Field cancerisation and polyclonal p53 mutation in the upper aero-digestive tract.

Field cancerisation of the aerodigestive tract is caused by chronic exposure to alcohol and tobacco, but the nature of the genetic alterations preceding overt malignancy is unknown. To identify potential field changes we have used a functional assay which tests the transcriptional competence of human p53 expressed in yeast. To increase the sensitivity and reliability of the technique for samples containing under 20% mutant p53, the 5' and 3'-ends of the p53 cDNA were examined separately. With this split form of the assay the tissue p53 mRNA acts as its own control for RNA quality. Mutations were detected in 87% (46/53) of tumours, reflecting the high sensitivity of the technique. Multiple biopsies of histologically normal tissue from the upper aero-digestive tract were tested and clonal p53 mutations were identified in 76% (38/50) of biopsies from patients presenting with multiple tumours compared with 32% (38/117) of biopsies from patients presenting with single tumours (P<0.000001). All patients (16/16) presenting with multiple tumours had at least one positive biopsy, compared with only 53% (19/36) of patients presenting with single tumours (P <0.001). This defines expansion of multiple clones of mutant p53-containing cells as an important biological mechanism of field cancerisation, and provides a means to identify patients likely to benefit from intensive screening for the development of new head and neck tumours.

Interstitial photodynamic therapy with tetra(m-hydroxyphenyl)chlorin: tumor versus striated muscle damage.

PURPOSE: The present study was initiated to determine the conditions under which a single photodynamic treatment would induce maximal damage to a tumor with no or at least minimal reversible damage to a normal striated muscle. METHODS AND MATERIALS: The technique of interstitial light delivery was used after prior 0.5 mg/kg tetra(m-hydroxyphenyl)chlorin administration in a hamster model. After having estimated the threshold light doses required for minimal muscle damage, the same light doses were applied to squamous cell carcinomas to evaluate the efficiency of interstitial photodynamic therapy. Sixteen and 96 h after the injection, irradiation at 650 nm was performed on the thigh muscle of the left hind leg. The applied light doses ranged between 0.3-15 J and were delivered at an intensity of 44 mW per cm of diffuser length. RESULTS: The threshold of muscle damage was obtained using light doses of 1.5-3 J at two drug-light intervals of 16 and 96 h, respectively. More than 85% of the tumor mass was destroyed when lesions were illuminated using these threshold conditions. In terms of immediate short-term tumor response, this means that for the given irradiation conditions, a relatively low threshold energy of only 1.5 or 3 J, depending on the drug-light interval, is sufficient to induce massive tumor destruction with minimal muscle damage. CONCLUSION: These results have implications for evaluating interstitial PDT for squamous cell cancers in unfavorable localization in the oral cavity or pharynx, such as at the base of the tongue.

Promoter methylation analysis on microdissected paraffin-embedded tissues using bisulfite treatment and PCR-SSCP.

Methylation-sensitive single-strand conformation analysis (MS-SSCA) is a new method of screening for DNA methylation changes. The combination of bisulfite modification and PCR results in the conversion of unmethylated cytosines to thymines, whereas methylated cytosines remain unchanged. This sequence conversion can lead to methylation-dependent alterations of single-strand conformation, which can be detected by SSCA. An analysis of mixtures of methylated and unmethylated DNA at known ratios revealed that the relative intensities of the corresponding bands following MS-SSCA were maintained. MS-SSCA was applied for methylation analysis of human p16 promoter region using genomic DNA obtained from either frozen, fixed, or microdissected fixed tissue sections. MS-SSCA is a rapid, specific, and semiquantitative approach that allows the detection of methylation of the p16 gene promoter. In reconstruction experiments, the method permits the detection of 10% or less of cells harboring a methylated p16 promoter. We have been successful in analyzing by MS-SSCA almost all (96%) tumor samples microdissected from archival paraffin-embedded fixed tissue sections and obtaining reproducible results. In addition, when microdissection was performed, the clonality of this genetic alteration could be identified.

Nuclear accumulation of beta-catenin is a common and early event during neoplastic progression of Barrett esophagus.

