Lethal cardiac amyloidosis: Modification of the Congo Red technique on a forensic case.

Congo Red staining is usually used in diagnosing amyloidosis, a pathology characterized by the storage of abnormal proteins in several human organs. When assessed on samples fixated in formalin and embended in paraffin, this staining can undergo several artefacts, causing diagnostic and interpretative difficulties due to its weak stainability and a consequent reduced visibility of the amyloid. These complications, in time, requested several variations of this staining technique, especially in clinical practice, while in the forensic field no protocols has ever been adapted to cadaveric samples, a material that is already characteristically burdened by a peculiar stainability. In our work, studying a sudden death caused by cardiac amyloidosis and diagnosed only with post-mortem exams, we present a modified Congo Red staining used with the purpose to demonstrate amyloid in cadaveric material after the unsuccessfully use of all standard protocols.

Technical note: Improvement of cadaveric skin samples (with severe morphological alteration connected to putrefaction or injury) by an extended histological processing.

INTRODUCTION: The microscopic study and the interpretation of skin samples with advanced post-mortal phenomena or with particular destructive injuries is problematic for the forensic pathologist. In an attempt of restoring the histological architecture of cadaveric skin and overcoming these types of problem, the Authors performed a histological processing that was longer than the standard: it was extended until 62 days to evaluate the improvement of the microscopic morphological aspect. MATERIALS AND METHODS: Cutaneous samples were taken from 25 cadavers (5 typologies of skin: charred, putrified, corifed, mummified and partially skeletonized), fixed with a 10%-buffered formalin and then processed in two different ways: one half of the samples was routinely addressed to the standard-time automatic technique, while the other half was manually processed with prolonged times. All the slides were then stained in Hematoxylin-Eosin. RESULTS: The standard-processed slides demonstrated marked morphological alterations and artefacts at the microscopic observation; conversely, those processed with the prolonged manual technique showed an improvement in the morphological structure, sometimes permitting the identification of the anatomical components. CONCLUSION: Though it is characterized by the inconvenience of protracted times, the application of a long-term manual histological processing to cadaveric skin samples with advanced post-mortal alteration permits to better observe the anatomical architecture of skin and it could be useful and helpful in the evaluation of such cases.

The risk of misinterpreting genital signs of sexual abuse in cadavers: a case report.

The significance of genital findings in a case of suspected child sexual abuse has been widely debated in the past decades, as shown by the different classifications available in literature. In the case of postmortem examination, the search for signs of sexual abuse is considerably more difficult because of the superimposition of postmortem modifications, which may determine tissue modifications that can be mistaken for traumatic lesions. This study aims at reporting a case where presumed findings of the first autopsy were denied by histological analysis; in detail, what looked like a possible bruise of the hymen was correctly recognized as hypostasis (livor) of the hymenal tissue by histological analysis. This case report suggests caution in the analysis and discussion of genital lesions found during postmortem examination since the superimposition of cadaveric modifications may radically modify the morphology of soft tissues.

Sudden death from systemic sarcoidosis: a case of legal medicine.

INTRODUCTION: The sarcoid condition of vital organs such as heart, lungs, liver and brain, may constitute, though rarely, a dangerous situation for survival. In forensic pathology, sudden death related to such disease represents an unusual event occurring in subjects who die unexpectedly in spite of their previous good health, and whose autopsy reveals, mainly, the involvement of heart or the central nervous system (CNS). THE CASE: The Authors describe a case of sudden death due to systemic sarcoidosis with atypical presentation in a young South American nulliparous woman showing, as the only symptom, occasional episodes of spotting during the last two months of her life. RESULTS: The only noteworthy finding from the autopsy resulted in secondary obstructive hydrocephalus. The subsequent forensic toxicological examination was negative, whereas the histopathological examination, conducted by means of post-fixation techniques and standard coloring methods on the viscera removed during autopsy, revealed useful data to determine the cause of death, consisting of a diffuse inflammatory reaction with giant cells and epithelioid cells of sarcoid type localized in the CNS and in the genitourinary apparatus. CONCLUSIONS: The case presented, ranking among deaths occurred unexpectedly, is a useful opportunity both for clinical remarks, given its inherent diagnostic difficulties, especially in the presence of atypical symptoms, and for legal medicine ones, given the inclusion of sarcoidosis in cases of sudden death that can give rise to criminal investigations.

