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Variability in the Expression of Immunohistochemical Markers: Implications for Biomarker Interpretation in Cutaneous T-Cell Lymphoma

  • Ziba Rahbar
    Affiliations
    Department of Dermatology, Stanford University School of Medicine, Palo Alto, California, USA
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  • Shufeng Li
    Affiliations
    Department of Dermatology, Stanford University School of Medicine, Palo Alto, California, USA
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  • Mahkam Tavallaee
    Affiliations
    Department of Pathology, Stanford University School of Medicine, Palo Alto, California, USA
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  • Roberto A. Novoa
    Affiliations
    Department of Dermatology, Stanford University School of Medicine, Palo Alto, California, USA

    Department of Pathology, Stanford University School of Medicine, Palo Alto, California, USA
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  • Author Footnotes
    4 These authors are senior authors and contributed equally to this work.
    Jinah Kim
    Footnotes
    4 These authors are senior authors and contributed equally to this work.
    Affiliations
    Department of Dermatology, Stanford University School of Medicine, Palo Alto, California, USA

    Department of Pathology, Stanford University School of Medicine, Palo Alto, California, USA
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  • Author Footnotes
    4 These authors are senior authors and contributed equally to this work.
    Youn H. Kim
    Correspondence
    Corresponding author
    Footnotes
    4 These authors are senior authors and contributed equally to this work.
    Affiliations
    Department of Dermatology, Stanford University School of Medicine, Palo Alto, California, USA

