinical variants of MF, and does not seem to have prognostic significance. Like CD4 + MF, the tumor cells represent memory T cells and in many cases express α/β TCR, but unlike CD4 + MF, they have a mostly cytotoxic phenotype. We suggest that CD4/CD8 DN MF should be recognized as another immunohistochemical variant of this lymphoma.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine tumor of the skin that is associated with a high incidence of recurrence and metastasis. The therapeutic arsenal for this malignancy is limited and once it spreads, there is no effective treatment. c-kit expression has been demonstrated previously in primary MCCs thus raising the possibility of treating MCCs with imatinib mesylate, the tyrosine kinase inhibitor that has shown promise in the management of c-kit expressing tumors. In this study we examine 25 additional primary MCCs and also 6 of their lymph node metastases. Formalin-fixed, paraffin-embedded tissues were stained immunohistochemically with an antibody directed against the KIT receptor. Percentage and intensity of staining were analyzed semiquantitatively using a three-tiered system. Twenty-one of the 25 (84%) primary tumors stained positively for KIT, of which 14 (67%) showed widespread positivity. Five of the 6 lymph nodes (83%) were similarly positive. High mitotic rate and vascular invasion in the primary tumors tended to be associated with prominent staining in the lymph node metastases. No association was found between c-kit expression and outcome. We confirm that the majority of primary MCCs express c-kit and further find that metastases are positive for the KIT receptor as well. Thus, c-kit expression may be an early event in the transformation of MCC, but not a marker for tumor progression.

Background and aim Sentinel lymph node biopsy (SLNB) is reportedly associated with insignificant morbidity. The study aims at documenting SLNB-related complications and identifying possible risk factors. Method Data of all melanoma patients who underwent SLNB in our medical center (1994–2002) were analysed. Procedure-related complications were recorded. Results Three hundred and nine lymphatic basins of 250 patients were explored for SLNB. Overall complication rate was 20%. Sensory morbidity was significantly associated with axillary SLNB ( p =0.04) and was more prevalent in younger patients. The use of blue dye alone or combined with a hand-held gamma probe had no statistically significant impact on the identification rate. There were six false-negatives (2.3%), for an overall false-negative rate of 18%. A positive sentinel node was significantly associated with shortened overall survival ( p =0.04). Conclusion Wound complications are more frequent than usually reported. Sensory morbidity occurs mostly in the axilla. Neck SLNB is associated with the highest rate of identification failure. Patient age, basin location, and number of excised nodes may serve as prognostic factors of morbidity.

The literature regarding mycosis fungoides in children is sparse. To shed further light on the characteristics of mycosis fungoides in the paediatric population we analysed the clinicopathological features of 10 patients in whom this malignancy was diagnosed before the age of 18 years. All were Jews and Arabs with histologically proven patch/early plaque stage disease: 4 in stage IA, 4 in IB and 2 with unilesional disease. Seven patients had hypopigmented lesions either constituting the sole manifestation (2 patients) or in combination with classic lesions (5 patients); of these, 3 had light skin and 4 pigmented skin. Most patients had immunohistochemical features characteristic of mycosis fungoides, with a predominance of CD4+ T cells. Some had deletion of CD7+ cells. In 3 patients, however, the epidermotropic cells were exclusively or predominantly CD8+ cells. All patients responded to conventional therapy and during an average follow-up of 3.4 years only one patient showed stage progression, but without extracutaneous involvement. It is concluded that juvenile mycosis fungoides is characterized by early stage disease, occasionally with unilesional disease, usually with hypopigmented lesions irrespective of skin colour, and a good response to therapy. On the basis of our experience and review of the literature, it appears that the CD8+ phenotype is over-represented in juvenile disease.

