Interdisciplinary management of EGFR-inhibitor-induced skin reactions: a German expert opinion.

BACKGROUND: Anti-epidermal growth factor receptor treatment strategies, i.e. monoclonal antibodies such as cetuximab and panitumumab, or epidermal growth factor receptor (EGFR) small molecule tyrosine kinase inhibitors, such as erlotinib and gefitinib, have expanded the treatment options for different tumor types. Dermatologic toxic effects are the most common side-effects of EGFR inhibitor therapy. They can profoundly affect the patient's quality of life. PURPOSE: The aim of this study was to provide interdisciplinary expert recommendations on how to treat patients with skin reactions undergoing anti-EGFR treatment. MATERIAL AND METHODS: An expert panel from Germany with expertise in medical oncology, dermatology or clinical pharmacology was convened to develop expert recommendations based on published peer-reviewed literature. RESULTS: The expert recommendations for the state-of-the-art treatment of skin reactions induced by EGFR inhibitor therapy include recommendations for diagnostics and grading as well as grade-specific and stage-adapted treatment approaches and preventive measures. It was concluded that EGFR-inhibitor-related dermatologic reactions should always be treated combining basic care of the skin and a specific therapy adapted to stage and grade of skin reaction. For grade 2 and above, specific treatment recommendations for early- and later-stage skin reactions induced by EGFR-inhibitor therapy were proposed. CONCLUSION: This paper presents a German national expert opinion for the treatment of skin reactions in patients receiving EGFR inhibitor therapy.

Quality of life in localised malignant melanoma.

BACKGROUND: The incidence of melanoma is still increasing in fair-skinned populations. At least 80% of patients have localised disease and expect a 5-year relative survival of >90%. PATIENTS AND METHODS: In 2003-2004, disease-free patients with localised melanoma were recruited from the Munich Cancer Registry to answer quality-of-life (QoL) questionnaires 2 years after treatment. RESULTS: A response rate of 72% was achieved from a total of 1085 distributed questionnaires. Hundred and seventeen questionnaires had to be excluded because of updated information about secondary tumour and progression events. Thus, questionnaires from 664 patients were evaluated. QoL scores in melanoma patients were essentially similar to those of a general population. Differences were detected between women and men concerning emotional and sexual functioning. Age and number of comorbidities were the strongest factors influencing most all aspects of QoL. Fifty percent of patients referred to deficits in communication with their doctors. CONCLUSIONS: Patients who overcome melanoma do not necessarily have a reduced QoL. Strategies used by these melanoma patients resulted in similar levels of coping as previous studies in comparable general populations. Nevertheless, doctor-patient communication was correlated with emotional and social functioning and should be emphasised in treatment and care of melanoma patients.

Distal and proximal interleukin (IL)-10 promoter polymorphisms associated with risk of cutaneous melanoma development: a case--control study.

Inherited promoter polymorphisms of the interleukin (IL)-10 gene resulting in altered IL-10 production may contribute to a genetic susceptibility for melanoma. We investigated the role of a haplotype from distal as well as proximal polymorphic sites [-7400InDel, -6752AT (rs6676671), -3538AT (rs1800890), -1087AG (rs1800896), -597AC (rs1800872)] of the IL-10 5'-flanking region in a hospital-based case-control study of 165 Caucasian patients with cutaneous melanoma from Germany in comparison with 162 healthy cancer-free Caucasian control participants from the same area matched by age. Using multivariate analysis for the number of nevi and skin type, the IL-10 'higher producing' haplotype ITAGC was found to be significantly associated with a reduced risk of developing melanoma (adjusted P=0.02). Although our findings need to be confirmed by independent and larger multicenter studies, we have described for the first time the association of distal gene variants of the IL-10 gene as an independent risk factor for melanoma.

Identification and characterization of 20 immunocompetent patients with simultaneous varicella zoster and herpes simplex virus infection.

