J. M. Theaker

EGF +61 gene polymorphism and susceptibility to and prognostic markers in cutaneous malignant melanoma.

CMM is the most serious cutaneous malignancy and is increasing in frequency among most Caucasian populations, where the most important risk factor is exposure to UV light. Relatively little is known of the genetic factors that mediate susceptibility to and prognosis in sporadic CMM, although a number of genes have been implicated. A striking association between EGF polymorphism and Breslow thickness of invasive CMM has been reported. We have sought confirmation of this finding in an independent study of 159 patients and 310 controls using TaqMan fluorescence‐based genotyping for EGF +61. In our study group, there were no significant differences in EGF genotype frequencies between patients and controls nor was EGF genotype associated with tumour growth phase, stage or mitotic count. However, correlation between EGF genotype and Breslow thickness showed a modestly significant increase in frequency of the EGF (G/G) genotype among tumours >3.5 mm thick (30.0% vs. 9.8%, p = 0.03). In summary, in our group, the EGF +61 polymorphism was not a risk factor for CMM susceptibility, but this polymorphism may play a role in disease progression.

The growing teratoma syndrome in a nongerminomatous germ cell tumor of the pineal gland

The growing teratoma syndrome is a recognized complication of metastatic nonseminomatous germ cell tumors of the testis and is managed surgically. It may also occur in intracranial nongerminomatous germ cell tumors.

Orchiectomy after chemotherapy in patients with metastatic testicular cancer. Is it indicated

Background. A small proportion of patients with testicular germ cell tumors present with widely metastatic disease and are treated initially with chemotherapy. Little is known about the efficacy of systemic chemotherapy in eradicating the primary testicular germ cell cancer; however, there is concern that the testis may act as a sanctuary site for germ cell cancer in these patients, and orchiectomy, is, therefore, recommended after chemotherapy.

The continued value of central histopathological review of testicular tumours

Aims :u2002Central histopathological review of testicular tumours prior to definitive treatment can have an important impact on patient management. This study was designed to assess the continued value of central review in the light of increasing subspecialization and increased numbers of consultant histopathologists.

Granuloma annulare-like tattoo reaction

To the Editor, Cutaneous reactions to tattoos are not uncommon and have been attributed to the metallic salts used in the preparation of the pigment. The histological patterns of tattoo reactions are diverse and include lichenoid reactions, pseudolymphomas (cutaneous lymphoid hyperplasia), local and generalized sarcoidal granulomas, pseudoepitheliomatous hyperplasia, morphea-like and spongiotic reactions. Here, we describe a patient with a granuloma annulare-like tattoo reaction to blue and black pigment. A 34-year-old man presented with a pruritic, inflamed and indurated area over a 7-month-old tattoo on the forearm. The referring clinician stated that the tattoo was red in colouration. The patient had no cutaneous manifestations of granuloma annulare or any evidence of systemic disease. Microscopic examination of a skin biopsy showed hyperkeratosis and parakeratosis with occasional intraepidermal vesicles that contained neutrophils. The dermis showed a dense superficial and deep perivascular and periappendageal infiltrate of lymphocytes and plasma cells (Fig. 1). Extraand intracellular deposits of tattoo pigment were noted in the dermis (Fig. 2). In addition, the superficial dermis showed numerous discrete areas of circumscribed necrobiosis with peripheral pallisading of macrophages and fibroblasts (Fig. 1). The necrobiotic areas showed a mild increase in dermal mucin on staining with alcian blue. Polarized microscopy did not show any foreign material within the lesions. There was also evidence of associated dermal fibrosis. Electron probe microanalysis showed that the tattoo pigment was a combination of blue pigment (copper and titanium compounds) and black pigment (carbon). Given the patients history, the biopsy was interpreted as an unusual reaction to tattoo pigment, with areas that histologically mimic granuloma annulare. Hypersensitivity reactions, although most common with red (mercuric sulphide) tattoos, have also been reported with yellow (cadmium sulphide), brown (iron oxide), blue (cobalt), purple (manganese), green (chromium) and black (carbon) tattoos. A lichenoid infiltrate is the most common reaction to permanent cosmetic tattoo, but other patterns are reported. These include granulomas, cutaneous lymphoid

Nonseminomatous germ cell tumor with very high serum human chorionic gonadotropin.

