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WO2009005816A1 - Procédés de diagnostic différentiel de lésions mélanocytiques - Google Patents

Procédés de diagnostic différentiel de lésions mélanocytiques Download PDF

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Publication number
WO2009005816A1
WO2009005816A1 PCT/US2008/008229 US2008008229W WO2009005816A1 WO 2009005816 A1 WO2009005816 A1 WO 2009005816A1 US 2008008229 W US2008008229 W US 2008008229W WO 2009005816 A1 WO2009005816 A1 WO 2009005816A1
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gploo
stain
nuclear compartment
monoclonal antibody
nuclear
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WO2009005816A8 (fr
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David L. Rimm
Christopher B. Moeder
Bonnie E. Gouldrothberg
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Yale University
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Yale University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/5743—Specifically defined cancers of skin, e.g. melanoma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96486—Metalloendopeptidases (3.4.24)
    • G01N2333/96491—Metalloendopeptidases (3.4.24) with definite EC number
    • G01N2333/96497—Enkephalinase (3.4.24.11)

Definitions

  • This invention relates to the field of diagnosing malignant melanocytic lesions.
  • the human homolog of the mouse silver protein (gplOO or Pmell7), is a melanocyte-specific type I membrane protein required for proper formation of melanosomal fibrils, which facilitates the maturation of stage I pre-melanosomes to stage II (1, 2) .
  • GplOO has long been used as a melanocyte/melanosome marker in the diagnosis of primary cutaneous melanoma and in the identification of melanoma cells in sentinel lymph node biopsies (3, 4) .
  • the recognition of gplOO is commonly performed with HMB45, a mouse monoclonal antibody that specifically reacts with the glycosylated form of gplOO restricted to the fibrillar matrix of stage II premelanosomes (5-7).
  • HMB45 staining patterns for benign and malignant melanocytic lesions has been characterized by multiple groups using routine techniques that employ 3-3 ' -diaminobenzidine (DAB) or 3'- aminoethyl-carbazole (AEC) as a chromogen.
  • DAB 3-3 ' -diaminobenzidine
  • AEC 3'- aminoethyl-carbazole
  • HMB45 staining is observed in over 95% of epithelioid cutaneous melanomas with most lesions yielding over 50% positive staining of the cytoplasmic portion of the cells (8-11).
  • Spindle cell and desmoplastic melanomas tend to be HMB45-negative under standard antigen retrieval and immunostaining techniques (12).
  • Aggressive antigen retrieval can induce HMB45 staining in spindle cell but not in desmoplastic melanomas (12, 13).
  • Evaluation of a series of metastatic lesions from 121 individuals with Stage IV disease revealed substantial heterogeneity in HMB45 staining patterns with no staining in 26 (21.5%) of the lesions, and one third of the remaining lesions demonstrating either weak, moderate or strong staining (14).
  • HMB45 staining patterns in benign melanocytic lesions is stratified by the histologic type of lesion and location of the specific urinary cells relative to the dermal-epidermal junction.
  • Compound melanocytic nevi are characterized by cytoplasmic HMB45 reactivity within the epidermal component of the lesion, focal positivity in the junctional regions and subsequent loss of stain in the dermal component (9, 12, 15). This corresponds to the traditional conception of maturation with the depth of melanocytes in nevi.
  • Ordinary dermal nevi are HMB45 negative (16) whereas dysplastic nevi (17) and blue nevi (18, 19) are positive.
  • Chromogenic stains suffer from several drawbacks including a limited dynamic range, lack of robust standardization methods and the reliance mostly on the subjective interpretation of a human observer to classify staining patterns into discrete ordinal categories, although automated readers have been developed (20) .
  • This limitation was addressed by the optimization of fluorescence-based immunohistochemistry techniques on tissue microarrays coupled with the development of automated methods of quantification of staining intensity as a continuous parameter.
  • the invention provides a method for determining whether a melanocytic lesion in a patient is malignant which comprises determining a quantity of gplOO present within a nuclear compartment in cells in a tissue sample from the melanocytic lesion; determining a quantity of gplOO present within a non-nuclear compartment in the cells in such tissue sample; obtaining a ratio of the quantity of gplOO present within the nuclear compartment relative to the quantity of gplOO present within the non-nuclear compartment; and comparing the ratio so obtained with a predetermined ratio associated with a melanocytic lesion that is malignant; wherein the melanocytic lesion is determined to be malignant if the ratio obtained is less than the predetermined ratio.