BACKGROUND: It has been shown that varicella zoster virus (VZV) and herpes simplex virus (HSV) can co-localize to the same sensory ganglion. However, only a few case reports on VZV/HSV co-infections exist. Objective To identify and characterize patients with concurrent VZV and HSV infection at the same body site. SUBJECTS/METHODS: In 1718 patients, the presence of VZV and HSV in suspicious skin lesions was investigated by polymerase chain reaction analysis. Clinical characteristics of co-infected patients were compared with matched control patients infected with either VZV or HSV. The data are discussed in the context of an extensive review of the literature. RESULTS: Twenty (1.2%) of 1718 patients were infected with both VZV and HSV at the same body site. The mean age was 54 years (range, 2-83). The clinical diagnosis was zoster in 65%, herpes simplex in 20%, varicella in 10% and erythema multiforme in 5% of cases. The trigeminus region was affected in 60% and the trunk in 25%. Involvement of the head was most commonly associated with a severe course of disease and with older age. CONCLUSION: Simultaneous VZV/HSV infection is rare but can occur in immunocompetent patients, which is often overlooked. The majority of cases is localized to the trigeminus region and affects elderly people.

Peripheral blood tyrosinase messenger RNA detection and survival in malignant melanoma.

BACKGROUND: The most widely accepted criteria for the evaluation of prognosis of malignant melanoma are histopathologic and clinical presentation. No currently available laboratory tests provide additional prognostic information. It has recently been suggested that reverse transcription and polymerase chain reaction (RT-PCR)-based detection of tyrosinase messenger RNA (mRNA) in peripheral blood might be useful in the early detection of circulating tumor cells, since tyrosinase is thought to be a melanocyte-specific marker. PURPOSE: To further evaluate the clinical relevance of this potential marker, we examined peripheral blood samples from patients with malignant melanoma in different stages of disease for the presence of tyrosinase mRNA. METHODS: Total cellular RNA was extracted from heparinized peripheral blood cells from 64 patients with malignant melanoma, from five healthy control subjects, and from four patients with other cancers using the RNAzol A method. For analysis of tyrosinase mRNA, RT-PCR was performed as previously described by Smith et al.; the sensitivity of this assay was tested using RNA extracted from human melanoma cells (SK-mel 1 and SK-mel 3 cell lines) serially diluted with peripheral blood obtained from healthy control subjects. Two additional human melanoma cell lines (SK-mel 30 and RPMI-7951) served as positive controls for RT-PCR detection of tyrosinase mRNA. Overall patient survival curves were constructed using Kaplan-Meier estimates. RESULTS: Tyrosinase mRNA was detected by RT-PCR assay of all four of the established melanoma cell lines tested. Nine of the 64 patients with malignant melanoma were found to have detectable tyrosinase mRNA in their peripheral blood cells (tyrosinase-positive patients). The 16 patients with localized primary melanoma did not have detectable tyrosinase mRNA in their peripheral blood cells. Among the 48 patients with metastatic disease, all 27 patients who exhibited no evidence of disease progression were tyrosinase negative. Notably, all nine tyrosinase-positive patients had visceral metastases and were found to exhibit disease progression at the time of the sampling. Four of the nine tyrosinase-positive patients were also found to test negative at times without evidence of progressive disease; one patient became negative after achieving stable disease and three became positive for tyrosinase transcripts on disease progression. The probability of survival from time of sampling was significantly lower in the nine tyrosinase-positive patients when tested versus the 23 patients with comparable disease but without detectable tyrosinase mRNA (two-sided; P < or = .05). CONCLUSIONS: The results of this study demonstrate that the detection of tyrosinase mRNA in cells in the peripheral blood by RT-PCR may be a useful prognostic marker for predicting tumor progression and poor clinical outcome in patients with malignant melanoma.

Quantitation of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase gene expression in human tumors using the polymerase chain reaction.

A polymerase chain reaction (PCR)-based method was used to quantitate the expression levels of low abundance genes relevant to cancer drug activity. RNA from tumor samples as small as 20 mg was isolated and converted to cDNA using random hexamers. The 5' primers for the PCR contained a T7 polymerase promoter sequence, allowing the PCR-amplified DNA to be transcribed to RNA fragments. In each sample, the linear ranges of amplification of each cDNA of interest were established. Relative gene expressions were calculated by extrapolating the amounts of PCR products generated within the linear amplification regions of each gene to equal volumes of the cDNA solution. The method was accurate to less than a 2-fold difference in expression levels. Using beta 2-microglobulin and beta-actin gene expressions as internal reference standards and cDNA from HT-29 cells as an external linearity standard, we measured the relative expressions of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase in a number of clinical tumor samples. The expressions of these genes varied from 50- to 100-fold among different tumors, although most of the values were grouped within about a 10-fold range. The amount of thymidylate synthase gene expression in tumor tissues was directly proportional to the content of thymidylate synthase protein. Those tumors with the lowest thymidylate synthase expression had the best response to both the 5-fluorouracil-leucovorin and 5-fluorouracil-cisplatin combinations.