Our aim was to characterize expression and mutation of beta-catenin in the progression of Barrett esophagus to adenocarcinoma. Immunohistochemical analysis of beta-catenin was performed on paraffin-embedded tissue from 30 cases with adenocarcinomas and premalignant lesions. To determine whether there is a correlation between beta-catenin nuclear accumulation and exon 3 mutation of this gene, mutational analysis by polymerase chain reaction-single-strand conformation polymorphism was performed on DNA extracted from the same 30 adenocarcinomas. As a result, the prevalence of reduced expression of beta-catenin on the membrane, with or without nuclear staining, increased significantly from low-grade (LG) to high-grade (HG) dysplasia. Focal nuclear staining for beta-catenin was present in 19 cases of adenocarcinoma, and nuclear staining was associated significantly with progression from metaplasia to LG dysplasia. In addition, in glands with clear histologic transition from metaplasia to LG dysplasia, nuclear accumulation of beta-catenin was found only in the LG dysplastic areas. No mutation in exon 3 of the beta-catenin gene was detected in adenocarcinomas. These results demonstrate that disturbance of the APC/beta-catenin pathway, as indicated by nuclear accumulation of beta-catenin, is a common and early event during neoplastic progression in Barrett esophagus.

Synchronous ileal and colonic adenocarcinomas associated with Crohn's disease: report of a case with a focus on genetic alterations and carcinogenesis.

Patients with Crohn's disease have an increased risk of developing intestinal tumours. However, the carcinogenic mechanisms remain poorly understood. To address this question, this report describes an unusual case of Crohn's disease complicated by synchronous small intestinal and colonic adenocarcinomas. Genetic events in both the tumours and their adjacent mucosae were evaluated and the tumorigenesis of these cancers is discussed.

Time-dependent biodistribution of tetra(m-hydroxyphenyl)chlorin and benzoporphyrin derivative monoacid ring A in the hamster model: comparative fluorescence microscopy study.

The pharmacokinetics of the photosensitizer used play a key role in the understanding of the mechanism of photodynamic therapy-induced damage. Fluorescence microscopy was used to compare time-dependent biodistribution of tetra(m-hydroxyphenyl)chlorin (mTHPC) and benzoporphyrin derivative monoacid ring A (BPD-MA) in different hamster tissues, including an early, chemically induced, squamous cell carcinoma. Following injection of 0.5 mg/kg body weight of mTHPC and 2.0 mg/kg BPD-MA, groups of three animals were sacrificed at different time points and a series of fluorescence micrographs from different excised organs were analyzed. The highest fluorescence intensities of mTHPC were observed at 96 h for squamous epithelia and skin and at 48 h for smooth muscle. There is no real peak of BPD-MA fluorescence between 30 min and 3 h in the basal epithelial layers, fibroconnective tissue, muscles or blood vessels. At 4 h after injection, the fluorescence level of BPD-MA decreased and at 24 h it had returned to background level in all observed tissues. The significantly faster clearance of BPD-MA is the principal advantage as compared to mTHPC. However, similar localization patterns in different tissues with essentially vascular affinity represent a possible disadvantage for treating early malignancies with BPD-MA as compared to mTHPC, which is mainly localized in various epithelia. For both photosensitizers no significant selectivity between early squamous cell carcinoma and healthy mucosae is seen. Pharmacokinetic studies of different photosensitizers in an appropriate animal model are essential for selecting new-generation photosensitizers with the most favorable localization for photodynamic therapy of early malignancies in hollow organs.

In vivo fluence rate effect in photodynamic therapy of early cancers with tetra(m-hydroxyphenyl)chlorin.

Several parameters affect clinical trials in photodynamic therapy and influence the therapeutic outcome. Beside drug dose, light dose, drug-light interval and other variables, the fluence rate is a parameter that can influence the therapeutic results. In this study we have evaluated the fluence rate effect with a second-generation photosensitizer, tetra(m-hydroxyphenyl)chlorin (mTHPC) using a 7,12-dimethylbenz(a)anthracene induced early squamous cell carcinoma of the Syrian hamster cheek pouch as a tumor model. Following injection of 0.5 mg/kg of mTHPC, irradiation tests were performed at two drug-light intervals, 4 and 8 days. Wavelength and light dose were adapted from those applied routinely in clinical trials. Irradiations at 652 nm were carried out with fluences ranging from 8 to 20 J/cm2 delivered at fluence rates of 15 and 150 mW/cm2. Similar tests were also performed at 514 nm with a fluence of 80 J/cm2 delivered at fluence rates ranging from 25 to 125 mW/cm2. At both wavelengths and drug-light intervals for a given fluence, the higher fluence rates resulted in less tissue damage in tumor and healthy mucosae. However, the lower fluence rates yielded slightly less therapeutic selectivity. This study confirms that the fluence rate is of major importance in clinical PDT.

Photodynamic therapy for early squamous cell carcinomas of the esophagus, bronchi, and mouth with m-tetra (hydroxyphenyl) chlorin.