The use of the anti-Glycophorin A antibody in the detection of red blood cell residues in human soft tissue lesions decomposed in air and water: a pilot study.

The distinction between antemortem and postmortem wounds is one of the most important medico-legal problems. In fresh cadavers the macroscopic examination of haemorrhagic infiltration can be sufficient to reveal the vitality of a wound but in more difficult cases (putrefied corpses) histological and histochemical analyses need to be performed. The scope of this study was to detect the vitality of soft tissue samples in an advanced state of putrefaction using a monoclonal anti-human Glycoforin A antibody in order to evaluate the presence of red blood cells or red blood cell residues. Samples of skin wounds were taken from cadavers with a known time survival between trauma and death, and then submitted to a simulated putrefaction procedure. The skin samples were left to decompose for 30 days in air and in water and analyzed at a time interval of 3-6-15-30 days. These samples were stained with haematoxylin eosin stain, trichrome stain and with immunohistochemical stains. Results showed that in the air red blood cells could be seen for up to six days and granular deposits of Glycophorin reactive material for up to 15 days whereas no red blood cells were recognizable after 30 days. In water red cell putrefaction (or non-detection) was faster than in the air: after six days only amorphous granular deposits slightly reactive to Glycophorin could be seen. Nonetheless results showed that extravasated red blood cell residues can still be detected in skin bruises left to decompose in air and water, for up to at least 15 days in the open air and one week in water.

PI3KCA/PTEN deregulation contributes to impaired responses to cetuximab in metastatic colorectal cancer patients.

BACKGROUND: It has been reported that KRAS mutations (and to a lesser extent KRAS mutations with the BRAF V600E mutation) negatively affect response to anti-epidermal growth factor receptor (EGFR) mAbs in metastatic colorectal cancer (mCRC) patients, while the biological impact of the EGFR pathway represented by PI3K/PTEN/AKT on anti-EGFR treatment is still not clear. PATIENTS AND METHODS: We analysed formalin-fixed samples from a cohort of 32 mCRC patients treated with cetuximab by means of EGFR immunohistochemistry, EGFR and PTEN FISH analysis, and KRAS, BRAF, PI3KCA, and PTEN genomic sequencing. RESULTS: Ten (31%) of 32 patients showed a partial response to cetuximab and 22 (69%) did not [nonresponder (NR)]. EGFR immunophenotype and FISH-based gene status did not predict an anti-EGFR mAb response, whereas KRAS mutations (24%) and PI3K pathway activation, by means of PI3KCA mutations (13%) or PTEN mutation (10%)/loss (13%), were significantly restricted to, respectively, 41% and 37% of NRs. CONCLUSION: These findings suggested that KRAS mutations and PI3KCA/PTEN deregulation significantly correlate with resistance to cetuximab. In line with this, patients carrying KRAS mutations or with activated PI3K profiles can benefit from targeted treatments only by switching off molecules belonging to the downstream signalling of activated EGFR, such as mammalian target of rapamycin.

Tissue localization of TGFalpha and apoptosis are inversely related in colorectal tumors.

Expression of TGFalpha and the EGF receptor was studied in relation to apoptosis in human colorectal mucosa and premalignant and malignant tumors. In normal mucosa the proteins colocalized both in the proliferation compartment and at the luminal pole of the crypts in cells committed to undergo apoptosis. While staining for the EGF receptor was increased in premalignant and malignant lesions, TGFalpha was undetectable in aberrant crypt foci as well as large areas of adenomas. Incidence of apoptosis (AI) was high in these areas ranging from 8.83-24.59. Adenomas did, however, contain islands of high TGFalpha expression where AI was decreased to a range of 0.76-4.00 (decreased at P=0.0027). In carcinomas TGFalpha expression was increased above both normal and adenoma levels corresponding to the decrease in apoptosis in the malignant tumors. Tissue localization of TGFalpha and AI were still inversely related ( P=0.022), but interpatient variability was much larger than for adenomas. The data indicate that TGFalpha is the main survival factor in premalignant tumor cells of the colon, while additional factors moderate its effect in carcinomas. This suggests the possibility of targeting the EGF receptor pathway not only for treatment but also for the reversal of adenoma growth and the prevention of malignant colorectal tumors.

