Sapienza Global Bedside Evaluation of Swallowing after Stroke: the GLOBE-3S study.

BACKGROUND AND PURPOSE: Dysphagia occurs in up to 50% of all patients with acute stroke. There is debate regarding which is the most effective screening tool in identifying aspiration in patients with acute stroke. We assessed the accuracy of the Sapienza Global Bedside Evaluation of Swallowing after Stroke (GLOBE-3S), which combines the Toronto Bedside Swallowing Screening Test (TOR-BSST©) with oxygen desaturation and laryngeal elevation measurement during swallowing. METHODS: We prospectively enrolled consecutive patients with stroke within 72 h of symptom onset. All patients with stroke firstly underwent a standard neurological examination, then the GLOBE-3S evaluation and finally the fiberoptic endoscopic evaluation of swallowing (FEES). Two different assessors, a neurologist and a speech pathologist, blind to both the clinical data and each other's evaluation, administered the GLOBE-3S and FEES examination. We assessed the accuracy of the GLOBE-3S in detecting post-stroke swallow impairment with aspiration using the FEES as the standard. RESULTS: We enrolled 50 patients with acute stroke, 28 of whom (56%) had swallowing impairment with aspiration at FEES evaluation. A total of 33 patients (66%) failed the GLOBE-3S evaluation. The GLOBE-3S reached a sensitivity of 100% and a specificity of 77.3% (negative predictive value, 100%; positive likelihood ratio, 4.34). The median time required for the GLOBE-3S to be performed was 297 s. CONCLUSIONS: GLOBE-3S is quick to perform at the bedside and can accurately identify aspiration in patients with acute stroke. By including the measurement of laryngeal elevation and monitoring of oxygen desaturation, it could represent a highly sensitive instrument to avoid the misdiagnosis of silent aspirators.

Unknown primary tumors.

An unknown primary tumor (UPT) is defined by the presence of a metastatic cancer without a known primary site of origin despite a standardized diagnostic workup. Clinically, UPTs show rapid progression and early dissemination, with signs and symptoms related to the metastatic site. The molecular bases of their biology remain largely unknown, with no evidence as to whether they represent a distinct biological entity. Immunohistochemistry remain the best diagnostic tool in term of cost-effectiveness, but the time-consuming "algorithmic process" it relies on has led to the application of new molecular techniques for the identification of the primary site of UPTs. For example, several microarray or miRNA classifications of UPTs have been used, with an accuracy in the prediction of the primary site as high as 90%. It should be noted that validating a prediction of tissue origin is challenging in these patients, since most of them will never have a primary site identified. Moreover, prospective studies to determine whether selection of treatment options based on such profiling methods actually improves patient outcome are still missing. In the last few years functional imaging (i.e. FDG-PET/CT) has gained a main role in the detection of the site of origin of UPTs and is currently recommended by the European Association of Nuclear Medicine. However, despite recent refinements in the diagnostic workup, the site of origin of UPT often remains elusive. As a consequence, treatment of patients with UPT is still empirical and inadequate.

Gene variants associated to malignant thyroid disease in familial adenomatous polyposis: a novel APC germline mutation.