OBJECTIVE: To clinically evaluate a new photosensitizer, m-tetra(hydroxyphenyl) chlorin (m-THPC), for the photodynamic therapy of early squamous cell carcinomas of the upper aerodigestive tract. DESIGN: Phase 1 included evaluation of the innocuousness of the compound after intravenous injection (control of vital parameters and blood analysis before and after injection) and evaluation of the duration of skin photosensitization. Phase 2 included assessment of optimal conditions for treatment (injected dose, drug-light interval, light dose, wavelength, etc), on 33 early squamous cell carcinomas of the mouth, esophagus, and bronchi, with a mean follow-up of 14 months; irradiation tests on healthy and neoplastic mucosae to determine the irradiation conditions that lead to tumor eradication with minimal damage to the surrounding normal mucosa and muscle layers; and localization of the dye in various tissue compartments and cells at different time intervals after the injection of the photosensitizer, by using a fluorescence microscope to analyze 46 biopsy specimens taken during the treatment sessions and 8 resected specimens of early cancers, excised with the carbon dioxide laser. SETTING: Endoscopic medical center of an otolaryngology-head and neck surgery department. PATIENTS: Twenty-five patients treated previously for a head and neck cancer with a synchronous or metachronous early second primary cancer. Patients with porphyria were excluded from the trial. RESULTS: The best results in the bronchi and mouth were obtained with an injected dose of 0.15 mg of m-THPC per kilogram of body weight 4 days before irradiation. The fluence was 7 to 16 J/cm2, and the fluence rate was between 100 and 150 mW/cm2 using red light at 652 nm. In the esophagus, green light at 514 nm is preferred to the red light to avoid fistulas. Optimal irradiation conditions at this wavelength, which was also used in the trachea, were found at a fluence of 75 to 100 J/cm2 and a fluence rate between 70 and 100 mW/cm2. Of 33 lesions treated thus far by photodynamic therapy with m-THPC, 28 show no recurrence with a mean follow-up of 14 months. Photosensitivity to sunlight does not exceed 6 weeks. CONCLUSIONS: m-Tetra(hydroxyphenyl) chlorin is a second-generation photosensitizer that has several significant advantages as compared with the first-generation porphyrin mixtures hematoporphyrin derivative and porfimer sodium (Photofrin II). It is a pure compound that is 100 times more phototoxic at 652 nm and 10 times more photoxic at 514 nm, has better selectivity for early carcinomas, and a shorter duration of skin photosensitivity. The therapeutic results indicate a recurrence rate that is similar to that obtained with Photofrin II, ie, about 15%.

Glomerulonephritis associated with Hemophilus aphrophilus endocarditis.

A 65 year old man developed endocarditis and septicemia due to Hemophilus aphrophilus, a Gram-negative coccobacillus. Renal rather than cardiac failure was the principal feature of his illness and renal biopsy was compatible with glomerulonephritis secondary to septicemia. Rapid recovery of renal function and improvement of the glomerular lesion followed antibiotic treatment of the septicemia. This case illustrates the renal damage that can occur in association with septicemia due to rarer infectious agents. As with more common organisms, specific antimicrobial therapy leads to rapid improvement of the nephropathy.

Localization of tetra(m-hydroxyphenyl)chlorin (Foscan) in human healthy tissues and squamous cell carcinomas of the upper aero-digestive tract, the esophagus and the bronchi: a fluorescence microscopy study.

To date, little is known about precise time-dependent distribution and histological localization of tetra(m-hydroxyphenyl)chlorin (mTHPC) in human healthy tissues and squamous cell malignancies in the upper aero-digestive tract. A fluorescence microscopy study was performed on 50 healthy tissue biopsies and on 13 tumors (graded from Tis to T1 SCC) from 30 patients. Tissue samples were taken between 4 h and 11 days following injection of 0.15 mg/kg mTHPC. A fairly comparable distribution pattern in various tissues was observed over time in different patients. Vascular localization of mTHPC fluorescence predominates at a short delay, whereas the dye is essentially located in the tumoral and healthy mucosa after longer delays. A much lower uptake and retention of mTHPC fluorescence was noted in striated muscle and cartilage as compared to neoplastic lesions. No significant selectivity was found between healthy and tumoral mucosa. The obtained data are important to confirm drug-light interval that have been selected for effective PDT for early SCC malignancies while minimizing the risks of over- or under-treatment. The low fluorescence level in striated muscle provides the opportunity to develop interstitial PDT as a treatment modality for invasive SCC of unfavorable locations in the oral cavity or pharynx, such as the base of the tongue.