Surgical treatment of gastric metastases from cutaneous melanoma: experience of the National Cancer Institute of Milan.

AIM: To evaluate the role of a surgical approach in patients affected with gastric metastases from cutaneous melanoma. METHODS: A retrospective review of our local melanoma database of 2100 patients identified 31 cases with gastric metastatic deposits. Nine of them were considered candidates for surgical resection. RESULTS: Median overall survival of the 9 patients who underwent surgery was 14.2 months. Six (67%) underwent a local radical resection of disease, and 3 (33%) had a simple exploratory laparotomy. The median survival was 21.6 months (range, 4-32 months) for the subset receiving radical surgery and 3.6 months (range, 2-6 months) for the patients who had no resection. Median follow-up was 14.2 months. No specific correlation of serologic LDH levels and final outcome, as documented elsewhere, was observed. A marked decreased or substantial remission of symptoms with an improvement in quality of life was observed in all radically resected patients. CONCLUSIONS: Patients with gastric metastases from melanoma may benefit from surgery if all macroscopic disease can be removed. In addition, gastric resection in patients with symptomatic melanoma spread to the stomach provides important symptomatic palliation.

Adenocarcinoma of the lower third of the rectum surgically treated with a <10-MM distal clearance: preliminary results in 35 N0 patients.

BACKGROUND: Recent reports suggest that a distal clearance (DC) of 10 mm at the lower surgical margin may be considered adequate in the surgical treatment of rectal cancer, but there are no data on the possible adequacy of a < 10-mm DC in N0 patients in whom a good prognosis can otherwise be expected, that is, those with negative surgical margins and negative lymph nodes. METHODS: Between November 1991 and December 1998, 154 consecutive patients with adenocarcinoma of the lower third of the rectum had a total rectal resection with total mesorectal excision and coloendoanal anastomosis. Among 76 N0 patients, there were 35 with <10-mm DC and 41 with > or =10-mm DC. Each group was divided into two subgroups depending on whether the surgical margins were involved or not, and the rate of local recurrence in the various categories was compared. All B2 Astler-Coller stage patients in the series received postsurgical chemoradiotherapy. RESULTS: The local recurrence rate in the 35 patients with DC < 10 mm was 11.4% and that of the 41 patients with DC > or =10 mm was 7.3%. When only patients with negative surgical margins were considered, the local recurrence rate was 3.4% for those with < 10-mm DC and 5.1% for those with > or =10-mm DC. CONCLUSIONS: Our results suggest that a radical surgery with <10-mm DC followed by chemoradiotherapy may be adequate in N0 patients, provided that a careful pathologic examination of the surgical specimen excludes the presence of lymph node metastases and that the distal rectal and mesorectal resection margins fall in healthy tissue.

Adenocarcinoma of the lower third of the rectum: metastases in lymph nodes smaller than 5 mm and occult micrometastases; preliminary results on early tumor recurrence.

BACKGROUND: The number of examined lymph nodes and metastases in lymph nodes smaller than 5 mm (small lymph nodes) are a determining factor in the stage of rectal cancer although the clinical significance of occult micrometastases is controversial. We are reporting our preliminary results on the identification and prognostic utility of metastases in small lymph nodes and occult micrometastases. METHODS: We searched small metastatic lymph nodes in 101 cases of adenocarcinoma of the lower third of the rectum. We used the manual technique to dissect mesorectal fat and occult micrometastases in the lymph nodes of 52 Dukes' A and B patients, using a pool of anticytokeratin antibodies. RESULTS: Forty-five percent of the metastatic lymph nodes were smaller than 5 mm in diameter and determined the Dukes' stage in 15 (30.6%) of 49 Dukes' C patients. Occult micrometastases were found in 21 (40.4%) patients: five recurred but vascular invasion, positive distal margin of the rectum, and positive circumferential margin of the mesorectum were present. CONCLUSIONS: Small metastatic lymph nodes, vascular invasion, positive distal margin of the rectum, and positive circumferential margin of the mesorectum were found to be more important than occult micrometastases in predicting early recurrence of rectal cancer.

