Establishment of patient-derived tumor organoids to functionally inform treatment decisions in metastatic colorectal cancer.

BACKGROUND: Metastatic colorectal cancer (mCRC) patients tend to have modest benefits from molecularly driven therapeutics. Patient-derived tumor organoids (PDTOs) represent an unmatched model to elucidate tumor resistance to therapy, due to their high capacity to resemble tumor characteristics. MATERIALS AND METHODS: We used viable tumor tissue from two cohorts of patients with mCRC, naïve or refractory to treatment, respectively, for generating PDTOs. The derived models were subjected to a 6-day drug screening assay (DSA) with a comprehensive pipeline of chemotherapy and targeted drugs against almost all the actionable mCRC molecular drivers. For the second cohort DSA data were matched with those from PDTO genotyping. RESULTS: A total of 40 PDTOs included in the two cohorts were derived from mCRC primary tumors or metastases. The first cohort included 31 PDTOs derived from patients treated in front line. For this cohort, DSA results were matched with patient responses. Moreover, RAS/BRAF mutational status was matched with DSA cetuximab response. Ten out of 12 (83.3%) RAS wild-type PDTOs responded to cetuximab, while all the mutant PDTOs, 8 out of 8 (100%), were resistant. For the second cohort (chemorefractory patients), we used part of tumor tissue for genotyping. Four out of nine DSA/genotyping data resulted applicable in the clinic. Two RAS-mutant mCRC patients have been treated with FOLFOX-bevacizumab and mitomycin-capecitabine in third line, respectively, based on DSA results, obtaining disease control. One patient was treated with nivolumab-second mitochondrial-derived activator of caspases mimetic (phase I trial) due to high tumor mutational burden at genotyping, experiencing stable disease. In one case, the presence of BRCA2 mutation correlated with DSA sensitivity to olaparib; however, the patient could not receive the therapy. CONCLUSIONS: Using CRC as a model, we have designed and validated a clinically applicable methodology to potentially inform clinical decisions with functional data. Undoubtedly, further larger analyses are needed to improve methodology success rates and propose suitable treatment strategies for mCRC patients.

Immune microenvironment characterisation and dynamics during anti-HER2-based neoadjuvant treatment in HER2-positive breast cancer.

Despite their recognised role in HER2-positive (HER2+) breast cancer (BC), the composition, localisation and functional orientation of immune cells within tumour microenvironment, as well as its dynamics during anti-HER2 treatment, is largely unknown. We here investigate changes in tumour-immune contexture, as assessed by stromal tumour-infiltrating lymphocytes (sTILs) and by multiplexed spatial cellular phenotyping, during treatment with lapatinib-trastuzumab in HER2+ BC patients (PAMELA trial). Moreover, we evaluate the relationship of tumour-immune contexture with hormone receptor status, intrinsic subtype and immune-related gene expression. sTIL levels increase after 2 weeks of HER2 blockade in HR-negative disease and HER2-enriched subtype. This is linked to a concomitant increase in cell density of all four immune subpopulations (CD3+, CD4+, CD8+, Foxp3+). Moreover, immune contexture analysis showed that immune cells spatially interacting with tumour cells have the strongest association with response to anti-HER2 treatment. Subsequently, sTILs consistently decrease at the surgery in patients achieving pathologic complete response, whereas most residual tumours at surgery remain inflamed, possibly reflecting a progressive loss of function of T cells. Understanding the features of the resulting tumour immunosuppressive microenvironment has crucial implications for the design of new strategies to de-escalate or escalate systemic therapy in early-stage HER2+ BC.

Fusobacterium nucleatum persistence and risk of recurrence after preoperative treatment in locally advanced rectal cancer.