BACKGROUND AND AIM: Familial adenomatous polyposis (FAP) is an autosomal inherited syndrome characterized by hundreds to thousands colorectal adenomatous polyps with oncological transformation lifetime risk of 100%. FAP is mainly associated with mutations in APC (autosomal dominant inheritance) or MUTYH (autosomal recessive inheritance) genes. Affected individuals are at increased risk of developing extra-intestinal tumors. Lifetime risk of developing thyroid carcinoma has been described in previous reports of about 2-12%, mainly in females, and the mean age is below 30 yr. About 95% of cancers are papillary thyroid carcinomas (PTC), mostly multifocal. The aim of this study was to evaluate the frequency of PTC among our series of FAP patients and to assess the type of gene mutation associated with the disease. METHODS: Fifty-four subjects from 36 FAP families were selected (29 females/25 males) and the mean age (±SD) at diagnosis was 28.8±10.8 yr. All patients underwent blood examination for thyroid hormones and antibodies, germline mutational analysis of APC and/or MUTYH genes, thyroid ultrasound, and endocrinological evaluation. RESULTS: In 13/54 (24.1%) subjects, an eumetabolic thyroid disease was found: plurinodular disease in 7/54 (13.0%); single nodule in 4/54 (7.4%); in 2/54 patients (3.7%), we found a malignant nodule characterized after total thyroidectomy as a classical PTC. Both patients were female and showed a classic FAP phenotype. Mutational analysis revealed in the first patient the APC germline mutation 3183_87del ACAAA and in the second patient the del9-10 (del9080dup11) novel APC variant; the first mutation has been already reported in association with PTC; to our knowledge the second mutation has never been previously reported in association with FAP. CONCLUSIONS: In the population examined, the estimated prevalence of thyroid malignant diseases was 3.7%. In both patients, the identified APC gene pathogenetic variants mapped within the 5' region of the gene, previously reported as a PTC-associated mutational hot spot. Both patients had classic FAP phenotype and genetic analysis revealed two pathogenetic APC mutations: c.3183_87delACAAA, a recurrent pathogenetic variant and del9-10 (del9080dup11), a novel, not previously described genomic rearrangement. In agreement with previous studies, the morpho-functional surveillance of thyroid in FAP series should be recommended. A better insight into the overall genotype-phenotype correlation of APC gene mutations would be helpful for the identification of at-risk individuals.

Hyperthermic isolated perfusion with tumor necrosis factor-alpha and doxorubicin for the treatment of limb-threatening soft tissue sarcoma: the experience of the Italian Society of Integrated Locoregional Treatment in Oncology (SITILO).

BACKGROUND: Tumor necrosis factor-alpha (TNFalpha)-based hyperthermic isolated limb perfusion (HILP) is routinely carried out at most oncological institutions in the treatment of locally advanced soft tissue limb sarcoma (STS), employing high TNFalpha dosages. After a phase I-II study, the SITILO (Italian Society of Integrated Locoregional Therapies in Oncology) centers began to employ the lower dose of 1 mg of TNFalpha. The aim of this paper is to report on the results obtained in 75 patients with limb-threatening STS treated with a low TNFalpha dose and doxorubicin (Dx). PATIENTS AND METHODS: HILP with TNFalpha (at a dosage of either 1 mg) and Dx was administered to 75 patients with limb-threatening STS: 37 males and 38 females; median age 50 years; tumor in the lower and upper limbs in 58 and 17 patients, respectively; primary and recurrent tumors in 45 and 30 patients, respectively. Most tumors (77%) were high grade. Tumor resection was carried out 6 to 8 weeks after HILP. RESULTS: The grade of limb toxicity was mild to moderate in the vast majority of patients (76%). Grades IV and V were observed, but only when high muscle temperatures were recorded and high TNFalpha dosages were employed. Systemic toxicity was also mild to moderate and there were no postoperative deaths. Complete and partial tumor responses were 34% and 48%, respectively, with an overall response of 82% . Limb sparing surgery was carried out in 85.3% of patients. At a median follow-up of 28 months, 16 recurrences (21.3%) were recorded, with a 5-year locoregional disease-free survival of 63% . The 5-year disease-free survival and overall survival were 36.7% and 61.6%, respectively. CONCLUSION: HILP with 1 mg of TNFalpha is an effective neoadjuvant therapy resulting in a high rate of limb sparing in limb-threatening STS, with acceptable local reactions and negligible systemic toxicity.

Prognostic factors influencing tumor response, locoregional control and survival, in melanoma patients with multiple limb in-transit metastases treated with TNFalpha-based isolated limb perfusion.