    Stanford Cancer Institute, Stanford University School of Medicine, Palo Alto, California, USA
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  • Author Footnotes
    4 These authors are senior authors and contributed equally to this work.
Open ArchivePublished:December 14, 2017DOI:https://doi.org/10.1016/j.jid.2017.11.035
      To the Editor
      Clinical development of targeted therapies in cutaneous T-cell lymphoma compels us to explore biomarkers that may correlate with clinical outcome. In contrast to the uniform distribution of Sézary cells in the blood, the skin compartment in mycosis fungoides (MF) and Sézary syndrome (SS) is often heterogeneous, with malignant T cells variably admixed with the resident and infiltrating non-malignant cells across the varying morphologic types of skin lesions, including patches, plaques, and tumors (
      • Olsen E.A.
      • Whittaker S.
      • Kim Y.H.
      • Duvic M.
      • Prince H.M.
      • Lessin S.R.
      • et al.
      Clinical end points and response criteria in mycosis fungoides and Sézary syndrome: a consensus statement of the International Society for Cutaneous Lymphomas, the United States Cutaneous Lymphoma Consortium, and the Cutaneous Lymphoma Task Force of the European Organisation for Research and Treatment of Cancer.
      ,
      • Swerdlow S.H.
      • Campo E.
      • Pileri S.A.
      • Harris N.L.
      • Stein H.
      • Siebert R.
      • et al.
      The 2016 revision of the World Health Organization classification of lymphoid neoplasms.
      ). Thus, collecting a single skin biopsy in a patient with MF/SS may lead to misinterpretation of biomarker information. Recently, the phase 3 trial of brentuximab vedotin (antibody drug conjugate against CD30) versus physician’s choice (methotrexate or bexarotene) in CD30-positive cutaneous T-cell lymphoma reported notable variability in CD30 expression among multiple biopsies collected at baseline for patients with MF (
      • Kim Y.H.
      • Prince H.M.
      • Whittaker S.
      • Horwitz S.M.
      • Duvic M.
      • Scarisbrick J.
      • et al.
      Outcomes by CD30 expression in patients with CTCL receiving brentuximab vedotin (BV) vs physician’s choice (PC) in the Phase 3 ALCANZA study.
      ). In that study, approximately one-third of patients with MF were excluded during screening using an arbitrary cutoff of 10% CD30 expression (
      • Kim Y.H.
      • Prince H.M.
      • Whittaker S.
      • Horwitz S.M.
      • Duvic M.
      • Scarisbrick J.
      • et al.
      Outcomes by CD30 expression in patients with CTCL receiving brentuximab vedotin (BV) vs physician’s choice (PC) in the Phase 3 ALCANZA study.
      ). Here, we conducted an in-depth evaluation of the variability of CD30 and other potential tissue biomarkers in the skin as assessed by immunohistochemistry (IHC), which was a planned exploratory objective in our phase 2 investigator-initiated trial of brentuximab vedotin in patients with MF/SS (
      • Kim Y.H.
      • Tavallaee M.
      • Sundram U.
      • Salva K.A.
      • Wood G.S.
      • Li S.
      • et al.
      Phase II investigator-initiated study of brentuximab vedotin in mycosis fungoides and Sézary syndrome with variable CD30 expression level: a multi-institution collaborative project.
      ). The primary goal was to assess the variability of IHC marker expression levels in different skin lesions in the same patient (intra-patient, inter-lesional) and within a lesion (intra-lesional). The secondary goal was to evaluate the variability of IHC marker expression in different clinical types of lesions (patch/plaque vs. tumor) and in those biopsy samples with and without large cell transformation.
      We evaluated CD30 and other potential tissue biomarker expression in 144 biopsy samples obtained from 36 patients at baseline (Supplementary Figure S1). The study was approved by the Institutional Review Board at Stanford University (NCT01396070; IRB21324). Written informed consent was obtained for each patient. Tissue IHC was performed using standard protocols (Supplementary Table S1). A board-certified dermatopathologist (J.K.) reviewed all final slides. We evaluated the variability in the biomarker expression using intraclass correlation coefficient (ICC) and delta. ICC reflects the degree of correlation and agreement between measurements, where values closer to 1.0 represent stronger agreement (
      • Koo T.K.
      • Li M.Y.
      A guideline of selecting and reporting intraclass correlation coefficients for reliability research.
      ). An ICC of <0.4 was considered poor, 0.4−0.7 was considered acceptable, while an ICC >0.7 was considered excellent correlation (
      • Shrout P.E.
      • Fleiss J.L.
      Intraclass correlations: uses in assessing rater reliability.
      ). The inter-lesional ICC demonstrated the agreement in the expression of biomarkers between two samples from different lesions in the same patient and intra-lesional ICC demonstrated the agreement in the expression levels between two samples within the same lesion. Delta was defined as the difference between maximum and minimum expression of the biomarker among all biopsy samples in the same patient. Of the 36 patients enrolled in the study, the majority had the diagnosis of MF (31 MF, 5 SS) and 86% of patients had advanced disease (20 stage IIB, 11 stage IV). The median number of skin biopsies per patient was 4 (range 2−7). Eighty-one percent of the biopsies were from plaque- or tumor-type (81 plaque, 35 tumor) lesions. Large cell transformation was detected in 21% (30 of 144) of the biopsy samples. We observed varying histomorphology in the biopsy samples with the tumor-type lesions more likely to show diffuse pattern of mononuclear cell infiltrate (100%) and large cell transformation (43%). Figure 1a highlights the inter- and intra-patient variability in the expression of selected IHC markers among multiple biopsies for each patient. Nondetectable CD30 expression (0%) was reported in at least one biopsy sample from a patient in 17% (6 of 36) of patients. In these patients, the maximum CD30 expression ranged from 1% to 40% (median 5%) among 4−7 biopsy samples. Table 1 summarizes the estimates for intra-lesional ICC, inter-lesional ICC, and the median delta (range) across all