  • the invention provides a method of diagnosing whether a patient's melanocytic lesion is malignant which comprises determining a quantity of gplOO present within a nuclear compartment in cells in a tissue sample from the melanocytic lesion; determining a quantity of gplOO present within a non-nuclear compartment in the cells in such tissue sample; obtaining a ratio of the quantity of gplOO present within the nuclear compartment relative to the quantity of gplOO present within the non-nuclear compartment; and comparing the ratio so obtained with a predetermined ratio associated with a melanocytic lesion that is malignant; wherein the patient's melanocytic lesion is diagnosed as malignant if the ratio obtained is less than the predetermined ratio.
  • the invention provides a method of diagnosing whether a patient's melanocytic lesion is benign which comprises determining a quantity of gplOO present within a nuclear compartment in cells in a tissue sample from the melanocytic lesion; determining a quantity of gplOO present within a non-nuclear compartment in the cells in such tissue sample; obtaining a ratio of the quantity of gplOO present within the nuclear compartment relative to the quantity of gplOO present within the non- nuclear compartment; and comparing the ratio so obtained with a predetermined ratio associated with a melanocytic lesion that is malignant; wherein the patient's melanocytic lesion is diagnosed as benign if the ratio obtained is greater than the predetermined ratio.
  • the invention provides a method for discriminating between a benign melanocytic lesion and a malignant melanocytic lesion which comprises determining a quantity of gplOO present within a nuclear compartment in cells in a tissue sample from the melanocytic lesion; determining a quantity of gplOO present within a non-nuclear compartment in the cells in such tissue sample; obtaining a ratio of the quantity of gplOO present within the nuclear compartment relative to the quantity of gplOO present within the non-nuclear compartment; and comparing the ratio so obtained with a predetermined ratio associated with a melanocytic lesion that is malignant; wherein the melanocytic lesion is determined to be malignant if the ratio obtained is less than the predetermined ratio and the melanocytic lesion is determined to be benign if the ratio obtained is greater than the predetermined ratio.
  • This invention provides for a kit comprising a first stain specific for a nuclear compartment of a cell in a tissue sample; a second stain specific for a non-nuclear compartment of a cell in a tissue sample; a gplOO-specific monoclonal antibody; a reagent for quantitatively determining the amount of the gplOO-specific monoclonal antibody bound to gplOO; and instructions for using the components of the kit.
  • Figure 1 Box plots showing the relative distributions of AQUA scores for A) nuclear compartment, B) non-nuclear compartment, and, C) for total area under the tumor mask for the superficial, mid-level and deep regions of the thick nevi (as indicated) . No significant differences were observed using mixed model ANOVA (p>0.20) .
  • Figure 2 Representative images of a thin nevus (A) , thick nevus at mid-level (B) , a thick primary (C) and a visceral metastasis (D) .
  • A thin nevus
  • B thick nevus at mid-level
  • C thick primary
  • D visceral metastasis
  • HMB45 The composite of the DAPI stain-positive nuclear (blue) and DAPI stain-negative non-nuclear (green) compartments within the tumor mask (Aii-Dii) , and the distribution of HMB45 within these compartments (images Aiii-Diii) is presented. In both thin and thick nevi, HMB45 is concentrated in the nuclear compartment whereas in the malignant lesions, HMB45 distribution is predominantly cytoplasmic.
  • FIG. 3 HMB45 staining of melanocytic lesions using conventional immunohistochemistry with a DAB chromogen.
  • hematoxylin counterstain a nevus from the Yale Melanoma boutique collection
  • A shows no appreciable HMB45 staining.
  • faint nuclear DAB stain can be observed in a serial section (B) .
  • a representative primary melanoma lesion from the same collection shows intense cytoplasmic staining that can be visualized in the presence (C) or absence (D) of hematoxylin counterstain, but no nuclear reactivity is seen, even in the absence of counterstain.