BACKGROUND: In isolated limb perfusion (ILP) with tumor necrosis factor-alpha (TNFalpha) and interferon (IFN)-gamma, pioneered by Lienard and Lejenne in 1988, TNFalpha was empirically employed at a dosage (3-4 mg) ten times higher than the systemic maximum tolerable dose (MTD). We previously conducted a phase I/II study in 20 patients with in-transit melanoma metastases, using a combination of melphalan and TNFalpha at dosages ranging from 0.5 to 3.3 mg. The dose of 1 mg of TNFalpha was identified as optimal in terms of both efficacy and toxicity. The aim of the present study was to describe our experience with 113 stage IIIA/IIIAB melanoma patients treated with a TNFalpha-based ILP and identify prognostic factors for response, locoregional control and survival. PATIENTS AND METHODS: Patients at stage IIIA-IIIAB (presence of in-transit metastases and/or regional node involvement) were considered eligible. The disease was bulky (>or=10 nodules3 cm) in 42.5% of the patients and unresectable in 33% . Forty patients were treated with a TNFalpha dosage of >1 mg and 73 with 1 mg. Patients with tumors in the upper and lower limbs were submitted to ILP via axillary and iliac vessels, respectively. TNFalpha was injected in the arterial line of an extracorporeal circuit at the pre-established dose, followed by melphalan (13 and 10 mg/l of limb volume for the upper and lower limbs, respectively) 30 minutes later. RESULTS: Complete responses (CR) and partial responses (PR) were 63% and 24.5%, respectively, with an objective response (OR) of 87.5%. No change (NC) was observed in only 12.5% of the patients. Upon multivariate analysis, only bulky disease maintained its independent value for tumor response with an odds ratio of 4.07 and a p-value of 0.02. The 5-year locoregional disease-free survival was 42.7%. Upon multivariate analysis, the only prognostic factors were stage, age and bulky disease. The 5-year overall survival was 49%. Multivariate analysis showed that only sex, stage and CR maintained their independent values. CONCLUSION: TNFalpha-based ILP was proven to be an effective treatment for melanoma patients with in-transit metastases. The TNFalpha dosage of 1 mg was as effective as 3-4 mg, with lower toxicity and cost. We propose that TNFalpha and melphalan-based ILP should be employed for bulky tumors or after failure of melphalan-based ILP.

Hearing and cognitive impairment: a functional evaluation of associative brain areas in patients affected by Alzheimer's disease.

Auditory dysfunction observed in patients with cognitive diseases is probably due to the alteration of some brain areas involved in sound stimulus processing. The present study aimed to investigate differences in such processing and in connectivity of the primary auditory cortex in patients affected by Alzheimer's disease (AD) and in normal subjects. We examined 131 diagnosed AD patients and a control group (CG) of 36 normal subjects. After a complete clinical investigation, focused on hearing function, all subjects underwent a brain FDG PET/CT. AD subjects vs CG showed reduced glucose consumption in BA 6,7,8,39, whereas we did not find differences in the primary auditory cortex. In AD, connectivity analyses showed a positive correlation of the primary auditory cortex with BA 6,8,21,31,39,40,42 and a negative correlation with BA 19, cerebellum and basal ganglia. Our findings suggest that neurological evaluation of patients with hearing loss might allow earlier (preclinical) identification of those affected by cognitive impairment.

Effectiveness of delayed-release dimethyl fumarate on patient-reported outcomes and clinical measures in patients with relapsing-remitting multiple sclerosis in a real-world clinical setting: PROTEC.

BACKGROUND: Patient-reported outcomes (PRO) and clinical outcomes give a broad assessment of relapsing-remitting multiple sclerosis (RRMS) disease. OBJECTIVE: The aim is to evaluate the effectiveness of delayed-release dimethyl fumarate (DMF) on disease activity and PROs in patients with RRMS in the clinic. METHODS: PROTEC, a phase 4, open-label, 12-month observational study, assessed annualized relapse rate (ARR), proportion of patients relapsed, and changes in PROs. Newly diagnosed and early MS (≤3.5 EDSS and ≤1 relapse in the prior year) patient subgroups were evaluated. RESULTS: Unadjusted ARR at 12 months post-DMF versus 12 months before DMF initiation was 75% lower (0.161 vs. 0.643, p < 0.0001) overall (n = 1105) and 84%, 77%, and 71% lower in newly diagnosed, ≤3.5 EDSS, and ≤1 relapse subgroups, respectively. Overall, 88% of patients were relapse-free 12 months after DMF initiation (84%, newly diagnosed; 88%, ≤3.5 EDSS; 88%, ≤1 relapse). PRO measures for fatigue, treatment satisfaction, daily living, and work improved significantly over 12 months of DMF versus baseline. CONCLUSION: At 12 months after versus 12 months before DMF initiation, ARR was significantly lower, the majority of patients were relapse-free, and multiple PRO measures showed improvement (overall and for subgroups), suggesting that DMF is effective based on clinical outcomes and from a patient perspective.Clinical trial: A Study Evaluating the Effectiveness of Tecfidera (Dimethyl Fumarate) on Multiple Sclerosis (MS) Disease Activity and Patient-Reported Outcomes (PROTEC), NCT01930708, https://clinicaltrials.gov/ct2/show/NCT01930708.