BACKGROUND: Accumulating evidence has identified Fusobacterium as an important pathogenic gut bacterium associated with colorectal cancer. Nevertheless, only limited data exist about the role of this bacterium in locally advanced rectal cancer (LARC). In this study, we quantified Fusobacterium nucleatum in untreated and post-neoadjuvant chemoradiotherapy (nCRT) samples from LARC patients and investigated its association with therapy response and survival. PATIENTS AND METHODS: A total of 254 samples from 143 patients with rectal adenocarcinomas were analyzed for the presence and abundance of F. nucleatum using RNA in situ hybridization and digital image analysis. Assay accuracy was determined using infected cell lines and tumor samples with available quantitative PCR data. We studied the impact of F. nucleatum load on pathologic complete response and relapse-free survival. Treatment-induced changes were evaluated in paired pre- and post-nCRT samples (n = 71). Finally, tumor microenvironment changes during nCRT were assessed in paired samples (n = 45) by immune contexture analysis. RESULTS: F. nucleatum tissue levels by RNA in situ hybridization strongly correlated with quantitative PCR (r = 0.804, P < 0.001). F. nucleatum abundance was higher in untreated [median, 7.4; 95% confidence interval (3.7-16.2)] compared with treated [median, 1.6; 95% confidence interval (1.3-2.4)] tumors (P <0.001) with 58% (73/126) and 26% (22/85) positive tumors, respectively (P < 0.001). Baseline F. nucleatum levels were not associated with pathologic complete response. F. nucleatum positivity after nCRT, but not baseline status, significantly increased risk of relapse [hazard ratio = 7.5, 95% confidence interval (3.0-19.0); P < 0.001]. Tumors that turned F. nucleatum-negative after nCRT had a strong increase in CD8+ T cells post-nCRT (P < 0.001), while those that persisted F. nucleatum-positive after nCRT lacked CD8+ T cells induction in post-nCRT samples compared with baseline (P = 0.69). CONCLUSION: F. nucleatum persistence post-nCRT is associated with high relapse rates in LARC, potentially linked to suppression of immune cytotoxicity.

RAD51 foci as a functional biomarker of homologous recombination repair and PARP inhibitor resistance in germline BRCA-mutated breast cancer.

Background: BRCA1 and BRCA2 (BRCA1/2)-deficient tumors display impaired homologous recombination repair (HRR) and enhanced sensitivity to DNA damaging agents or to poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi). Their efficacy in germline BRCA1/2 (gBRCA1/2)-mutated metastatic breast cancers has been recently confirmed in clinical trials. Numerous mechanisms of PARPi resistance have been described, whose clinical relevance in gBRCA-mutated breast cancer is unknown. This highlights the need to identify functional biomarkers to better predict PARPi sensitivity. Patients and methods: We investigated the in vivo mechanisms of PARPi resistance in gBRCA1 patient-derived tumor xenografts (PDXs) exhibiting differential response to PARPi. Analysis included exome sequencing and immunostaining of DNA damage response proteins to functionally evaluate HRR. Findings were validated in a retrospective sample set from gBRCA1/2-cancer patients treated with PARPi. Results: RAD51 nuclear foci, a surrogate marker of HRR functionality, were the only common feature in PDX and patient samples with primary or acquired PARPi resistance. Consistently, low RAD51 was associated with objective response to PARPi. Evaluation of the RAD51 biomarker in untreated tumors was feasible due to endogenous DNA damage. In PARPi-resistant gBRCA1 PDXs, genetic analysis found no in-frame secondary mutations, but BRCA1 hypomorphic proteins in 60% of the models, TP53BP1-loss in 20% and RAD51-amplification in one sample, none mutually exclusive. Conversely, one of three PARPi-resistant gBRCA2 tumors displayed BRCA2 restoration by exome sequencing. In PDXs, PARPi resistance could be reverted upon combination of a PARPi with an ataxia-telangiectasia mutated (ATM) inhibitor. Conclusion: Detection of RAD51 foci in gBRCA tumors correlates with PARPi resistance regardless of the underlying mechanism restoring HRR function. This is a promising biomarker to be used in the clinic to better select patients for PARPi therapy. Our study also supports the clinical development of PARPi combinations such as those with ATM inhibitors.

A predictive model of pathologic response based on tumor cellularity and tumor-infiltrating lymphocytes (CelTIL) in HER2-positive breast cancer treated with chemo-free dual HER2 blockade.

