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Diagnosis Of Cutaneous Melanoma Biology Essay

Introduction

Malignant Melanoma is a malignancy arising from cells called melanocytes which produce a pigment known as melanin. Melanin is responsible for skin, hair and eye colour. 90% of invasive melanoma arises in the skin, 5% in the eye and the remainder on the mucosa of the oral and genital regions (www.ncri.ie). The incidence of melanoma is rapidly increasing, 5443 cases were reported by the National Cancer Registry Ireland between 1994 and 2004. Melanoma may be benign or malignant in nature. A number of risk factors exist in the aetiology of the disease including sun exposure, family history, presence of melanocytic nevi and immune compromised patients. Although the definitive causes of melanoma are not yet known with certainty, epidemiological studies suggest that intense exposure to ultraviolet A light contributes to the development of melanoma. Genetic alteration also has been attributed to the disease, with the loss of tumour suppressor gene function. About 40-50% of melanomas develop from pigmented moles (merckmanuals.com) although the condition can arise from melanocytes in normal skin.

Melanoma may be categorised into 4 main types of the disease: Lentigo maligna melanoma most often arises from lentigo maligna in older patients. Superficial spreading melanoma accounts for approximately two thirds of melanoma and is most often asymptomatic in nature. Although it is now believed that such information does not provide significant prognostic difference, classification remains importance to epidemiological studies and clinical diagnosis. Atypical melanocytes characteristically invade the dermis and epidermis in superficial melanoma. Nodular melanoma may occur at any site, unless ulceration occurs, this type of melanoma may be asymptomatic. Acral-lentiginous melanoma presents similar characteristics to lentigo melanoma and accounts for approximately 5-10% of melanomas. (merckmanuals.com)

Diagnosis of cutaneous melanoma may occur upon presentation of a new or notably changed lesion on the skin. In concerns of melanoma, a total skin examination must be conducted including unexposed skin areas and mucous membranes. Asymmetry, Border integrity, Colour and Diameter indicating size are often used in the examination of lesions in addition to noted changes in the lesion.

Melanoma must be staged in order to determine specific diagnosis and to establish prognosis. Malignant Melanoma is said to be in situ or invasive. The American Joint Committee on Cancer (AJCC) is the most often described means of staging melanoma. Also referred to as the TNM system, T denotes the tumours thickness and ulceration, N category describes the spread of melanoma to the lymph nodes in close proximity to the melanocytic lesion, the M category indicates metastasis to distant organs. The size and invasion of the tumour may be described using the Breslow and Clarke classifications, Clarke levels may be indicated as Level I (confined to epidermis), II (tumour present but does not fill and expand papillary dermis), III (tumour fills and expands papillary dermis), IV (tumour invades reticular dermis) and V (invasion of the subcutaneous fat). The Breslow thickness was first reported by Dr. Alexander Breslow in 1970, it may be defined as a measure of the maximum tumour thickness determining the penetration of the tumour. This measurement appears to be more easily reproducible among institution and more objective than Clarke levels. The Royal College of Pathologists regard the tumour thickness of melanoma as the most important prognostic feature, ideally reported in millimetres, tumour thickness may be measured using an ocular micrometre, vernier scale or an eye-piece measuring-graticule (Royal College of Pathology).