Effect of lifestyle, smoking, and diet on development of intestinal metaplasia in H. pylori-positive subjects.

OBJECTIVES: This study aimed to evaluate the influence of environmental and sociodemographic factors and the effect of smoking, alcohol, and dietary habits on the risk of gastric intestinal metaplasia (IM) in Helicobacter pylori-infected subjects. METHODS: The investigation was based on 2598 consecutive volunteer blood donors tested for the presence of antibodies against H. pylori from March 1995 to March 1997. Endoscopy with multiple biopsies was offered to all H. pylori-positive, symptomatic subjects. The presence or absence of IM was diagnosed by gastric biopsies. A serologically H. pylori-positive subject with gastric IM was defined as a case, whereas serologically H. pylori-positive subjects without IM were used as controls. All patients answered a detailed questionnaire collecting sociodemographic characteristics and smoking, alcohol drinking, and dietary habits. Odds ratios (ORs) and their 95% CIs were estimated by unconditional logistic regression, including terms for age and sex, to assess the association between the data collected and IM. RESULTS: Three hundred forty-four subjects with serological H. pylori infection and upper-GI symptoms underwent GI endoscopy, during which biopsies were taken for histological diagnosis. Histology revealed metaplasia in 74 subjects (21.5%). Incomplete IM was found in 37.8% of these cases. No significant associations were found between IM and anthropometric or sociodemographic factors. There was a significant association between age and IM (chi2 for trend, 6.67; p value, 0.009). Current smokers of over 20 cigarettes per day had a 4-fold risk of IM (OR, 4.75, 95% CI, 1.33-16.99). A 2-fold increased risk was found for high butter consumers (OR, 2.17; 95% CI, 1.14-4.11). No significant specific associations were found between the variables studied and complete or incomplete IM. CONCLUSIONS: This study found that smoking and high butter consumption may increase the risk of having gastric IM in H. pylori-positive subjects.

Double-contrast barium enema and computerised tomography in the pre-operative evaluation of rectal carcinoma: are they still useful diagnostic procedures?

PURPOSE: In modern management of rectal carcinoma, the preoperative evaluation of disease parameters is important for selection of therapeutic options. Such parameters are currently defined through endorectal ultrasonography or endoscopic ultrasonography. A retrospective analysis of the parameters obtained with double-contrast barium enema (DCBE) and endorectal balloon computed tomography (CT) was conducted to verify the diagnostic reliability of the radiological techniques and to establish whether there is still an indication for their use. METHODS: 53 consecutive patients with adenocarcinoma of the distal half of the rectal ampulla underwent double contrast barium enema examination and CT of the pelvis with endorectal balloon. On the basis of the DCBE and CT assessment we evaluated: 1) the distance between the cranial extremity of the anal canal and the distal margin of the neoplasm; 2) the radial diffusion of the tumor; 3) the metastatic involvement of the perirectal and inferior mesenteric lymph nodes. RESULTS: 1) CT and DBCE measurements of the distal margin tended to coincide, but both tended to overestimate the measurement when compared to the pathologic examination; 2) in the identification of neoplastic infiltration of perirectal fat (T3) CT had 100% sensitivity, 78.7% specificity and 86.8% accuracy; 3) the CT sensitivity for detecting lymph node metastasis was 52.6%, specificity 85.3% and accuracy 73.6%. CONCLUSIONS: The diagnostic information provided by the radiological examinations is comparable to that of clinical and instrumental methods currently employed for staging of rectal carcinoma, although the latter are preferred because they are more readily accessible and less costly. DCBE and CT can therefore be usefully employed for staging of cancer of the rectum in those cases in which there are limitations of the current standard methods.