The hump columellar strut: a reliable technique for correction of nasal tip underprojection.

Nasal tip under projection is often found in rhinoplasty cases both for congenital or post-traumatic deformity. Nasal trauma may result in alteration of the external and internal nasal structures with following aesthetic impairment and difficulties in breathing. Post-traumatic surgery is frequent, but restoration of pre-traumatic form and function remains a challenge. The present paper describes a new method to increase tip projection by a columellar strut harvested from the autologous nasal bone and cartilage of the resected hump. A total of 15 cases (11 women/4 men, mean age 32.6 ± 12.3 years) of major tip projection/misalignment abnormalities to be corrected by increased nasal tip projection were drawn, and all underwent closed or open rhinoplasty with the placement of a bony columellar strut harvested from the resected hump of the patient. Short and long-term advantages of this procedure are to be underlined. Harvesting is routinely performed during dorsal resection and preparation of the graft is easy. Differently from bone of the vomer or the inferior turbinate, this is cortical bone straight in shape and rigid in framework, and therefore ideal to gain reliable tip support overtime. No additional harvesting areas are needed. Placement of this bony strut is carried out in the standard fashion without additional dissection or further procedures. Long-term follow-up shows maintained projection over time. This graft can be combined with various grafting or suturing techniques usually applied according to each surgeon's experience and the needs of each patient.

Functional outcome of auditory implants in hearing loss.

The auditory implant provides a new mechanism for hearing when a hearing aid is not enough. It is the only medical technology able to functionally restore a human sense i.e. hearing. The auditory implant is very different from a hearing aid. Hearing aids amplify sound. Auditory implants compensate for damaged or non-working parts of the inner ear because they can directly stimulate the acoustic nerve. There are two principal types of auditory implant: the cochlear implant and the auditory brainstem implant. They have common basic characteristics, but different applications. A cochlear implant attempts to replace a function lost by the cochlea, usually due to an absence of functioning hair cells; the auditory brainstem implant (ABI) is a modification of the cochlear implant, in which the electrode array is placed directly into the brain when the acoustic nerve is not anymore able to carry the auditory signal. Different types of deaf or severely hearing-impaired patients choose auditory implants. Both children and adults can be candidates for implants. The best age for implantation is still being debated, but most children who receive implants are between 2 and 6 years old. Earlier implantation seems to perform better thanks to neural plasticity. The decision to receive an implant should involve a discussion with many medical specialists and an experienced surgeon.

Approach to the correction of drooping tip: common problems and solutions.

The drooping tip deformity is both a bothersome aesthetic feature and functional impairment of the nose. Both static and dynamic factors may affect tip appearance and it seems logical to take into account these factors when planning correction of drooping tip. Many studies have examined this topic, but its treatment remains controversial. In order to make nasal tip surgery successful, it is useful to identify the keystone anatomical characteristics of the tip itself. Naso-labial angle, nostril axis, tip rotation angle according to Frankfort plane and columellar-facial angle may be measured to assess nasal tip position. The present study focuses on the authors' personal experience on the key anatomic changes of the nose that deserve correction and on the main surgical steps needed to achieve consistent results when dealing with a drooping tip. Pre- and post-operative nasal tip rotation and projection were studied. Correction of the drooping tip was accomplished by an open or closed septorhinoplasty approach according to patient's needs. The surgical techniques mostly employed for tip repositioning was septum straightening (41/41) and tongue-in-groove (36/41 cases) (87.8%). A columellar strut was used in 8/41 (19.51%) cases. LLC cephalic resection was applied in 29/41 patients (70.73%), LLC re-orienting sutures were made in 18/41 cases (43.9%) and lateral crural overlay was needed in 2/41 (4.8%). The key anatomic changes of the nose that deserve correction and the surgical steps needed to ease the often intriguing pre-operative decision-making process are reviewed.

Ionic mechanisms underlying differential vulnerability to ischemia in striatal neurons.