  • Figure 4 A) Box plot describing the distributions of In (nuclear/non- nuclear compartment AQUA scores) for the 6 levels of disease spotted on the melanoma SPORE progression array. Thin and thick nevi display elevated ratios that are significantly different from each category of malignant lesions (mixed model ANOVA pairwise comparison p-values all ⁇ 0.0001). B) Box plot describing the distribution of In (nuclear/non-nuclear compartment AQUA scores) for nevi, thick primaries and metastatic lesions in the Yale boutique Array (fixed effects ANOVA pairwise comparison p-values all ⁇ 0.01).
  • Figure 5 Receiver-operator characteristic curve describing the predictive capabilities of the In (nuclear/non-nuclear compartment AQUA score ratio) for distinguishing a nevus from a malignant lesion.
  • HMB45 is not a Significant Predictor of Core Depth in Thick Nevi A.
  • HMB45 is not a Significant Predictor of Core Depth in Thick Primaries A.
  • the thin and thick nevi and thin primaries all group together (p>0.10) and are significantly different from thick primaries and both classes of metastasis (p ⁇ 0.002).
  • Thin nevus Mean ⁇ Standard Deviation is 18.91 ⁇ 9.16 Thick nevus : Mean ⁇ Standard Deviation is 21.69 ⁇ 9.58 Thin primary: Mean ⁇ Standard Deviation is 26.27 ⁇ 15.35 Thick primary: Mean ⁇ Standard Deviation is 36.96 ⁇ 21.30 Lymph node met . : Mean ⁇ Standard Deviation is 32.62 ⁇ 17.39 Visceral met. : Mean ⁇ Standard Deviation is 31.53 ⁇ 16.53
  • Thin nevus Mean ⁇ Standard Deviation is 23.79 ⁇ 11.75 Thick nevus : Mean ⁇ Standard Deviation is 21.69 ⁇ 9.58 Thin primary: Mean ⁇ Standard Deviation is 25.45 ⁇ 12.64 Thick primary: Mean ⁇ Standard Deviation is 32.31 ⁇ 16.95 Lymph node met . : Mean ⁇ Standard Deviation is 30.56 ⁇ 15.23 Visceral met. : Mean ⁇ Standard Deviation is 31.29 ⁇ 15.21
  • Thin nevus Mean ⁇ Standard Deviation is 14.70 ⁇ 6.98 Thick nevus : Mean ⁇ Standard Deviation is 16.74 ⁇ 8.27 Thin primary: Mean ⁇ Standard Deviation is 26.56 ⁇ 17.97 Thick primary: Mean ⁇ Standard Deviation is 39.87 ⁇ 24.74 Lymph node met . : Mean ⁇ Standard Deviation is 33.77 ⁇ 19.03 Visceral met. : Mean ⁇ Standard Deviation is 32.08 ⁇ 18.32
  • Ln (nuclear/non-nuclear) AQUA scores defines nevus from malignancy
  • Thin and thick nevi group together (p>0.32) Thin and thick nevi are significantly different from each malignant class at p ⁇ 0.0001.
  • Thin nevus Mean ⁇ Standard Deviation is 0.450 ⁇ 0.253 Thick nevus : Mean ⁇ Standard Deviation is 0.513 ⁇ 0.227 Thin primary: Mean ⁇ Standard Deviation is 0.036 ⁇ 0.318 Thick primary: Mean ⁇ Standard Deviation is -0.128 ⁇ 0.275 Lymph node met . : Mean ⁇ Standard Deviation is -0.055 ⁇ 0.186 Visceral met . : Mean ⁇ Standard Deviation is 0.027 ⁇ 0.234 Figure 12 :
  • HMB45 staining when present, adopts a primarily non-nuclear pattern. Silhouttes of nuclear spaces can be visualized as dark circles on the Cy5 image. In contrast, among the nevi, staining is not visualized in the non-nuclear regions. Addditionally, low-level staining is present in their nuclear compartment.
  • the invention provides a method for determining whether a melanocytic lesion in a patient is malignant which comprises determining a quantity of gplOO present within a nuclear compartment in cells in a tissue sample from the melanocytic lesion; determining a quantity of gplOO present within a non-nuclear compartment in the cells in such tissue sample; obtaining a ratio of the quantity of gplOO present within the nuclear compartment relative to .the quantity of gplOO present within the non-nuclear compartment; and comparing the ratio so obtained with a predetermined ratio associated with a melanocytic lesion that is malignant; wherein the melanocytic lesion is determined to be malignant if the ratio obtained is less than the predetermined ratio.
