1
Lack of correlations between the immunohistochemical detection of indoleamine 2,3
dioxygenase in skin melanoma and in draining lymph node from patients with
primary skin melanoma.
Ana Maria Abreu Velez, MD, phD.
Corresponding author: Ana Maria Abreu Velez Institute for Molecular Medicine and
Genetics, Medical College of Georgia, CB 2803, 1120 15th Street, Augusta, GA, 309122630, USA. Fax: (+ 1) 706 721-7915.E-mail: aavelez@mail.mcg.edu
Abbreviations: The sentinel lymph node (SLN), tumor (T), sentinel node biopsy (SNB),
Immunohistochemistry (IHC), reverse-transcriptase polymerase chain reaction (RTPCR), Hematoxylin and Eosin (H&E), 2,3-dioxygenase human enzyme (IDO), dendritic
cells (DCs), lymph nodes.
Conflict of interest: None.
Key words: IDO, melanoma, sentinel lymph node.
Number of tables:3
Number of figures:6
Introduction:
Postdoctoral fellows often face severe retaliation form their bosses for exposing
wrongdoing in their work. Retaliations include firing, removing of visas, bad mouth to
get other jobs. This study shows the real data of misrepresented by others in the lack of
relationship with 2,3-dioxygenase human enzyme, in skin melanoma and they lymph
nodes. Some universities and institutions have created Over the past few decades, the
incidence of cutaneous malignant melanoma has been rising in both sexes in almost all
developed countries, notably those with fair-skinned populations. Presently, the
improvement of survival over time is most likely due to earlier detection of skin
melanoma. In the 1990s, about 30% of all newly diagnosed skin melanoma had stage of
tumor (T) T3 or T4, implying that further improvement in survival by earlier detection is
feasible.1-3 Furthermore depth of invasion and stage of the disease are accepted
established prognostic indicators in cutaneous malignant melanoma. It is accepted that
increasing Breslow thickness, Clark level of more than III, the presence of ulceration, and
patient age of 60 or less are the most important independent prognostic factors associated
2
with the finding of melanoma metastasis.1-8 The sentinel lymph node (SLN) is the first
lymph node in the regional nodal basin to receive metastatic cells. In-transit nodes are
found between the primary melanoma site and regional nodal basins. SLN biopsy has
become a standard method of staging patients with cutaneous melanoma. There has, for a
long time, been an ongoing discussion on whether the prophylactic removal of lymph
nodes ("elective lymph node dissection") is of benefit for melanoma patients. More
recently, "selective" lymphadenectomy ("sentinel node biopsy", SNB) has been proposed
to evaluate the status of the first draining lymph node ("sentinel node") of the regional
basin. Several studies now demonstrate that the sentinel node evaluation for underlying
metastatic disease reflects the status of the entire lymph node region and is therefore a
useful prognostic factor. A multi-institutional study highlighted SNB status as the most
significant prognostic factor, superior to measurement of tumor thickness in primary
melanoma. Different techniques to detect micrometastasis within the lymph node are
under current evaluation.5-8 Histology and immunohistology using antibodies against
melanoma-associated antigens are routinely performed in SLN. The clinical value of
reverse-transcriptase polymerase chain reaction (RT-PCR) based search for minimal
melanoma disease in lymph nodes remains unclear.
The long-term survival of some patients with metastatic melanoma and the correlation
with presence of primary or metastatic tumor into the lymph node is not understood. It
may be attributable to many factors such as cellular immune responses to melanoma
antigens or the presence of dendritic cells (DCs) at the site of a tumor could correlate
with an improved prognosis similar to that which occurs in patients with a variety of
histological tumor types.9-11
Another problem in melanoma studies is the ability to make an accurate histopathologic
diagnosis on skin and lymph nodes when a variety of cytomorphological features,
architectural patterns and stromal changes may be observed in malignant melanomas.
Hence, melanomas may mimic carcinomas, sarcomas, benign stromal tumors,
lymphomas, plasmacytomas and germ cell tumors. For improving diagnosis, a series of
typically melanomas are S100 protein although is nonspecific for melanoma, NKIC3,
HMB-45 that seem to have a sensitivity of 67-93%, Melan-A and tyrosinase positive but
some melanomas may exhibit an aberrant immunophenotype and may express
cytokeratin’s, desmin, smooth muscle actin, KP1 (CD68), CEA, EMA and VS38. Very
rarely, neurofilament protein and GFAP positivity may be seen.
