Hi, I'm Dr. Alina Bridges, and I'm going to talk to you about diagnostic and prognostic tests predominantly for the evaluation of pigmented lesions in the molecular area of dermatopathology. I'm the Director of Dermatopathology at the Zucker School of Medicine at Hofstra Northwell and the Director of Cutaneous Pathology at the Northwell Cancer Center in New York. Before I begin, I'll let you think about what you typically do in your practice regarding molecular testing for melanocytic neoplasms. Molecular testing of Spitz neoplasms is now the standard of care. More than 70 years ago, Sophie Spitz, who was a brilliant pathologist ahead of her time because she raised the questions that are still relevant today, and hopefully I will help resolve some of these quandaries in this presentation. I will also review which molecular tests to use and when. Here I'm also reviewing the terminology that we use when diagnosing Spitz neoplasms. A Spitz nevus is benign. Then you have atypical Spitz nevi versus atypical Spitz tumors and Spitz melanoma, which differs from a Spitzoid melanoma, which is just a conventional melanoma with Spitzoid features. We've had a changing paradigm in our understanding of atypical Spitz neoplasms. While melanomas are predominantly associated with chromosomal aberrations, Spitz tumors are associated with chimeric fusion proteins and, unlike traditional nevi, do not have a BRAF mutation, so they will be BRAF negative. Neuropromoter mutations are strongly associated with melanoma and are a marker for a more aggressive clinical behavior. As I mentioned, kinase fusions are detected in atypical Spitz tumors. They're detected across the entire spectrum from benign to malignant and are thought to represent an early oncogenic event. For the most part, they're felt to be indolent. We're also now able to correlate the gene fusion abnormality with the clinical pathological features. For example, an atypical Spitz tumor with an ALK fusion is an exophytic polyploid lesion that has fusiform and plexiform nests. As specific kinase fusions become more readily identified and better characterized, it's possible to determine if specific histologic features align with the presence of a particular kinase, and I'm going to show you examples of that. As I mentioned here, common and congenital nevi are associated with BRAF mutations. White nevi are not. They're associated with various fusion protein rearrangements, and blue nevi are associated with GNAC and GNA11 mutations. So this is a patient who is a 24-year-old woman who had this lesion on the right posterior arm that was favored to be a dermatofibroma, but as you can see, it was not a dermatofibroma. There's an atypical compound melanocytic proliferation that on low power appears asymmetric. You can see that there is epidermal hyperplasia, kamino bodies, and nests of large pleomorphic epithelioid melanocytes with amphiphilic cytoplasm. This looks like a spitz neoplasm. Here's a close-up of the spitz cells that are atypical. There's pagetosis going up into the granular layer, and the pagetosis is not only located centrally but at the edge of the specimen. Here's a close-up of the kamino bodies, and here are the spitz cells. So I think there's too much atypia for this to be a spitz nevus or even a severely atypical spitz nevus. This could be an atypical spitz tumor or a spitz melanoma, but I think additional evaluation is necessary, including molecular testing, which was performed. The melanocytic proliferation shows pagetoid spread throughout the epidermis. This is the HMB45 positive staining. There is a loss of P16, which is concerning, and the KI67 shows slightly increased proliferative index. This biopsy was sent for chromosomal microarray, although I was worried that there was not enough tissue, which is why I did not order a lot of immunohistochemical stains. More than four chromosomal abnormalities were detected. If you have three or more, then it's best to call this a spitz melanoma. This had a Breslo thickness of at least 0.6, so this was at least T1A. This shows you that as you go and develop melanoma, you have more copy number alterations and point mutations, as this case illustrated. Here's a biopsy that was taken from an 18-year-old male on the right superior upper back. This was a consultation. You can see that the lesion is asymmetric. It's compound, and it's severely atypical. There's a pleomorphism. Unlike the previous biopsy, the cells do not have the same morphology as spitz cells do. There are mitoses, and there was pagetosis. You can see this with the melan A stain, so the tumor cells are melan A and HB45 positive. Again, in this case, there's loss of P16, and the PRAME stain, which is a nuclear stain, is positive in the same area where there's loss of P16 staining. This is worrisome for being melanoma. What's the best