—  SPECIALTY CONFERENCE  —

Dermatopathology

Case 4 - Malignant Melanoma with Vertical Growth Phase (VGP) Heterogeneity and Lymph Node Metastasis with Rhabdoid Features

George F. Murphy
Harvard Medical School, Brigham and Women's Hospital
Boston, MA





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Clinical History
A 58-year-old male presented with a changing lesion of the left calf. Physical exam revealed a pink-tan, dome-shaped cutaneous lesion with foci of irregular brown pigmentation. A shave biopsy was obtained. Sixteen months later he presented with left inguinal adenopathy. A partial lymphadenectomy was performed, revealing extensive nodal replacement by malignant cells with rhabdoid cytologic features and foci of S100 positivity, but no reactivity for Mart-1, HMB-45, or an extensive panel screening for epithelial, mesenchymal, and hematopoietic lineages. Images 4a-4g = skin biopsy; images 4h-4j = lymph node [4j = S100 stain]).


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Key Histologic Findings
  • Metastatic malignant tumor in lymph node with rhabdoid cytology

  • Rare foci of S100 positivity, but without other melanocytic markers

  • Skin lesion with nested junctional and expansile dermal proliferation of variably atypical cells

  • Foci of pagetoid intraepidermal spread

  • Cytologic heterogeneity, with epithelioid, nevoid, and anaplastic components

  • Rare foci of incomplete melanization

Differential and Final Diagnosis
The differential diagnosis of the lymph node metastasis includes origin from a primary lesion that may have rhabdoid features. Rhabdoid, literally indicating rod-like, refers to cytologic similarity within various tumors to rhabdomyoblasts, such as those encountered in malignant rhabdoid tumors of the kidney. Such features may be observed in a variety of primary tumors, including carcinomas of the colon, kidney/renal pelvis, and stomach, among other sites. In the case presented, a malignant melanoma in the vertical stage of growth and with cytologic heterogeneity was identified in a cutaneous site that drained to the involved nodal basin. Although the primary lesion did not show rhabdoid differentiation identical to the metastasis, the latter expressed focal S-100 positivity as well as some areas where more conventional melanoma cytology was represented (not shown). As will be discussed further below, melanoma may rarely give rise to a rhabdoid phenotype characterized by large hyaline-like cytoplasmic inclusions and restricted melanocytic lineage marker expression by immunohistochemistry. The diagnosis, therefore, is malignant melanoma with vertical growth phase (VGP) heterogeneity and lymph node metastasis with rhabdoid features (annotated with appropriate AJCC microstaging for the primary melanoma). The ability of both primary and metastatic melanoma to exhibit profound cytologic diversity not only poses diagnostic challenges, but provides tantalizing biologic clues to its inherent pathobiology.

Diagnostic and Pathobiologic Implications of Melanoma VGP Heterogeneity

Introduction
Malignant melanoma evolves in a step-wise manner from an in situ component whereby malignant cells grow initially along and above the surface of a laminin-rich basement membrane to produce an irregular flat disk, to an invasive intradermal component that enlarges in three dimensions as an expanding tumorigenic nodule with the potential for metastasis. [1, 2] Although there exist distinctive clinicopathological types of melanoma that often correlate with biological behavior, [3] these are largely predicated on architectural and cytological characteristics of the most superficial growth phase. These more curable earlier phases of melanoma development frequently exhibit characteristic clinical manifestations in color, contour, and size, facilitating life-saving early detection and surgical intervention. [3] Recognition that most melanomas arise from a characteristic phase of flat, clinically-distinctive and surgically-curable 'radial' growth [1, 2, 3, 4], followed by eventual development of invasion with accompanying expansile or 'vertical' growth, has provided a platform for the concept of tumor progression that has proven to be broadly applicable to a variety of neoplastic systems. Moreover, there exists validation of this concept at antigenic and genomic levels. [4, 5, 6] Because tumorigenic vertical growth is of key importance to understanding mechanisms of metastatic spread, major recent interest has focused on the cellular constituents of this invasive component.