The main aim of this study was to examine if the presence of the 2,3-dioxygenase human
enzyme (IDO) could be a predictive of SLN metastasis and primary skin melanoma. For
this purpose, we use IDO antibody an anti-human rabbit raised against part of IDO
peptide sequence. Hematoxilin-Eosin (H & E) and the immunohistochemical preparations
3
were examined separately by three different investigators. As a control, tonsils from 12
young patients that had a routine tonsillectomy for clinical tonsillitis were used, for the
difficulty to obtain normal human lymph nodes.
Methods:
Samples: IRB approval form was obtained for performing this study.
Primary skin melanoma: Nine paraffin fixed samples from patients with primary skin
melanoma were tested. The diagnosis of skin melanoma was based on clinical and
histopathological criteria of H&E-stained sections. Skin samples that fulfilled the
histopathologic criteria for lentigo maligna melanoma, superficial spreading melanoma,
nodular melanoma and acral letiginous type were scrutinize. When using H & E, all cases
showed the downward streaming of the typical melanoma tumor cells. The epidermis
showed considerable irregular junctional activity with downward streaming of tumor
cells possessing anaplastic nuclei from the epidermis into the dermis. The epidermis
showed considerable irregular downward proliferation of its rete ridges. The tumor cells
in the dermis showed great variation in morphology; however, the two most common
major types of cells were recognized as epitheliod (8/9) and a spindle-shape cell
malignant melanoma (1/9). The malignant melanoma shows a band-like appearance of
inflammatory infiltrate, often intermingled with melanophages, at the base of the tumor,
but also in some cases the band-like inflammatory infiltrate often extended beyond the
tumor along the normal-appearing epidermis as classically described for melanoma.
Lymph nodes from patients with melanoma, (H&E). 37 paraffin fixed lymph nodes
(LN) samples from patients with primary skin melanoma were tested. No information
was available if the skin samples and the LN belonged to the same patient or not.
Sections of all lymph nodes were examined to detect possible metastatic cells as routinely
described.
Human tonsils: Paraffin fixed tonsils from 12 young patients that had a routine
tonsillectomy for clinical tonsillitis were stained with the IDO polyclonal antibody. Table
1 summarizes the histopathologic diagnosis that the pathologist reported in the tonsils
observed by stain H&E.
Classification of the patterns and grading observed in the tonsils: The classification
of the pattern as well as the grading scale use in this study are summarize in table 1.
Immuno histochemistry studies: All antibodies used in this study works on paraffin
sections. IDO antibody was used for immunohistochemistry. The antibody was obtained
4
from two rabbits immunized 3 times using the 18 aminoacid peptide sequence AcDLIESGQLRERVEKLNMC-amide of the indoleamine 2,3-dioxygenase human enzyme.
Source of human sequence: Genbank M34455 (IDO.}).12 The antibody was affinity
purified. The peptide that contains the above-mentioned sequence was conjugate to KLH
for immunization purposes with 90% purification quality using HPLC and mass
spectroscopic verification of the peptide. The antibody was tested in an ELISA using
BSA-coupled peptide on the solid phase (Biosurce, MA, USA).
Melanin Bleaching technique: All lymph nodes (LN) and primary skin melanoma were
subject to a melanin bleaching technique to improve the cell structure detection as well as
to improve the visualization of the immune stain. Briefly, pre coated slides were bleached
using a standard protocol of acidification with potassium permanganate, 0,3% sulfuric
acid and 1% oxalic acid. 13-15
Staining protocol used against the IDO antibody: Briefly, paraffin processed formalin
fixed tissues were sectioned 4 um, hydrated, the endogenous peroxidase blocked, a
proteinase K enzyme added, and the slides rinsed. The slides were blocked for
nonspecific staining using normal human sera. As primary tracer, the IDO antibody was
added and after two hours incubation, the slides were washed and rinsed in PBS-Tween
0,05% buffer. The slides were then incubated with a diluted secondary biotinylated
mouse anti-rabbit antibody (DAKO, CA, USA).16 After washing, a further incubation
with Streptavidin was performing subsequently. Incubation with another chromogen Fast
Red was performed. Hematoxilin was used as a counter stain. Finally, the slides were
covered with mountain media and examined under light microscopy.12
Other immunohistochemistry markers used in this study. Two lymph nodes cases that
had shown presence of IDO, follicular pattern, complete metastasis infiltration of
melanoma cells and classical pattern of IDO were simultaneously stained using a set of
commercially available antibodies. Monoclonal mouse anti-human CD 68, macrophage
monoclonal mouse anti-human macrophage HAM 56, rabbit anti-cow S-100 (Dako
Corporation, Carpinteria, CA, USA), and monoclonal antibody IgG2 mouse anti-human
CD83 (Coulter Corporation, Miami, USA), were used all following manufactures
recommendations. (Table 2).