diagnosis? At this point, I did order molecular testing. The outside dermatopathologist was hedging on this lesion and gave a differential that this could be melanoma in situ, arising with an atypical nevus, or it could be an atypical spitz tumor or spizoid melanoma. This was sent for a chromosomal microarray, which showed multiple chromosomal aberrations. In order to do this chromosomal microarray, they had to use the entire specimen, which had a Breslow thickness of at least one millimeter. This was superficial spreading, malignant melanoma without ulceration, at least one millimeter Breslow thickness, and at least T1B. And there were five mitoses per millimeter squared as an adverse finding for this T1B lesion. So how many abnormalities do you need to diagnose melanoma? And the magical number is at least three, so three or more chromosomal abnormalities. And the other thing that was significant in the previous two cases is that there was loss of P16 staining. And that tends to be associated with mutations in the CDKN2A and 2B on chromosome 9. So this patient underwent a wide excision. Residual malignant melanoma was found with a Breslow thickness of 1.9 millimeters. A sentinel lymph node biopsy was performed, and it was positive, showing subcapsular and intramedullary nodal metastasis. So regarding molecular testing for melanocytic tumors, which tests should you use in what lesions, if you are looking to make a diagnosis between melanoma or nevus, then the gold standard is what I've just showed you, the chromosomal microarray or SNP array, which is based on detection of the chromosomal abnormalities. Sometimes you can't perform that test if you don't have enough tissue, in which case a fish is done. But keep in mind that a fish can miss about 20% of melanomas. A TERT promoter can be added on as, if it's positive, it's strongly associated with melanoma, particularly a more aggressive melanoma. Next generation sequencing does whole exome sequencing, and it's DNA and RNA based. And it's best used for classification of melanocytic tumors, as well as to predict prognosis and response to treatment. But keep in mind that these studies, this molecular testing now is considered the standard of care. This is just going over the differences between the chromosomal microarray or SNP array or CGH testing versus the fish. And sometimes there's just not enough tissue for the chromosomal microarray. And I'll get a call from the lab telling me that they feel that there's insufficient tissue, they can't run it, or the test is invalid because there's just not enough tissue. In those cases, you can do a fish, which has a faster turnaround time. And anytime I order the chromosomal microarray, I have to tell the clinicians that I'm ordering this test, but I'm not going to have a result for 21 days. But it's well worth it. So the American Society of Dermatopathology has an appropriate use criteria committee that I am part of. And they have an app that you can download, which is under development as we add more and more evidence-based research in the development of the app. But here you can see that with regards to melanocytic lesions, particularly the diagnosis of melanoma versus nevus, the gold standard is the chromosomal microarray with the fish coming in second. And gene expression testing for diagnosis is not recommended. And here's another example showing you the high sensitivity of the chromosomal microarray compared to the fish and the gene expression profile testing. And the TERT promoter also has a high sensitivity and specificity if it's positive. Our next case is this 63-year-old woman that had a birthmark on her left ear that was mostly stable since childhood, but recently developed an enlarging left occipital mass and new retroarricular swelling and tenderness for about a month. This was confirmed with a PET CT. She underwent an excision that I'm showing you here, first from the postauricular area where you can see melanophages, pigmented dendritic melanocytes extending from the dermis down into the subcutaneous tissue. There's not much of a cellular component. So it looks like a blue nevus. Here's the deeper portion with the pigmented dendritic melanocytes and melanophages. But again, not much cellularity. Here's another area from the same region that's showing some, the area of cellularity in this lesion. But it does not look overtly pleomorphic, but it is highly melanocytic. Here I'm showing you the excision of the parotid mass, which shows some features that were seen with the excision from the postauricular area. So again, melanophage is dendritic, pigmented, melanocytes, and now more cellularity here and more pleomorphism and crowding in the parotid mass. And then this was the excision from the suboccipital mass. The surgeon was just overwhelmed when he saw the extent of the pigment