Brain cells express extremely different sensitivity to ischemic insults. The reason for this differential vulnerability is still largely unknown. Here we discuss the ionic bases underlying the physiological responses to in vitro ischemia in two neostriatal neuronal subtypes exhibiting respectively high sensitivity and high resistance to energy deprivation. Vulnerable neostriatal neurons respond to ischemia with a membrane depolarization. This membrane depolarization mainly depends on the increased permeability to Na+ ions. In contrast, resistant neostriatal neurons respond to ischemia with a membrane hyperpolarization due to the opening of K+ channels. Interestingly, in both neuronal subtypes the ischemia-dependent membrane potential changes can be significantly enhanced or attenuated by a variety of pharmacological agents interfering with intracellular Ca2+ entry, ATP-dependent K+ channels opening, and Na+/Ca2+ exchanger functioning. The understanding of the ionic mechanisms underlying the differential membrane responses to ischemia represents the basis for the development of rational neuroprotective treatments during acute cerebrovascular insults.

Antigenic heterogeneity of skin tumors of nonmelanocyte origin: analysis with monoclonal antibodies to tumor-associated antigens and to histocompatibility antigens.

Surgically removed benign and malignant human skin lesions of nonmelanocyte origin have been tested with monoclonal antibodies to la antigens, to the HLA-A,B antigenic molecular complex, and to melanoma-associated antigen(s) (MAA). MAA include a high-molecular-weight (HMW) MAA, a 115,000-molecular-weight MAA, a 94,000-molecular-weight MAA, and a cytoplasmic MAA. Indirect immunofluorescence was used as the assay system because of the limited amount of tissue available. When the amount of tissue available was sufficient, double determinant immunoassays (DDIA) were used to quantitate the level of the HMW MAA and of the cytoplasmic MAA. The results of the DDIA were in agreement with those of indirect immunofluorescence in more than 75% of the cases. Malignant skin tumors of various histiotypes displayed three types of changes: 1) appearance of la antigens and cytoplasmic MAA, 2) increase in the level of the HMW MAA, of a 115,000- and a 100,000-molecular-weight MAA, and 3) reduction in the level of HLA-A,B antigens and beta 2-microglobulin. A significant heterogeneity was found in the antigenic profile among various lesions of a given histiotype as well as among tumor cells within a given lesion.

Expression of tumor antigen correlated with metastatic potential of Lewis lung carcinoma and B16 melanoma clones in mice.

Expression of a tumor-associated antigen, recognized by a monoclonal antibody (MoAb 135-13C) to lung carcinoma cells, has been studied in cloned Lewis lung carcinoma (3LL) and in B16 melanoma (F1 and F10) tumor lines endowed with different metastatic potentials. MoAb 135-13C recognizes a protein complex (tumor-specific Mr 180,000 protein) that appears on the cell surface of several murine lung carcinomas but is not detected on normal cells in culture. Standard metastatic variants of B16 melanoma (F1 and F10) and two variant sublines of 3LL (M1087 and BM21548) together with the parental line of 3LL have been used for these experiments. The two cloned variant lines derived from 3LL have been shown to retain high (M1087) and low (BM21548) metastatic phenotypes during in vivo passaging. We found that all three cell lines of 3LL bind monoclonal antibody specifically, but one cell variant with higher metastatic potential shows a higher capacity to bind MoAb 135-13C than did the other variant. Similarly we found that B16 F10 cells bind higher amounts of MoAb 135-13C than did B16 F1 cells. In addition the analysis of the amounts of MoAb 135-13C bound to the cell surface of several other in vitro and in vivo tumor lines with different metastatic capacity demonstrates that all tumor lines which express high ability to colonize to the lung also express, on the cell surface, higher amounts of tumor-specific Mr 180,000 protein. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiograms of immunoprecipitates from cell lysates of 3LL and B16 tumor lines demonstrate that MoAb 135-13C specifically precipitated three proteins banding at molecular weights of 204,000, 134,000, and 116,000. We conclude that MoAb 135-13C recognizes a surface protein complex which is present in higher amounts in 3LL and B16 cells which possess higher capacity to metastasize to the lung.

A cytoplasmic human melanoma associated antigen as a marker of activation in lymphoid cells.