Results:
IDO expression in tonsils: 2/12 tonsil contained IDO positive material. This was mostly
located at the sub-epithelial area bordering the cortical tissue of the LN (Figure 1). The
degree and the presence of inflammatory cells correlated with the degree of presence of
the polyclonal antibody positive stain. (Table 1).
5
IDO expression in primary skin melanoma: Eight from nine primary skin melanoma
showed a positive stain in some of the melanoma cells in clustered areas (Figure 2). One
primary skin melanoma was negative. This sample seems to correspond to the melanoma
spindle-shape cell type instead of the epitheliod. Other types of positive were observed.
The cells that stain with this antibody have a condensed nucleus and a small cytoplasm,
resembling lymphocytes. The pattern of staining is cytoplasmic, condensed around the
large nucleus (Figure 3). The cells that showed this pattern were mostly observed in the
superficial blood vessels of the skin. The correlation of presence of inflammatory cells
and those that were IDO positive was similar. A mild degree of inflammation as observed
in all skin melanoma samples as classically described for melanoma. In one case the
primary skin melanoma shows co-migration of IDO positive cells with some melanoma
cells that seem to be migrating through the hair follicle.
IDO expression in lymph node from patients with skin melanoma: The most common
pattern of distribution of cells (90%) that were recognized by the antibody in the LN
seems to be in the lymph endothelium (Figure 4). These positive cells were mostly
located in the interface of the marginal and medullary sinus, bordering the cortex and the
medullary sinus. Most of the positive cells were observed in the medullary cord zone but
were also seen in the periphery of the cortex area of the LN. The cells stained by the
antibody seem to be invading the LN with other inflammatory cells. Similar cell
populations were observed through the afferent lymphatic vessels and the hilum. These
three patterns, seems to overlap with the circulation pathway of a normal LN. In about
17% of the LN, some degree of presence of metastatic cells and cells that recognize the
antibody was observed. This comigration pattern was mostly observed in the subcapsular area.
In 356 slides from 37 cases, 205 of them showed some degree of IDO positive material.
Non correlation of replacement of LN by metastatic melanoma cells and presence of
positive cells that recognize this antibody was observed. We observed one case of
probable Paget ‘s disease metastasizing into the LN, that could be wrongly identified as a
melanoma but may correspond to a pigmented Paget’s disease? or other malady.
In about 20% of the LN, the cells that were recognized by the antibody tended to be
clustered in small areas. Anecdotical in two cases when the metastatic melanoma cells
invaded the LN over more than 90% of it’s surface a few IDO positive cells were also
found (Figures 6 a and b). In 4 patients, 6 LN showed a very well define follicular IDO
positive pattern (Figure 5 and 6).
6
A peculiar pattern was observed in LN, when a “compact” metastasis of melanoma-cells
was found. The presence of a ring of cells that stained with the antibody was observed,
but also visualized using HAM 56 antibody (Figures 6a and b).
A positive IDO cells were commonly seen in relation to blood vessels of the lipid tissue
that surrounds the LN.
IDO expression: Skeletal muscle fibers seemed to be recognized by the antibody, but
the specificity and sensitivity needs to be further tested. The reactivity observed in this
tissue may also be due to the counterstain when using Hematoxilin. Another cell types
that seemed to be IDO positive were the secretory cells of the mammary ductus as well as
a few eccrine glands of the skin.
Immunohistochemistry of lymph node from patients with melanoma: Ham 56 reacts
clearly with two morphologically distinct cell types. One of these types of cells resembles
macrophages structures (“foamy” cytoplasm) and the other type resembles dendritic cells.
In this study the pattern of distribution of HAM 56 resembled that seen using IDO
antibody i.e., the peri-cortical zone bordering the sinuses areas. These two antibody
patterns (HAM 56 and IDO) seem to show affinity for the medullar as well as the
follicular interfaces. However, a slight affinity for the medullar area where macrophages
are located was seen.
