Four different programmed death ligand 1 immunohistochemical assays are approved or in development as companion or complementary diagnostics to different immunotherapeutic agents in lung carcinoma. ...We sought to determine whether these assays are technically equivalent and whether one antibody can be used on an alternate staining platform.
Serial sections of tissue microarrays constructed from 368 cases of resected lung cancer were stained for 22C3 and 28-8 on the Dako Link 48 platform (Dako, Carpinteria, Ca) and for SP142 and SP263 on the Ventana Benchmark Ultra platform (Ventana Medical Systems, Tucson, AZ) strictly as per product insert. A protocol was developed to use the 22C3 antibody on the Ventana Benchmark Ultra platform.
Differences in mean tumor cell and immune cell staining were observed between the four assays (p < 0.001). Differences between 22C3 and 28-8 were not statistically significant. Concordance of tumor cell scores was good (intraclass correlation coefficient ICC = 0.674), particularly when SP142 was excluded as an outlier (ICC = 0.755). The highest concordance was seen between 22C3 and 28-8 (ICC = 0.812). Concordance was poor for immune cell staining (ICC = 0.212). When dichotomized according to clinically relevant cutoffs, pairwise comparisons showed poor to moderate concordance (κ = 0.196–0.578), with positive percent agreement ranging from 15.1% to 90.0%. The 22C3 antibody performed comparably on the Dako Link 48 platform and the alternate Ventana Benchmark Ultra platform (ICC = 0.921, κ = 0.897).
Concordance between the four programmed death ligand 1 immunohistochemical assays when performed and scored as intended show that apart from 28-8 and 22C3, they cannot be used interchangeably in clinical practice. A protocol was successfully developed to use 22C3 on an alternate platform, which may help to overcome some barriers to implementation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Purpose: Melanoma cells express antigens that can induce T-cell and antibody responses. Obtaining a detailed understanding of antigen
expression in primary and metastatic melanoma is essential if ...these molecules are to be useful targets for immunotherapy of
melanoma.
Experimental Design: Malignant melanomas ( n = 586) from 426 patients were typed for antigen expression. Multiple samples were available from 86 individuals, enabling
analysis of antigen expression patterns over time. Paraffin-embedded samples were tested by immunohistochemistry for the presence
of the differentiation antigens: gp100, Melan-A, tyrosinase, and the “cancer/testis” antigens MAGE-A1, MAGE-A4, and NY-ESO-1.
Results: Samples were primary tumors ( n = 251), lymph node metastases ( n = 174), s.c. metastases ( n = 71), and distant metastases ( n = 90). The differentiation antigens were strongly expressed in 93% to 95% of tumors regardless of stage. In contrast, the
frequency of cancer/testis antigen expression in primary tumors for MAGE-A1, MAGE-A4, and NY-ESO-1 was lower (20%, 9%, and
45%, respectively). MAGE-A1 and MAGE-A4 were acquired with advancing disease (to 51% and 44% in distant metastases, respectively)
but not NY-ESO-1, which remained positive in 45%. MAGE-A1 expression was twice as prevalent in ulcerated primaries as in nonulcerated
primaries (30% versus 15%; P = 0.006) and in thicker as opposed to thin melanomas (26% versus 10%; P = 0.1).
Conclusions: This large series describes patterns of antigen expression in melanoma and their evolution over time. This will help inform
decisions about selection of patients and target antigens for melanoma immunotherapy clinical trials.
Background: Histological interpretation of testicular biopsies in the investigation of infertility in men with azoospermia requires adequate tissue fixation to preserve the nuclear and cytoplasmic ...detail, as well as the architectural organisation of germ cells in different phases of maturation within seminiferous tubules.
Research Aims/Objectives: The aim of the study was to assess the histomorphological quality of testicular biopsies using Davidson’s fluid (DF) as fixative and compare it to standard 10% neutral buffered Formalin.
Methodology: Concurrent testicular biopsies from the same testis from patients undergoing microsurgical testicular sperm exploration (m-TESE) were separately fixed in DF and Formalin and processed for histological examination. Histological parameters including sloughing of cells, cytoplasmic shrinkage of seminiferous tubular cells, nuclear chromatin detail, cytoplasmic graininess and overall clarity of morphological detail were graded on a scale of 0 to 4 (0=none, 1=minimal, 2=slight, 3=moderate, 4=marked). The effect of DF on biopsy diagnoses was assessed by comparison with corresponding Formalin fixed biopsy diagnoses.