Squamous cell carcinomas of the head and neck (SCCHN) are excellent candidates for local immunotherapy owing to their accessibility and their infiltration by mononuclear cells that are susceptible to immunomodulation. A response rate of 25-60% has been reported for treatment with natural IL-2 or a mixture of natural lymphokines. In the present study, biopsies and posttreatment excision specimens from nine patients with operable SCCHN treated systemically with a variety of immunomodulators and locally with natural lymphokines (multikine, CelSci) were analyzed in an attempt to correlate clinical response to histopathological and immunohistochemical changes. Formalin-fixed, paraffin-embedded tissues were stained with antibodies against lymphocytes (CD45, CD3, CD4, CD8, CD20), macrophages (CD68) including dendritic cells (S-100), markers for lymphocyte activation (CD30, HLA-DR), natural killer cells (CD56 and CD57), beta-2-microglobulin and keratin. One patient showed a complete response to treatment and two a partial response. Tumor size was significantly smaller after therapy. Clinical and pathological regression were more prominent in the smaller tumors. Numerous macrophages, both mononucleated and multinucleated, were present along the tumor-stroma interface in the posttreatment specimens of seven patients, most prominently in the three patients with tumor regression. The increase in the number of CD68+ and S-100+ macrophages after treatment was statistically significant. Lymphocytic infiltrates, which showed some increase following treatment, were composed of a mixture of T and B lymphocytes, the former mostly in contact with the tumor and the latter placed more peripherally. CD8+ lymphocytes extended into the tumors, whereas CD4+ lymphocytes showed minimal extension. Intensity of beta-2-microglobulin staining in tumors was significantly higher following therapy and associated with a better outcome. The marked increase in macrophages following treatment may indicate that the macrophage plays a major role in tumor recognition, destruction and clearance. An increase in the number of macrophages in a posttreatment specimen may indicate immunoresponsiveness.

Inflammatory myofibroblastic tumor is a rare solid tumor that most often affects children and young adults. Although benign, the tumor may be very aggressive locally. We describe a 9-year-old boy with primary inflammatory myofibroblastic tumor of the left upper lobe involving the left atrium.

Keratinocytes influence the number, morphology, and proliferation of melanocytes. An interference in the melanocyte-keratinocyte relationship may contribute to melanoma development. This study examined the expression of apoptotic and proliferative markers in keratinocytes in lentigo maligna to characterize the epidermis permissive to these lesions. Formalin-fixed and paraffin-embedded tissues from 25 samples of lentigo maligna, 20 samples of solar keratoses, and 5 samples each of normal sun-exposed and non-sun-exposed skin (controls) were immunostained with antibodies directed against the proapoptotic markers bax and p53, the antiapoptotic marker bcl-2, and the proliferation marker ki-67. Eight percent of the lentigo maligna samples were positive for keratinocyte expression of bcl-2, 24% were positive for p53, and 76% were positive for bax; respective findings for solar keratoses were 35%, 85%, and 90%. Comparison with normal sun-exposed skin yielded lower rates of keratinocyte proliferation in 56% of the lentigo maligna samples, similar rates in 36%, and higher rates in 8%; for solar keratoses, proliferation was higher than controls in 60% of samples, similar in 35%, and lower in 5%. All these differences were statistically significant. These findings indicate that there are variable patterns of epidermal reaction to chronic sun exposure. The epidermis in lentigo maligna shows overall low proliferation and an apparently low apoptotic tendency. The dysfunctional epidermis may be permissive to aberrant melanocyte proliferation in the early stages of melanoma development.

Calciphylaxis is a rare disorder in patients with chronic renal failure that is characterized by ischemic necrotic skin lesions. The prognosis is grave and mortality is high (80%). The precise mechanism of calciphylaxis is still unknown, but in addition to chronic renal failure, elevated parathyroid hormone levels appear to play a role. The role of parathyroidectomy in treating affected patients is questionable. In this article, we describe the case of a patient with chronic renal failure who developed rapidly progressive subcutaneous calcifications and ulcerations in the lower extremities. These lesions regressed following subtotal parathyroidectomy. We also review the literature on calciphylaxis, with a focus on treatment options.

Hypertrophic scars cause great discomfort to the patient and pose a challenge for the reconstructive surgeon. This is particularly true in the facial area. Optimal function and aesthetic appearance are the main goals of reconstruction. We suggest an adjunct to the surgical management of facial hypertrophic scars which involves abrasion of the subdermal plane. The technique consists of removal of all scarred skin from the aesthetic unit, dermabrasion of subcutaneous tissues, including the muscular surface (subdermabrasion), and a full thickness skin grafting. This technique was applied in a young patient with hypertrophic burn scars of the chin. Biopsy confirmed our basic assumption that hypertrophic scars extend into the muscular plane.