  • the method may further comprise staining the nuclear compartment with a first stain and the non-nuclear compartment with a second stain wherein the second stain is detectably differentiated from the first stain.
  • Each of the first stain and the second stain may be a fluorophor.
  • the quantity of gplOO present within the nuclear compartment may be determined by a procedure employing a gplOO-specific monoclonal antibody.
  • the quantity of gplOO present within the non-nuclear compartment may be determined by a procedure employing a gplOO-specific monoclonal antibody.
  • the monoclonal antibody may be the monoclonal antibody designated
  • HMB45 which is commercially available, for example, from GeneTex, Inc., San Antonio Texas USA or BioGenex, San Ramon, California USA.
  • the monoclonal antibody may also refer to a number of other gplOO- specific monoclonal antibodies, for example, 3F272 and SPM142, which are commercially available, for example from Abeam, Cambridge, Massachusetts USA.
  • Each of the quantity of gplOO present within the nuclear compartment and the quantity of gplOO present within the non-nuclear compartment may be determined by a quantitative image analysis procedure.
  • AQUA® procedures as described in issued U.S. Patent No. 7,219,016, which is incorporated by reference into this application in its entirety, the Bliss system, the ACIS system, the IVision and GenoMx system, the ScanScope Systems, the Ariol SL-50 System, and the
  • LSC system which are available from the following respective manufacturers: Bacus Laboratories, Inc., Clarient, Inc., BioGenex,
  • the melanocytic lesion may be, for example, a thin nevus, a thick nevus, a thick primary, a lymph node metastasis, or a visceral metastasis .
  • the invention provides a method of diagnosing whether a patient's melanocytic lesion is malignant which comprises determining a quantity of gplOO present within a nuclear compartment in cells in a tissue sample from the melanocytic lesion; determining a quantity of gplOO present within a non-nuclear compartment in the cells in such tissue sample; obtaining a ratio of the quantity of gplOO present within the nuclear compartment relative to the quantity of gplOO present within the non-nuclear compartment; and comparing the ratio so obtained with a predetermined ratio associated with a melanocytic lesion that is malignant; wherein the patient's melanocytic lesion is diagnosed as malignant if the ratio obtained is less than the predetermined ratio.
  • the method may further comprise staining the nuclear compartment with a first stain and the non-nuclear compartment with a second stain wherein the second stain is detectably differentiated from the first stain.
  • Each of the first stain and the second stain may be a fluorophor.
  • the quantity of gplOO present within the nuclear compartment may be determined by a procedure employing a gplOO-specific monoclonal antibody.
  • the quantity of gplOO present within the non-nuclear compartment may be determined by a procedure employing a gplOO-specific monoclonal antibody.
  • the monoclonal antibody may be the monoclonal antibody designated HMB45, which is commercially available, for example, from GeneTex, Inc., San Antonio Texas USA or BioGenex, San Ramon, California USA.
  • the monoclonal antibody may also refer to a number of other gplOO- specific monoclonal antibodies, for example, 3F272 and SPM142, which are commercially available, for example from Abeam, Cambridge, Massachusetts USA.
  • Each of the quantity of gplOO present within the nuclear compartment and the quantity of gplOO present within the non-nuclear compartment may be determined by a quantitative image analysis procedure.
  • the melanocytic lesion may be, for example, a thin nevus, a thick nevus, a thick primary, a lymph node metastasis, or a visceral metastasis .
  • the invention provides a method of diagnosing whether a patient's melanocytic lesion is benign which comprises determining a quantity of gplOO present within a nuclear compartment in cells in a tissue sample from the melanocytic lesion; determining a quantity of gplOO present within a non-nuclear compartment in the cells in such tissue sample; obtaining a ratio of the quantity of gplOO present within the nuclear compartment relative to the quantity of gplOO present within the non- nuclear compartment; and comparing the ratio so obtained with a predetermined ratio associated with a melanocytic lesion that is malignant; wherein the patient's melanocytic lesion is diagnosed as benign if the ratio obtained is greater than the predetermined ratio.
  • the method may further comprise staining the nuclear compartment with a first stain and the non-nuclear compartment with a second stain wherein the second stain is detectably differentiated from the first stain.
  • Each of the first stain and the second stain may be a fluorophor.
  • the quantity of gplOO present within the nuclear compartment may be determined by a procedure employing a gplOO-specific monoclonal antibody.
