Pedram Gerami

Fluorescence In Situ Hybridization (FISH) as an Ancillary Diagnostic Tool in the Diagnosis of Melanoma

Although the clinical and pathologic diagnosis of some melanomas is clear-cut, there are many histopathologic simulators of melanoma that pose problems. Over-diagnosis of melanoma can lead to inappropriate therapy and psychologic burdens, whereas under-diagnosis can lead to inadequate treatment of a deadly cancer. We used existing data on DNA copy number alterations in melanoma to assemble panels of fluorescence in situ hybridization (FISH) probes suitable for the analysis of paraffin-embedded tissue. Using FISH data from a training set of 301 tumors, we established a discriminatory algorithm and validated it on an independent set of 169 unequivocal nevi and melanomas as well as 27 cases with ambiguous pathology, for which we had long-term follow-up data. An algorithm-using signal counts from a combination of 4 probes targeting chromosome 6p25, 6 centromere, 6q23, and 11q13 provided the highest diagnostic discrimination. This algorithm correctly classified melanoma with 86.7% sensitivity and 95.4% specificity in the validation cohort. The test also correctly identified as melanoma all 6 of 6 cases with ambiguous pathology that later metastasized. There was a significant difference in the metastasis free survival between test-positive and negative cases with ambiguous pathology (P=0.003). Sufficient chromosomal alterations are present in melanoma that a limited panel of FISH probes can distinguish most melanomas from most nevi, providing useful diagnostic information in cases that cannot be classified reliably by current methods. As a diagnostic aid to traditional histologic evaluation, this assay can have significant clinical impact and improve classification of melanocytic neoplasms with conflicting morphologic criteria.

Folliculotropic Mycosis Fungoides: An Aggressive Variant of Cutaneous T-Cell Lymphoma

OBJECTIVES To study the clinical features, therapeutic responses, and outcomes in patients with folliculotropic mycosis fungoides (FMF) and to compare our single-center experience of 43 patients with the findings from the Dutch Cutaneous Lymphoma Group. SETTING A single-center experience from the Northwestern University Multidisciplinary Cutaneous Lymphoma Group. PATIENTS Forty-three patients with FMF were included in the study and compared with 43 age- and stage-matched patients with classic epidermotropic mycosis fungoides (MF) with similar follow-up time. RESULTS Folliculotropic mycosis fungoides showed distinct clinical features, with 37 patients having facial involvement (86%) and only 6 having lesions limited to the torso (14%). The morphologic spectrum of lesions is broad and includes erythematous papules and plaques with follicular prominence with or without alopecia; comedonal, acneiform, and cystic lesions; alopecic patches with or without scarring; and nodular and prurigolike lesions. Sixty-five percent of patients had alopecia, which in 71% of cases involved the face. Severe pruritus was seen in 68% of patients. In general, patients responded poorly to skin-directed therapy and in almost all cases required systemic agents to induce even a partial remission, including patients with early-stage disease. Overall survival was poor. Patients with early-stage disease (< or =IIA) had a 10-year survival of 82%, which took a steep drop off to 41% by 15 years. Patients with late-stage disease (> or =IIB) had an outcome similar to those patients in the control group with conventional epidermotropic MF of a similar stage. CONCLUSIONS The morphologic spectrum of clinical presentation for FMF is broad and distinct from those in conventional MF. This is at least partially attributed to the ability of FMF to simulate a variety of inflammatory conditions afflicting the follicular unit. The disease course is aggressive, and many patients, including those with early disease, show a poor outcome particularly between 10 and 15 years after the initial onset of disease. Response to skin-directed therapy is poor even in early-stage disease, and our best results were seen with psoralen plus UV-A (PUVA) therapy with oral bexarotene or PUVA with interferon alfa. These findings corroborate those of the Dutch Cutaneous Lymphoma Group and further validate the classification of FMF as a distinct entity.