Molecular studies of advanced-stage neuroblastoma (NBL) have revealed a marked genetic heterogeneity. In addition to MYCN amplification and chromosome 1 short-arm deletions/translocations detected by conventional cytogenetics, application of fluorescence in situ hybridization has disclosed a high prevalence of 17q gain, whereas allelotyping and comparative genomic hybridization techniques also have revealed loss of 11q and of other chromosomal material. Using the recently developed technique of spectral karyotyping (SKY), we sought to refine the cytogenetic information, identify hidden recurrent structural chromosomal abnormalities, and compare them to the molecular findings. Thirteen samples of metaphase spreads from 11 patients with advanced-stage NBL were analyzed by SKY. Most of them were found to have complex karyotypes (more than three changes per metaphase) and complex unbalanced rearrangements. Recurrent aberrations leading to 17q gain, deletion of 1p, MYCN amplification, and loss of 11q appeared in 7, 4, 4, and 5 patients, respectively, in simple and complex karyotypes. Chromosome 3 changes and gain of 1q and 7q appeared in 6, 5, and 4 patients, respectively, in complex karyotypes only, reflecting later changes. A strikingly high prevalence of the unbalanced translocation der(11)t(11;17), leading to concomitant 11q loss and 17q gain in 4 patients, delineated a distinct cytogenetic group, none having 1p deletion and/or MYCN amplification. der(11)t(11;17) was associated with complex karyotypes with changes in chromosomes 3 and 7q. The 17q translocations with partners other than 11q were associated with 1p deletion and/or MYCN amplification. The distinct cytogenetic subgroups identified by SKY confirm and extend the recent molecular observations, and suggest that different genes may interact in the der(11)t(11;17) pathway of NBL development and progression. © 2002 Wiley-Liss, Inc.

We describe four patients with mycosis fungoides (MF) in whom depigmentation, and also leucotrichia in one, occurred following the resolution of the eruption during phototherapy (psoralen plus ultraviolet A treatment in three patients, climatotherapy in one). In all cases, the depigmentation was localized to the area of the pre-existing MF lesions. There was no clinically obvious phototoxicity. Biopsy study including S100 staining in all cases, and electron microscopy in one case, demonstrated the total absence of melanocytes, with no evidence of MF. It is suggested that the phototherapy may have activated a cell-mediated immunity leading to destruction of the melanocytes. We recommend that vitiligo-like leucoderma be added to the list of untoward effects of phototherapy in MF.

Adrenal masses are discovered on 1% to 3% of abdominal computed tomograms. Most are nonsecretory benign adenomas. Diagnosis is established by different imaging methods combined with hormonal evaluation. Complete surgical resection is the preferred treatment for large masses. The authors previously described an infant with an adrenocortical carcinoma that was Ga-67 avid. The English-language literature contains few reports of Ga-67 uptake by these malignant tumors. The authors describe a patient in whom a benign adrenocortical tumor was disclosed by Ga-67 scintigraphy. This indicates that large adrenocortical masses, both malignant and benign, may be Ga-67 avid.

BACKGROUND Merkel cell carcinoma (MCC) has been associated with a high incidence of other skin tumors and hematological malignancies. The purpose of this study was to analyze data from the Israel Cancer Registry regarding the incidence of second neoplasms in patients with MCC and their impact on survival. METHODS Sixty-seven patients in whom MCC was diagnosed between 1983 and 1999 were included. Data were collected on age, gender and ethnic origin, dates of diagnosis of MCC and any other neoplasm, and date and cause of death, if applicable. Comparison of MCC-specific survival, estimated by the Kaplan–Meier product limit method, between patients with no other neoplasm and those with second primary tumors was performed by log rank test. Age-specific standardized incidence ratio (SIR) was calculated using 5751 age- and ethnic-matched malignant melanoma patients as a control group. RESULTS Seventeen patients (25%) had a second neoplasm before, concomitant with, or after the diagnosis of MCC; 2 of them also had a third primary tumor. The SIR was 2.8 (95% CI; range, 1.38–4.22), significantly higher than the control group. Almost half the tumors were squamous cell carcinomas, either skin or head and neck, and most of the remainder were hematological malignancies or breast and ovarian adenocarcinomas. On univariate analysis, the presence of another neoplasm, regardless of its chronology, was associated with higher MCC-specific mortality (65% vs. 40% for patients with MCC only; P = 0.022). Analysis of only those patients in whom a second neoplasm developed during follow-up after treatment for MCC yielded an estimated actuarial risk of developing a second primary of 2.1% for each year of observation. CONCLUSIONS There is a high incidence of second neoplasms, including noncutaneous solid tumors, in patients with MCC. The presence of these neoplasms, whether they appear before, after, or simultaneously with MCC, is associated with a higher MCC-specific mortality. Cancer 2001;91:1358–62. © 2001 American Cancer Society.

A 44-year-old woman presented with erythema multi forme minor followed by disseminated gramuloma nnulare 4 weeks later: The patient was not taking any medication and had no history of herpes simplex infec tion. Involvement of a delayed-type hypersensitivity reaction in the pathogenesis of these two well known disorders, as suggested by immunological investigations, may explain their concurrence in our patient. The substitution of the erythema multiforme minor lesions by an eruption of disseminated granuloma annulare at the same sites suggests the possibility of a at the same sites suggests the possibility of a Koebner phenomenon or an isotopic response.

