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
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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]).
Key Histologic Findings
- Metastatic malignant tumor in lymph node with
- 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
Diagnostic and Pathobiologic Implications of Melanoma VGP Heterogeneity
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.
there exist distinctive clinicopathological types of melanoma that often correlate with biological
behavior,  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.  Recognition that most melanomas arise from a
characteristic phase of flat, clinically-distinctive and surgically-curable 'radial'
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.
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
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,  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
and also potentially from an associated nevus when this is present.  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
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
or mitochondria and dilated rough endoplasmic
reticulum containing microtubular arrays.  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,
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,  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.  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
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.  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.
cancers have been recognized for some time to consist of phenotypically heterogeneous cell
and functional heterogeneity related to in vitro growth and proliferation
has also been documented.  There also exist differences in the ability of various
subpopulations of cancer cells to initiate and sustain tumorigenic growth in vivo.  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
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.  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.
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.  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
a known determinant of tumor growth.  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
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 . 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.  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.
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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