A highly specific and discriminatory FISH assay for distinguishing between benign and malignant melanocytic neoplasms

The diagnosis of certain melanocytic proliferations remains one of the most challenging areas in pathology. In recent times, fluorescence in situ hybridization (FISH) has emerged as a promising diagnostic aid to conventional microscopy. We previously showed that a 4-probe FISH assay targeting 6p25 (RREB1), 6q23 (MYB), Cep6 (centromere 6), and 11q13 (CCND1) could discriminate between histologically unequivocal melanomas and benign nevi with a sensitivity of 86.7% and specificity of 95.4%. However, the sensitivity of the assay is approximately 70% in melanomas with spitzoid morphology. Furthermore, differentiating true gains from tetraploidy may cause difficulties in interpretation by inexperienced examiners. Here we refine the current probe set to better target spitzoid melanomas and more easily distinguish cells with imbalanced copy number aberrations from tetraploid cells. Using FISH data from 3 training sets of 322 tumors, including 152 melanomas and 170 nevi, we identified 9p21, 6p25, 11q13, and 8q24 as a probe set with improved discriminatory power in differentiating melanomas from nevi. In a validation set of 51 melanomas and 51 nevi this probe set had a sensitivity of 94% and specificity of 98%, compared with the original probe set that had a sensitivity of 75% and specificity of 96% in the same validation cohort. We propose that by incorporating 9p21 into the 4-probe FISH assay, with a new diagnostic algorithm, this new probe set would have improved discriminatory power in melanocytic neoplasms and improved sensitivity for detecting spitzoid melanomas, as demonstrated by our previous studies.

Fluorescence in situ hybridization for distinguishing nevoid melanomas from mitotically active nevi

Nevoid melanoma may resemble benign compound or intradermal nevi by their silhouette and profile on low power. Higher power usually reveals nuclear atypia, confluence of cells, incomplete maturation and dermal mitotic activity. However, to some extent all of these features maybe seen in benign compound or intradermal nevi and no single criteria can be used to differentiate nevoid melanoma from a benign nevus. The distinction can be particularly problematic in nevi that show mitotic activity and we have noted a recent trend in diagnosis of melanocytic neoplasms with dermal mitosis as nevoid melanoma despite the presence of normal maturation in the dermis and lack of significant nuclear atypia. Therefore in this study we evaluated 10 nevoid melanomas, 4 of which resulted in metastasis and 10 mitotically active nevi with fluorescence in situ hybridization targeting key chromosomal loci previously shown to effectively discriminate been malignant and benign melanocytic neoplasms. All 10 nevoid melanomas showed copy number abnormalities by fluorescence in situ hybridization in either chromosome 6 or 11 while none of the 10 mitotically active nevi did. The results demonstrate that fluorescence in situ hybridization targeting key chromosomal loci on chromosomes 6 and 11 can be effective in discriminating nevoid melanomas from mitotically active nevi. Additionally, our study presents further evidence that dermal mitoses alone without other diagnostic features such as nuclear atypia and lack of maturation does not constitute sufficient evidence alone for a diagnosis of melanoma.

Sensitivity of fluorescence in situ hybridization for melanoma diagnosis using RREB1, MYB, Cep6, and 11q13 probes in melanoma subtypes.

OBJECTIVE To evaluate the diagnostic sensitivity of fluorescence in situ hybridization (FISH) using probes targeting 6p25, 6q23, 11q13, and Cep6 in melanoma subtypes. DESIGN Blinded comparison of chromosomal copy number changes detected using FISH targeting 6p25, 6q23, 11q13, and Cep6 in benign nevi and melanoma subtypes. SETTING Dermatopathology Laboratory, Department of Dermatology, Northwestern University, Chicago, Illinois. PARTICIPANTS One hundred ten individuals with benign nevi and 123 with melanoma (70 superficial spreading, 28 lentigo maligna, 22 nodular, and 3 acral lentiginous melanomas). MAIN OUTCOME MEASURES Sensitivity of previously developed criteria using FISH using probes targeting 6p25, 6q23, 11q13, and Cep6 in the melanoma subtypes. RESULTS Overall, sensitivity was 83.0% and specificity was 94.0%. The 6p25 gain criterion had the highest sensitivity overall and in each subtype. The assay was most sensitive in the subgroups of nodular and acral melanomas and least sensitive in the superficial spreading subtype. The 11q13 gain was more commonly seen in chronically sun-damaged skin and infrequently in non-chronically sun-damaged skin. CONCLUSIONS Heterogeneous changes in melanoma occur at the molecular level, and the changes are different among melanoma subtypes. Clonal abnormalities in chromosome 6 with increased copies of the short arm relative to the long arm are common in all melanoma subtypes, suggesting that isochromosome 6 is common in all variants of cutaneous melanoma subtypes. An increase in copy number of 11q13 is most frequent in chronically sun-damaged melanomas.