Lymph nodes from melanoma patients stained with mostly the sinuses areas of the nodes
both CD 68 and less strong by with CD 63. The CD 68 antibody recognized foamy cells
with big cytoplasm. These two antibodies were not seen in the primary and secondary
lymphoid follicles, with few exceptions. The stain pattern of them was commonly
observed in the medullar cord’s macrophages and plasma cells areas. This pattern differs
to that one observed with Ham 56 and IDO antibodies.
Discussion:
The immunoregulatory antigen-presenting cells (APC) play an important role in
maintaining T cell homeostasis and self-tolerance. Recent evidence demonstrates a role
for inhibition of T cell proliferation by macrophage tryptophan catabolism involving the
activity of the enzyme IDO. Dendritic cells (DC) have also been shown to exert
immunoregulatory effects mediated by tryptophan catabolism and to cause T cell
apoptosis. In this study, it was trying to correlate the presence of IDO stain and metastatic
melanoma cells in skin and in SLN.
7
By conventional histopathology using H & E stain, skin melanomas may be composed of
large pleomorphic cells, small cells, spindle cells and may contain clear, signet-ring,
pseudolipoblastic, rhabdoid, plasmacytoid or balloon cells. Various inclusions and
phagocytosed material may be present in their cytoplasm. Nuclei may show bi- or multinucleation, lobation, inclusions, grooving and angulation. Architectural variations include
fasciculation, whirling, nesting, trabeculation, pseudo glandular/ pseudopapillary/
pseudofollicular, pseudorosetting and angiocentric patterns. Myxoid or desmoplastic
changes and very rarely pseudoangiosarcomatous change, granulomatous inflammation
or osteoclastic giant cell response may be seen in the stroma. The stromal blood vessels
may exhibit a haemangiopericytomatous pattern, proliferation of glomeruloid blood
vessels and perivascular hyalinization. Occasionally, differentiation to nonmelanocytic
structures (Schwannian, fibro-/myofibroblastic, osseocartilaginous, smooth muscle,
rhabdomyolysis, ganglionic and ganglioneuroblastic) may be noted. From all skin
melanoma samples in this study, the only non positive had the features of the spindle
shaped cells-type. It is difficult to know if this degree of tumor differentiation can derive
from the lack of expression of the structures that are recognized for the other cell types of
melanoma. To address this question many primary skin melanoma with spindle shaped
cells-type and epitheliod ones is needed.
As regards the lymph nodes, it is generally accepted that the SLN node is in theory the
first node on the direct lymphatic drainage pathway from a tumor. This is because
melanoma-associated solitary nodes seem to be the most likely site of early metastases.4-6
In this study no specific association was observed between degree of metastasis and
presence of IDO positive stain material. In order to improve the accuracy of detecting
melanoma metastatic cells some strategies that could be used are to perform a lymphatic
melanoma map using lymphoscintigraphy studies injecting a radioactive material and a
blue dye (isosulfan blue).17 The LN sections should be stained with hematoxylin and
eosin and with antibodies generated against tumor-associated markers (S-100, HMB-45,
and Melan-A/MART-1) in the case of melanoma.13-15 These other immunohistochemical
markers might increase the accuracy of the histopathological diagnosis of melanoma
cells. This is because it is accepted that H&E stain is not the most accurate method for
detecting metastatic melanoma cells.18-20 In-transit SLNs may harbor micrometastasis.
About 10% of the time, micrometastasis may involve the in-transit and not the regional
SLN. Therefore, both in-transit and regional SLNs should be examined.
To improve the isotyping studies on the cells that are positive at the lymphendothelium
interface of the LN, it might be useful to test more LN and skin, with other antibodies.
Some of the following antibodies could be used: HAM 56, CD 86, S-100, CD 68, and CD
83. By using these antibodies, better insight regarding the co-localization of the
melanoma tumor cells and the presence of IDO positive cells not only on skin but also in
8
LN might be obtained. When using H&E to describe the presence of metastatic
melanoma cells, some discrepancies were observed by the different investigators. Based
on these discrepancies and to try to improve the accuracy of detecting melanoma
metastasis cells, the use of other immunohistochemistry markers matrix
metalloproteinase 2 (MMP-2), S-100, vascular endothelia growth factor and its receptor
(FLT 1, KDR) is suggested. 18-20
The Clark level and Breslow thickness classification of the primary melanoma should be
used to obtain a better definition of invasiveness of melanoma.