Interlaboratory evaluation of a new reverse transcriptase polymerase chain reaction-based enzyme-linked immunosorbent assay for the detection of circulating melanoma cells: a multicenter study of the Dermatologic Cooperative Oncology Group.

PURPOSE: Reverse transcription-polymerase chain reaction (RT-PCR)-based detection of tyrosinase mRNA is the most frequently used laboratory method for the detection of circulating tumor cells in melanoma patients. However, previously published results showed considerable variability in the PCR positivity rates. MATERIALS AND METHODS: We designed a collaborative study to assess the sensitivity, specificity, and clinical relevance of a new standardized RT-PCR-based enzyme-linked immunosorbent assay (ELISA) for the detection of circulating melanoma cells. Blood samples of healthy donors mixed with cells of a melanoma cell line were prepared in a blinded fashion, and aliquots were sent to seven participating laboratories experienced in RT-PCR. RESULTS: The results demonstrate a high sensitivity (1 melanoma cell/mL blood) and specificity (no false-negatives and 7.4% [2 of 28] false-positives) of the assay and a satisfactory rate of interlaboratory reproducibility. The analysis of aliquots of blinded samples derived from 60 melanoma patients identified tyrosinase mRNA in 17 of 60 (28.3%): three (20%) of 15 stage I patients, two (13.3%) of 15 stage II patients, five (35.7%) of 14 stage III patients, and seven (43.8%) of 16 stage IV patients. The interlaboratory reproducibility of positive samples, however, was extremely low and indicates the presence of low amounts of target mRNA. CONCLUSION: Reverse transcriptase-PCR ELISA has a high sensitivity and specificity for the detection of tyrosinase mRNA in peripheral blood cells. The low interlaboratory reproducibility for the detection of tumor cells in blood samples of melanoma patients, however, raises the question of relevance of this assay for clinical use.

Molecular detection and characterization of clonal cell populations in acute lymphocytic leukemia by analysis of conformational polymorphisms of cRNA molecules of rearranged T-cell-receptor-gamma and immunoglobulin heavy-chain genes.

Junctional regions of rearranged T-cell-receptor-gamma (TCR) and immunoglobulin heavy-chain (IgH) genes represent an idiotypic DNA sequence for an individual lymphocytic cell, and any clone or clonal disease developing from this cell. In this study, a novel methodology for detection and characterization of clone specific DNA sequences in patients with acute lymphocytic leukemia (ALL) was developed. Junctional regions of rearranged TCR-gamma IgH genes in specimens of bone marrow aspirates of patients with ALL (precursor-B-ALL ten, T-ALL two, null-ALL one; ALL not classified one), of a patient with lymphoid blast crisis of chronic myeloid leukemia, of a B-cell chronic lymphocytic leukemia, and in DNA from peripheral blood mononuclear cells of ten healthy volunteers were amplified by polymerase chain reaction (PCR). The PCR products were transcribed into complementary RNA (cRNA). Conformational polymorphisms of cRNA molecules were analyzed by non-denaturing polyacrylamide gel electrophoresis. A specific cRNA banding pattern for rearranged TCR-gamma or IgH genes was observed in all patients with lymphocytic leukemia. In contrast, analysis of DNA from healthy volunteers yielded a smear of confluent polymorphisms representing multiple different cRNA molecules. In two patients with precursor-B-ALL, cRNA banding patterns of junctional regions of rearranged TCR-gamma genes were analyzed in sequential bone marrow aspirates. The banding patterns disappeared after chemotherapy and achievement of blast clearance. This novel and rapid molecular assay offers several advantages as compared to Southern blot analyses and previous PCR based methodologies for the detection of clonal lymphocytic populations. With this methodology, studies on the clonal evolution of lymphoproliferative disorders (e.g. the prognostic significance of the emergence of additional clones) can be performed more easily than with any other traditional molecular method.

Molecular evidence for the existence of disseminated zoster as a distinct entity in an immunosuppressed renal transplant patient.

