Hans-Peter Fischer

Loss of DNA mismatch repair proteins in skin tumors from patients with Muir-Torre syndrome and MSH2 or MLH1 germline mutations: establishment of immunohistochemical analysis as a screening test.

Muir-Torre syndrome (MTS) is a rare autosomal-dominant disorder characterized by the predisposition to both sebaceous skin tumors (or multiple keratoacanthomas) and internal malignancies. A subtype of MTS is allelic to hereditary nonpolyposis colorectal cancer and is caused by germline mutations in the DNA mismatch repair genes MSH2 or MLH1. In these cases both internal and skin tumors show characteristic microsatellite instability (MSI). The aim of the present study was to determine whether immunohistochemical examination of MSH2 or MLH1 protein expression in MTS-associated skin tumors can be used as a diagnostic screening tool to identify patients with germline mutations in MSH2 or MLH1. In the present study 28 skin lesions from 17 patients (20 sebaceous gland tumors, 4 sebaceous hyperplasias, 3 keratoacanthomas, and 1 squamous cell carcinoma) were tested immunohistochemically with antibodies against MSH2 and MLH1. Eighteen of these tumors were from eight patients with known MSH2 germline mutations, two tumors were from a patient with a germline mutation in MLH1, and eight microsatellite stable sporadic skin tumors served as controls. One sample had to be excluded because of a lack of immunoreactivity. All eight microsatellite stable tumors expressed both DNA repair proteins. In 15 of the tumors from MSH2 germline mutation carriers, loss of MSH2 expression was observed, one tumor showed reduced MSH2 expression, and one tumor displayed positive immunoreactivity to MSH2. Both tumors of the MLH1 germline mutation carrier showed loss of the MLH1 protein. In conclusion, our findings demonstrate that immunohistochemical testing of MTS-related skin tumors is a reliable screening method with high predictive value for the diagnosis of the DNA mismatch repair-deficient MTS.

Carcinoma of the ampulla of Vater: Comparative histologic/immunohistochemical classification and follow-up

A broad histomorphologic spectrum of ampullary carcinomas of Vater make a reproducible histologic classification difficult. Using cytokeratin immunohistochemistry, we present a new classification of ampullary carcinomas and analyze their clinical significance. Fifty-five invasive carcinomas of Vater’s ampulla were histologically classified into pancreaticobiliary, intestinal, and other types. Serial sections of all carcinoma specimens were additionally stained with antibodies to cytokeratins (CK7, CK20), apomucins (MUC1, MUC2, MUC5AC), CEA, CA19-9, Ki67, and p53. Follow-up of patients from 4 months to 22 years after surgery (mean interval, 51.6 months) was evaluated. Most carcinomas of the ampulla of Vater were of immunohistochemically pancreaticobiliary type (iPT, CK7+, CK20−; 54.5%) or intestinal type (immunohistochemically intestinal type [iIT], CK7−, CK20+; 23.6%). Some carcinomas of immunohistochemically “other” type (iOT both CK7+ and CK20+ or CK7− and CK20−; 21.8%) had precursor lesions of iIT or iPT. Carcinomas positive for MUC2 or CEA were associated with iIT (MUC2, P < 0.001; CEA, P = 0.003), whereas MUC5AC-positive carcinomas were related to iPT (P = 0.005). Our classification based on cytokeratin-immunohistochemistry correlated well with the histologic classification according to published criteria (κ-coefficient = 0.398; P < 0.001). Furthermore, histologically unusual types could be histogenetically related to pancreaticobiliary duct mucosa or intestinal mucosa. Therefore, all 4 signet-ring cell carcinomas were iIT carcinomas. Thus, cytokeratin immunohistochemistry allows a reproducible, histogenetically based categorization of ampullary carcinomas. However, neither histopathologic nor immunohistochemical subgroups significantly correlated with clinical outcome in our German collective. The overall survival was significantly shorter in males (P = 0.032) and patients with positive nodal stage (N1 < N0; P = 0.0025).

c-kit mutations in gastrointestinal stromal tumors occur preferentially in the spindle rather than in the epithelioid cell variant.