Alternatively, melanin bleaching prior to the application of primary antibody can alter the
immunoreactivity of several antigens. It is known that melanin bleaching using
permanganate/oxalate on subsequent immunohistochemical staining of heavily
pigmented melanocytic neoplasms can alter immune reactivity of some antibodies. It is
known that oxalate preclude the use of some antibodies but allows much faster bleaching
times. The IDO antibody does not cause problems with this technique. The antigens
(HMB-45, S-100, factor VIII have been previously shown to be unaffected by bleaching.
It is not known if the CD 68, CD 83, and HAM 56 antibodies can be affected by this
melanin bleaching technique.
In the few cases where a well define follicular pattern was observe in LN from patients
with melanoma, the germinal centers of these positive stained follicles had the
histological appearance of larger and more mitotically active cells with round nuclei. This
part of the follicle is where B-cell proliferation occurs by somatic hyper-mutation, and
selection for antigen binding. These rapidly proliferating B cell areas in germinal centers
are also better known as centroblasts follicular centers. It could be speculated that the
few but clear positive stains observed with the IDO antibody and with a HAM 56, CD 86,
positive cells for antibody production or to other immunological process nonrelated with
this malady. It will be interesting to study more cases of SLN and skin primary melanoma
comparing IDO and Ham 56 expression hypothesize a difference.
In Table 3 we summarize the correlation or not with IDO, the skin melanoma and the
lymph nodes.
Melanoma cells, and melanocytic nevus cells as well, are rarely found within the
epithelial structure of sweat glands, yet they are frequently found in hair follicles.
Comigration of metastatic and IDO positive in same cells site of the melanoma structure
could be seen. Hair follicles are well recognized to be one of the most important
pathways for metastasizing.
9
Other recommendations to determine the precise histopathologic pattern of distribution of
the cells that are recognized by the IDO antibody in the LN, include that all lymph nodes
are cut in the same plane. In this study we only could observe two lymph node that were
cut sagitally. This plane allows to improve anatomical localization of the cell type in the
LN. By improving this, a better distribution pattern of the micro-metastasis, the IDO
positive cells, and the relationship between them and CD 68, CD 83, Ham, S-100
metastatic melanoma cells can be determining. Another observation derived from this
study is the fact that we might recommend a more precise anatomical area since the term
cortex is overly broad.
Retaliation against whistleblowers is widespread and poses a significant barrier to the
accountability and transparency of government, Universities, and corporate conduct. only
a small fraction of employees who filed retaliation claims prevail through the legal
process.23 Caught Between Conscience and Career is intended to help employees
encountering these difficult ethical issues in their workplaces and to empower them to
make the best choices. It is a survival guide for whistle-blowers. It walks through the
difficulties that may be encountered when blowing the whistle, the path of disclosing
information.23 One person against a principal investigator and universities and or entities
that bring a lot of money falsifying data is quite common government agency is
inherently a David-versus- Goliath struggle. The organization holds most of the cards.
People who speak out loudly and publicly against their organization can face repercussions in their jobs.23 Not all these repercussions are immediately obvious.23 But
human lives are sacred, and the scares minimal resources devoted to find better
medications and cures for people suffering devastating disease as cancers including
melanoma are worth to battle.
Figure legends
Table 1. Presence of IDO in tonsils.
Table 2. Antibodies used in this study.
Table 3. Summary of correlation or not with IDO, the skin melanoma and the lymph
nodes.
Figure 1. IHC stain of IDO in a tonsil from a patient with tonsillitis. The cells that were
recognize by the IDO antibody were mostly observed in the sub-epithelial region (yellow
arrow), but few were seen inside the follicle (black arrow, reddish stain), (200X)
Figure 2. IHC stain using the IDO antibody on primary skin melanoma. A possible
constitutive expression on some malignant melanoma cells on skin or cross-reactivity
10
with this antibody can be seen in some cluster’s areas (black arrows, reddish stain),
(400X).
Figure 3. IHC stain using the IDO antibody on primary skin melanoma. Cells in similar
appearance to the melanoma, recognized by the antibody are inside the superficial skin
blood vessel. (Possible primary melanoma in skin or cross-reactivity with the antibody)
(black arrow). Cells that also stain their cytoplasm resembling the structure of
lymphocytes positive for this antibody inside the vessel were also seen (blue arrow),
(400X).