Immunosuppressed renal transplant recipient are at substantially increased risk for the development of varicella zoster virus infections. They are also more prone than immunocompetent patients to develop atypical zoster and to experience a protracted course, and among them there is a higher frequency of generalized infections with possible fatal outcome. While establishing the diagnosis is essential to provide adequate therapy, conventional laboratory methods frequently fail to confirm the suspected infection. We report on a 47-year-old renal transplant recipient who developed multiple necrotic cutaneous ulcers under immunosuppressive treatment. While electron-microscopic analysis (negative staining) revealed no viral structures, varicella zoster virus specific DNA was detected by polymerase chain reaction in material obtained by a swab from these ulcers. Atypical herpetic infection should also be considered as a cause of disseminated ulcerative or necrotic skin lesions in immunosuppressed patients. Assays based on polymerase chain reaction are useful for the rapid confirmation or rejection of the suspected diagnosis of atypical herpetic infection.

Recombinant adeno-associated virus for the generation of autologous, gene-modified tumor vaccines: evidence for a high transduction efficiency into primary epithelial cancer cells.

To explore the potential of recombinant vectors based on recombinant adeno-associated virus (rAAV) for cancer vaccination, we investigated the transduction efficiency of rAAV into cancer cells ex vivo. Infection of human epithelial cancer cell lines with rAAV carrying reporter genes encoding beta-galactosidase (rAAV/LacZ) or luciferase (rAAV/Luc) resulted in high levels of reporter gene expression (>90% positive cells). In marked contrast, rAAV poorly transduced all murine tumor cell lines, as well as human hematopoietic cell lines. Either irradiation or adenovirus infection of tumor cells prior to rAAV infection induced a 10- to 100-fold increase of reporter gene expression. To determine the transduction efficiency of rAAV into primary cancer cells, freshly isolated, irradiated tumor cells from malignant melanoma and ovarian carcinoma patients were infected with rAAV/Luc, resulting in up to 6.9-fold higher levels of gene expression than in a HeLa tumor cell line. Time course experiments with freshly isolated tumor cells infected with rAAV/Luc showed maximal levels of luciferase expression between days 3 and 9 posttransduction. Simultaneous infection of primary tumor cells with up to three rAAV vectors containing genes encoding the immunostimulatory proteins B7-2 (CD86), p35 subunit of IL-12, and p40 subunit of IL-12 resulted in high expression of B7-2 in more than 90% of the tumor cells and in the secretion of high levels of IL-12. Taken together, our results demonstrate that rAAV efficiently transduces freshly isolated human, epithelial tumor cells and might therefore be a potent tool to produce improved, gene-modified cancer vaccines.

Development of a highly specific and sensitive molecular probe for detection of cutaneous lymphoma.

Using DNA from formalin-fixed paraffin-embedded tissue of a patient with mycosis fungoides, a highly specific and sensitive molecular probe for the malignant lymphoid cells of this patient was developed. Polymerase chain reaction (PCR) was used to selectively amplify the region of the rearrangement of TCR-gamma genes. Randomly inserted nucleotides between rearranged segments (N-region) were used as a specific marker for the malignant lymphoid clone of this patient. Clonal cells in paraffin-embedded tissue of previous and later biopsies were detected. This technique provides a new and unique tool for retrospective detection of early evolving cutaneous lymphoma, as well as for detection of minimal residual and systemic disease.

bcl-2 protein expression and correlation with the interchromosomal 14;18 translocation in cutaneous lymphomas and pseudolymphomas.