Gastrointestinal stromal tumors (GISTs) coexpress CD34 and the Kit tyrosine-kinase receptor (CD117). A subset of GISTs carry gain-of-function mutations of the c-kit proto-oncogene in its juxtamembrane domain. The relationship between the mutational status and histological as well as immunohistochemical features has not been assessed in detail. 36 GISTs and 14 other gastrointestinal mesenchymal tumors were investigated for their morphology and immunophenotype as well as for the presence of c-kit mutations. DNA was extracted from formalin-fixed, paraffin-embedded tissue. Exons 9, 11, 13, and 17 of c-kit were analyzed by SSCP. Bands with altered mobility were excised, reamplified, and sequenced. C-kit mutations in Exon 11 encoding the juxtamembrane domain were identified in 19 cases (52.8%), with deletions in 12 cases, insertions in 3 cases (2 of these as duplications), and point mutations in 4 cases. The mutations clustered between Codons 553 and 561, pinpointing the critical region for deregulated Kit receptor activation. In both Exons 9 and 13, single mutations could be identified, whereas no mutations were found in Exon 17. There were c-kit mutations in 66.6% of benign GISTs (14/21), 83.3% of the malignant (5/6), and 40% of the cases of intermediate malignancy (2/5). A low frequency of mutations in benign GISTs, as reported previously by other researchers, could not be observed in our panel. Interestingly, all GISTs with c-kit mutations displayed a spindle cell phenotype, whereas mutations were absent in all 7 tumors with an epithelioid component (P = .03). This finding suggests a relationship between c-kit mutation and histological subtype in GISTs.

INTERFERON/ANTIOXIDANT COMBINATION THERAPY FOR CHRONIC HEPATITIS C--A CONTROLLED PILOT TRIAL

The effects of two forms of antioxidative co-therapy were analyzed in 24 interferon-alpha (IFN)-naive patients with chronic hepatitis C who were randomized to either receive IFN monotherapy (3 x 4.5 million units IFN-alpha 2a per week), (group A), or IFN and N-acetylcysteine (N-acetylcysteine (NAC) 1.800 mg/day) plus sodium selenite (400 microg/day) supplementation (group B), or treatment as in group B plus vitamin E (544 IU/day) (group C), over 24 weeks. Changes in histology, normalization of ALT, reduction of viral RNA, serum levels of glutathione, selenium, vitamin E, erythrocyte glutathione peroxidase, trolox equivalent antioxidative capacity (TEAC), thiobarbituric acid reactive substances (TBARS) and protein carbonyl groups were measured. Low baseline TEAC and elevated TBARS indicated increased oxidative stress before therapy, which was not affected by antioxidant supplementation. At the end of treatment complete responses were found in 3/8, 2/8 and 6/8 patients in groups A, B and C, respectively, but liver histology had not significantly improved. Vitamin E treated patients had a 2.4 greater chance (95% CI: 1.05-5.5) of obtaining a complete response and had significantly greater reduction in viral load (P = 0.028) than patients without vitamin E. Relapses, i.e. re-appearance of detectable hepatitis C virus (HCV) RNA and/or re-elevation of ALT-activity occurred in 7 out of the 11 responders within 6 months after termination of therapy (group A: 2/3, group B: 1/2 and group C: 4/6). Thus, no overall beneficial effect of antioxidant/IFN therapy was detected. However, the apparent trend towards a more favorable outcome with vitamin E supplementation warrants to further study this substance as an adjuvant to IFN therapy in chronic hepatitis C.

Tumours from MSH2 mutation carriers show loss of MSH2 expression but many tumours from MLH1 mutation carriers exhibit weak positive MLH1 staining

Microsatellite analysis (MSA) in tumour tissue is useful for pre‐selection of hereditary non‐polyposis colorectal cancer (HNPCC) patients for mutation screening, but is time‐consuming and cost‐intensive. Immunohistochemistry (IHC) for expression of MLH1 and MSH2 proteins is simple, fast, and indicates the affected gene. IHC has therefore been proposed as an alternative pre‐screening method. However, some authors report a lower sensitivity of IHC compared with MSA. The present study reports IHC results for MSH2 and MLH1 performed in 82 tumours with high microsatellite instability (MSI‐H) from 81 carriers of pathogenic mutations in MSH2 or MLH1. One hundred per cent (38/38) of the tumours from MSH2 mutation carriers showed loss of MSH2 staining; in all cases, the affected MSH2 gene was predicted correctly by IHC. Complete loss of MLH1 expression was observed in 66% (29/44) of MLH1 mutation carriers. Weak positive MLH1 staining was observed in 14 (32%) cases and, in one case, normal MLH1 staining was seen. The pathologist was aware of the weak staining pattern as an indicator of an MLH1 mutation; 98% of the MLH1 mutations were predicted correctly. To evaluate whether weak positive MLH1 staining is observed more often with in‐frame or missense mutations, IHC data from 23 MSI‐H tumours from carriers of unspecified variants were added and mutations were grouped into truncating mutations, large non‐truncating deletions, and small non‐truncating mutations. Weak MLH1 staining was observed in all three categories and it is postulated that other factors, such as mutation of the second allele, also influence protein expression. In conclusion, IHC can be regarded as a very useful method for selecting HNPCC patients for mutation analysis, as long as it is interpreted by an experienced pathologist. The high specificity of IHC in terms of indicating the affected gene is useful for evaluating unspecified variants. However, the staining pattern does not predict whether the underlying germ‐line mutation is truncating or not. Copyright