A cytoplasmic glycoprotein, originally identified by the monoclonal antibody 465.12S in melanoma tumors, is significantly increased in epithelial cells of different histotype following transformation. In the present study we show that the cytoplasmic melanoma associated antigen (cyt-MAA) is drastically enhanced in lymphoid cells by polyclonal and allogeneic stimulation, as well as by transformation. Normal T-cells with helper and suppressor phenotype are far more susceptible than B-cells to this enhancement. However, among transformed lymphoid cells, the expression of the cyt-MAA does not correlate with lineage, but rather with stage of differentiation. Acute lymphoblastic leukemias represent the only exception, since in these lymphoid malignancies cyt-MAA levels are highly heterogeneous even within groups of phenotypically similar lesions. Thus, the expression of the cyt-MAA is shared by cells of distant embryological origin in early stages of their differentiation and/or during proliferation. Quantitation of the cyt-MAA may provide useful information for the classification of some lymphoid malignancies.

Level of a membrane-bound high-molecular-weight melanoma-associated antigen and a cytoplasmic melanoma-associated antigen in surgically removed tissues and in sera from patients with melanoma.

Utilizing double-determinant immunoassays (DDIAs), the high-molecular-weight melanoma-associated antigen (HMW-MAA) was detected only in fetal skin and in one nipple of 54 normal tissues from adults tested, while the cytoplasmic MAA recognized by the monoclonal antibody 465. 12S was found in most of the normal tissues tested. Among malignant lesions, the HMW-MAA was found in melanomas, astrocytomas, and skin carcinomas; the cytoplasmic MAA was found in all of the malignant lesions tested, even those which originated from normal tissues without detectable cytoplasmic MAA. The levels of the HMW-MAA and of the cytoplasmic MAA showed marked variations in malignant lesions removed from various patients, as well as in autologous metastatic lesions removed from four patients with melanoma. No relationship was found between the degree of expression of the two MAA analyzed and the clinical stage of the disease. Both types of MAA were found in sera from patients with melanoma or other types of cancers, as well as in sera from healthy donors. The level of the HMW-MAA tended to be higher in patients with Stage IV melanoma.

Heterogeneity in the expression of HLA and tumor-associated antigens by surgically removed and cultured breast carcinoma cells.

Surgically removed normal and malignant mammary tissues and human breast carcinoma cell lines were tested in binding assays with monoclonal antibodies to HLA-A,B,C antigens, beta 2-microglobulin, HLA-DR antigens, and tumor-associated antigens; the latter included a Mr 280,000, a Mr 94,000, and a Mr 85,000 membrane-bound glycoprotein and a cytoplasmic antigen. HLA-A,B antigens, beta 2-microglobulin, HLA-DR antigens, and the cytoplasmic antigen are expressed by normal mammary cells. Their malignant transformation may be associated with quantitative changes in the expression of these antigens and with the appearance of Mr 94,000 and Mr 85,000 glycoproteins. The Mr 280,000 glycoprotein was detected on only one of the breast carcinoma cell lines tested. Analysis of primary tumors and autologous axillary lymph node metastasis from 13 patients has shown differences in the expression of all the antigens tested between primary and metastatic lesions.

Effect of recombinant human leukocyte, fibroblast, and immune interferons on expression of class I and II major histocompatibility complex and invariant chain in early passage human melanoma cells.

Twenty-five early-passage (less than or equal to 8) melanoma cell lines, isolated from ten patients with metastatic melanoma, were analyzed by a combination of serological, immunochemical, and molecular methods for mRNA levels, synthesis, and surface expression of MHC class I and class II antigens prior to and following exposure to recombinant human leukocyte (IFN-alpha A), fibroblast (IFN-beta), and immune (IFN-gamma) interferon. All the cell lines expressed variable levels of HLA class I gene products that were up-regulated to different extents upon exposure to specific interferons (IFNs). HLA class II antigens were expressed in 22 of the 25 melanoma lines and IFN-gamma increased the levels of class II mRNA, protein synthesis, and surface expression in all cultures displaying baseline expression. A significant up-regulation of class II antigen expression by IFN-alpha or -beta, associated with higher levels of class II transcripts and enhanced synthesis, was found only in two early-passage human melanoma cell lines. In three lesions from the same patient which did not constitutively express class II antigens, no expression of these glycoproteins could be induced with any of the IFNs. These results indicate that IFN-gamma does not act as a de novo inducer of class II antigen expression in early-passage human melanoma cell lines. This hypothesis is further supported by analysis of class II-associated invariant chain (Ii) expression, which is expressed and induced by IFNs in a manner similar to that of class II antigens. The present study also indicates that early-passage metastatic melanoma lesions from the same patient are heterogeneous in their de novo expression of major histocompatibility antigens and in their modulation by IFNs.

