Heterogeneity of kinase inhibitor resistance mechanisms in GIST.

Most GIST patients develop clinical resistance to KIT/PDGFRA tyrosine kinase inhibitors (TKI). However, it is unclear whether clinical resistance results from single or multiple molecular mechanisms in each patient. KIT and PDGFRA mutations were evaluated in 53 GIST metastases obtained from 14 patients who underwent surgical debulking after progression on imatinib or sunitinib. To interrogate possible resistance mechanisms across a broad biological spectrum of GISTs, inter- and intra-lesional heterogeneity of molecular drug-resistance mechanisms were evaluated in the following: conventional KIT (CD117)-positive GISTs with KIT mutations in exon 9, 11 or 13; KIT-negative GISTs; GISTs with unusual morphology; and KIT/PDGFRA wild-type GISTs. Genomic KIT and PDGFRA mutations were characterized systematically, using complementary techniques including D-HPLC for KIT exons 9, 11-18 and PDGFRA exons 12, 14, 18, and mutation-specific PCR (V654A, D820G, N822K, Y823D). Primary KIT oncogenic mutations were found in 11/14 patients (79%). Of these, 9/11 (83%), had secondary drug-resistant KIT mutations, including six (67%) with two to five different secondary mutations in separate metastases, and three (34%) with two secondary KIT mutations in the same metastasis. The secondary mutations clustered in the KIT ATP binding pocket and kinase catalytic regions. FISH analyses revealed KIT amplicons in 2/10 metastases lacking secondary KIT mutations. This study demonstrates extensive intra- and inter-lesional heterogeneity of resistance mutations and gene amplification in patients with clinically progressing GIST. KIT kinase resistance mutations were not found in KIT/PDGFRA wild-type GISTs or in KIT-mutant GISTs showing unusual morphology and/or loss of KIT expression by IHC, indicating that resistance mechanisms are fundamentally different in these tumours. Our observations underscore the heterogeneity of clinical TKI resistance, and highlight the therapeutic challenges involved in salvaging patients after clinical progression on TKI monotherapies.

Expression of platelet-derived growth factor receptor in low-grade endometrial stromal sarcomas in the absence of activating mutations.

AIMS: To investigate platelet-derived growth factor receptor (PDGFR)alpha and PDGFRbeta expression and a mutational analysis of PDGFRalpha (exons 11, 12, 17 and 18) and PDGFRbeta (exon 12) genes in endometrial stromal sarcomas (ESS). Gastrointestinal stromal tumours (GISTs), which have somatic mutations of the transmembrane tyrosine kinase receptor, respond to tyrosine kinase inhibitors, which act through an inhibitory effect on class 3 receptor tyrosine kinase members such as PDGFRalpha, PDGFRbeta and c-kit. METHODS AND RESULTS: The immunohistochemical expression of PDGFRalpha and PDGFRbeta was investigated in 37 archival c-kit- ESS. Staining was scored as negative (0-10% positive tumour cells) and positive (weakly positive 11-50% positive cells; strongly positive > 50% positive cells). PDGFRalpha was expressed in 24/37 ESS [65%; strongly by 19/37 (51.5%) and weakly by 5/37 ESS (13.5%)]. ESS tumour cells were negative for PDGFRbeta, but endothelial cells stained positive. A mutational analysis of PDGFRalpha (exons 11, 12, 17 and 18) and PDGFRbeta (exon 12) genes on frozen metastatic ESS from three patients detected no mutations leading to amino acid changes in the mature protein. CONCLUSIONS: Patients with PDGFRalpha+ ESS may benefit from treatment with tyrosine kinase inhibitors by blocking autocrine and paracrine stimulation loops, blocking neovascularization and enhancing the effects of chemotherapy.

Mammary and extramammary Paget's disease: an immunohistochemical study of 83 cases.

AIM: Mammary Paget's disease (MPD) and extramammary Paget's disease (EMPD) are rare neoplasms. The aim of this study was, by the use of immunohistochemistry, to derive further information about the cell(s) of origin, find a diagnostically useful immunohistochemical panel and investigate candidates for possible targeted therapy. MATERIAL AND RESULTS: Sixty MPD and 23 EMPD cases were studied using antibodies to cytokeratin (CK) 34betaE12, CK8/18, CK7, CK5/6, CK20, gross cyctic disease fluid protein (GCDFP)-15, MUC1-8, epidermal growth factor receptor (EGFR) (HER1), HER3 and HER4. In all MPD cases CK7 and MUC1 were positive. CK8/18 was positive in 59/60 cases. GCDFP-15, MUC2, MUC3, MUC4, MUC7, MUC8 were positive in 29/60, 3/60, 35/47, 4/40, 3/43 and 2/45 cases, respectively. In all EMPD cases CK8/18 and CK7 were positive. MUC1, GCDFP-15, MUC5AC, MUC3, MUC8 and CK20 were positive in 22/23, 19/23, 8/19, 3/19, 1/19 and 3/23 cases, respectively. With the remaining antibodies no immunoreactivity was observed. CONCLUSION: MUC1 and low-molecular-weight CKs in conjunction with immunonegativity for high-molecular-weight CKs are the most diagnostically useful markers. MPD is caused by the epidermotropic spread of underlying tumour cells, whereas EMPD probably arises from intraepithelial cells of sweat gland origin. Targeted therapy with antibodies against EGFR (HER1), HER3 or HER4 is unlikely to prove of clinical value.