Results/Findings: 87 testicular biopsies from 27 patients were examined. DF fixation resulted in significantly less luminal sloughing of cells (1.59±1.34 vs. 3.44±0.83, p =
<
0.00001), less cytoplasmic shrinkage of seminiferous tubular cells (1.58±1.11 vs. 3.11±1.07, p =
<
0.00001), better nuclear chromatin detail (3.06±0.91 vs. 1.92±0.48, p =
<
0.00001), less cytoplasmic graininess (2.11±0.96 vs. 2.86±0.87, p = 0.0014) and better overall clarity of morphological detail than Formalin fixation (3.14±0.69 vs. 2.14±0.58, p =
<
0.00001). The diagnostic concordance between DF fixed and Formalin fixed biopsies was 90.8%.
Conclusions: This study supports the use DF as a superior alternative fixative to Formalin for histological assessment of testicular biopsies.
NY-ESO-1 is a "cancer-testis" antigen expressed in many cancers. ISCOMATRIX is a saponin-based adjuvant that induces antibody and T cell responses. We performed a placebo-controlled clinical trial ...evaluating the safety and immunogenicity of recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant. Forty-six evaluable patients with resected NY-ESO-1-positive tumors received three doses of vaccine intramuscularly at monthly intervals. The vaccine was well tolerated. We observed high-titer antibody responses, strong delayed-type hypersensitivity reactions, and circulating CD8+ and CD4+ T cells specific for a broad range of NY-ESO-1 epitopes, including known and previously unknown epitopes. In an unplanned analysis, vaccinated patients appeared to have superior clinical outcomes to those treated with placebo or protein alone. The vaccine is safe and highly potent immunologically.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Purpose: NY-ESO-1 and LAGE-1 are homologous cancer-testis antigens, which are expressed in many different cancers. It is essential
to type tumors accurately to assess patient suitability for clinical ...trials which target these. This study evaluates typing
strategies used to distinguish these two homologous but distinct antigens and to characterize and quantitate expression of
each in clinical samples.
Experimental Design: We typed 120 malignant melanomas for the expression of NY-ESO-1 and LAGE-1 with immunohistochemistry, reverse transcription-PCR
(RT-PCR), and quantitative real-time (qRT-PCR), which was also used to explore the relationship between NY-ESO-1 and LAGE
expression.
Results: The two monoclonal antibodies ES121 and E978 had very similar immunohistochemistry reactivities. Both were specific for NY-ESO-1
because neither bound to homologous LAGE-1 peptides despite 84% overall amino acid homology. Of 120 melanomas tested by immunohistochemistry,
NY-ESO-1 was expressed in >50% of cells in 23 melanomas (19%), between 11 and 50% cells in 15 (12.5%), <11% cells in 16 (13.5%),
and negative in 66 (55%). Although specific for both antigens, the PCR methods did not provide this information about microheterogeneity.
Polymorphisms in the LAGE-1 gene resulted in false negative LAGE-1 typing by qRT-PCR by inhibiting binding of oligonucleotide
primers, thereby showing the exquisite specificity of qRT-PCR as a typing method.
Conclusions: For NY-ESO-1 typing, immunohistochemistry compared favorably with the RT-PCR, with the added advantage of being able to characterize
heterogeneity of antigen expression. Because neither mAb bound LAGE and because there was no coordinate expression LAGE and
NY-ESO-1, separate typing for each is required.
Histological interpretation of testicular biopsies in the investigation of infertility in men with azoospermia requires adequate tissue fixation to preserve the nuclear and cytoplasmic detail, as ...well as the architectural organisation of germ cells in different phases of maturation within seminiferous tubules. The aim of the study was to assess the histomorphological quality of testicular biopsies using Davidson's fluid (DF) as fixative and compare it to standard 10% neutral buffered formalin. Concurrent testicular biopsies from the same testis from patients undergoing microsurgical testicular sperm exploration (m-TESE) were separately fixed in DF and formalin and processed for histological examination. Histological parameters including sloughing of cells, cytoplasmic shrinkage of seminiferous tubular cells, nuclear chromatin detail, cytoplasmic graininess and overall clarity of morphological detail were graded on a scale of 0–4 (0, none; 1, minimal; 2, slight; 3, moderate; 4, marked). The effect of DF on biopsy diagnoses was assessed by comparison with corresponding formalin fixed biopsy diagnoses. Eighty-seven testicular biopsies from 27 patients were examined. DF fixation resulted in significantly less luminal sloughing of cells (1.59±1.34 vs 3.44±0.83, p≤0.00001), less cytoplasmic shrinkage of seminiferous tubular cells (1.58±1.11 vs 3.11±1.07, p≤0.00001), better nuclear chromatin detail (3.06±0.91 vs 