Aims—To examine the expression of HLA-DR and s2 microglobulin in medullary carcinoma and atypical medullary carcinoma of the breast to determine if the effective presentation of tumour antigens to the immune system can differentiate between these two histopathologically similar entities. Methods—Expression of HLA-DR and s2 microglobulin was examined by immunohistochemical methods in five samples of medullary carcinoma of the breast, which has a relatively favourable prognosis, six samples of atypical medullary carcinoma of the breast, which has a prognosis closer to that of regular invasive duct carcinoma, and 20 samples of invasive duct carcinomas, 10 with an accompanying lymphocytic infiltrate. Results—A positive and significant correlation was found between tumour type and both HLA-DR and s2 microglobulin expression. Expression was most prominent in medullary carcinoma, followed by atypical medullary carcinoma and invasive duct carcinoma with and without lymphocytic infiltrates. The mean intensity and percentage of HLA-DR tumour immunostaining were significantly higher in medullary carcinoma than in the other three tumour groups, as was the mean intensity of s2 microglobulin immunostaining. Mean percentage of s2 microglobulin immunostaining was significantly higher in medullary carcinoma than in invasive duct carcinoma without lymphocytic infiltrates, and showed a trend to increase from invasive duct carcinoma with lymphocytic infiltrates to atypical medullary carcinoma and medullary carcinoma. Conclusions—Medullary carcinoma and atypical medullary carcinoma of the breast differ in their expression of HLA-DR and s2 microglobulin. The relatively favourable prognosis of medullary carcinoma of the breast may be related to effective tumour antigen presentation to the immune system through MHC-I and MHC-II expression. Immunotherapy aimed at MHC-I and MHC-II induction might have a beneficial effect in breast cancer. Key Words: medullary carcinoma of the breast • HLA-DR expression • s2 microglobulin expression

Skin ulcers are commonly described in the juvenile form of dermatomyositis; however, in the adult form they are rarely described. We report on a patient with adult dermatomyositis, deep cutaneous ulcers in the skin folds and livedo reticularis eruption.

Background: Kaposi's sarcoma (KS) accounts for more than 5% of malignancies in immunosuppressed organ transplant patients (OKS). A new herpesvirus (HHV-8) was identified with high prevalence in biopsy specimens of AIDS-KS, endemic KS, and classic KS and in OKS. KS has also been associated with other underlying diseases in patients treated with corticosteroids, but this subset of KS has been reported to contain HHV-8 in only a few case reports. Objective: In this larger study, we determined the prevalence of HHV-8 in seven patients of Jewish origin in whom KS developed during immunosuppressive therapy for different primary diseases (ISKS). Methods: The study included HHV-8 DNA detection by polymerase chain reaction (PCR) coupled with Southern blot and sequence analysis as well as by in situ hybridization. Results: HHV-8 sequences were detected by PCR with confirmation by Southern blot and sequence analysis in 100% of the ISKS samples. Direct sequencing revealed several previously unknown base changes within the 208 bp region from open reading frame 26 (ORF26 208 ) of HHV-8 in ISKS. Conclusion: Ours is the largest known study describing the presence of HHV-8 in iatrogenic KS from immunosuppressed nontransplant patients and provides data of previously unknown sequence variations within the ORF26 of HHV-8 DNA. (J Am Acad Dermatol 1998;38:429-37.)

The modulating effects of mesoderm on ectoderm during the embryonic period have been well documented. Some cutaneous conditions such as dermatofibroma with basaloid proliferation are examples of such a relationship. Seven cases of basal cell carcinoma (BCC) developing in port-wine stains and one case developing over an arteriovenous (AV) malformation have been reported. We report 3 elderly patients with BCC developing over AV malformations of the lower extremities. The association between BCC and AV malformation range from a chance association to a complex interaction between mesenchyme and epithelium, in which mesenchyme induces and modulates epithelial growth through the elaboration of specific growth factors (GF).

Lobular capillary hemangioma and bacillary angiomatosis due to Bartonella infection share several clinical and histopathologic characteristics. We sought to determine whether lobular capillary hemangioma is caused by the same agent as bacillary angiomatosis. Forty-five pathology specimens with a histologic diagnosis of lobular capillary hemangioma obtained from patients with the same clinical diagnosis were tested by immunohistochemistry and polymerase chain reaction for the presence of DNA elements of Bartonella spp. None of the 45 lobular capillary hemangioma specimens tested positive for Bartonella spp. We conclude that lobular capillary hemangioma is not associated with Bartonella spp infection. Further research is required to determine the etiologic agent.