Immunohistochemical and mutational analysis of PDGF and PDGFR in desmoid tumours: is there a role for tyrosine kinase inhibitors in c-kit-negative desmoid tumours?

AIM: To determine the platelet-derived growth factor (PDGF) alpha and beta status of desmoid tumours. Desmoid tumours are rare monoclonal neoplasms that appear to have no metastatic potential. Surgical resection and radiotherapy in the event of a positive surgical margin is the first-line treatment. Recurrences are frequent. Treatment results using non-steroidal anti-inflammatory agents, anti-oestrogen compounds and other agents such as Imatinib mesylate have been published. Therapy with Imatinib has been proposed as a therapeutic option, although in most reports desmoid tumours are reported to be c-kit-. METHODS AND RESULTS: We performed immunohistochemical analysis on 124 archived samples (85 patients) of desmoid tumours using antibodies to PDGFalpha, PDGFbeta, PDGFRalpha and PDGFRbeta. All desmoid tumours showed immunoreactivity with antibodies to PDGFalpha and PDGFRalpha, whereas with antibodies to PDGFbeta and PDGFRbeta no specific reaction could be detected. Mutational analysis of PDGFRalpha (exons 11, 12, 17 and 18) and PDGFRbeta (exon 12) on frozen material from 14 patients was performed, but no mutations leading to amino acid changes in the mature protein were identified. CONCLUSION: The absence of an activating mutation in a protooncogene does not exclude the efficacy of tyrosine kinase inhibitors through other possible mechanisms, and these might be a therapeutic option for patients with desmoid tumours in whom established local and systemic approaches fail to control the disease.

p53 immunostaining in lichen sclerosus is related to ischaemic stress and is not a marker of differentiated vulvar intraepithelial neoplasia (d-VIN).

AIM: To analyse p53 immunoreactivity in 207 biopsy specimens of lichen sclerosus (LS) and "differentiated vulvar intraepithelial neoplasia" (d-VIN), a postulated precursor lesion for LS-associated vulvar squamous cell carcinoma (SCC), which is characterized by atypical basal keratinocyte proliferations with p53+ basal/suprabasal keratinocyte nuclei. METHODS AND RESULTS: Forty early, 78 classic, 30 hypertrophic vulvar LS, 26 paediatric vulvar and penile LS, 33 vulvar LS-associated SCC and 30 vulvar/penile control specimens were examined for p53 expression and the presence of d-VIN. Nuclear p53 staining was observed in 175/207 LS biopsy specimens. Eighty percent of early and 69% of paediatric LS showed discontinuous/continuous p53 staining in basal keratinocytes. Classic LS showed no p53 staining in 17%, discontinuous basal keratinocyte staining in 20%, continuous basal keratinocyte staining in 58%, basal/suprabasal staining in 5%. Hypertrophic LS revealed basal keratinocyte staining in 32% and basal/suprabasal staining in 61%. p53 staining was associated with sclerosis of blood vessels and dermis, lymphoid infiltrates, vasculitis and hypertrophic LS. d-VIN was seen in 2% of LS alone and in 24% of LS-associated SCC. CONCLUSION: d-VIN in LS is rare, while p53 staining is common and best explained as an ischaemic stress response due to poor oxygenation, vasculitis and inflammation rather than as a marker of a precancerous lesion in LS.

Early vulvar lichen sclerosus: a histopathological challenge.

Vulvar lichen sclerosus (LS), a lymphocyte-mediated chronic skin disease, begins with uncharacteristic symptoms and progresses undiagnosed to atrophy and destructive scarring. Some patients with longstanding advanced LS have an increased risk of vulvar carcinoma. Early LS is treatable, although not curable, if diagnosed early. Therefore, patients with persistent vulvar symptoms should be biopsied to establish the diagnosis. In contrast to advanced LS, the histological features in early LS are quite subtle and often more prominent in adnexal structures than in interfollicular skin. Adnexal structures show acanthosis, luminal hyperkeratosis and hypergranulosis with/without dystrophic hair and basement membrane thickening. The epidermis/mucosa shows mild irregular, occasionally psoriasiform acanthosis and focal basement membrane thickening. Early dermal changes are homogenized collagen and wide ectatic capillaries in dermal papillae immediately beneath the basement membrane. The lymphocytic infiltrate can be sparse or dense, lichenoid or interstitial with epidermal lymphocyte exocytosis and lymphocytic/lymphohistiocytic vasculitis. Dermal melanophages indicate preceding keratinocyte/melanocyte destruction. Biopsy specimens of early LS rarely display all features. Therefore, serial sections and periodic acid-Schiff reactions are necessary for their identification. Recognition and treatment of these early stages of LS may result in longstanding remission. Progression to atrophic stages with their associated morbidity and even to squamous cell carcinoma may be prevented.