Most patients with disseminated nonseminomatous germ cell tumor (NSGCT) have an excellent prognosis with modern chemotherapy, although certain subgroups with a worse prognosis have been described. One such subgroup includes patients with high serum levels of the tumor marker, human chorionic gonadotropin (HCG). Sixteen patients of 104 treated for NSGCT at the CRC Wessex Medical Oncology Unit (Southampton, UK) presented with serum HCG > 25,000. Most of these patients exhibited features of the “choriocarcinoma syndrome” with bulky, rapidly progressive disease; frequent pulmonary, hepatic, and central nervous system complications; and a generally poorer response to standard NSGCT chemotherapy. Histologic identification of trophoblastic tumor was not made in all patients and is not essential for the diagnosis of the syndrome; indeed, closed biopsy may be contradicted in some circumstances because of the risk of hemorrhage. The NSGCT patients with poor prognosis, including patients with the choriocarcinoma syndrome, must be clearly identified in order to improve management and, eventually, cure rates.

Investigation of specimen mislabelling in paraffin-embedded tissue using a rapid, allele-specific, PCR-based HLA class II typing method.

Mislabelling of surgical specimens can seriously affect the accuracy of histopathology reports. We describe two cases in which clinically suspected mislabelling was investigated by polymerase chain reaction (PCR)‐based HLA DRB and DQB tissue typing of paraffin biopsy‐derived DNA, using sequence specific primers (PCR‐SSP HLA typing). In the first case, two patients underwent surgery for breast carcinoma. A subcutaneous lymph node containing metastatic carcinoma was received with the breast specimen from one patient, but was clinically considered more likely to originate from the other patient who underwent breast surgery on the same day. In the second case, histological examination of retained products of conception from a young woman revealed adenocarcinoma, but a repeat curettage specimen consisted of secretory phase endometrium. In case 1, PCR‐SSP HLA typing confirmed the clinical suspicion that the subcutaneous lymph node received with tissue from one patient originated from the other patient. This result converted the first patient from lymph node positive breast carcinoma to lymph node negative disease. In case 2, there was no evidence from PCR‐SSP HLA typing that the endometrial samples had originated from different patients. PCR‐SSP HLA typing requires fewer steps than methods based on PCR amplification followed by oligonucleotide probing (PCR‐SSOP HLA typing), and relies on the amplification of shorter sequences of DNA. Therefore, this technique can produce more rapid results than PCR‐SSOP HLA typing, and is ideally suited to typing partially degraded DNA derived from formalin‐fixed and paraffin‐embedded tissue.

Observer agreement comparing the use of virtual slides with glass slides in the pathology review component of the POSH breast cancer cohort study

Aims (1) To compare the use of scanned virtual slide images (virtual microscopy) with glass slides (conventional microscopy) in the assessment of morphological characteristics of breast cancers within the setting of the Prospective study of Outcomes in Sporadic versus Hereditary breast cancer (POSH), involving a cohort of women under 40 years of age, presenting with breast cancer. (2) To assess the acceptability to histopathologists of the use of virtual slide images. Methods 13 histopathologists from the UK and Australia participated in the POSH pathology review. The observers were asked to assess multiple morphological features such as tumour grade and type. Comparisons were made for a single observer using both virtual images and glass slides. Intra- and inter-observer variability was calculated using the κ statistic and a comparison was made between the use of each image modality. Results Diagnostic performance with virtual slides was comparable to conventional microscopic assessment, with the measurement of agreement best for vascular invasion, necrosis and the presence of a central scar (κ=0.37–0.78), and poor for more subjective parameters such as pleomorphism, stroma, the nature of the tumour border and the degree of lymphocytic infiltrate (κ=0.1). Conclusion Virtual slides represent an acceptable methodology for central review of breast cancer histopathology and can circumvent the need for either travel to view material, or the potential problems of sending it by post.