Figure 4. Immunohistochemistry on LN from patients with skin melanoma. The dotted
red cells show the most common pattern of distribution of the cells that were IDO was
positive (black arrows, reddish stain), (200X).
Figure 5. IHC stain using the IDO antibody on a lymph node from a patient with skin
melanoma. The dotted red cells are IDO positive with a follicular pattern (black arrow,
reddish stain). A few scatter cells were also positive in the parenchyma (white arrow),
(100X). b. positive IDO cells around the endothelial cells (black arrows).
Figure 6 a. A peculiar staining pattern was observed in one lymph node. When a
“compact” metastasis of melanoma-cells was found, the presence of a ring of IDO
positive cells were seen (black arrow). Other IDO positive cells were seen inside the
tumor (yellow arrow). b. Invasion of the melanoma tumor cells in the epidermis being
positive with IDO antibody, (black arrows) as well as into the deep subcutaneous tissue
especially around the upper dermal vessels (blue arrow) (400X).
Table 1
Specimen
Tonsils (12)
Histopathologic
Diagnosis
Lymphoid hyperplasia
(9/12).
Lymphoid hyperplasia
with focal acute
inflammation (1/12)
Follicular hyperplasia
(1/12)
Normal tonsil (1/12)
Presence of IDO Pattern of IDO
positive stain
staining
(2/12)
Sub-epithelial and
nonspecific pattern
(2/12)
(0/12)
(0/12)
(0/12)
11
Table 2. Antibodies used in this study.
MARKER
CD 68
IMMUNOGEN
A 110 glycosylated transmembrane
glycoprotein which is mainly located
in the lysosomes, that seems to
belong to the family of glycosomal
glycoproteins/plasma membrane
shutting proteins playing a role in
lysosomal trafficking and
endocytosis, including as lysosomal
associated membrane proteins –1 and
–2 (LAMP-1 and 2). The functions
of this CD 68 are not known.
CD 83
A 43 Kda protein belonging to the
immunoglobulin superfamily
S-100
Cow brain extracted with 2.5mm
EDTA and 0.1mM 2mercapthethanol
HAM 56
Sonicated human alveolar
macrophages
IDO
Synthetic peptide coupled to KLH
SPECIFICITY
Macrophages and APC could be
weakly positive, peripheral blood
monocytes are positive with a
granular staining pattern. Reaction
with Plasmacytoid T cells which are
present in many reactive lymph
nodes of the cytoplasm and which
are to believe to be of
monocyte/macrophage origin.
Malignant cells in chronic and acute
myeloid leukemia giving strong
granular staining of the cytoplasm of
many cells and reacts with rare cases
of true histiocytic neoplasia
Circulating and Interdigitatim
reticulum cells in the T cell zones of
lymphoid organs and weakly
expressed by some germinal center
lymphocytes. Also, by L.C. cells
In the brain glial and ependymal
cells. L.C.; Schwan cells; in skin
melanocytes and LC; in lymph nodes
Interdigitatim reticulum cells,
malignant melanocytic tumors
(cytoplasmic as well as nuclei)
Macrophages and Interdigitatim
reticulum cells; and small population
of endothelial cells most prominently
those of the capillaries of the smaller
blood vessels
SOURCE
Mouse anti
human
Rabbit anticow
Mouse antihuman
Rabbit antihuman
L.C: Langerhans cells
APC: Antigen presenting cells.
TABLE 3. Summary of correlation or not with IDO, the skin melanoma and the lymph
nodes.
Tissue
Total number
of cases tested
IDO positive
Tonsil
13 tonsil
3 from 13
IDO Positive
strength of
positivity
2 were grade 2
IDO + and pattern
3/3 at sub-epithelial and in
the cortex area mainly and
12
1 was grade 3.
Skin primary
melanoma
Lymph node from
patients with
melanoma
9
8 from 9
37
225 from 356
slides
Lymph node blocks
from patients with
melanoma
Lymph node blocks
from patients with
melanoma with
metastasis detected
by H&E stain
176
356
200 grade 1
100 grades 2
20 grade 3
5
grade 4
Total: 215
Cortical: 201
Hiliar: 24
1/3 one follicle was
positive also.
8/9 at superficial blood
vessels of the skin.
6 slides follicular from 4
cases.
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