The process of metastasis comprises a number of phases although the actual initiating event of metastasis is not known. Melanoma is known to be more aggressive than many other cancers; it can follow different patterns of metastatic spread although melanoma is considered to metastasise mainly via the lymphatic system. Primary melanoma tumours therefore most frequently metastasise to regional lymph nodes. The concept of Sentinel Lymph Node biopsy was initially introduced in 1991 by Morton et al. The course of the disease most often involves the orderly progression from invasion at the primary site to regional lymph nodes via dermal lymphatic systems and then to distant sites. The Sentinel Node may be defined as the lymph node at the most of metastasis in the lymphatic basin draining the tumour, or the first lymph node to which cancer is likely to spread. The application of sentinel node biopsy in patients with melanoma is based on the concept that the sentinel node is representative of all lymph nodes in the basin. One or more sentinel nodes may exist to primary tumours, the location of which may be achieved through intraoperative mapping techniques through the use of vital blue dye or radiolabelled trace substances (Glass et al, Lymphatic mapping and sentinel biopsy in the management of high risk melanoma, 1998). Vast research and debate has been focused on establishing the optimal diagnostic and prognostic strategy for melanoma. Sentinel node biopsy has become a significant measure of prognosis, allowing for the detection of metastasis. Sentinel node biopsy was initially introduced into the management of cancer patients more than twenty years ago. The technique is complex and numerous details need to be determined and assessed to provide reliable diagnostic and prognostic information. The Sentinel node must first be identified during surgery by use of blue marker stain or radiolabelled compounds. Lymph from a given area of the body drains directly into a lymph node before passing through other regional lymph nodes. This usually indicates the sentinel node as the one closest to the site of the primary skin tumour that receives direct lymphatic drainage. As such, the sentinel node is the most likely to contain metastatic cells and thus detection of metastasis determines the need for complete lymphadenectomy. The manipulation and demonstration of the sentinel node is therefore of upmost importance as a prognostic factor, noting the presence or absence of metastasis. Haematoxylin and Eosin staining of paraffin embedded material is sensitive enough to detect one melanoma cell in a background of 10000 normal cells. This is generally accepted as sufficient in the demonstration of malignant melanoma in sentinel node sections. Immunohistochemistry can aid the differentiation between melanoma cells and other benign cells present in the lymph node. The histological procedure implemented in the detection of melanoma is fundamental to the investigation of melanoma and its evaluation by the pathologists. It is important that skin excisions and biopsies are adequately fixed in order to stabilise proteins and prevent tissue decay. The fixative of choice is 10% neutral buffered formalin solution. Frozen sections are not The main goal in examining the Sentinel Node remains the determination of accurate staging of early lesions to provide more consistent grouping. The most current protocols in the approach to sentinel node biopsy require more than one Haematoxylin and Eosin section and usually include the use of immunohistochemistry. Sections are taken at varying depths to provide more comprehensive tissue assessment. Protocols most often involve the review of Haematoxylin and Eosin stained slides from a multiple of levels through serially sectioned lymph nodes. This is believed to increasing the sensitivity of detecting microscopic melanoma metastasis. Routine analysis of sections from cut side sections of bisected lymph nodes may lead to a false negative rate of 10 to 15%. The incidence of sentinel node metastasis is about 4 to 6% in thin melanomas versus 15% for melanomas greater or equal to 1 mm thick, according to the College of American Pathologists.

Melanocytes appear as randomly dispersed cells within the basal cell layer. They exhibit a small dark staining nucleus and large clear cytoplasm. Several special stains facilitate the light microscopy of melanocytes and their products. Silver stains indicate the presence of melanin which can be detected through both the argentaffin and argyrophilic reactions. The Haematoxylin and Eosin has been accepted as routine for the detection of melanoma. Immunohistochemical staining is often employed in the investigation also; the most commonly utilised include S-100, HMB-45 and MART-1/Melan-A. S-100 protein was originally isolated from bovine brain extract and is present in the cytoplasm of a variety of cells including melanocytes. It is an acidic protein that binds Ca²+ and Zn2+. The polyclonal antibody to S-100 protein works well on paraffin embedded sections. Although S-100 has a high sensitivity, the specificity of the antibody for melanoma is low. HMB-45 antibody reacts with a cytoplasmic epitope with most melanomas and in few nevi conditions. HMB-45 is a monoclonal antibody that was initially generated from an extract of metastatic melanoma. This antibody reacts with melanosomal protein gp-100 and is mostly expressed on immature or proliferating cells. HMB-45 is not however reliable as a differential diagnosis between malignant melanoma and a benign nevus. Melan-A is a melanosome associated marker that has a high specificity for melanocytes. Melanoma Antigen Recognised by T cells (MART-1) is quite new in its application to melanocytic differentiation. The antigen is expressed on normal melanocytes, common nevi, spitz nevi and malignant melanoma. MART-1 and Melan A are commercially available antibodies. Melan A mRNA has been detected in melanocytic skin lesions and this antibody is a useful component to the evaluation of intraepidermal melanocytes as well as amelanotic melanoma (Elder D. E, Lever’s Histopathology of the Skin, 2005).