Thyrocyte HLA-DR expression in association with lymphocytic infiltration has been demonstrated in autoimmune diseases of the thyroid. HLA-DR expression has been noted both in association with lymphocytic infiltration and in its absence. However, no prognostic significance was attributed to these findings. The purpose of this study on papillary carcinoma of the thyroid was to examine how tumour behaviour relates independently to HLA-DR expression and lymphocytic infiltration. Fifty patients with papillary carcinoma with known metastatic status were examined for HLA-DR expression utilizing the immunoperoxidase conjugated indirect method. The patients were divided into three groups: no metastases (n = 16), regional lymph node metastases only (n = 25), and distant haematogenous metastases (n = 9). Overall HLA-DR expression was noted in 6, 36 and 55% of the respective groups. Tumour HLA-DR expression without lymphocytic infiltration was noted in 0, 4 and 33% of the respective groups. Tumour HLA-DR expression with lymphocytic infiltration occurred in 6, 32 and 22% of the respective groups. From these findings we conclude that HLA-DR expression with lymphocytic infiltration occurs more frequently than HLA-DR expression in the absence of infiltration. The concurrence of HLA-DR expression and lymphocytic infiltration is most commonly associated with regional lymph node metastasis. HLA-DR expression in the absence of lymphocytic infiltration occurs in tumours with distant haematogenous metastases and is an uncommon feature of tumours that have not metastasized or have metastasized only to lymph nodes. Tumour HLA-DR expression in the absence of lymphocytic infiltration may indicate the potential for more aggressive behaviour.

Tall cell carcinoma of the thyroid gland is an aggressive variant of papillary carcinoma. A high nuclear DNA content has been associated with aggressive clinical behavior and an unfavorable prognosis in several malignant human tumors. It was also described in differentiated thyroid carcinoma. Therefore, analysis of the nuclear DNA content may yield information predictive of aggressive behavior. Accordingly, the DNA content of the tall cell variant was measured and compared with that of the usual form of papillary carcinoma by flow cytometry. Although all the aneuploid tumors were in the tall cell variant group, the difference in nuclear content was not statistically significant. We conclude that differences in the clinical behavior of these neoplasms are not related to alterations in DNA ploidy.

10528 Background: Lung cancer is the leading cause of cancer deaths in the US. Treatment options are determined by tumor subtyping, for which there is lack of standardized, objective, and highly accurate techniques. In 20%-30% of cases significant limitations of tumor quantity and quality prevent full classification of the tumor using traditional diagnostic methods. Using microRNA microarray data generated from over a hundred formalin-fixed, paraffin-embedded (FFPE) primary lung cancer samples, we have identified microRNA expression profiles that differ significantly for the main lung cancer types. Based on these findings, we have developed and validated a microRNA-based qRT-PCR assay that differentiates primary lung cancers into four types: squamous cell carcinoma, non-squamous non-small cell lung cancer, carcinoid and small cell carcinoma. Methods: Over 700 primary tumor samples from different histological types of lung cancer were collected. Samples included FFPE blocks from resection or biopsies and cell blocks from cytology specimens including fine needle aspiration, bronchial brushing and bronchial washing. High-quality RNA was extracted from the samples using proprietary protocols. Expression levels of potential microRNA biomarkers were profiled using microarrays followed by a sensitive and specific qRT-PCR platform. An assay for lung tumors classification using 8 microRNAs on qRT-PCR was developed based on data from 261 samples. This assay was validated on an independent blinded set of 451 cytological and pathological samples. Results: Using the expression levels of 8 microRNAs measured in qRT-PCR, accurate classification of the primary lung tumors into the four main cancer types is obtained. The microRNA-based assay reached an accuracy of 94%. Moreover, cytological samples composed over 50% of the validation set and reached an accuracy of 95%. Conclusions: We present here a new microRNA-based assay for the classification of the four main types of lung cancer based only on the expression of 8 microRNAs. This assay displays very high levels of accuracy for both pathological and cytological samples. The assay comprises a standardized, well-tested and objective tool which can assist physicians in the diagnosis of lung cancer.

10575 Background: Cancer of unknown primary (CUP) constitutes 3%-5% of all newly diagnosed cancer cases. It presents a major diagnostic challenge as knowing the tumor tissue of origin (ToO) of the ...