Risk Assessment for Atypical Spitzoid Melanocytic Neoplasms Using FISH to Identify Chromosomal Copy Number Aberrations

Risk assessment for atypical Spitz tumors remains an enigma for physicians. Many prognosticators including sentinel lymph node biopsy fail to show the same prognostic significance in these tumors as seen in conventional melanoma. We conducted a case-controlled collaborative study involving multiple major melanoma treatment centers in the United States and Australia. Sixty-four atypical Spitz tumors with 5 years of uneventful follow-up and 11 atypical Spitz tumors resulting in advanced locoregional disease, distant metastasis, or death were evaluated by fluorescence in situ hybridization using 2 probe sets targeting 6 chromosomal loci. Predetermined criteria were utilized to detect the presence or absence of copy number aberrations for each locus. Logistic regression analysis, Fisher exact test, and multivariate analysis were performed to determine chromosomal copy number aberrations with statistically significant association with aggressive clinical behavior. Gains in 6p25 or 11q13 and homozygous deletions in 9p21 had statistically significant association with aggressive clinical behavior with P-values of 0.02, 0.02, and <0.0001, respectively. In multivariate analysis, homozygous 9p21 deletion was highly associated with clinically aggressive behavior (P<0.0001) and death due to disease (P=0.003). Fluorescence in situ hybridization detecting a limited number of chromosomal copy number aberrations can provide clinically useful and statistically significant risk assessment for atypical Spitz tumors. Cases with homozygous 9p21 deletions have the greatest risk. Cases with 6p25 or 11q13 gains also have higher risk for aggressive clinical behavior than FISH-negative atypical Spitz tumors or cases with 6q23 deletions.

The spectrum of histopathologic and immunohistochemical findings in folliculotropic mycosis fungoides

BackgroundSince the original designation of folliculotropic mycosis fungoides (FMF) as a distinct entity, there has been an increasing appreciation of the broad clinical and histopathologic spectrum with which this disease can present. However, there have been few large histologic studies characterizing the various histopathologic patterns. ObjectiveIn this study, we attempt to describe the histopathologic and immunohistochemical features of 47 biopsy specimens from 34 patients with FMF. MethodsWe searched our lymphoma database for patients with FMF in which detailed histopathologic information and slides as well as clinical information was available for review. Additionally, immunohistochemical studies for CD4, CD8, and CD1a were performed in all cases in which the block was available. ResultsIn addition to the prototypical pattern of a folliculotropic lypmphoid infiltrate with or without mucinosis, the histologic features of follicular mycosis fungoides may include a granulomatous reaction, cystic and comedonal changes, an eosinophilic folliculitis pattern and basaloid folliculolymphoid hyperplasia as well as pustular changes, interface dermatitis and an interstitial dermatitislike pattern. Unlike conventional mycosis fungoides, eosinophils and plasma cells are conspicuous within the accompanying reactive infiltrate. We have also noted an exceedingly high number of Langerhans cells within the follicular epithelium. The CD4:CD8 ratio frequently is 10:1 or greater and the follicles show abundant CD1a positive cells. ConclusionsFMF may present with a broad spectrum of histopathologic changes including interstitial, granulomatous, fibrotic and acneiform reactions that may lack the typical histologic attributes of a cutaneous T-cell lymphoma. Recognition of these myriad of histologic presentations can be of great diagnostic utility.

Enhanced detection of spitzoid melanomas using fluorescence in situ hybridization with 9p21 as an adjunctive probe.