extending around the neurovascular bundles in these areas. But here you can see it's predominantly cellular, no mitoses in any of the tissue from the excisions. But it looks the most pleomorphic. But there are still some areas that resemble a blue nevus. But again, no mitoses. Immunohistochemistry was performed, and blue nevi tend to be strongly HMB45 positive, which this was. And these are stains I'm showing you from the suboccipital mass. The lesional cells were melan A positive, P16 was retained, ABAP1 was performed and should be performed for these melanocytoses and was retained, ALK1 was positive, PDL1 was negative, and KI67 showed no mitotic. All three excisions were submitted for chromosomal microarray testing. And the excisions from the postauricular area, the parotid area showed three chromosomal abnormalities, and the suboccipital mass had 14 abnormalities. So what's the most likely diagnosis? And it's actually all of these things. The patient had a nevus of Oto, which in her case was clinically a large plaque-type blue nevus with subcutaneous cellular nodules. The excisions from the postauricular area and from the parotid were atypical dermal melanocytic proliferations in a background of blue nevus-like changes with early transformation to blue nevus-like melanoma. And the suboccipital mass was now a blue nevus-like melanoma. And I guess an all-encompassing term for this entity is diffuse mucocutaneous dermal melanocytoses because dermal melanocytoses represent a confusing heterogeneous group of pigmented lesions characterized by the presence of dendritic melanocytes. Classification is challenging for cases with poorly demarcated clinical and pathologic features. This patient clinically presented with large plaque-type blue nevus with subcutaneous nodules. This presentation is associated with the development of nodules years later and has a high risk of melanoma development when involving large areas. We felt that diffuse mucocutaneous melanocytoses may be a unifying clinical pathologic term to describe spindled and dendritic melanocytic proliferations involving more than one anatomic site under more than one microscopic region. And management of these cases is very challenging because this is not resectable. It's similar to uveal melanoma in terms of risk stratification. This patient underwent treatment with checkpoint inhibitors. And they're looking at ALK1 inhibitors because this is getting progressively larger. What other entities could be considered as a dermal melanocytosis? And we're going to go through these, depenetrating nevus, pigmented epithelioid melanocytoma, and BAP1-inactivated melanocytic tumors. These, for the most part, behave in an indolent fashion and are considered to be of low to intermediate risk because they can transform to melanoma, as in the case of the patient who had that nevus of OTA that I presented. So depenetrating nevus looks concerning clinically because it is a darkly pigmented lesion. It tends to arise in younger patients on the head and neck, trunk, and upper extremities. Histologically, you have this dermal wedge-shaped melanocytic proliferation that's heavily pigmented. And this is of indeterminate intermediate risk because it also has a low risk of transformation to melanoma. So molecular testing can assist with diagnosis and prognosis because depenetrating nevi are characterized by mutations in beta-catenin and MAP kinase. And if the fish or the chromosomal microarray is abnormal, then this can represent transformation to depenetrating nevus-like melanoma. This is a core biopsy from a large nodule that a surgeon sent. So it was only a partial biopsy from a larger lesion. This is an atypical melanocytic proliferation with a small epidermal component. The dermal component extends to all of the biopsy margins. There are mitoses here, pleomorphic melanocytes, pigmented melanocytes, and melanophages. The tumor cells were strongly HMB45 positive and weakly melan A positive. MAP1 was retained, and the KS67 showed a low proliferative index. This was sent for fish, which was positive for melanoma. It was suspected that this was either a malignant depenetrating nevus-like melanoma or a malignant blue nevus melanoma. I signed this case out as portion of malignant melanoma with a Bresol thickness of approximately 5 millimeters, a T4A. And when I got the results of the Next Generation Sequencing Melanoma Panel, which showed mutations in BRAF, beta-catenin, and NRAS, I said that these findings supported the diagnosis of depenetrating nevus-like melanoma. Another important tumor is the BAP1-inactivated melanocytic tumor. Clinically, it does not look like a pigmented lesion. It's a skin-colored, dome-shaped papule. There's often a lot of lymphocytes admixed with the melanocytic lesion. So even histologically, sometimes a melanocytic lesion could