Measurements by fluorescence microscopy of the time-dependent distribution of meso-tetra-hydroxyphenylchlorin in healthy tissues and chemically induced "early" squamous cell carcinoma of the Syrian hamster cheek pouch.

The influence of the time interval between dye administration and detection by fluorescence microscopy was assessed in "early" squamous cell carcinomas of the cheek pouch mucosa and different healthy tissues of the Syrian hamster. Following intracardiac injection of 0.15 mg (kg bodyweight)-1 of meso-tetra-hydroxyphenylchlorin (m-THPC), groups of three animals were sacrificed at different time intervals up to 30 days. A group of three non-injected animals was used to detect the endogene fluorescence of the corresponding normal tissues for autofluorescence subtraction. The following excised organs (oesophagus, trachea, liver, spleen, kidney, skin, striated muscle, healthy and tumoral cheek pouch mucosae) were fast frozen in liquid nitrogen and stored at -70 degrees C for fluorescence microscopy. The results show significant differences in the detectable m-THPC levels in different tissue layers (for instance, the epithelia and muscle of the oesophagus, trachea and cheek pouch) at different time intervals. These data indicate that pharmacokinetic studies may be useful for selecting the optimal time for the photodetection and phototherapy of cancer.

The histological patterns of chronic active hepatitis--excluding cirrhosis--and their prognostic significance.

In a retrospective clinico-pathological analysis of 30 cases of chronic active hepatitis, no significant correlation could be demonstrated between any histological feature and the end result of the evolution. The so-called "bridging" lesion, in particular, has no prognostic value. A possible effect of the therapy (cortico-steroids or not) on the final outcome could not be established. There is however a clearcut difference in the rate of development, be it favorable or not, between the slow progress of chronic aggressive hepatitis, and the rapidly changing subacute hepatitis. We therefore feel that such a distinction should be maintained, and question the nomenclature proposed by the International Association for Study of the Liver.

Abnormalities of cilia and chronic sinusitis.

An ultrastructural study was performed on 34 biopsy samples of the sinusal mucosa in 28 patients who were investigated for chronic sinusitis. Twelve specimens with normoplastic sinusitis and eighteen with hyperplastic sinusitis were studied. Four normal specimens served as controls. The incidence of ultrastructural ciliary abnormalities was 2%. Morphological changes of dynein arms have not been observed in the present study. Compound cilia were found in approximately 2/3 of the studied cases. Microtubular abnormalities occurred in roughly 50%. The observed abnormalities, also seen in the control group, were independent on the sinusitis type and could not been correlated with mucociliary transport. Their significance is discussed.

Resection of metastases to the alimentary tract from malignant melanoma.

Metastases to the celio-mesenteric organs from malignant melanoma are studied retrospectively in 22 autopsied cases and in seven surgically treated patients. Metastatic disease to one or more celio-mesenteric organs was found in 86.3% of postmortem examinations: the liver was the most frequently involved organ, followed by the pancreas, peritoneum, small bowel, biliary tract spleen, colon and stomach, in that order. Surgery only resulted in long-term asymptomatic survival in those patients with a long relapse-free interval and a single metastasis. It is concluded that, apart from cases with intestinal obstruction or massive GI bleeding, resection should be reserved for patients with a relapse-free interval of at least 15 months and with a single "alimentary" metastasis. It is suggested that diagnosis of systemic spread at the asymptomatic stage would improve prognosis of stage IV malignant melanoma.

[Multicentric intestinal angiosarcoma. Report on a case]. / Angiosarcome multicentrique intestinal. A propos d'un cas.

The authors describe the case of a seventy-four year old female with no past medical history, in whom a duodenal angiosarcoma was found during investigation for anemia. Despite adequate surgical excision, hemorrhage persisted and the patient died. Autopsy disclosed multiple angiosarcomatous tumours in the colon and pulmonary micrometastases. Angiosarcoma is a rare tumour in the digestive tract. Multiple tumors occur but are often difficult to detect. In the present case, radiologican and angiographic investigations did not confirm the diagnosis, which was made on endoscopic and surgical findings and substantiated by histological examination of the surgical specimen.

[Magnetic resonance imaging and x-ray computed tomography in advanced cancer of the oral cavity. A comparative clinical, radiological and morphological study]. / Apport de l'imagerie par résonance magnétique et de la tomodensitométrie dans les cancers avancés de la cavité buccale. Etude comparative clinique, radiologique, morphologique.