Nested polymerase chain reaction-based HLA class II typing for the unique identification of formalin-fixed and paraffin-embedded tissue

Human leukocyte antigen (HLA) genotyping is routinely performed prior to organ transplantation using peripheral blood leukocyte‐derived DNA. In addition, polymerase chain reaction (PCR)‐based methods have permitted HLA genotyping using DNA extracted from formalin‐fixed and paraffin‐embedded tissue, with proven applications in HLA–disease association studies and surgical biopsy identification. The utility of current techniques may be limited by the poor yield of intact DNA from such paraffin biopsies. This paper describes a new nested PCR‐based HLA class II genotyping method which reliably detects HLA DRB alleles within DNA extracted from even extremely small paraffin biopsies. This method comprises initial PCR amplification of exon II sequences of the HLA DRB1, 3, 4, and 5 genes using generic PCR primers. Identification of the HLA DRB1 alleles and detection of the DRB3, 4, and 5 genes is then performed using a series of separate individual second‐round PCR reactions, each of which contains PCR primer pairs detecting a single HLA DRB allele or group of alleles (PCR‐SSP). The ability of this method to detect 19 individual HLA DRB1 alleles or groups of alleles, covering all common DRB1 specificities, was confirmed via concordant results when compared with ‘direct’ (single amplification step) PCR‐SSP analysis of one cell line‐derived and nine peripheral blood DNA samples, and with five DNA samples extracted from paraffin biopsies. The technique was then successfully applied to 11 further paraffin biopsy‐derived DNA samples, of which ten were untypable by ‘direct’ PCR‐SSP analysis, from five cases in which doubt existed as to the individual origin of the tissues.

Genetic analysis of hydatidiform moles in paraffin wax embedded tissue using rapid, sequence specific PCR-based HLA class II typing.

AIMS: To determine the applicability of rapid, sequence specific polymerase chain reaction (PCR)-based HLA class II genotyping for the distinction of complete from partial hydatidiform moles (HM) using DNA extracted from formalin fixed and paraffin wax embedded tissue. METHODS: Nine HM were studied. DNA was extracted from formalin fixed and paraffin wax embedded tissue after mechanical separation of decidual and molar components. HLA class II DRB (DRB1, -3, -4, and -5) and DQB1 genotyping was performed using a parallel series of PCR reactions, each of which contained sequence specific primers designed to amplify different HLA DRB and DQB1 alleles or allele groups (PCR-SSP analysis). In each case the HLA DRB and DQB1 genotypes identified within the decidua and HM were compared. RESULTS: Within the decidual tissue, HLA DRB genotypes were assignable in all nine cases, and HLA DQB1 genotypes were identified in seven cases. Within the molar tissue, HLA DRB genotypes were assignable in seven cases, and at least one HLA DQB1 allele was identified in seven cases. Interpretation based on HLA class II genotyping was therefore possible in two cases classified on histological appearances as complete HM, in four classified as partial HM, and in one HM of uncertain type. Different HLA DRB and DQB1 haplotypes were identified within the decidual and molar tissue from both complete HM, consistent with a solely paternal origin and supporting the histological diagnosis. HLA DRB and DQB1 alleles common to the decidual and molar tissue were present within the four partial HM and the HM of histologically uncertain type, consistent with combined maternal and paternal genetic input to these HM, supporting the histological diagnosis in four cases and suggesting that the histologically equivocal case was also a partial HM. CONCLUSION: PCR-SSP HLA class II DRB and DQB1 typing is reliably applicable to DNA extracted from formalin fixed and paraffin wax embedded tissue. Therefore, in a suitably equipped HLA typing laboratory, this technique provides a useful adjunct to histological examination for differentiation of complete from partial HM.