  • the quantity of gplOO present within the non-nuclear compartment may be determined by a procedure employing a gplOO-specific monoclonal antibody.
  • the monoclonal antibody may be the monoclonal antibody designated HMB45, which is commercially available, for example, from GeneTex, Inc., San Antonio Texas USA or BioGenex, San Ramon, California USA.
  • the monoclonal antibody may also refer to a number of other gplOO- specific monoclonal antibodies, for example, 3F272 and SPM142, which are commercially available, for example from Abeam, Cambridge, Massachusetts USA.
  • Each of the quantity of gplOO present within the nuclear compartment and the quantity of gplOO present within the non-nuclear compartment may be determined by a quantitative image analysis procedure.
  • the melanocytic lesion may be, for example, a thin nevus, a thick nevus, a thick primary, a lymph node metastasis, or a visceral metastasis .
  • the invention provides a method for discriminating between a benign melanocytic lesion and a malignant melanocytic lesion which comprises determining a quantity of gplOO present within a nuclear compartment in cells in a tissue sample from the melanocytic lesion; determining a quantity of gplOO present within a non-nuclear compartment in the cells in such tissue sample; obtaining a ratio of the quantity of gplOO present within the nuclear compartment relative to the quantity of gplOO present within the non-nuclear compartment; and comparing the ratio so obtained with a predetermined ratio associated with a melanocytic lesion that is malignant; wherein the melanocytic lesion is determined to be malignant if the ratio obtained is less than the predetermined ratio and the melanocytic lesion is determined to be benign if the ratio obtained is greater than the predetermined ratio.
  • the method may further comprise staining the nuclear compartment with a first stain and the non-nuclear compartment with a second stain wherein the second stain is detectably differentiated from the first stain.
  • Each of the first stain and the second stain may be a fluorophor.
  • the quantity of gplOO present within the nuclear compartment may be determined by a procedure employing a gplOO-specific monoclonal antibody.
  • the quantity of gplOO present within the non-nuclear compartment may be determined by a procedure employing a gplOO-specific monoclonal antibody.
  • the monoclonal antibody may be the monoclonal antibody designated HMB45, which is commercially available, for example, from GeneTex, Inc., San Antonio Texas USA or BioGenex, San Ramon, California USA.
  • the monoclonal antibody may also refer to a number of other gplOO- specific monoclonal antibodies, for example, 3F272 and SPM142, which are commercially available, for example from Abeam, Cambridge, Massachusetts USA.
  • Each of the quantity of gplOO present within the nuclear compartment and the quantity of gplOO present within the non-nuclear compartment may be determined by a quantitative image analysis procedure.
  • AQUA® procedures as described in issued U.S. Patent No. 7,219,016, which is incorporated by reference into this application in its entirety, the Bliss system, the ACIS system, the IVision and GenoMx system, the ScanScope Systems, the Ariol SL-50 System, and the
  • LSC system which are available from the following respective manufacturers: Bacus Laboratories, Inc., Clarient, Inc., BioGenex,
  • the melanocytic lesion may be, for example, a thin nevus, a thick nevus, a thick primary, a lymph node metastasis, or a visceral metastasis .
  • the benign melanocytic lesion may be a Spitz nevus and the malignant melanocytic lesion may be a Spitzoid melanoma.
  • This invention provides for a kit comprising a first stain specific for a nuclear compartment of a cell in a tissue sample; a second stain specific for a non-nuclear compartment of a cell in a tissue sample; a gplOO-specific monoclonal antibody; a reagent for quantitatively determining the amount of the gplOO-specific monoclonal antibody bound to gplOO; and instructions for using the components of the kit.
  • the first stain, the second stain, or both may be fluorophors.
  • the gplOO-specific monoclonal antibody may be HMB45.
  • the gplOO-specific monoclonal antibody may also refer to a number of other gplOO-specific monoclonal antibodies, for example, 3F272 and SPM142, which are commercially available, for example from Abeam, Cambridge, Massachusetts USA.
  • the reagent may be a fluorescently labeled antibody.
  • HMB45 is a mouse monoclonal antibody raised against Pmell7/gpl00, a melanoma-specific marker, which is routinely used in the diagnosis of primary cutaneous malignant melanoma.