T cell responses in colorectal cancer patients: evidence for class II HLA-restricted recognition of shared tumor-associated antigens.

Few cases of anti-colon cancer specific T lymphocytes have been described so far. Moreover, the majority of these effectors were generated in vitro by stimulating PBMC from patients or healthy donors with peptides that were derived from proteins expressed and/or secreted by colon cancer tissue such as CEA, Mucin or Her-2/neu. The aim of our study was to evaluate the immunogenicity of colorectal carcinomas in an autologous setting. We exploited the antigen processing and presentation capacity of dendritic cells (DC) to establish an in vitro autologous system that can bypass the need of obtaining cultured tumor cells. DC were generated from the adherent monocyte fraction of PBMC taken from stage II/III colorectal cancer patients. A single cell suspension was prepared by mechanical and enzymatic disruption of the surgical specimens immediately after resection. DC were loaded with autologous tumor lysate, obtained by repeated freezing and thawing, before being used as stimulators for autologous PBL. HLA-class II restricted T cells that recognize the autologous tumor could be generated in a proportion of patients. The fine specificity of the anti-tumor T cells indicates that differentiation as well as tumor restricted antigens are expressed in colon cancer and that these antigens can evoke a class II HLA-restricted response in an autologous setting. Altogether these findings may open a new perspective for a DC based vaccination of colon cancer patients.

Preoperative endoscopic ultrasonography in patients with gastric cancer.

AIMS AND BACKGROUND: There is a need to assess the accuracy of endoscopic ultrasonography (EUS) in the diagnosis and staging of gastric cancer, especially in the early and very advanced stages of the disease when the therapeutic approach is still controversial. METHODS: A retrospective study was performed on 79 patients with gastric cancer in order to compare the stage defined by preoperative EUS with that assessed histopathologically. All patients underwent laparotomy for final diagnosis, staging, and eventually treatment. The results of EUS were correlated with the histologic findings of the resected specimens. RESULTS: In the uT1 group, which corresponds to early gastric cancer, the diagnosis was histologically confirmed in 85.7% of the cases. In patients with advanced tumors defined as uT3-uT4, i.e., tumors infiltrating the serosa or neighboring structures, the diagnostic concordance was 91.1%. In contrast, concordance for less advanced lesions confined to the muscular layer was only 31.2%. As regards the lymph nodes, they were defined metastatic in 31 patients and confirmed to be histologically involved in 77.4%. In contrast, when the lymph nodes were assessed as negative at EUS, they proved to be metastatic in more than half the cases. CONCLUSIONS: From the data it appears that EUS has proven to be valuable in correctly staging most of the patients. EUS shows not only tumor depth and local spread but also the passage from a pathologic to a normal wall and lymph node metastasis. EUS appears to represent an important advance in the staging and follow-up of patients with gastric cancer. Instruments and techniques will continue to evolve, but the next level of research should be designed to show that the improved staging provided by EUS has clinical utility and can affect patient outcome. It is noteworthy that the highest accuracy of EUS has been shown in those conditions (uT1 and uT3-4) which currently are under consideration for a therapeutic approach that differs from the standard one.

Total rectal resection and complete mesorectum excision followed by coloendoanal anastomosis as the optimal treatment for low rectal cancer: the experience of the National Cancer Institute of Milano.

BACKGROUND: At present, abdominoperineal resection remains the most diffuse method of treatment of very low rectal cancer. Today, we can avoid this method in some patients by using a sphincter-saving procedure. METHODS: From March 1990 to January 1999, 273 consecutive total rectal resections and coloendoanal anastomoses were performed at our Institute; this study concerns 141 consecutive patients treated for a primary adenocarcinoma of the distal rectum, from 3.5 to 8 cm from the anal verge. Patient stratification, based on definitive pathological report, was 31 Dukes' stage A (T2N0), 44 stage B (T3N0), and 66 stage C (T2N+-T3N+). RESULTS: Overall recurrence rate was 9.2%; postoperative morbidity attributable to the procedure was low. A perfect continence was documented in 61% of cases. The only pathological factor related to local recurrence rate is peritumoral lymphocytic reaction inside and around the tumor (P = .0005 and .031) independently from the number of metastatic lymph nodes, depth of fatty tissue infiltration, and lymphatic and venous neoplastic emboli. The minimum follow-up time is 12 months. CONCLUSIONS: Our data, in accordance with other authors, seem to highlight the relevant role that a well-practiced surgery, together with accurate information on the spreading of this disease, has in achieving an optimal local control of cancer.