1.92±0.48, p≤0.00001), less cytoplasmic graininess (2.11±0.96 vs 2.86±0.87, p=0.0014) and better overall clarity of morphological detail than formalin fixation (3.14±0.69 vs 2.14±0.58, p≤0.00001). The diagnostic concordance between DF fixed and formalin fixed biopsies was 90.8%. This study supports the use of DF as a superior alternative fixative to formalin for histological assessment of testicular biopsies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Immune responses to cancer vaccines are commonly tested by measuring cutaneous reactions to intradermal (i.d.) antigen. When well-characterized peptide epitopes are injected i.d., infiltrates of CD4+ ...and CD8+ T lymphocytes are frequently seen. In this study, we have further characterized T cells derived from vaccine-infiltrating lymphocyte (VIL) responses. We found that the infiltrates capable of producing IFN-gamma and cytolytic activity could recognize vaccine peptide, as well as antigen-positive melanoma cells. We studied antigen-specific T cell responses from VILs and peripheral blood in 10 patients who participated in a clinical trial. All patients received systemic Flt3 ligand (20 microg/kg/d) and i.d. peptides: Three NY-ESO-1 peptides, SLLMWITQCFL (157-167), SLLMWITQC (157-165), QLSLLMWIT (155-163); tyrosinase internal peptide YMDGTMSQV (368-376); Melan-A/MART-1 analogue peptide ELAGIGILTV (26-35, E27L substitution); and influenza matrix peptide GILGFVFTL (58-66). In 54 paired VIL and peripheral blood analyses, a good correlation was found between responses in skin and in blood. These cells could be rapidly expanded in a short-term assay and thus appear to be memory T cells. The demonstrated presence of antigen-specific T cells at vaccination sites validates this method of assessing the immune response to i.d. vaccines.
Preclinical studies have shown that low dose IL-12 can potentiate cytotoxic lymphocyte responses. Since previous trials have demonstrated significant toxicity from high dose recombinant human IL-12 ...(rhIL-12), we sought to determine an optimal biological dose for rhIL-12 at lower doses when combined with peptide antigens. Two studies were undertaken. The rhIL-12 was administered at doses of 0 (placebo), 10, 30 and 100 ng/kg, subcutaneously in one study and intravenously in the other. Apart from IL-12 dosing, the studies were identical. Subjects had evaluable stage III or IV melanoma which expressed Melan-A by RT-PCR or immunohistochemistry. Melan-A (26-35) (EAAGIGILTV) and influenza matrix (58-66) (GILGFVFTL) peptides were administered intradermally on weeks 1, 2, 3, 4 and 9. Twenty-eight subjects were enrolled, of whom 24 were evaluable for clinical and immunological responses. Therapy was well tolerated, the main adverse event being influenza-like symptoms. Immunological monitoring included the evaluation of cutaneous reactions and assays for antigen-specific T-cells. Clinical responses included a complete response in a subject with small volume subcutaneous disease, a partial response in a subject with hepatic metastases, and mixed responses in pulmonary, pleural and nodal disease. Biopsies of accessible tumors showed infiltration with CD4+ and CD8+ lymphocytes capable of lysing Melan-A peptide-pulsed targets in vitro. No clear dose-dependent effect of rhIL-12 could be determined. The rhIL-12 given either s.c. or i.v. was well tolerated at doses of 10-100 ng/kg. Clinical and immunological activity has been observed in this study where peptides were administered either with or without low dose rhIL-12.
Thirty-five pediatric lymphomas were categorized as either Burkitt 's lymphoma (BL), lymphoblastic lymphoma (LL), or large cell anaplastic lymphoma (LCAL) by histological and immunophenotypic ...methods. They were further characterized by molecular analysis of their antigen receptor genes. Southern blot (SB) and polymerase chain reaction (PCR) techniques were compared in the detection of immunogloblin heavy chain gene (IgH) rearrangement. T cell receptor β (TCRβ) rearrangements were analyzed by SB and TCRγ gene rearrangements by PCR. The PCR method of IgH and TCRγ gene analysis was preferred to the SB methods, because there were fewer equivocal results in IgH gene analysis, TCRγ rearrangement was more frequently detected than TCRβ in both lymphoblastic and large cell anaplastic lymphomas, and the PCR technique was more rapid, required less DNA, and could be used with archival material. In addition, analysis of IgH gene rearrangement by PCR was more specific for assessing B cell lineage. Although most of the molecular data were easily interpreted, occasional ambiguous results were seen due to genetic events other than antigen receptor gene rearrangement affecting the genetic, analysis. Thus, it is imperative to interpret these genetic data in the context of adequate morphological and immunophenotypic analysis.
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