Some authors recommend the use of frozen sections to examine SLNs, particularly in breast carcinoma, to try to render an immediate diagnosis of metastatic disease during the surgical procedure. Patients with positive SLNs by frozen section then undergo completion of the lymphadenectomy in the same surgical procedure. However, frozen sections provide a suboptimal morphology and may lack the subcapsular region of the lymph node (which is the area most likely involved in microscopic

metastases). Furthermore, because processing of the frozen tissue requires embedding in paraffin, facing off the block, and new sectioning of the tissue, micrometastases may be lost in the discarded, unexamined sections. 9 Therefore, at least for SLN from melanoma patients, most authors consider the gold standard to be the examination of routinely processed material (formalin fixed, paraffin embedded) and discourage the use of frozen sections.

(http://www.nzmu.co.nz/data/media/images/snb_2.jpg)

Methodology

The Laboratory Information Management System, APEX, was used to retrieve case reports from case numbers provide by pathologist Dr. Zsolt Orosz. These case numbers described the examination of sentinel node specimens in the investigation of cutaneous melanoma. Once reports were obtained a random “study number” was subsequently assigned. This was achieved through random number function on casio calculator until every case had been assigned a coded number.

The reports were subsequently analysed and data was recorded regarding the patient diagnosis. The main focus of the case analysis involved establishing the presence or absence of metastatic deposits. Metastatic deposits were regarded as isolated tumour cells, micro metastasis or macro metastasis indicating the spread of melanocytic cells to the lymphatic system.

Results:

Gross

Initial Level

Futher Level

Melan A

Notes

Medical Codes

1

SN (B1) Fat (B2)

NEM

NEM

Neg

Prior C IV M.M.

T0800

M09450

2

SS (AE)

RC (NEM)

EM (One Slide 0.17mm)

NC

pN (sn) 1

T01000

M49060

3

FT (32x30x10mm)

RC (NEM)

NC

Neg

 

T01000 / T0800

M45020

4

SN (AE)

NEM

NEM

Neg

 

T01000

M49060

5

SS (AE)

NEM

NEM

Neg

 

T01000

M87203

6

2x SN

2 x RC (NEM)

1 x SN(B) SMD (0.51mm)

Pos

pN1(sn)

T01000 / T08000

M49060 / M00100

7

SN (AE)

NEM

EM (1.8mm)

Pos

pN1(sn)

T01000 / T08000

M45020

8

SN (7x5mm)

RC (NEM)

NEM

Neg

 

T01000 / T08000

M49060 / M00100

9

SS (AE)

RC (NEM)

NEM

Neg

pT2apN0 (sn)

T01000 / TY7000

M87203 / M00100

10

SS (AE)

NEM

EM (0.3mm)

Pos

pT2apN1a (sn)

T01000 / T08000

M87203

11

15x9x2mm (A1E)

NEM

NEM

Neg

 

T01000 / T08000

M49060 / M72020

12

11x7x4mm SS (AE)

NEM

NEM

Neg

 

T01000 / T08820

M49060 / M00100

13

4x4x1mm (B1E)

NEM

NEM

Neg

 

T01000 / T08000

M49060 / M72020

14

2x SN

NEM

NEM

SN(B) ?

Cell Nests Pos with Melan A represent naevus cell inclusions

T01000

M45020

15

2x SN

1 xRC / 1x NEM

? NR

? NR

 

T01000

M49060

Initial Sections H&E

Observation

No. Cases

Evidence of Metastasis

0

Mo Evidence of Metastasis

15

Further Sections H&E

Observation

No. Cases

Evidence of Metastasis

4

Mo Evidence of Metastasis

11

Further Sections Melan A

Observation

No. Cases

Evidence of Metastasis

4

Mo Evidence of Metastasis

11

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