be missed, and this could be considered to be an inflammatory lesion. But it tends to have this pleomorphic, nevoid appearance. And here, I'm showing the loss of nuclear immunoreactivity with BAP1-IHC stain. There are several reasons why this tumor is significant. One is that it may be the earliest sign of BAP1 hereditary cancer predisposition syndrome, which is inherited in an autosomal dominant fashion. It's more likely to be part of the syndrome if there is an extensive junctional component. If the patient has a history of melanoma or prior or multiple BAP1-inactivated melanocytic tumors. Patients with this cancer predisposition syndrome have an increased lifetime risk for cancers such as uveal melanoma, cutaneous melanoma, basal cell carcinoma, malignant mesothelioma, and clear cell renal cell carcinoma. So it is recommended that these patients have eye exams with imaging by ocular oncologists starting at 11 years of age, along with annual skin exams and physical exams with appropriate imaging and are educated about the importance of sun protection. So having a BAP1-inactivated tumor should trigger a complete history, including family history, physical examination, and if suspicious, then genetic studies for germline genetic deficiency or investigation of BAP1 deficiency-associated tumors. I saw me order a lot of BAP1 IHC stains on lesions, and that is because malignant melanomas in the category of blue nevus, these diffuse melanocytoses, melanocytomas, melanoma of uveal and CNS often have BAP1 expression loss. And also, sometimes the BAP1-inactivated tumors can have a spitz morphology. So in those types of lesions, I will order the BAP1 immunostain. You have to keep in mind that melanoma can have a loss of BAP1, and that not all BAP1s are indolent. And if a tumor shows the right morphology with BAP1 loss, it's better to call it melanoma. But we need longer-term prospective studies of these types of lesions. Here's a patient that had multiple nevi. She looks like she has dysplastic nevus syndrome, 32-year-old, and multiple nevi were biopsied. I'm showing you the histology from this lesion on the left upper arm that was submitted rule out melanoma. There's no junctional component. This is a dermal proliferation. It looks nevoid. But then when you look closely, it's pleomorphic, as there is cell-to-cell heterogeneity. BAP1 was autored, which showed loss. Interestingly, in addition, the P16 was lost. The KI67 showed a low proliferative index. These melanocytes were melan A positive and HMB45 negative. Chromosomal microarray testing showed only BAP1 loss. But given the loss of P16 and the pleomorphic cells, this was a diagnosis atypical BAP1-inactivated melanocytic tumor. Patient underwent complete excision of this lesion and consideration of referral to medical genetics. This is an older gentleman. This was an outside consultation that was called, diagnosed as nodular malignant melanoma. And there is malignant melanoma here with overlying melanophages. There's this adjacent area that looks very monotonous and not as atypical. That area showed loss of BAP1 staining and retention of P16. P16 was lost in the melanoma. The melanoma also was BRAF positive. KI67 was low in the areas where there was BAP1 loss. Chromosomal microarray showed more than three chromosomal aberrations. This patient was diagnosed as having malignant melanoma with loss of BAP1. Here, we have a 19-year-old female with a blue pigmented papule that looked concerning, similar to the deep penetrating nevus on the left posterior shoulder that recently appeared. It was noticed two weeks prior to her visit. Sequencing was performed, showed this heavily melanized melanocytic proliferation. Here on close-up, you can actually see that there are melanocytes there, which stain with the melanate red. Next-generation sequencing was performed, which showed a Parker mutation and BRAF mutation consistent with a pigmented epithelioid melanocytoma. So here's an example where the molecular testing was very helpful in supporting the clinical and histologic findings, excluding an animal type or equine melanoma and confirming a pigmented epithelioid melanocytoma. Here are the next three cases that I'm going to present. Case A is a 3-year-old girl that had this 5- to 6-millimeter brown papule increasing in size. It was sent in as rule-out congenital nevus. Patient B is a 31-year-old female with this firm erythematous brown papule with telangiectasias on the left buttock, sent in as rule-out dermatofibroma, folliculitis, BCC scar, and C is a 32-year-old gentleman with a pink nodule on the mid-back sent in as rule-out dysplastic nevus. So this is the histology from the 3-year-old on the hand. So here you can see that this is an asymmetric, atypical compound melanocytic proliferation. It kind of has this bilobed appearance. And there's