Early gastric cancer: unrecognized indicator of multiple malignancies.

The aim of this study was to evaluate the postoperative outcome and occurrence of second primary malignancies in patients who underwent radical resection for early gastric cancer. Altogether 105 patients with early gastric cancer underwent radical resection and were followed up for a median period of 71 months. Overall lethality was analyzed with regard to mortality due to gastric cancer or other causes. Standardized mortality and morbidity ratios were calculated using data from the Tumor Registry of Lombardy. The 5-year survival rate was 82.8% (operative mortality excluded), which was not different from that expected from an age- and sex-matched Italian population. Ten second primary malignancies were identified, giving a standardized mortality ratio of 1.12 and a standardized morbidity ratio of 1.50. Patients undergoing surgery for early gastric cancer (especially the intestinal type) may have a circa 50% higher risk of a second tumor and should be periodically checked for other tumors, mainly in the supramesocolic area.

Microsatellite instability in colorectal-cancer patients with suspected genetic predisposition.

Hereditary non-polyposis colorectal cancer (HNPCC) is a dominantly inherited syndrome linked to DNA-mismatch-repair (MMR) gene defects, which also account for microsatellite instability (MSI) in tumour tissues. Diagnosis is based mainly on family history, according to widely accepted criteria (Amsterdam Criteria: AC). Aim of this work was to assess MSI in colorectal-cancer patients with suspected genetic predisposition, and to verify whether MSI represents a tool to manage MMR gene (hMSH2 and hMLH1) mutation analysis. We investigated 13 microsatellites (including the 5 NCI/ICG-HNPCC markers) in 45 patients with suspected hereditary predisposition (including 16 subjects from HNPCC families fulfilling the AC). We found MSI-H (high frequency of instability, i.e., in > or =30% of the markers) in 85% of the HNPCC patients and in 16% of the non-HNPCC subjects. The 5 NCI/ICG-HNPCC microsatellites proved to be the most effective in detecting MSI, being mononucleotide repeats the most unstable markers. We investigated the association between hMSH2- and hMLH1 gene mutations and MSI. Our results indicate that AC are highly predictive both of tumour instability and of MMR-gene mutations. Therefore, as the most likely mutation carriers, HNPCC subjects might be directly analyzed for gene mutations, while to test for MSI in selected non-HNPCC patients and to further investigate MMR genes in MSI-H cases, appears to be a cost-effective way to identify subjects, other than those from kindred fulfilling AC, who might benefit from genetic testing.

Prognostic effects of lymph node micrometastases in patients undergoing curative gastrectomy for cancer.

AIMS & BACKGROUND: It is not known whether the presence of micrometastases in the regional lymph nodes has an impact on the oncologic outcome of patients undergoing a curative (R0) gastrectomy for cancer. The aim of the study was to assess the effects of the presence of micrometastases on survival. METHODS: We reviewed 29 patients operated on for curative (R0) gastrectomy, with a final diagnosis of pN0, 16 or more lymph nodes in the specimen, and a follow-up of at least 4 years. The original hemotoxylin and eosin slides were reviewed, and a new section was cut from the lymph nodes and immunostained with a pool of antibodies against different types of cytokeratins. Micrometastases were detected in 5 patients (27.5% of the series) and 11 lymph nodes (1.51% of all removed lymph nodes). RESULTS: Mortality due to cancer progression occurred in 3 patients from the pN0 group (14.2%) and 1 patient from the pN1 group (12.5%). CONCLUSIONS: There was no suggestion from the data that the presence of micrometastases carries an ominous prognosis in terms of survival.