  • the standard expression pattern for a positive HMB45 staining result on immunohistochemistry is based upon the results of chromogenic-based methods.
  • SPORE melanoma progression array containing lesions representing the spectrum of melanocytic lesions ranging from thin nevus to visceral metastasis using the fluorescence-based staining technique and Automated Quantitative Analysis (AQUA) of the obtained digital
  • the fluorescence-based approach revealed a nuclear gplOO localization present in the dermal component of all nevi that was not seen before. This nuclear localization could not be observed on routine chromogenic stains because the standard hematoxylin nuclear counterstain overwhelms the weak nuclear HMB45 stain.
  • the SPORE Progression Array was constructed by the Harvard Tissue Microarray facility using the standard method (22) and included a series of randomly-selected, formalin-fixed, paraffin-embedded melanocytic lesions retrieved from the archives at Harvard University, the University of Pennsylvania and the MD Anderson Cancer Center.
  • the array includes 21 thin ( ⁇ 1 mm) nevi, 15 thick (> 1 mm) nevi, 38 radial growth phase ( ⁇ 1 mm) primary melanomas, 20 vertical growth phase (>1 mm) primary melanomas, 28 lymph node metastases and 45 metastases to visceral organs.
  • Duplicate 0.6 mm cores were obtained from each thin lesion as well as from the set of metastatic lesions. For the thick lesions, three depths of invasion relative to the skin edge were defined (superficial, mid-level and deep) and duplicate 0.6 mm cores were taken at each of these depths to produce a total of 6 cores from each tumor .
  • the Yale Melanoma boutique Array was constructed by the Yale Tissue Microarray facility using formalin-fixed, paraffin-embedded archival material. It included single 0.6 mm cores from 20 benign nevi, 20 vertical growth phase primary melanomas and 20 metastases, the latter representing lesions from subcutaneous, lymph node and visceral sites. In addition, the tissue microarray included as controls 0.6 mm cores from paraffin-embedded pellets of 16 melanoma and 18 non-melanoma cell lines grown in culture to confluency and harvested into 10% neutral-buffered formalin.
  • BSA bovine serum albumin
  • TBS IM tris-buffered saline
  • Fluorescence-based immunohistochemical staining was performed by multiplexing the pre-diluted HMB45 mAb (BioGenex, San Ramon, CA) with rabbit polyclonal anti-S100B (Dako Cytomation, Carpenteria, CA) at 1:200 dilution. The latter was used to define the "tumor mask" which discriminates the regions corresponding to melanoma from the surrounding tissue within the histospot in the absence of simultaneous H&E staining. Primary antibodies were incubated at 4 0 C overnight. Visualization was accomplished using fluorescent probes.
  • the secondaryfluorescent antibodies Alexa-546-conjugated goat anti-rabbit (1:100 dilution, Molecular Probes, Eugene OR) diluted into Envision anti-mouse (neat; DAKO) were applied for one hour at room temperature.
  • the slides were washed three times for 10-minute with IM TBS, incubated with cyanine-5-conjugated tyramide (Perkin-Elmer, Wellesley, MA) for 10 minutes, and then with 0.01 mg/mL 4 ' , 6-diamidino-2- phenylindole (DAPI) for 20-minute for nuclear compartment visualization.
  • DAPI 6-diamidino-2- phenylindole
  • the slides were mounted with Prolong-Gold antifade reagent (Invitrogen) , sealed with a nylon-based lacquer and stored in the dark until scoring.
  • Prolong-Gold antifade reagent Invitrogen
  • the pre-diluted HMB45 antibody was applied neat for 1 hr at room temperature and visualized using EnvisionTM with a 3-3 ' -diaminobenzidine chromogen. Counterstaining was achieved using Meyer's hematoxylin. Evaluation of chromogen-stained arrays and conventional imaging was done by a single pathologist (DLR) .
  • DLR single pathologist
  • the gplOO staining patterns were captured by a 65 ms exposure time in the cyanine-5 channel on the SPORE progression array and 57 ms for the Yale Melanoma boutique array.
  • the Alexa-546 signal representing SlOOB staining was binary gated to indicate whether a pixel is within the tumor mask ("on") or not ( "off” ).
  • Further subcellular compartmentalization defined nuclear from non-nuclear regions within the tumor.