Background: Lung cancers have traditionally been subdivided into two main groups: Small Cell Lung Cancer (SCLC) and Non Small Cell Lung Cancer (NSCLC). Due to limited tumor sample amounts and an abundance of poor quality samples, accurate subclassification of primary lung cancer using small pre-operative biopsies presents a diagnostic challenge using current tools (H&E and immunohistochemical stains). For patients with primary lung cancer, accurate determination of the tumor type significantly influences treatment decisions. Our previous studies have shown that microRNAs are differentially expressed in different subtypes of lung cancer; hence microRNA expression profiling holds a promising new strategy for the precise subclassification of lung cancers. Based on these findings, we present here the development of a microRNA-based qRT-PCR diagnostic assay that uses pathology and cytology samples in order to classify primary lung cancers into four subtypes: squamous, non-squamous, SCLC and carcinoid. Methods: FFPE tissue blocks and cell blocks from different histological subtypes of lung cancer, taken from primary lung cancer resection or pre-operative diagnostic procedures, were collected. High-quality RNA, including the well-preserved microRNA fraction, was extracted from the samples using proprietary protocols. Expression levels of potential microRNA biomarkers were profiled using a sensitive and specific qRT-PCR platform. Results: We find that combinations of a few microRNAs can successfully differentiate between different histological types of lung tumors. A classification algorithm was developed that reached high accuracy in the identification of the four main subtypes of lung tumors. Conclusion: We developed a highly accurate microRNA-based diagnostic assay that classifies primary lung cancer types with very high sensitivity and specificity. This assay is a simple and reliable diagnostic assay which will offer an accurate and standardized tool for primary lung cancer cytological samples, including those that presently fail pathological evaluation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4943. doi:10.1158/1538-7445.AM2011-4943

Background: Cancer of unknown primary (CUP) constitutes 3%-5% of all newly diagnosed cancer cases. It presents a major diagnostic challenge due to the therapeutic implications of the tissue origin of the cancer. MicroRNAs are a family of non-coding, regulatory RNA genes that are involved in development, differentiation and cell cycle and were shown to be involved in oncogenesis. MicroRNAs are highly tissue-specific and therefore ideal candidates for CUP diagnosis. We have previously presented a biologically-motivated classification assay to identify the tissue of origin using a set of 48 microRNAs measured on a qRT-PCR platform. This assay can differentiate 25 different tumor types from 17 origins. We present here the development and validation of a second generation assay that can identify 42 different tumor types using a custom array platform. Methods: Over 1200 primary and metastatic tumor FFPE samples were used for the training and development of the assay. High-quality RNA, including the well-preserved microRNA fraction, was extracted from the FFPE blocks using proprietary protocols. Expression levels of all known and additional Rosetta's proprietary microRNAs were profiled using a custom array platform. A combination of a decision tree classifier with a KNN classifier was developed to identify the tissue of origin based on the microRNA expression patterns. A validation set of more than 500 independent FFPE samples of primary tumors and metastases was analyzed blindly and classified using the developed assay. Results: The assay is able to identify 42 tumor types based on the expression of only several dozens microRNAs with very high accuracy. Based on the validation set, the sensitivity (positive agreement) for identifying the tumor origin is 85%. In more than 80% of the cases, a single answer was reported, and for these cases, the sensitivity is 90%. Conclusions: Previous studies have highlighted the tissue-specificity of microRNA expression, and have demonstrated their potential use for classification of human malignancies. An assay utilizing microRNA expression in paraffin-embedded tissue for identification of the tissue of origin was previously developed. Here we present the development and the performance of a second generation assay, designed to identify 42 tumor types. This assay will provide an important new tool for diagnosing tumor tissue origin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1148. doi:10.1158/1538-7445.AM2011-1148

Background: Cancer of unknown primary (CUP) constitutes 3%~5% of all newly diagnosed cancer cases. It presents a major diagnostic challenge due to the therapeutic implications of the tissue origin of the cancer. Accurate tumor of origin (ToO) assignment could help guide use of specific therapy which may impact survival. MicroRNAs are a family of noncoding, regulatory RNA genes that are involved in development, differentiation and cell cycle and were shown to be involved in oncogenesis. MicroRNAs are highly tissue specific and therefore ideal candidates for CUP diagnosis. We have previously presented a biologically-motivated classification assay to identify the tissue of origin using a set of 48 microRNAs measured on a qRT-PCR platform. This assay can differentiate 25 different tumor types from 17 origins. We present here the development and validation of a second generation assay that can identify 43 different tumor types using a custom array platform. The assay is based on the normalized expression of less than 70 microRNAs. We will present performance data including inter-laboratory concordance from the validation of the assay. Methods: Over 1200 primary and metastatic tumor FFPE samples were used for the training and development of the next generation assay. High-quality RNA, including the well-preserved microRNA fraction, was extracted from the FFPE blocks using proprietary protocols previously described. Expression levels of all known and additional Rosetta's proprietary microRNAs were profiled using a custom array platform. MicroRNAs containing information about the ToO were identified and two classifiers were developed to utilize the information of the microRNA expression patterns for the diagnosis of ToO. The assay reports a ToO based on these two classifications, where in most cases the assay result is a single ToO reported. Results: The developed assay is able to accurately identify more than 40 tumor types based on the expression of only several dozens microRNAs. The results of a comprehensive validation study using hundreds of independent tumor and metastases FFPE samples will be presented, including inter-laboratory concordance data. Conclusions: Previous studies have highlighted the tissue-specificity of microRNA expression, and have demonstrated their potential use for classification of human malignancies. An assay utilizing microRNA expression in a biopsy for identifying the tissue of origin was developed. Here we present the development and the performance of a second generation assay, designed to identify over 40 tumor types. This assay will provide an important new tool for diagnosing tumor tissue origin.