The interchromosomal 14;18 translocation occurs in approximately 70-80% of follicular lymphomas and in a lower proportion of high-grade non-Hodgkin lymphomas of the lymph nodes. This translocation results in the fusion of the bcl-2 oncogene on chromosome 18 with immunoglobulin heavy chain genes on chromosome 14, and in the expression of higher amounts of normal bcl-2 protein. We studied bcl-2 expression in biopsies of 108 patients with benign and malignant cutaneous lymphoproliferative diseases (B-cell lymphoma, primary cutaneous, 42; secondary cutaneous, 21; primary cutaneous T-cell lymphoma, 21; B-cell pseudolymphoma, 24), using a monoclonal anti-bcl-2 antibody on paraffin-embedded tissue sections, bcl-2 protein was detected immunohistochemically in 16 of 63 cases of cutaneous B-cell lymphoma, whereas cutaneous T-cell lymphomas and B-cell pseudolymphomas were negative. The proportion of bcl-2 protein expression was significantly higher in secondary (11/21) than in primary cutaneous B-cell lymphomas (5/42; chi 2 test, p < 0.001). Biopsies from 25 of these patients (B-cell lymphoma, 22; B-cell pseudolymphoma, three) were analyzed previously on the molecular level for the t(14;18), using polymerase chain reaction amplification of DNA obtained from paraffin-embedded sections. In four of 11 cases of bcl-2 protein-positive B-cell lymphoma (primary, one; secondary, three) the t(14;18) was detected by polymerase chain reaction. All other cases of B-cell lymphoma, including seven cases where bcl-2 protein was detected by immunohistology, and B-cell pseudolymphoma were negative. These results demonstrate: 1) bcl-2 protein is expressed in a small portion of cutaneous B-cell lymphomas; 2) bcl-2 protein expression is significantly more frequent in secondary than in primary cutaneous B-cell lymphoma; 3) only approximately one-third of cases expressing the bcl-2 protein are characterized also by the t(14;18). bcl-2 protein expression might indicate that the cutaneous manifestation of the lymphoma represents a secondary spread from a node-based lymphoma.

Molecular subtyping of Borrelia burgdorferi in erythema migrans and acrodermatitis chronica atrophicans.

Recently, three subtypes of Borrelia burgdorferi have been identified: Borrelia burgdorferi sensu stricto, Borrelia garinii, and the VS 461 group of Borrelia burgdorferi. These subtypes differ by nucleotide sequence variations within several Borrelia burgdorferi specific genes and most likely by their pathogenetic potential. To assess whether different subtypes of Borrelia burgdorferi might be associated with different cutaneous manifestations and clinical courses of Lyme disease, lesional skin biopsies from 35 patients with erythema migrans and 18 patients with acrodermatitis chronica atrophicans were analyzed. A Borrelia burgdorferi specific gene segment encoding a 26-kD protein with subtype specific nucleotide sequence variations was amplified by a nested polymerase chain reaction technique. For molecular subtyping, the products were transcribed into complementary RNA. Upon polyacrylamide gel electrophoresis, complementary RNA molecules separate into several metastable conformational forms resulting in patterns of bands highly specific for the nucleotide sequence of the transcribed molecules. In biopsy specimens of erythema migrans, the VS 461 subtype was detected in 28 of 35 and the Borrelia garinii subtype in six of 35 cases. In one of 35 cases of erythema migrans Borrelia burgdorferi sensu stricto as well as Borrelia garinii was detected. In contrast, in all 18 biopsies of acrodermatitis chronica atrophicans, only the VS 461 subtype was identified. This subtype is rarely found in the USA, where acrodermatitis chronica atrophicans is almost unknown. These data indicate that acrodermatitis chronica atrophicans might be closely associated with the VS 461 group of Borrelia burgdorferi.

No evidence for Borrelia burgdorferi-specific DNA in lesions of localized scleroderma.

A possible association of Borrelia burgdorferi with localized scleroderma is currently the focus of intense research and discussion. Skin biopsies from 30 patients with localized scleroderma (28 of the plaque type/morphea; two linear scleroderma) were analyzed for the presence of Borrelia burgdorferi using three different polymerase chain reaction systems for amplification of segments of borrelial genes. Formalin-fixed, paraffin-embedded biopsies of 14 patients and fresh-frozen, cryo-conserved biopsies of 16 patients with localized scleroderma were obtained. Lesions of all patients showed clear signs of scleroderma and disease progression at the time of biopsy. Fresh-frozen as well as formalin-fixed biopsies from patients with erythema migrans or acrodermatitis chronica atrophicans were used as positive controls. With all three polymerase chain reaction systems, borrelial DNA was detected in none of the 30 specimens of localized scleroderma. In contrast, with one polymerase chain reaction system, Borrelia burgdorferi-specific DNA was found in 24 of 27 frozen biopsies from patients with erythema migrans and in all 5 analyzed frozen biopsies of patients with acrodermatitis chronica atrophicans. In approximately half of the paraffin-embedded biopsies from patients with erythema migrans (nine of 23) and acrodermatitis chronica atrophicans (13 of 27), Borrelia burgdorferi-specific DNA was identified. These results question the association of localized scleroderma with known subtypes of Borrelia burgdorferi.