The use of molecular diagnostic methods such as fluorescence in situ hybridization (FISH) for challenging melanocytic neoplasms is becoming more widespread. In light of the diagnostic difficulty they pose, spitzoid melanocytic neoplasms constitute an area of greatest potential utilization. In this study we wished to evaluate the sensitivity of the currently used melanoma FISH probe assay in a group of unambiguous spitzoid melanomas. On the basis of comparative genomic hybridization data, copy number losses at chromosome 9 have long been recognized as a frequent event in melanoma. In this study we wished to evaluate the efficacy of 9p21 as a potential FISH target and then evaluate the added benefit of reflexing the standard melanoma FISH assay, with FISH targeting 9p21 and the centromere of chromosome 9 (Cep9). Cep9 was included as a control to exclude inadequate hybridization or truncation as a source of homozygous deletions. We first studied a training set of 85 melanomas and 58 nevi with FISH targeting 9p21 and Cep9. As per previous methodology, 30 cells were enumerated. In the training set, the nevi had a mean number of cells with homozygous 9p21 loss of 0.97, with a standard deviation of 1.26. The melanomas had a mean of 7.1 and a standard deviation of 6.76. This difference was significant (P=2×10−12). On the basis of the training set, we identified a cutoff of 10 homozygous deletions to distinguish between melanoma and nevi. In a subsequent validation set consisting of 76 melanomas and 88 nevi, we found this cutoff to have a sensitivity of 33% and a specificity of 100%. Finally, in our group of 43 unequivocal spitzoid melanomas, standard FISH against chromosomes 6 and 11 was 70% sensitive. Homozygous 9p21 loss was present in 11 of 27 (41%) cases tested. By combining the standard melanoma FISH assay with the 9p21 FISH assay, a combined sensitivity of 85% was found. Among these 27 cases tested with 9p21, there were 7 cases that were negative by the standard melanoma FISH assay but that were positive by 9p21, suggesting that the 9p21 assay may be highly complementary to the standard melanoma FISH assay. Hence, in this study, we validated the efficacy of 9p21/Cep9 as a diagnostic FISH assay in melanoma, and demonstrated its complementary effect to the standard FISH assay. 9p21 may be particularly helpful in lesions with spitzoid morphology.

Distinguishing epithelioid blue nevus from blue nevus-like cutaneous melanoma metastasis using fluorescence in situ hybridization.

Blue nevus (BN)-like cutaneous melanoma metastasis is a well-recognized variant of melanoma metastasis. These lesions may clinically and histologically simulate benign blue nevi. The histologic changes may be indistinguishable from conventional blue nevi or epithelioid blue nevi (EBN), a benign dermal-based melanocytic neoplasm with epithelioid morphology and heavily pigmented cytoplasm. Distinguishing BN-like cutaneous melanoma metastasis from benign conventional or EBN is important for staging and treatment. We evaluated a fluorescence in situ hybridization (FISH) assay using probes targeting 6p25 (RREB1), 6q23 (MYB), 11q13 (CCND1), and centromere 6 (Cep6) with previously determined criteria, to distinguish EBN and BN-like melanoma metastasis. Ten BN-like cutaneous melanoma metastatic lesions and 10 EBN were blindly evaluated with the above mentioned FISH probes. FISH enumeration and criteria for diagnosis of melanoma was as previously described. Nine of 10 BN-like cutaneous metastatic lesions showed significant aberrations and met previously established criteria for melanoma. None of the EBN cases showed evidence of significant copy number changes or met FISH criteria for a diagnosis of melanoma. FISH is an important diagnostic adjunct for melanocytic neoplasms. In this study, we show that a FISH assay targeting 6p25, 6q23, 11q13, and centromere 6 can distinguish EBN from BN-like metastatic melanoma with high accuracy. The test and the parameters previously established can perform with high sensitivity and specificity when dealing with this differential diagnosis.

Alemtuzumab for relapsed and refractory erythrodermic cutaneous T-cell lymphoma: a single institution experience from the Robert H. Lurie Comprehensive Cancer Center

We present the results of an open-label clinical trial and the clinical use of alemtuzumab in 19 heavily pretreated patients with advanced erythrodermic cutaneous T-cell lymphomas (CTCL) (erythrodermic mycosis fungoides and Sézary syndrome). Ten patients received alemtuzumab intravenously using an escalating dose regimen with a final dose of 30 mg three times weekly for 4 weeks followed by subcutaneous administration for 8 weeks. Nine patients were treated with only the SQ or IV dosing. The overall response rate was 84%, with 9 (47%) complete and 7 (37%) partial remissions. The median follow-up was 24 months (range, 6 to 62+ months). Median overall survival was 41 months whereas median progression free survival was 6 months. Minimal residual disease by T-cell gene rearrangement studies was detected in 11 patients who achieved complete response and partial response. Toxicities included myelosuppression and infections; however, the majority of side effects were of Grade 2 in severity and transient. One patient was diagnosed with a concurrent lymphoma (mantle cell lymphoma) 6 months after completing alemtuzumab therapy. Alemtuzumab is particularly effective in patients with erythrodermic CTCL with acceptable toxicities. Combined strategies with alemtuzumab may achieve molecular remissions with longer response durations.