Background: Diffuse large cell lymphoma (DLBCL) is heterogeneous in its clinical sequel. Approximately 50% of patients relapse after treatment and many succumb to the disease. Risk stratification of these patients is based mainly on the International Prognostic Index (IPI), while molecular profiling is not widely used for prognostication. Thus, there exists a need for identification of additional biomarkers that can be used as prognostic indicators for patients with DLBCL. Here we propose microRNAs, a family of small non-coding regulatory RNAs involved in cancer development, as an emerging class of potential biomarkers for risk stratification of DLBCL. Methods: Expression profiling of microRNAs was performed on RNA extracted from biopsy specimens of 83 DLBCL patients diagnosed and treated at Rabin Medical Center, using custom microRNAs microarrays. All patients were treated with CHOP or CHOP like therapy and were dichotomized into a “good prognosis” group (patients that achieved complete remission and no relapse within 5 years) and a “bad prognosis” group (patients that were either resistant to treatment or relapsed within 9 months). Expression levels were compared between the two groups of samples and statistically analyzed. The expression of differential microRNAs was verified using a microRNA qRT-PCR platform. Results: We found that several microRNAs are significantly differentially expressed in tumors from patients with bad prognosis compared with patients with good prognosis. Proprietary microRNA qRT-PCR showed a high correlation to microarrays and similar separation into prognosis groups based on miR expression. Conclusion: In summary, according to the results of this study, miRNA expression can serve as a novel tool for risk stratification and prognosis prediction of DLBCL patients. This approach can potentially be used to tailor treatment and further management to specific patient needs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4054.

11069 Background: Recent advances in biologically directed therapies for non-small cell lung carcinoma (NSCLC) emphasize the need for more accurate sub-classification of NSCLC, as treatment may be dictated by histologic subtype. In particular, squamous histology can be a counter-indication for treatment by VEGF inhibitors. MicroRNAs are highly tissue-specific biomarkers with potential clinical applicability for defining cancer type and origin. MicroRNAs are well preserved in formalin fixed tissue, making them ideal candidates for molecular markers for use in routinely processed material. Here we report on the development and performance of a microRNA-based assay for the differential diagnosis of squamous from non-squamous NSCLC. Methods: We developed protocols for extraction of high-quality RNA that retain the microRNA fraction from FFPE archival tissue samples. MicroRNA microarrays were used to profile more than a hundred NSCLC samples. Specific microRNA qRT-PCR was used to validate results, and to develop a diagnostic assay. Results: We identified a microRNA biomarker that is strongly overexpressed in squamous cell NSCLC. A diagnostic assay (miRview squamous) was developed, that utilizes qRT-PCR measurement of this microRNA, normalized by an additional microRNA and a small nuclear RNA. This assay was validated on a blinded test set of 64 tumor samples, and had sensitivity of 97% and specificity of 91%. More than ¾ of the samples were classified with high confidence, and these classifications were accurate in 96% of the cases. Conclusions: MicroRNAs are becoming an important tool for classification of cancers. A diagnostic assay based on the specificity of a single microRNA accurately identifies squamous from non-squamous NSCLC. This assay provides an important new tool for the classification of NSCLC. [Table: see text]

8547 Background: Maintenance of telomeres, in the majority of cases by reactivation of telomerase, is obligatory for the indefinite proliferation of tumor cells. In some tumors, telomeres are maint...