Liver histopathology in patients with concurrent chronic hepatitis C and HIV infection.

To investigate the influence of human immunodeficiency virus (HIV) coinfection on preexisting long-term chronic C hepatitis (HCV) 68 liver biopsies from 22 HIV/HCV-coinfected, 13 HIV and 33 HCV-monoinfected patients and 71 livers obtained at autopsy from 26 HIV/HCV-coinfected and 45 HIV-monoinfected patients were studied by histo- and immunohistochemistry. All HIV patients had reached the advanced stage of immunodeficiency (stage III CDC), except for 3 haemophiliacs (stage II CDC). HCV infection was associated with a higher degree of portal, periportal and lobular inflammation — regardless of whether there was concurrent HIV infection. HIV/HCV coinfection was associated with a significantly higher rate of granulocytic cholangiolitis than HCV and HIV monoinfection (P < 0.05), a histological feature uncommon in C hepatitis. In HIV/HCV coinfection cholestasis was a predominant histological feature. HCV monoinfection and HCV/HIV coinfection were associated with the highest fibrosis index. In HIV/HCV coinfection centrilobular fibrosis was significantly more marked than in HCV monoinfection (P < 0.05), suggesting an HIV associated fibrogenic effect. Patients with chronic C hepatitis showed a significantly increased rate of posthepatitic cirrhosis compared with the patients without HCV infection (P < 0.05). At autopsy, 10 of the 20 HIV/HCV-coinfected haemophiliacs had developed cirrhosis because of chronic C hepatitis, whereas cirrhosis was found in only 2 of 6 HIV/HCV-coinfected non-haemophiliacs (1 case of chronic B and C hepatitis, and 1 case of chronic alcohol abuse). No cirrhosis was observed in the 45 autopsy patients with HIV monoinfection. The findings suggest that HIV coinfection aggravates the course of preceding long-term chronic C hepatitis by a more marked (centrilobular) fibrosis. HIV/HCV-coinfected patients are threatened by a higher rate of posthepatitic cirrhosis —particularly in multitransfused haemophiliacs — and cholestatic hepatopathy.

Chronic liver disease in heterozygous α1-antitrypsin deficiency PiZ

Abstract Background/Aims: The contribution of the heterozygous state PiZ of α1-antitrypsin deficiency (AATD) to the pathogenesis of chronic liver disease is debated. We analyzed whether patients with this genetic defect carrying a single PiZ gene are at increased risk for developing chronic liver disease. Methods: 1847 consecutive biopsy cases and 1030 autopsy cases of Caucasian adults were screened immunohistochemically for PiZ deposits. The zygosity status was analyzed by single-strand conformational polymorphism (SSCP) and by sequencing DNA extracted from paraffin-embedded tissue. Results: All analyzed biopsy cases were heterozygous for the PiZ mutation. The biopsy group revealed a significantly higher rate of PiZ-positive cases (3.4%) than the autopsy group (1.8%) ( p =0.019). PiZ deposits ranged from scarce granules to extensive globular inclusions as in homozygous AATD of PiZ type. The extent of PiZ deposits correlated well with the inflammatory activity and stage of fibrosis. Cirrhotic livers contained globular PiZ deposits significantly more often than the biopsies with minor fibrosis. PiZ-positive biopsies from patients without concurrent liver disease ( n =26) revealed only minor fibrosis in the age group between 20 and 39 years, but significantly more severe fibrosis and significantly more PiZ deposits in the older age groups. Biopsies with concurrent liver disease ( n =28) presented with significantly more severe inflammation and fibrosis, and more PiZ deposits than the cases without concurrent liver disease. Conclusions: Patients with heterozygous AATD of PiZ type bear an increased risk for chronic liver disease. If at all, this genetic defect will become clinically relevant only in middle-aged or old adults. It rarely causes liver cirrhosis even without concurrent liver disease. It can aggravate or can be aggravated by advanced coexistent chronic liver diseases. PiZ immunohistochemistry is an easy, highly specific method to detect this metabolic defect on liver biopsies.