pseudoepitheliomatous hyperplasia, Camino bodies. And this was quite mitotically active. I believe that there were 8 to 10 mitoses, including at least half of them being deep mitoses in the dermal nodule. So these appear to be spit cells. The mitoses were in that deeper nodule. So this is an atypical spits tumor versus severely atypical spits tumor versus spits melanoma in a 3-year-old. The melanocytes are melan A positive. P16 is biphasic with some loss, particularly in that deeper nodule. The ALK immunostain is positive. The chromosomal microarray showed no copy number variations and only an ALK fusion. So I diagnosed this as severely atypical spits tumor with an ALK fusion, 4 millimeters thick, 8 mitoses per millimeter squared. This was historically called spits melanoma of childhood. And with regards to a sentinel lymph node biopsy, the studies have not shown prognostic benefit of a sentinel lymph node biopsy, as having a positive sentinel lymph node biopsy doesn't seem to predict a poor outcome for patients with atypical spits tumor, particularly in the pediatric population. So in this case, I recommended that the lesion be completely removed. Sentinel lymph node biopsy was not necessary. Close follow-up was necessary. And that the biological behavior could not be predicted, but was felt to be indolent. Part B was the lesion on the buttock, which was also dermal-based melanocytic proliferation that had this fibrosis or sclerosis. And for all these lesions, I want to tell you that the clinical and histologic findings correlate with the genomic alteration. So here, some people might not even think that this is a melanocytic lesion, but it has some desmoplastic features. I guess some people might have even diagnosed this as being a desmoplastic spits nevus in the past. And the cells are large, like spits cells are, but they are atypical, pleomorphic. And in this case, next-generation sequencing was performed for classification, which showed an NTRK gene rearrangement. So I diagnosed this as an atypical spits tumor with NTRK rearrangement. It had a low tumor mutational burden. Complete excision was recommended. Close follow-up was recommended. And for all of these, I mentioned that the biological behavior is felt to be benign, but we could not predict the exact behavior of these lesions. This was the lesion on the lower back, C, sent in as desmoplastic nevus. And again, similar to the previous case, you can see that there is some fibrosis, sclerosis. There is melanocytes here, but it could easily be mistaken for not being a melanocytic tumor. Here I'm showing you the only junctional nest I could find. So it was compound. It was pleomorphic. It looked like it had some characteristics of a spits, which was supported by the negative BRAF staining. The melanocytes were melan A positive, HMB45 positive. P16 was retained. BAP1 was retained. KS67 showed a low proliferative index. Transmission sequencing was performed, showing an H-RAS mutation, a diagnosis as atypical spits tumor with H-RAS mutation, and this had a low tumor mutational burden. This was an interesting case, showing you the clinical here from 38-year-old female who had this irritated, itchy papule on the mid-back. It for the most part looks like it could be a congenital compound nevus. You can even see this, what could be considered to have findings that resemble an inverted type A nevus. Here you can see this area that's more atypical, and you don't want to diagnose this as melanoma. There is some pigment and some dendritic melanocytes here. These cells look atypical. There's prominent nucleoli. They're epithelioid. They don't look like spits cells. There's some findings here that kind of resemble a combined nevus, and that's supported by the fact that that area with the pigmented melanocytes is HMB45 positive. That area also showed loss of P16 staining. Next-generation sequencing was performed, and I diagnosed this as an atypical melanocytic tumor with concomitant NRAS and IDH1 mutation. The paper that I referenced there with the six cases, this is exactly what the histomorphology looked like in these cases. There was a background architecture that resembled a congenital type nevus with superimposed biphasic pattern that was formed by dendritic pigmented melanocytes surrounding areas of more nevoid melanocytes, so kind of had that combined look to it. Here, again, in this, I said the combination could represent melanocytoma of intermediate prognosis, which could result in malignant transformation. Biological behavior of these lesions is not completely known. Recommend complete excision with clear margins and careful clinical follow-up. Here, I'm showing you a 25-year-old female that had a 1-centimeter pink firm nodule on the left buttock. The clinical was rule-out dermatofibroma versus amelanotic melanoma. This is an atypical