  • the nuclear compartment was defined as the subset of pixels that demonstrated any DAPI staining within in the plane of focus. This was required to compensate for the 3-dimensional thickness of the tumor sections, which can blur the discrimination of the nuclear boundary.
  • the non- nuclear compartment was then defined as all pixels assigned to the tumor mask but are not included within the nuclear compartment.
  • gplOO expression levels were determined in an automated fashion, blinded to any a priori clinical information, from the images acquired under the Cy5 channel to obtain a relative pixel intensity restricted to the signal emanating from the plane of focus.
  • the final AQUA score for the entire tumor mask or any of its subcellular compartments was calculated as the average AQUA score for each of the individual pixels included in the selected compartment and was reported on a scale of 0 to 255.
  • HMB45 staining patterns for each class of lesion spotted on the tissue microarray are shown in Figure 2, A-D.
  • HMB45 most strongly- stained the cytoplasmic regions (non-nuclear compartment) of the cells although some degree of nuclear staining could be observed.
  • AQUA score of 20 as the maximal cut-off level designating moderate to strong HMB45 staining in the non-nuclear compartment, cytoplasmic staining was observed in 16/25 radial growth phase primaries, 14/19 vertical growth phase primaries, 16/24 lymph node metastases and 24/40 visceral metastases.
  • Cytoplasmic staining was present in none of the thin nevi and only in one thick nevus. Interestingly, definitive nuclear staining was observed in the dermal component of all thin and thick nevi, whereas only three metastatic melanoma lesions demonstrated any nuclear staining and weak cytoplasmic HMB45 staining. Focal staining was observed in two of these cases, with the third lesion displaying a punctuated nuclear staining pattern throughout the histospot.
  • the ratio of nuclear/non-nuclear AQUA scores is a significant discriminator of benign melanocytic lesions
  • the ratio of nuclear to non-nuclear AQUA scores was evaluated as a possible discriminator of benign melanocytic lesions.
  • the natural log transformation of the ratio was selected as the preferred parameter.
  • both the thin (0.450 ⁇ 0.253) and thick (0.513 ⁇ 0.227) nevi had strongly positive mean In (nuclear/non-nuclear ratios) that were significantly different (p ⁇ 0.0001) from the group of malignant lesions whose means ⁇ standard deviations range from 0.128 ⁇ 0.285 to 0.055 ⁇ 0.186 ( Figure 4A).
  • Figure 4A To determine the reproducibility of this finding on an independent collection of benign and malignant melanocytic lesions, the Yale Melanoma boutique array was stained with the HMB45 antibody using fluorescence-based methods. Twenty benign nevi, 20 vertical growth phase primaries and 18 metastases yielded histospots with sufficient material for analysis.
  • a receiver-operator characteristic curve was constructed from these data across all possible dichotomizing cut points covering the full range of the In (nuclear/non-nuclear ratio) scores, which produced 0.93 units of area and, at the shoulder of the curve, created by designating an In (nuclear/non-nuclear HMB45 AQUA score ratio) >0.154 as positively identifying a nevus, a maximized simultaneous sensitivity of 0.92 and specificity of 0.80 for discriminating benign nevi from malignant melanocytic lesions ( Figure 5) .
  • HMB45 reactivity is a cornerstone for the pathologic identification of melanocytic lesions.
  • the cytoplasm of epithelioid cutaneous melanomas characteristically stains positively for HMB45, a diagnostic feature frequently used when pathologists evaluate malignancies of unknown primary (4).
  • HMB45 was first described in 1986 (23) , the body of literature on the patterns of HMB45 staining among distinct classes of melanocytic lesions has been largely derived from nonfluorescent-based immunohistochemical techniques with brown or red colored chromogens . As such, their results are limited by the usual issues associated with any chromogen evaluation, including lack of dynamic range, lack of standardization, the reliance on subjective scoring methods and the categorization of scores into discrete categories. In this analysis, we reconsidered patterns of HMB45 reactivity through the application of immunofluorescence-based staining techniques. The increased sensitivity, combined with the lack of obscuring counterstains , revealed an interesting expression patterns that have not previously been described
  • the SPORE progression array was constructed by the Harvard TMA facility with melanocytic lesions from Harvard, University of Pennsylvania and MD Anderson Cancer Center. It contains 480 0.6 mm cores including 35 nevi represented in 130 cores (superficial , mid, and deep areas of each when available) , 60 primary melanomas represented in 200 cores (radial and vertical growth phases of each, when available) and 75 metastases represented in 150 cores.