Vasculitis in lichen sclerosus: an under recognized feature?

AIMS: To analyse 90 vulvar and 72 penile cases of lichen sclerosus (LS) on haematoxylin and eosin sections for vascular changes and the vascular infiltrates immunohistochemically with antibodies to T cells, B cells and antigen-presenting dendritic cells. LS is a skin disease of presumed autoimmune origin. Autoimmune diseases are mediated by lymphocytes which occasionally produce a lymphocytic vasculitis. METHODS AND RESULTS: Three types of lymphocytic infiltrates were identified: (i) perivascular lymphocytic infiltrates without damage to vessel walls; (ii) lymphocytic vasculitis in three forms: (a) concentric lymphohistiocytic infiltrates with lamination of the adventitia by basement membrane material which was typical for penile LS; (b) lymphocytic vasculitis with dense perivascular lymphocytic cuffing with occasional fibrin deposition in vessel walls and subendothelial lymphocyte infiltration, quite common in vulvar LS; and (c) intramural lymphocytic infiltrates in large muscular vessels; (iii) leukocytoclastic vasculitis in LS was exceptionally rare. In lymphocytic vasculitis, CD20+ B cells, CD4+ T cells and dendritic cells were the principal infiltrating cells. CONCLUSIONS: Dendritic cells capture (foreign) antigens after entry into the affected tissues and initiate immune responses acting as a matrix on which antigen-specific T and B cells interact. The described vascular features are indicative of antigen-mediated vasculitic changes in LS.

Malignant transformation within benign adnexal skin tumours.

AIMS: To report five malignant trichogenic tumours arising in longstanding, previously benign adnexal neoplasms through malignant transformation. Malignant trichogenic adnexal tumours are extremely rare neoplasms. METHODS AND RESULTS: The patients were between 55 years and 79 years of age. Three of the tumours were located on the arms, two on the face. Three of our patients had a history of chronic lymphocytic leukaemia, one patient had a history of colonic adenocarcinoma. The duration of the tumour nodules was reported as between 20 and 40 years before sudden changes occurred. These changes included rapid growth, pain, itching, ulceration and bleeding. Histologically, all tumours were well circumscribed and encapsulated. There was a residual benign tumour component and morphological signs such as bone formation, dystrophic calcification and sclerosis suggesting long duration of the lesions. All patients except for one, who refused further clinical investigation due to her advanced age of 79 years, had an underlying systemic malignancy. CONCLUSIONS: The growth stimulus in these benign adnexal neoplasms resulting in malignant transformation may be attributed to the acquisition of additional genetic events or to immunosuppression due to an underlying neoplastic disease. Therefore, patients with systemic diseases or malignancy should be carefully examined and followed for sudden changes in pre-existing benign cutaneous tumours.

[Vulvar lichen sclerosus. The importance of early clinical and histological diagnosis]. / Lichen sclerosus vulvae. Die besondere Bedeutung der klinischen und histopathologischen Früherkennung.

Vulvar lichen sclerosus (LS) is a chronic progressive skin disease of unclear etiology. It is often overlooked in early stages, but progresses to destructive atrophy and is associated with an increased risk of vulvar squamous cell carcinoma. The classical symptoms are pruritus and pain, but they are often not distinctive, so that unclear vulvar problems often lead to a biopsy. The histological picture of early LS is quite different from that of late LS with an atrophic epidermis, markedly sclerotic dermis and stiff dilated vessels. The epidermis in early LS is usually normal with only minor irregularities in the rete pattern. The basement membrane is normal or focally widened, while the edematous dermis has only scattered ectatic vessels. The often dense lichenoid and intraepidermal infiltrate explains the spongiosis and vacuolization of the basal layer keratinocytes. Very early cases may only have a sparse lymphocytic infiltrate and hyper-/parakeratosis of the follicular ostia. Early topical therapy can dampen the progression to atrophic, irreversible LS.

Occurance of apoptosis during ischemia in porcine pancreas islet cells.

PURPOSE: Pancreas islet transplantation is a potential treatment of diabetes mellitus and porcine organs provide an easily available source of cells. Unfortunately quality and quantity of isolated islets are still not satisfactory. Apoptosis occurs in freshly isolated islets and plays a significant role in early graft loss. We evaluated the influence of four storage solutions on porcine pancreas islets. METHOD: After warm ischemia of 15-20 minutes 12 organs were stored in 4 cold preservation solutions: Histidine-Tryptophan-Ketoglutarate solution (HTK), Hank's buffered saline solution (HBSS), University of Wisconsin (UW) solution and Ringer-Lactate (R). After cold ischemia for 100 minutes, organs were fixed in 3% formalin. Apoptotic cells were counted on hematocylin-eosin stainings. RESULTS: Most apoptotic cells were found in organs stored in R. Low numbers were found in the other groups. The difference between organs stored in R and organs stored in UW, HTK, or HBSS was highly significant. No significant difference could be found between UW, HTK and HBSS. CONCLUSION: Cold and warm ischemia of the pancreas seems to induce apoptosis in islet cells. Preservation solutions cause less apoptosis than electrolyte solution. No significant differences could be found among the preservation solutions.