Vertical Growth Defined
Radial growth phase (RGP) melanoma cells have proven to have essentially no ability to metastasize [7, 8, 9], although most observers qualify them as malignant cells or "melanoma cells" based upon morphology and their biological potential for further progression. Vertical growth phase (VGP) melanoma, in contrast, is characterized by the essential feature of tumor nodule formation with potential for metastatic spread. This concept, as advanced by Elder and coworkers, [10] implies that mitotically-active cells as well as any cells forming an intradermal nodule that exceeds in diameter that of any nest of similar cells distributed along the dermal-epidermal junction, represent the earliest equivalent of vertical growth. Such 'mitogenic' and 'tumorigenic' patterns of growth portend the development of a clone now capable of progressive expansile growth. Recent molecular evidence supports the notion that the cells of VGP melanoma are clonally-derived from cells of the radial growth phase [10, 11, 12], and also potentially from an associated nevus when this is present. [13] VGP melanoma cells in the dermis show a mean Ki-67 expression rate of 13.9% in tumorigenic VGP and only 3.5% in RGP melanoma cells in the dermis, while the corresponding mitotic rates were 1.4 and 0. Such results support the notion that proliferation rates are higher in vertical than in radial growth phase melanomas. [14]

Heterogeneity within Vertical Growth and Metastases: "Polyclonism" and the Rhabdoid Phenotype
An important clue to the underlying nature of melanoma VGP is its cellular composition. The term VGP 'polyclonism' has been used to indicate the frequent occurrence of morphologically distinctive subpopulations comprising the tumorigenic component of primary human melanomas. VGP polyclonism may be manifested by distinctive zones of divergent cell size, shape, pigmentation, or stromal reaction. For example, tumorigenic invasive melanomas may be composed of both epithelioid and fusiform cells; epithelioid and small (nevoid) cells; pigmented and non-pigmented 'sub-clones'; and regions of highly variable stromal desmoplasia, angiogenesis, or tumor-infiltrating lymphocyte responses. VGP heterogeneity may also be evidenced upon immunostaining, where discrete patterns of varying reactivity for S100 protein, Mart-1, or other biomarkers are observed. Melanoma metastases not infrequently also display such polymorphic and antigenic profiles, indicating their kinship to cells in the VGP of the primary melanomas from which they originated.

One interesting example of melanoma polyclonism is found in the so-called "rhabdoid" phenotype, demonstrated in the lymph node in the case under discussion. Interestingly, although the primary lesion was prone to VGP heterogeneity, showing an admixture of larger epithelioid cells, some quite anaplastic, and smaller more nevoid cells, overtly rhabdoid features were lacking. The transition of larger more superficial melanoma cells to smaller, more nevoid ones in a primary lesion may raise the possibility of a co-existing nevus, and has been termed 'pseudomaturation'. The table below provides several differences between primary melanoma with epithelioid/nevoid VGP heterogeneity and exclusively epithelioid lesions arising in pre-existing nevi.

Epithelioid/Nevoid VGP Epithelioid VGP + Nevus
Gradual transitions Present Absent
Sharp demarcations Absent Present
Coarse chromatin in smaller cells Present Absent
Nuclear variability in smaller cells Present Absent
Nucleoli in smaller cells Present Absent
Pigment sometimes in smaller cells Absent Present

Rhabdoid heterogeneity in melanoma, either in the VGP or in a metastasis, refers to malignant cells with eccentric nuclei displaced by abundant cytoplasm containing hyaline filamentous inclusions. Ultrastructurally, such tumors have been found to contain whorls of paranuclear intermediate filaments with entrapped organelles [15, 16, 17] or mitochondria and dilated rough endoplasmic reticulum containing microtubular arrays. [18] Of interest, the immunophenotypic profile of melanomas with rhabdoid differentiation is highly variable and often depleted of melanocyte-associated antigens. In previously reported cases, including that of Abbott, Amirkhan, and Hoang, [16, 18, 19, 20] tumor cells were positive for S100 protein, but negative for HMB-45, Melan-A, Mart-1, tyrosinase, and microphthalmia transcription factor. At least one case of melanoma with rhabdoid features without S100 reactivity has been described, [16] and the relative absence of lineage markers is often attributed to the poorly differentiated state of the rhabdoid phenotype.