Conformational polymorphisms of cRNA of T-cell-receptor genes as a clone-specific molecular marker for cutaneous lymphoma.

A novel molecular assay for the detection and characterization of monoclonal lymphoid populations in clinical specimens was developed. The assay is based on the principle that upon non-denaturing polyacrylamide gel electrophoresis RNA molecules separate into several metastable conformational forms. These conformational polymorphisms strictly depend on the nucleotide sequence of the individual molecule. Using DNA from formalin-fixed, paraffin-embedded tissue of patients with mycosis fungoides, highly variable junctional sequences of rearranged T-cell receptor gamma genes were amplified by polymerase chain reaction. Subsequently, the polymerase chain reactions products were transcribed into complementary RNA and analyzed by non-denaturing polyacrylamide gel electrophoresis. In clinical specimens with a monoclonal lymphoid population, a clone-specific pattern of bands was identified representing conformational polymorphisms of cRNA molecules of rearranged T-cell receptor gamma genes of the predominant lymphoid clone. Three biopsies from one patient taken from different sites of the body over 3 years yielded an identical pattern of bands. This methodology provides a novel and rapid tool for the molecular identification and characterization of clonal lymphoid populations in clinical specimens. It is likely to be of special value for studies on the clonal evolution of lymphoid disorders of the skin.

Transfection with a cDNA encoding a Ser31 or Ser34 mutant human dihydrofolate reductase into Chinese hamster ovary and mouse marrow progenitor cells confers methotrexate resistance.

Chinese hamster ovary (CHO) DHFR- cells were converted into the DHFR+ phenotype when they were transfected with a mammalian expression vector carrying human dihydrofolate reductase-encoding cDNAs (DHFR) containing a Ser31 or a Ser34 mutation. Furthermore, transfection of these mutants into wild-type CHO cells resulted in resistance to high levels of methotrexate (MTX), indicating that these human variants can act as dominant selectable markers. Southern blot analysis and polymerase chain reaction amplifications confirmed that the transfected plasmids were integrated into the CHO DNA. Gene copy number analysis revealed that both the Ser3 1 and the Ser3.4 mutants amplifiable when grown in increasing concentrations of MTX. Retrovirus-mediated gene transfer of the Ser31 mutant into mouse marrow progenitor cells also resulted in MTX-resistant CFU-GM (colony-forming unit-granulocyte macrophage) cells.

Chemoimmunotherapy of advanced malignant melanoma: sequential administration of subcutaneous interleukin-2 and interferon-alpha after intravenous dacarbazine and carboplatin or intravenous dacarbazine, cisplatin, carmustine and tamoxifen.

Both chemotherapy and interleukin-2 and/or interferon-alpha produce objective responses in a proportion of advanced malignant melanoma patients. While duration of response to chemotherapy is short, i.e. usually below 4 months, immunotherapy has resulted in a small number of long-lasting remissions in patients with metastatic melanoma. In two consecutive phase II trials in a total of 67 patients, we assessed the potential synergism between both modalities, i.e. chemo- and immunotherapy. Treatment consisted of intravenous (i.v.) carboplatin (CBDCA, 400 mg/m2) and dacarbazine (DTIC, 750 mg/m2) given twice (i.v. bolus over 30 min) at 3-week intervals, or 4 cycles of DTIC (220 mg/m2 i.v. 3 days), cisplatin (DDP, 35 mg/m2 i.v. 3 days), carmustine (BCNU, 150 mg/m2 i.v. cycles 1 and 3) and tamoxifen (TAM, 20 mg oral/daily) at 3-week intervals. Chemotherapy was followed by immunotherapy with combined subcutaneous (s.c.) interleukin-2 (rIL-2) and SC interferon-alpha 2 (rIFN-alpha). Among 40 patients who received a full cycle of chemotherapy with CBDCA/DTIC and sequential immunotherapy, there were 3 (7.5%) complete remissions (CRs) with a median duration of 19 months (range 13-26+). Partial remissions (PRs) were noted in 11 (27.5%) patients with a median response duration of 8 (range 5-14) months. Among 27 patients who received DTIC/DDP/BCNU/TAM and rIL-2/rIFN-alpha, there were 3 (11%) complete remissions and 12 (44.5%) partial remissions. Duration of complete and partial remissions ranged from 9+ to 13+ (median, 11+), and 5 to 15+ (median, 7+) months, respectively. Chemotherapy produced mostly moderate toxicity. Thrombocytopenia was common with the nadir after a median time of 18 days following start of CBDCA/DTIC and DTIC/DDP/BCNU, respectively. 10 patients required transfusion of thrombocytes. Nausea and vomiting due to chemotherapy were well tolerated using concomitant ondansetrone (8 mg i.v.). Immunotherapy was self-administered at home with mild to moderate side effects; malaise, fever, chills, nausea/vomiting, diarrhoea, anorexia and arthralgias were most frequent, but were spontaneously reversible after ending rIL-2/IFN-alpha. A mean 87 and 88% of the projected doses of rIL-2 and rIFN-alpha were administered on either protocol. There were no life-threatening complications and no treatment-related deaths. The sequential combination of chemotherapy and rIL-2 plus rIFN-alpha had at least additive therapeutic activity against metastatic malignant melanoma. The schedules produced long-lasting remissions and were tolerated well overall. These trials substantiate a potential role for low to intermediate dose immunotherapy in maintaining and consolidating therapeutic effects of chemotherapy in metastatic melanoma.