  • the cases were stained with Ki-67 (BD Pha ⁇ ningen, mouse monoclonal antibody 1:500) and HMB-45 (BioGenex, a mouse monoclonal antibody to pmell7/gpl00, prediluted neat) and quantified using the AQUA technology for quantitative assessment of immunofluorescence. Data was analyzed using Statview, JMP5 and SAS software. ( Figure 12)
  • results The mean expression levels for Ki-67 showed progression that directly correlated with the morphologic selection, where the lowest mean AQUA scores were seen in thin nevi, followed by thick nevi, thin primaries, thick primaries, lymph node, and visceral metastases. The progression was significant for trend (p ⁇ 0.0001) and the malignant cases (excluding thin primaries) were significantly higher than the nevi (p ⁇ 0.05 adjusted for multiple comparison) .
  • Overall HMB-45 antibody staining for pmell7/gpl00 showed no significant differences between benign and malignant, nor was any significant difference seen between superficial, intermediate and deep levels of thick lesions ( Figures 6, 7, 8) . However, an interesting subcellular distribution was observed.
  • AQUA-based analysis of the SPORE progression array showed progressive increases in Ki-67 expression corresponding to morphologic stage.
  • HMB-45 analysis revealed low level nuclear expression in nevi that may have been previously missed on chromogenic stains due to hematoxylin counterstaining. It is also possible that we are detecting a protein other than pmell7/gpl00 due to uncharacterized cross-reactivity of the HMB-45 antibody. However, the ratio of nuclear over non-nuclear expression may be valuable in distinguishing benign nevi from malignant melanoma.
  • Figures 9 and 10 illustrate the distribution of gplOO AQUA® Scores in the nuclear compartment and in the non-nuclear compartment of the melanocytic lesion samples of thin nevus, thick nevus, thin primary, thick primary, lymph node metastases, and visceral metastases.
  • nevi displayed significantly lower non-nuclear staining and similar nuclear staining.
  • Figure 11 illustrates how In (nuclear/non-nuclear) AQUA® scores defines nevus from malignancy.

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Abstract

L'invention concerne un procédé de détermination de la malignité ou non d'une lésion mélanocytique chez un patient, qui consiste à déterminer une quantité de gp 100 présente dans un compartiment nucléaire de cellules colorées d'un échantillon de tissu de la lésion mélanocytique; à déterminer une quantité de gp 100 dans un compartiment non nucléaire de cellules colorées d'un tel échantillon; à obtenir un rapport de quantité de gp 100 présente dans le compartiment non nucléaire; et à comparer le rapport ainsi obtenu avec un rapport prédéfini associé à la lésion mélanocytique maligne; cette lésion étant déterminée maligne si le rapport obtenu est inférieur au rapport prédéfini.
PCT/US2008/008229 2007-07-03 2008-07-03 Procédés de diagnostic différentiel de lésions mélanocytiques Ceased WO2009005816A1 (fr)

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US7873480B2 (en) 2001-04-20 2011-01-18 Yale University Systems and methods for automated analysis of cells and tissues
US8036833B2 (en) 2001-04-20 2011-10-11 Yale University Systems and methods for automated analysis of cells and tissues
US8121794B2 (en) 2001-04-20 2012-02-21 Yale University Systems and methods for automated analysis of cells and tissues
US8185320B2 (en) 2001-04-20 2012-05-22 Yale University Systems and methods for automated analysis of cells and tissues
US8639450B2 (en) 2001-04-20 2014-01-28 Yale University Systems and methods for automated analysis of cells and tissues
US10217219B2 (en) 2001-04-20 2019-02-26 Yale University Systems and methods for automated analysis of cells and tissues
US11315250B2 (en) 2001-04-20 2022-04-26 Yale University Systems and methods for automated analysis of cells and tissues
US8367351B2 (en) 2006-05-05 2013-02-05 Historx, Inc. Methods for determining signal transduction activity in tumors
US8497080B2 (en) 2006-05-05 2013-07-30 Historx, Inc. Methods for determining signal transduction activity in tumors
US7709222B2 (en) 2006-07-13 2010-05-04 Yale University Methods for making cancer prognoses based on subcellular localization of biomarkers

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