Although VGP heterogeneity was prominent in the primary melanoma in the case presented, and a minority population of anaplastic cells could be detected, the rhabdoid cytology of much of the metastasis was dramatically different. Nonetheless, VGP melanoma cells in many instances, in contrast to RGP cells, are known to resemble those forming metastases with respect to overall morphology, in vitro plating efficiency, in vivo tumorigenicity, and chromosomal abnormalities. [21] Because the rhabdoid differentiation is so rarely encountered in primary or metastatic melanomas, it remains an open question as to whether the lymph nodal microenvironment may further influence the cytologic phenotype of cells that originate from a VGP prone to heterogeneity.

Had a primary melanoma not been implicated in the present case, the immunohistochemical evaluation would be critical to determination of histogenesis. For example, rhabdoid tumors of the kidney are often positive for vimentin, glial fibrillary acidic protein, desmin, actin, and AE1.AE3, [22, 23, 24] but not for S100, and rhabdomyosarcoma should express desmin and myoglobin. Malignant peripheral nerve sheath tumor, particularly the epithelioid variant with rhabdoid features, is generally positive for epithelial membrane antigen and synaptophysin, with focal S100 positivity. [25] Plasmacytoma/plasmablastic lymphoma and large cell anaplastic lymphoma may appear somewhat rhabdoid but will be reactive for CD138 and CD30, respectively. And rhabdoid variants of carcinoma and mesothelioma may also be encountered, although screening for low and high molecular weight cytokeratins should aid in identifying most such variants.

The notion that not all melanoma cells are created equally has enormous implications with respect to designing chemotherapies and immunotherapies that target only those subpopulations responsible for tumorigenic progression, and not those cells destined for eventual demise independent of treatment. It is possible that the recognition of heterogeneity ('polyclonism') within those subpopulations of melanoma lesions capable of virulence (i.e. cells comprising the VGP) may be one of the historically earliest observations relevant to the current concept that cancers are composed of dichotomous cell populations with either stem or non-stem like properties.

Pathobiology of VGP Heterogeneity: A Role for Melanoma Stem Cells?
The cancer stem cell model predicated on both the seminal concepts of tumor progression and heterogeneity. At the genomic level, cancer progression has been linked to sequential yet cumulative alterations in oncogenes, tumor suppressor genes, and repair/stability genes. [26, 27] Human cancers have been recognized for some time to consist of phenotypically heterogeneous cell populations. [28, 29] and functional heterogeneity related to in vitro growth and proliferation has also been documented. [30] There also exist differences in the ability of various subpopulations of cancer cells to initiate and sustain tumorigenic growth in vivo. [31] The cancer stem cell model suggests that a minority (albeit not necessarily 'rare') subpopulation of pluripotent cells within the tumor drive tumorigenic growth. The most important attribute of such cells is self-renewal while at the same time remaining capable of giving rise to differentiating progeny. This is related to the unique ability of the cancer stem cell to divide asymmetrically, producing different daughters, one a stem cell and one destined to differentiation usually culminating in eventual programmed cell death.