Sequence analysis of a human gene responsible for drug resistance: a rapid method for manual and automated direct sequencing of products generated by the polymerase chain reaction.

We developed a system for rapid, manual and automated sequence analysis by utilizing and modifying methods used in conjunction with the polymerase chain reaction (PCR). We are using these techniques to detect single base mutations in the dihydrofolate reductase (DHFR) gene giving rise to methotrexate (MTX) resistance of tumor cells obtained from patients with malignancies. Amplifying in vitro both genomic DNA and transcripts of the human DHFR we are able to reproducibly generate single-stranded templates. Utilizing [alpha-35S]dATP and both the universal and reverse sequencing primers we obtain sequence information from either strand. The methods described have been successfully used for automated sequencing with the Applied Biosystems Model 370A Sequencer using both modified T7 DNA polymerase and Taq I. DNA polymerase for dideoxy-termination sequencing. The use of this methodology to detect a single base change in a human colon carcinoma cell line, HCT-8, is illustrated.

Molecular evidence for a clonal relationship between lymphomatoid papulosis and Ki-1 positive large cell anaplastic lymphoma.

Currently, considerable controversy surrounds questions about the clonal evolution of lymphomas in patients with lymphomatoid papulosis. In order to analyze a possible clonal relationship between lesions of lymphomatoid papulosis and a Ki 1+ large cell anaplastic lymphoma in the same patient, a highly specific molecular probe for the malignant lymphoid clone of the large cell anaplastic lymphoma was developed. As a clone specific molecular marker, highly variable junctional sequences of rearranged T-cell receptor-gamma genes were used. An oligonucleotide primer complementary to these sequences was synthesized and, using the polymerase chain reaction, clone specific DNA was detected in all lesions of lymphomatoid papulosis of the patient. These results provide evidence for a clonal relationship between lesions of lymphomatoid papulosis and large cell anaplastic lymphoma developing in the same patient.

Sequence analysis of polymerase chain reaction amplified t(14;18) chromosomal breakpoints in formalin fixed, paraffin wax embedded follicular lymphoma.

AIMS: To determine whether junctional sequences of rearranged chromosomes can be amplified by use of the polymerase chain reaction (PCR) and whether direct sequence analysis of the PCR products is possible, using DNA from formalin fixed, paraffin wax embedded biopsy specimens. METHODS: DNA was extracted from paraffin wax embedded, formalin fixed lymphoma specimens, and junctional sequences of rearranged chromosomes were amplified by the PCR. The products were used as templates for asymmetrical PCR. Subsequently, direct sequence analysis was performed using the chain termination method. RESULTS: Formalin fixed, paraffin wax embedded biopsy specimens and PCR amplification could be used to determine the nucleotide sequences of junctional regions of rearranged chromosomes t(14;18) from patients with follicular lymphoma. CONCLUSION: The identification of junctional sequences of the translocation in follicular lymphoma provides a molecular "fingerprint" of t(14;18) of the lymphoma of an individual patient and can be used for the detection of clone specific DNA in any biopsy tissue obtained from the patient. The strategy used for rapid sequence analysis of PCR amplified DNA sequences will be useful in many areas of molecular pathology.