Class I major histocompatibility complex enhancement by recombinant leukocyte interferon in the peripheral blood mononuclear cells and plasma of melanoma patients.

In vivo administration of escalation doses of recombinant alpha-interferon (IFN-alpha) during a phase I trial in malignant melanoma patients caused dose-dependent increases in the mRNA accumulation, synthesis, steady state cellular content, and plasma membrane expression of class I major histocompatibility complex molecules in peripheral blood mononuclear cells. In addition, circulating levels of class I molecules were also enhanced. These findings show that (a) antigenic enhancement by biomodifiers may occur in vivo, in humans and (b) the mechanism of class I major histocompatibility complex enhancement by IFN-alpha is similar in vitro and in vivo. Furthermore, because peripheral blood mononuclear cells of different melanoma patients display different susceptibility to IFN-alpha, the entity of their antigenic modulation may represent a useful parameter to evaluate the efficacy of different therapeutic regimens and/or assess the individual susceptibility to the molecular changes induced by IFN-alpha.

Double determinant immunoassay to measure a human high-molecular-weight melanoma-associated antigen.

Using monoclonal antibodies to distinct determinants of a human high-molecular weight melanoma-associated antigen (HMW-MAA), a double determinant immunoassay has been developed. The assay is specific and reproducible. Its sensitivity is influenced by the incubation time of antibodies with antigen sources and the combination of antibodies, as well as by the pH of the buffer and the incubation time used to coat plates with antibodies. Testing with the double determinant immunoassay of Nonidet P-40 extracts of human cell lines and of surgically removed normal and malignant tissues has confirmed the restricted tissue distribution of the HMW-MAA. In addition, significant differences have been found in the level of HMW-MAA in melanoma cell lines, as well as in melanoma lesions removed from different patients and from different sites of a given patient. The amount of HMW-MAA shed by various melanoma cell lines does not correlate with their cell surface expression and with their level in Nonidet P-40 extracts. Interferon and hyperthermia increase the shedding of the HMW-MAA by melanoma cells.

Low prevalence of selective human leukocyte antigen (HLA)-A and HLA-B epitope losses in early-passage tumor cell lines.

The down-regulation of human leukocyte antigen (HLA) class I molecules, especially the selective down-regulation of certain allelic products, is believed to represent a major mechanism of tumor escape from immune surveillance. In the present report, an original approach is described to precisely evaluate and classify HLA class I epitope losses in 30 cancer patients with malignant melanoma and lung, breast, endometrium, ovary, and colon carcinoma tumors. Early-passage tumor cell lines were established in culture from the corresponding metastatic tumor lesions obtained in each patient. Both the cell lines and the tumor lesions were compared, in their HLA-A and -B expression, to the peripheral blood mononuclear cells (PBMCs) obtained from the same patient (autologous PBMCs). On the basis of HLA-genotyping data, the appropriate monoclonal antibodies identifying mono- and poly-morphic HLA-A and HLA-B epitopes were selected from a panel of 34 antibodies for a total of 24 testable alleles. The selected antibodies were used not only in immunohistochemical assays on cryostatic tumor sections and cytospins of PBMCs but also in quantitative, sensitive flow cytometry assays on early-passage tumor cells and PBMC suspensions. With this latter method, a low overall HLA expression was detected in 26 tumor cell explants and a complete, generalized HLA-A, HLA-B, HLA-C loss in the remaining 4 cases. However, no complete, selective loss of any of the 45 tested HLA-A and HLA-B allomorphs was observed. Sequences from all of the HLA class I alleles could be detected at the genomic DNA level in tumor cells and tissues. At variance from the literature and the results of immunohistochemical experiments performed in parallel on the corresponding tumor lesions, the relative proportions of the various HLA epitopes were relatively preserved in each early-passage cell line/PBMC pair, and selective increases, rather than decreases, in the expression of polymorphic HLA epitopes had the highest prevalence and greatest magnitude. Our data suggest an alternative tumor stealth strategy in which up- and down-regulation are equally important. This alternative model of tumor-host interaction better fits the available models of tumor cell recognition by CTLs and natural killer cells bearing activatory and inhibitory receptors for HLA-A, HLA-B, HLA-C molecules.