patients for the selected IHC markers. Intra-lesional and/or inter-lesional ICC estimates <0.40 (poor agreement or greater variability) were established for most IHC markers, including CD8, CD20, CD30, CD163, and PD-1. In addition, intra-lesional ICCs (level of agreement) were not necessarily higher than inter-lesional ICCs. The range of deltas in Table 1 demonstrates the variability in the IHC scores across the patients. Figure 1b demonstrates the variability of biomarkers among biopsies from different clinical types of lesions and between biopsy samples with and without large cell transformation. Figure 1c shows an example of notable inter- and intra-lesional variability in CD30 expression where biopsies were taken from two different lesions and within the same lesion with the corresponding CD30 immunostain from a patient. In order to assess the rater consistency, the dermatopathologist (J.K) rescored a set of representative biopsies of different levels of CD30 after 3 weeks, blinded to the initial review. The excellent intra-rater ICC for CD30 implies that the inconsistency was less likely due to the rater (Table 1).
      Figure 1
      Figure 1Variability in expression levels of immunohistochemical markers in mycosis fungoides/Sézary syndrome. (a) Distribution of marker expression demonstrates notable inter- and intra-patient variability. Ends of whisker represent maximum and minimum expression. Ends of box represent 25th and 75th percentiles. Dashed red line represents median expression of all biopsy samples. (b) Marker expression by the clinical type of lesions and presence of large cell transformation (LCT was defined as presence of large cells in ≥25% of the neoplastic lymphoid infiltrate). P < 0.05, ∗∗P < 0.01. (c) CD30 expression in two different lesions (inter-lesional) and within the same lesion (intra-lesional) obtained in the same patient. Scale bars = 50 μm.
      Table 1Variability in biomarker expression measured by inter-lesional ICC, intra-lesional ICC, and delta
      IHC MarkerTotalInter-Lesional ICCIntra-Lesional ICCDelta
      Delta was calculated as the difference of maximum and minimum of the expression level of each biomarker in the same patient. The median and range represent that of all deltas.
      (Max−Min)
      Median (Range)
      Each value is the percentage of the total number of mononuclear cell infiltrate.
      Samples (n)ICC (95% CI)Patients (n)ICC (95% CI)Paired Biopsies (n)Median (Range)
      Each value is the percentage of the total number of mononuclear cell infiltrate.
      Patients (n)
      CD490 (0−95)1220.01 (−0.42 to 0.42)
      Negative values denoted less than agreement by chance. ICC < 0.4 was considered poor agreement, 0.4−0.7 was considered acceptable, and >0.7 was considered excellent, respectively (Shrout and Fleiss 1979). The less the ICC, the greater the variability.
      230.35 (0.07 to 0.57)495 (0−95)29
      CD85 (0−80)1230.60 (0.29 to 0.80)250.33 (0.06 to 0.56)485 (0−79)30
      CD201 (0−40)1230.20 (−0.22 to 0.55)250.20 (−0.07 to 0.45)485 (0−39)30
      CD2525 (1−95)1260.57 (0.25 to 0.78)270.52 (0.29 to 0.70)4930 (0−90)31
      CD30
      Intra-rater ICC (95% CI) for CD30 was 0.86 (0.64, 0.95) (n = 14).
      5 (0−100)1410.21 (−0.12 to 0.50)360.49 (0.26 to 0.67)535 (0−95)36
      CD163
      Intra-rater ICC (95% CI) for CD163 was 0.87 (0.66, 0.96) (n = 14).
      20 (0−80)102−0.06 (−0.49 to 0.37)220.48 (0.13 to 0.72)2715 (0−70)30
      PD-10 (0−80)1150.36 (−0.06 to 0.66)24−0.03 (−0.35 to 0.29)391 (0−80)31
      Ki-6710 (0−90)580.12 (−0.61 to 0.69)100.91 (0.79 to 0.97)1920 (0−80)17
      Abbreviations: CI, confidence interval; ICC, intraclass correlation coefficient; IHC, immunohistochemistry.
      1 Delta was calculated as the difference of maximum and minimum of the expression level of each biomarker in the same patient. The median and range represent that of all deltas.
      2 Each value is the percentage of the total number of mononuclear cell infiltrate.
      3 Negative values denoted less than agreement by chance. ICC < 0.4 was considered poor agreement, 0.4−0.7 was considered acceptable, and >0.7 was considered excellent, respectively (
      • Shrout P.E.
      • Fleiss J.L.
      Intraclass correlations: uses in assessing rater reliability.
      ). The less the ICC, the greater the variability.
      4 Intra-rater ICC (95% CI) for CD30 was 0.86 (0.64, 0.95) (n = 14).
      5 Intra-rater ICC (95% CI) for CD163 was 0.87 (0.66, 0.96) (n = 14).
      We found notable variability of inter- and intra-lesional biomarker expression levels by IHC in MF/SS, as supported by the overall poor (low) ICC values. These findings may be partly accountable by the heterogeneous clinical, histological, and biological characteristics of MF/SS and the unique structure of the skin. In addition, improved methods of evaluation for tissue biomarkers with greater sensitivity and specificity than standard IHC, such as multispectral imaging (
      • Kim Y.H.
      • Tavallaee M.
      • Sundram U.
      • Salva K.A.
      • Wood G.S.
      • Li S.
      • et al.
      Phase II investigator-initiated study of brentuximab vedotin in mycosis fungoides and Sézary syndrome with variable CD30 expression level: a multi-institution collaborative project.
      ,
      • Salva K.A.
      • Wood G.S.
      Epigenetically enhanced photodynamic therapy (ePDT) is superior to conventional photodynamic therapy for inducing apoptosis in cutaneous T-cell lymphoma.
      ) or multiplexed ion beam imaging with tissue microarrays (
      • Angelo M.
      • Bendall S.C.
      • Finck R.
      • Hale M.B.
      • Hitzman C.
      • Borowsky A.D.
      • et al.
      Multiplexed ion beam imaging of human breast tumors.
      ), may enable us to better characterize sample heterogeneity and allow expansion of biomarker platforms. Our findings may influence the design and conduct of clinical trials for targeted therapies by discouraging arbitrary IHC cutoffs for eligibility and encouraging caution when interpreting biomarker data or establishing selection guidelines for targeted therapies in cutaneous T-cell lymphoma. Utilizing multiple biopsies from varying clinical lesions and/or different sites and applying newer technology may improve the assessment and interpretation of biomarkers in MF/SS.