compound melanocytic proliferation. It's asymmetric. It looks like it has features of spits, although it's atypical. There's pagetosis here. Here's a close-up of the cells that look like a spits. Histochemistry showed that the melanocytes were melan A-positive, BRAF negative, ALK negative with a low proliferative index, and retention of P16. The next-generation sequencing helped clarify the diagnosis because that showed BRAF gene fusion with AGK, not ALK, and a low tumor mutational burden. And these lesions are now referred to as BRAF-mutated and morphologically spitsoid tumor or spitsoid melanocytoma. So you can see how important, with these intermediate or ambiguous melanocytic lesions of uncertain malignant potential, how molecular testing has become the standard of care and has quite an impact on the final diagnosis. I mentioned earlier that you can use next-generation sequencing, melanoma panel for prognosis, and therapeutic targets, clinical trials. And that's what we've implemented. So there's a reflex testing protocol for deep primary melanomas that are T2 or greater, as well as metastatic melanoma. So those are sent for next-generation sequencing. This is one of the panels that I have used, so you can take a look at the results that we get when we send for this testing. And it's completed by the time the patient sees the medical oncologist. In summary, molecular testing is not only appropriate, but is the standard of care when a diagnosis of melanoma is in question and also in these indeterminate or intermediate melanocytic neoplasms, such as atypical spits neoplasms, melanocytic tumors of uncertain malignant potential, the group of dermal melanocytoses, deep penetrating nevi. It can assist in diagnosis, prognosis, and potentially treatment, but you always have to consider health care costs. This is my workhorse, as I mentioned, the chromosomal microarray versus the FISH, and then the next-generation sequencing. Now let's talk about gene expression profile testing for prognosis and clinical practice for malignant melanoma patients, focusing specifically on the CPGEP model or Merlin assay. As you can see from the graph here, there's an increased incidence of cutaneous malignancies and melanoma is the deadliest form of skin cancer. Sentinel lymph node biopsy is currently the gold standard for prognosis. And when you receive a diagnosis of invasive melanoma, both the physician and patient foremost on their mind is, what is the prognosis? Sentinel lymph node biopsy is crucial for melanoma staging, assessment of potential metastasis, and also is essential to see if a patient qualifies for adjuvant therapy and clinical trials. What patients should be referred for a sentinel lymph node biopsy? If a patient has an invasive melanoma that's greater than or equal to T2, then those patients should be referred for a sentinel lymph node biopsy. For thinner melanomas, referral for a sentinel lymph node biopsy is not as clear-cut. You would not recommend a sentinel lymph node biopsy for patients who have an invasive melanoma that is T1A without any adverse features, such as ulceration, increased mitotic activity, regression, brisk tumor-infiltrating lymphocytes, because the probability of having a positive sentinel lymph node biopsy in those patients is very low. You could consider a sentinel lymph node biopsy in patients who have T1A disease with adverse features and T1B. But keep in mind that 8 out of 10 patients that undergo a sentinel lymph node biopsy do not have a positive sentinel lymph node, and there are complications that need to be considered in patients who undergo a sentinel lymph node biopsy. So getting a sentinel lymph node biopsy may not be without an increase in morbidity, particularly. The CPGEP, a Merlin assay, helps to reduce sentinel lymph node biopsy surgeries in these T1 to T2 melanoma patients. What is this assay? This model uses patient age, Breslow thickness, and an eight-gene expression profile to classify melanoma as either low-risk or high-risk. This is the first diagnostic test developed for sentinel lymph node biopsy referral that incorporates clinical pathologic variables. In the discovery cohort, all of the clinical variables were looked at, and all of the pathologic variables were looked at. And the only clinical variable of significance was age, and the only pathologic variable of significance was Breslow thickness. The CP is done in combination with this eight-gene expression profile, and you have to keep in mind that the starting gene discovery set was comprised of over 16,000 genes linked to primary cutaneous melanoma, and only eight were found to be significant, and these eight are involved in metastasis, particularly the integrins. The CP-GEP test has been shown to outperform the nomograms, the Melanoma Institute of Australia and Memorial Sloan Kettering