Role of cannabinoid receptors in alcoholic hepatic injury: steatosis and fibrogenesis are increased in CB2 receptor-deficient mice and decreased in CB1 receptor knockouts

Background: Alcohol is a common cause of hepatic liver injury with steatosis and fibrosis. Cannabinoid receptors (CB) modulate steatosis, inflammation and fibrogenesis. To investigate the differences between CB1 and CB2 in the hepatic response to chronic alcohol intake, we examined CB knockout mice (CB1−/−, CB2−/−).

Expression of xenobiotic and steroid hormone metabolizing enzymes in human breast carcinomas

The potential to metabolize endogenous and exogenous substances may influence breast cancer development and tumor growth. Therefore, the authors investigated the protein expression of Glutathione S‐transferase (GST) isoforms and cytochrome P450 (CYP) known to be involved in the metabolism of steroid hormones and endogenous as well as exogenous carcinogens in breast cancer tissue to obtain new information on their possible role in tumor progression. Expression of GST pi, mu, alpha and CYP1A1/2, 1A2, 3A4/5, 1B1, 2E1 was assessed by immunohistochemistry for primary breast carcinomas of 393 patients from the German GENICA breast cancer collection. The percentages of positive tumors were 50.1 and 44.5% for GST mu and CYP2E1, and ranged from 13 to 24.7% for CYP1A2, GST pi, CYP1A1/2, CYP3A4/5, CYP1B1. GST alpha was expressed in 1.8% of tumors. The authors observed the following associations between strong protein expression and histopathological characteristics: GST expression was associated with a better tumor differentiation (GST mu, p = 0.018) and with reduced lymph node metastasis (GST pi, p = 0.02). In addition, GST mu expression was associated with a positive estrogen receptor and progesterone receptor status (p < 0.001). CYP3A4/5 expression was associated with a positive nodal status (p = 0.018). Expression of CYP1B1 was associated with poor tumor differentiation (p = 0.049). Our results demonstrate that the majority of breast carcinomas expressed xenobiotic and drug metabolizing enzymes. They particularly suggest that GST mu and pi expression may indicate a better prognosis and that strong CYP3A4/5 and CYP1B1 expression may be key features of nonfavourable prognosis.

Vaccination with dendritic cells transfected with mRNA-encoded folate-receptor-α for relapsed metastatic ovarian cancer

A 61-year-old woman was diagnosed in December, 2001, with advanced serous papillary ovarian cancer IIIc (International Federation of Gynaecology and Obstetrics classifi cation), widespread peritoneal carcinomatosis, and a raised cancer antigen (CA)-125 concentration (>3000 U/mL). Because optimum cytoreduction seemed impossible, a platinum and taxane-based chemotherapy (three cycles of carboplatin [AUC5] and paclitaxel [175 mg/m]) was given before debulking surgery in April, 2002. Postoperative residual disease was less than 1 cm. After three additional cycles of the same regimen, CA-125 concentration returned to normal (<30 U/mL). After 12 months, the patient presented with relapsed intra-abdominal ovarian cancer. Secondary tumour debulking was done in June, 2003, with no macroscopic residual tumour left. Postoperatively, a platinum-based reinduction treatment was done and CA-125 decreased to a normal concentration (20 U/mL). However, 4 months later, the patient presented again with a raised CA-125 concentration (234 U/mL; fi gure 1A), and para-aortic and axillary lymph-node (LN) metastases. The bulky axillary LN metastases, which impaired activities in her normal daily routine, were dissected, and a vaccination regimen with autologous dendritic cells engineered with mRNAencoded folate-receptor type alpha (FR-α) was initiated. Generation of the vaccine involved the reverse transcription of full-length FR-α-cDNA from total RNA from the human ovarian cancer-cell line, IGROV1, amplifi ed with specifi c primers (FR-α-F:[5 -ATGGCTCAGCGGATGACAACA-3 ]; FR-α-R:[5 -TCAGCTGAGCAGCCACAGCA-3 ]), and the Lancet Oncol 2007; 8: 451–54