The objective of this prospective study is to assess the impact of magnetic resonance imaging (MRI) and computed tomography (CT) as compared to physical examination in the choice of type of surgery for advanced intraoral cancers (with or without resection of the mandibula). From 1990 to 1993, we operated on 21 intraoral malignant tumors with segmental resection of the mandibula followed by a histological examination. The preoperative evaluation consisted of an MRI (n = 8), a CT (n = 8) or both (n = 5). MRI suspected an infiltration of the bone in 9 cases, CT in 4 and physical examination in 16. This was histologically confirmed in 6 of the 21 patients only. MRI and CT both have a high sensitivity, as does physical examination, but neither have a good specificity (physical examination: 5 true positive, 4 true negative, 11 false positive, 1 false negative; MRI: 4 true positive, 4 true negative, 5 false positive, 0 false negative; CT: 3 true positive, 7 true negative, 3 false positive, 0 false negative). In conclusion, the decision of a mandibular resection can only be taken after a careful physical examination, including palpation under general anesthesia in a fully relaxed patient. This is best accomplished during the pretherapy bronchoesophagoscopy, routinely performed for the detection of synchronous second primary tumors using toluidin blue as a vital staining method. If this initial evaluation gives a suspicion of a massive infiltration of the mandible, an MRI, rather than a CT, should be performed to determine the extent of the resection because of a high rate of artefacts with CT.

[Y-loop gastrojejunostomy in postoperative gastritis caused by reflux]. / Gastro-jéjunostomie sur anse en Y pour gastrite post-opératoire par reflux.

This prospective study involving 24 patients operated for postoperative reflux gastritis spanned over the period between 1974 and 1987. Diagnosis by exclusion was based on clinical, endoscopic and histological findings collected a few months to 16 years following surgery, the outcome of which was either alteration or destruction of the antropyloric sphincteral mechanism. All patients had Y-loop gastrojejunostomy performed according to Roux's procedure (at 50 cm). The clinical results as assessed on the basis of Visick's score and by gastroscopy were good. Anatomopathological examination of pre- and postoperative bioptic specimens blind-collected by a single operator indicated that foveolar hyperplasia is a good indicator of enterogastric reflux.

A long-term study of experimental duodenogastric reflux on the vagotomized stomach in dogs.

The possibility has been explored in dogs that the effects of chronic enteral reflux on the gastric mucosa may be modified by highly selective vagotomy (HSV). In both the innervated and the vagotomized stomachs, Roux-en-Y duodenogastrostomy induced mucosal hyperaemia and foveolar hyperplasia; the mucosa regained normal morphology after the elimination of reflux. Duodenogastrostomy without HSV did not influence basal serum gastrin but resulted in a significant increase in mucosal histamine. After duodenogastrostomy with HSV basal serum gastrin decreased significantly and mucosal histamine was not affected. It is concluded that foveolar hyperplasia is a reversible reaction of the mucosa to potentially damaging agents like enteral contents. It is also suggested that the combination of chronic enterogastric reflux, abnormal gastrointestinal motility and HSV might have an inhibitory effect on gastrin release.

Modifications of the gastric mucosal barrier induced by experimental duodenogastric reflux: an electron microscopy study.

The present study was undertaken to examine the ultrastructure of antral foveolar epithelium of dogs submitted to long term duodenogastric reflux. Minimal ultrastructural alterations (swelling of the cellular organelles, shortening of the microvilli, irregularity of the secretory granules) were observed. Total necrosis of isolated cells was rare. The lesions appeared in all dogs including the controls, and were not always reversed by the elimination of reflux. The significance of these findings is not clear and is probably not dependent on continuous exposure to duodenogastric reflux.

A long-term study of different types of experimental alkaline reflux and the effects of its suppression in dogs.

The gastric mucosa of 19 mongrel dogs was submitted to a bilio-pancreatic, isolated biliary or isolated pancreatic reflux. With an isolated biliary reflux, there is a more rapid and more severe hyperaemia and foveolar hyperplasia of the mucosa of the fundus than with an isolated pancreatic reflux. There was no significant change in the basal serum level of gastrin with any of these different types of alkaline reflux, but we observed a statistically significant increase in the level of histamine in the gastric mucosa. Hyperaemia and foveolar hyperplasia of the fundic mucosa both disappeared when the alkaline reflux was suppressed, and there was a statistically significant decrease in the basal serum level of gastrin and in the level of histamine in the gastric mucosa.