A recent finding that supports tumorigenic heterogeneity within the melanoma VGP is the discovery of a defined subpopulation of human melanoma cells that express the MDR transporter known as ABCB5. [32] ABCB5 is an ATP-binding cassette (ABC) transporter belonging to the superfamily of integral membrane proteins. It functions as a chemoresistance mediator such that, when blocked, is renders in vitro normally resistant melanoma cells vulnerable to the toxic effects of chemotherapeutic agents such as doxorubicin. [33, 34] A minority of cells in the melanoma VGP express the ABCB5 cell membrane-associated transporter. Such cells display a primitive molecular profile and correlate with clinical melanoma progression as determined by high-density tissue microarrays. [32] ABCB5-expressing melanoma subpopulations injected into immunodeficient mice induce formation of tumors that contain both ABCB5-positive and negative subsets, whereas tumorigenesis is markedly diminished to absent in the ABCB5-negative fraction. The association of an ABCB5-expressing melanoma subset with tumorigenic growth, as is typical of the VGP and metastatic melanoma, is supported by findings indicating that ABCB5 is co-regulated with Melanoma Tumor Antigen p97 (melanotransferrin [MTf]), a known determinant of tumor growth. [35] Lineage tracking approaches reveal the ABCB5 tumorigenic melanoma subpopulation to both self-renew and differentiate, as ABCB5-positive cells give rise to both positive and negative progeny, while ABCB5-negative cells produce homogeneously negative offspring.

Evaluation of human biopsies as well as in humanized melanoma xenografts in which intradermally injected melanoma cells form tumors in the grafted human skin remarkably similar to naturally occurring VGP lesions indicates that ABCB5 shows several patterns of labeling. One, for example, correlates with selective expression in subpopulations that form expanding rims of VGP micronodules. In a potentially related study, the embryonic neural crest stem cell transcription factor, SOX2, has been identified in melanomas with preferential distribution to more spindle, less epithelioid, regions of the VGP [36]. Such data thus begin to link morphological heterogeneity ('polyclonism') with genomic expression patterns. Although further and more comprehensive studies are required, the ability to now detect putative melanoma stem cells in both primary VGP lesions as well as in metastatic deposits holds promise that this new tool for detecting tumor heterogeneity may lead to improved and translationally-relevant prognostic and therapeutic advances.

Translational Significance of Functional VGP Heterogeneity
While cellular heterogeneity represents an important indicator that the traditional stochastic model that all melanoma cells are created equally may not be correct, only through demonstration of functional heterogeneity with regard to tumorgenicity and metastasis can we begin to examine the possibility that this information ultimately may yield clinical benefit. As a marker for melanoma tumorigenic potential, ABCB5-expression by melanoma stem/initiating cells raises the possibility that selective targeting of this minority cellular component may be of therapeutic benefit. As proof of principle, administration of anti-ABCB5 antibody to partially immunodeficient mice harboring human melanoma xenografts results in selective binding of antibody to melanoma cells in vivo, and elicits an antibody-dependent immune response that inhibited tumorigenic growth. [32] Thus, heterogeneous melanoma metastases also driven by a targetable stem cell component may be similarly vulnerable. It is interesting to speculate that the efficacy of host immune responses, such as tumor-infiltrating lymphocytes, and existing forms of immunotherapy may also relate to how precisely they affect the melanoma stem cell (versus the non-stem cell) components of the VGP or its metastases.

Conclusion
A 'cure' for certain melanomas and their precursors is already at hand as a result of prevention before they become clinically manifest (avoidance of environmental triggers, such as UV exposure) and early detection during the pre-tumorigenic RGP. Regrettably, all too many lesions will evade these approaches and present in the VGP either with metastases or with the potential for their future clinical development. The study of heterogeneity within tumorigenic melanoma, both in primary lesions and in metastases, may represent an important key to the eventual eradication of this highly virulent neoplasm, one that may disseminate widely when only occupying the volume of a grain of rice. Morphology provides the first insight into structure and function. In the case of tumorigenic VGP melanoma, cellular heterogeneity, or polyclonism, is a clue to the possibility of underlying functional heterogeneity within tumor nodules. Using meticulous and inclusive criteria to define more virulent cells, like tumor stem cells, in the context of clinically-relevant models, it is an intriguing possibility that melanoma heterogeneity may in part relate in a small subpopulation with the ability to self-renew to form tumors despite the presence of majority of significantly less virulent, more differentiated cells. Thus, recognition of VGP and metastatic heterogeneity at a morphological level may not only refine our approach to diagnosis and determination of histogenesis, but also provides the basis for better biological understanding of this most virulent of human cancers.

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