Background There is cumulative evidence suggesting that cells of the immune system recognize and may participate in eradicating neoplastic cells. As a result, immune modulation, first with interleukin 2 and later with other cytokines, has been tried in the clinical setting as part of antitumor therapy. Objective To examine the effectiveness and toxicity of a combination of natural interleukins in patients with squamous cell head and neck cancer. Methods Twelve previously untreated patients with various head and neck cancers were treated by peritumoral injection of a combination of cytokines (Multikine), in addition to zinc sulfate, indomethacin, and a single dose of cyclophosphamide, which were administered systemically. Response was evaluated clinically and histopathologically. T-lymphocyte determinants were studied by fluorescence-activated cell sorter analysis (against controls). Results Two patients showed complete regression and another 2 showed partial regression. There were no serious adverse effects of treatment. Pathological study results showed tumor fragmentation and the appearance of multinucleated macrophages. Fluorescence-activated cell sorter analysis showed lymphocyte activation, reflected by an unusually high number of cytotoxic T-lymphocyte activation 4 cells and natural killer cells. Conclusion Multikine warrants further investigation for inclusion in the pharmacotherapeutic armamentarium of head and neck cancer.

We report the light microscopic, transmission and scanning electron microscopic features of the adrenal cortices in rats bearing a medullary thyroid carcinoma cell line transfected with a corticotrophin-releasing hormone (CRH) cDNA expression vector. The animals had elevated CRH, ACTH and corticosterone blood levels, involuted thymuses and markedly enlarged adrenal glands with prominent lipid-depleted cortices and dilated congested capillaries, similar to those of animals treated with ACTH. Using electron microscopy it was found that the enlarged fasciculata and reticularis zones were composed of large, compact cells with abundant smooth endoplasmic reticulum, prominent Golgi complexes, increased number of large mitochondria with focal loss of cristae and cavitation of the internal compartments, numerous lysosomes and prominent elongated microvilli. In addition, small cytoplasmic fragments were seen within the capillary lumina; these structures resembled microvilli that were apparently detached from adrenocortical cells and entered the blood stream via discontinuous endothelium of dilated capillaries. By scanning electron microscopy it was found that the cells had bulging surfaces with scattered pits and numerous long microvilli pointing in different directions. This animal model allows analysis of the effects of protracted CRH excess resembling tumoural CRH-dependent Cushing's syndrome in human patients. Our findings call attention to the role of microvilli in adrenocortical secretion. The increased number and size of microvilli has been thought to lead to an increase in the surface area of adrenocortical cells, thereby facilitating hormone discharge. The detachment of microvilli from adrenocortical cells may represent a form of apocrine secretion and may contribute to hypercorticosteronaemia in CRH excess. Journal of Endocrinology (1992) 135, 271–277

Three hundred sixty-nine infertile couples were followed for 2 to 5 years in a study designed to determine the clinical long-term predictive value of the zona-free hamster ova sperm penetration assay (SPA). Semen analysis (SA), SPA, and a full infertility workup were done in all cases, and only couples in whom the female had no evident cause of infertility were included in the study. During the follow-up period, 106 couples (29%) achieved a pregnancy. Sixteen percent of 131 men who had an SPA of 0%, 23% of 120 men with 1% to 19%, and 48% of 118 men who had a penetration of greater than 19% impregnated their wives 2 to 5 years after the assays. Significant difference in fertility prognosis was found between those who had an SPA greater than 19% and those with an SPA less than 20% (48% versus 20%). Sperm penetration assay greater than 19% was predictive of higher pregnancy rates in both oligospermic (41% versus 17%) and unexplained infertile couples (52% versus 24%). The specificity and positive predictive values of the SPA were higher than those of the SA (77% versus 57% and 48% versus 37%). These findings emphasize the value and importance of the SPA in determining the long-term fertility potential of men.

The tumor microenvironment hosts antibody-secreting cells (ASCs) associated with a favorable prognosis in several types of cancer. Patient-derived antibodies have diagnostic and therapeutic potential; yet, it remains unclear how antibodies gain autoreactivity and target tumors. Here, we found that somatic hypermutations (SHMs) promote antibody antitumor reactivity against surface autoantigens in high-grade serous ovarian carcinoma (HGSOC). Patient-derived tumor cells were frequently coated with IgGs. Intratumoral ASCs in HGSOC were both mutated and clonally expanded and produced tumor-reactive antibodies that targeted MMP14, which is abundantly expressed on the tumor cell surface. The reversion of monoclonal antibodies to their germline configuration revealed two types of classes: one dependent on SHMs for tumor binding and a second with germline-encoded autoreactivity. Thus, tumor-reactive autoantibodies are either naturally occurring or evolve through an antigen-driven selection process. These findings highlight the origin and potential applicability of autoantibodies directed at surface antigens for tumor targeting in cancer patients.