Cancer Center nomograms. CP-GEP is a test that helps you rule in or rule out a cutaneous melanoma patient for sentinel lymph node biopsy. If a patient is low-risk, you can forgo the procedure because the risk of metastasis is low. If a patient is high-risk, you recommend the procedure. So again, easy. You run CP-GEP, the patient is low-risk, you forgo the sentinel node biopsy. If on the other hand, the patient is high-risk, the sentinel lymph node is recommended. In addition, the test provides prognostic information. For example, if CP-GEP is low-risk, relapse risk is low. If CP-GEP is high-risk and the sentinel nodes are positive, relapse risk is high. If the CP-GEP is high-risk and the sentinel nodes are negative, then the relapse risk is in between, and these patients will need close follow-up. The Merlin test results are available five days after sample receipt. Generally, five paraffin-embedded sections of tissue are needed, while it generally takes about three weeks to provide sentinel lymph node results to patients. I'm happy to tell you that the results of the first multicenter prospective trial that was performed in Europe was published recently. The cohort were newly diagnosed melanoma patients that were sentinel lymph node biopsy eligible and had a median Breslo thickness of 1.4. There were 260 patients, and 38% of the patients were risk stratified as CP-GEP low-risk and could forgo a sentinel lymph node biopsy. These results are in line with previous retrospective validation studies. This model accurately identified patients at low risk for sentinel lymph node biopsy and the implementation and clinical practice is feasible based on current turnaround time. This Merlin assay may be used to deselect patients for sentinel lymph node biopsy surgery. So again, a quick summary. CP-GEP low-risk patients can forgo sentinel lymph node biopsy and have excellent relapse-free survival. CP-GEP high-risk patients should be considered for sentinel lymph node biopsy, and their relapse risk is increased. The Merlin test is reported as either low-risk or high-risk, low-risk for nodal metastasis or high-risk for nodal metastasis, determining whether or not the patient should forgo or should be considered for sentinel lymph node biopsy, as well as what the risk is of developing recurrence, a five-year relapse-free survival, five-year distant metastasis-free survival, and five-year overall survival. Keep in mind that more than 50% of sentinel lymph node biopsy procedures could have been avoided based on CP-GEP results while also maintaining oncologic safety with very high negative predictive values greater than 95%. The test also requires a total of 5 microns of tissue, so 5 10-micron sections per cent. OK, great. So you might ask, has this test been validated? And yes, of course, the test has been validated on three continents, in seven countries, in 12 publications, and with the help of 27 global academic partners and based on more than 4,700 patient samples processed. The test is Medicare reimbursed. The multicenter prospective validation study in the United States has just been completed. That's Merlin-001. It was conducted at these highly reputable cancer centers shown here. The data is currently being analyzed and I'm certainly interested in learning about the results and so is the NCCN. You can see here that the Merlin 001 trial got a shout out from the Guidelines Committee in their last melanoma update from January. Let's say you want to order the Merlin test for a Sentinel lymph node biopsy eligible patient of yours. How could you do it? Well at this point there are several distribution channels available. For example, CEPGEP is now available through DermPath Diagnostics where it's sold under the test name Melanodal Predict. However, if you prefer, you can also order through Tempest or Skyline Diagnostics. The test name for CEPGEP with these two companies is Merlin. What's on the horizon is liquid biopsies that detect circulating tumor biomarkers for patients that have malignant melanoma and Merkel cell carcinoma. Liquid biopsies detect blood biomarkers of cancer. These are circulating tumor cells, circulating tumor DNA, circulating cell-free messenger RNA and exosomes. The biomarkers can be used for molecular diagnosis, monitoring disease progression, evaluation of treatment response, prognosis, personalized treatment, and therapeutic resistance. There are limited trials looking at liquid biopsy and melanoma and Merkel cell carcinoma with promising data in predicting early metastasis and potential for targeted therapy and disease monitoring. Thank you for your time and attention.

Dr. Alina Bridges

pigmented lesions

dermatopathology

molecular testing

Spitz neoplasms

Sophie Spitz

atypical Spitz neoplasms

chromosomal microarray

next-generation sequencing

CP-GEP Merlin assay