      Conflict of Interest

      Youn H. Kim is a member of the Advisory Board for Seattle Genetics; Principal Investigator on clinical trials, and correlative research is funded by Seattle Genetics.
      The remaining authors state no conflict of interest.

      Acknowledgments

      The preliminary result was presented at the 3rd World Congress of Cutaneous Lymphoma, October 2016, New York, NY. We would like to thank Uma Sundram for the histopathological review of the preliminary slides, and Samhita Kadiyala for organizing slides for IHC review. The study was co-funded by Seattle Genetics, Bothell, WA, USA and the Haas Family Foundation.

      Supplementary Material

      References

        • Angelo M.
        • Bendall S.C.
        • Finck R.
        • Hale M.B.
        • Hitzman C.
        • Borowsky A.D.
        • et al.
        Multiplexed ion beam imaging of human breast tumors.
        Nat Med. 2014; 20: 436-442
        • Kim Y.H.
        • Prince H.M.
        • Whittaker S.
        • Horwitz S.M.
        • Duvic M.
        • Scarisbrick J.
        • et al.
        Outcomes by CD30 expression in patients with CTCL receiving brentuximab vedotin (BV) vs physician’s choice (PC) in the Phase 3 ALCANZA study.
        J Clin Oncol. 2017; 35 (7517−7)
        • Kim Y.H.
        • Tavallaee M.
        • Sundram U.
        • Salva K.A.
        • Wood G.S.
        • Li S.
        • et al.
        Phase II investigator-initiated study of brentuximab vedotin in mycosis fungoides and Sézary syndrome with variable CD30 expression level: a multi-institution collaborative project.
        J Clin Oncol. 2015; 33: 3750-3758
        • Koo T.K.
        • Li M.Y.
        A guideline of selecting and reporting intraclass correlation coefficients for reliability research.
        J Chiropr Med. 2016; 15: 155-163
        • Olsen E.A.
        • Whittaker S.
        • Kim Y.H.
        • Duvic M.
        • Prince H.M.
        • Lessin S.R.
        • et al.
        Clinical end points and response criteria in mycosis fungoides and Sézary syndrome: a consensus statement of the International Society for Cutaneous Lymphomas, the United States Cutaneous Lymphoma Consortium, and the Cutaneous Lymphoma Task Force of the European Organisation for Research and Treatment of Cancer.
        J Clin Oncol. 2011; 29: 2598-2607
        • Salva K.A.
        • Wood G.S.
        Epigenetically enhanced photodynamic therapy (ePDT) is superior to conventional photodynamic therapy for inducing apoptosis in cutaneous T-cell lymphoma.
        Photochem Photobiol. 2015; 91: 1444-1451
        • Shrout P.E.
        • Fleiss J.L.
        Intraclass correlations: uses in assessing rater reliability.
        Psychol Bull. 1979; 86: 420-428
        • Swerdlow S.H.
        • Campo E.
        • Pileri S.A.
        • Harris N.L.
        • Stein H.
        • Siebert R.
        • et al.
        The 2016 revision of the World Health Organization classification of lymphoid neoplasms.
        Blood. 2016; 127: 2375-2390