Background: The presence of stromal tumor-infiltrating lymphocytes (TILs) is associated with increased pathologic complete response (pCR) and improved outcomes in HER2-positive early-breast cancer (BC) treated with anti-HER2-based chemotherapy. In the absence of chemotherapy, the association of TILs with pCR following anti-HER2 therapy-only is largely unknown. Patients and methods: The PAMELA neoadjuvant trial treated 151 women with HER2-positive BC with lapatinib and trastuzumab [and hormonal therapy if hormone receptor (HR)-positive] for 18 weeks. Percentage of TILs and tumor cellularity were determined at baseline (N = 148) and at day 15 (D15) of treatment (N = 134). Associations of TILs and tumor cellularity with pCR in the breast were evaluated. A combined score based on tumor cellularity and TILs (CelTIL) measured at D15 was derived in PAMELA, and validated in D15 samples from 65 patients with HER2-positive disease recruited in the LPT109096 neoadjuvant trial, where anti-HER2 therapy-only was administer for 2 weeks, then standard chemotherapy was added for 24 weeks. Results: In PAMELA, baseline and D15 TILs were significantly associated with pCR in univariate analysis. In multivariable analysis, D15 TILs, but not baseline TILs, were significantly associated with pCR. At D15, TILs and tumor cellularity were found independently associated with pCR. A combined score (CelTIL) taking into account both variables was derived. CelTIL at D15 as a continuous variable was significantly associated with pCR, and patients with CelTIL-low and CelTIL-high scores had a pCR rate of 0% and 33%, respectively. In LPT109096, CelTIL at D15 was found associated with pCR both as a continuous variable and as group categories using a pre-defined cut-off (75.0% versus 33.3%). Conclusions: On-treatment TILs, but not baseline TILs, are independently associated with response following anti-HER2 therapy-only. A combined score of TILs and tumor cellularity measured at D15 provides independent predictive information upon completion of neoadjuvant anti-HER2-based therapy. Clinical trial number: NCT01973660.

Dual MET and ERBB inhibition overcomes intratumor plasticity in osimertinib-resistant-advanced non-small-cell lung cancer (NSCLC).

BACKGROUND: Third-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) such as osimertinib are the last line of targeted treatment of metastatic non-small-cell lung cancer (NSCLC) EGFR-mutant harboring T790M. Different mechanisms of acquired resistance to third-generation EGFR-TKIs have been proposed. It is therefore crucial to identify new and effective strategies to overcome successive acquired mechanisms of resistance. METHODS: For Amplicon-seq analysis, samples from the index patient (primary and metastasis lesions at different timepoints) as well as the patient-derived orthotopic xenograft tumors corresponding to the different treatment arms were used. All samples were formalin-fixed paraffin-embedded, selected and evaluated by a pathologist. For droplet digital PCR, 20 patients diagnosed with NSCLC at baseline or progression to different lines of TKI therapies were selected. Formalin-fixed paraffin-embedded blocks corresponding to either primary tumor or metastasis specimens were used for analysis. For single-cell analysis, orthotopically grown metastases were dissected from the brain of an athymic nu/nu mouse and cryopreserved at -80°C. RESULTS: In a brain metastasis lesion from a NSCLC patient presenting an EGFR T790M mutation, we detected MET gene amplification after prolonged treatment with osimertinib. Importantly, the combination of capmatinib (c-MET inhibitor) and afatinib (ErbB-1/2/4 inhibitor) completely suppressed tumor growth in mice orthotopically injected with cells derived from this brain metastasis. In those mice treated with capmatinib or afatinib as monotherapy, we observed the emergence of KRAS G12C clones. Single-cell gene expression analyses also revealed intratumor heterogeneity, indicating the presence of a KRAS-driven subclone. We also detected low-frequent KRAS G12C alleles in patients treated with various EGFR-TKIs. CONCLUSION: Acquired resistance to subsequent EGFR-TKI treatment lines in EGFR-mutant lung cancer patients may induce genetic plasticity. We assess the biological insights of tumor heterogeneity in an osimertinib-resistant tumor with acquired MET-amplification and propose new treatment strategies in this situation.

Concordance of blood- and tumor-based detection of RAS mutations to guide anti-EGFR therapy in metastatic colorectal cancer.

BACKGROUND: Circulating tumor DNA (ctDNA) is a potential source for tumor genome analysis. We explored the concordance between the mutational status of RAS in tumor tissue and ctDNA in metastatic colorectal cancer (mCRC) patients to establish eligibility for anti-epidermal growth factor receptor (EGFR) therapy. PATIENTS AND METHODS: A prospective-retrospective cohort study was carried out. Tumor tissue from 146 mCRC patients was tested for RAS status with standard of care (SoC) PCR techniques, and Digital PCR (BEAMing) was used both in plasma and tumor tissue. RESULTS: ctDNA BEAMing RAS testing showed 89.7% agreement with SoC (Kappa index 0.80; 95% CI 0.71 - 0.90) and BEAMing in tissue showed 90.9% agreement with SoC (Kappa index 0.83; 95% CI 0.74 - 0.92). Fifteen cases (10.3%) showed discordant tissue-plasma results. ctDNA analysis identified nine cases of low frequency RAS mutations that were not detected in tissue, possibly due to technical sensitivity or heterogeneity. In six cases, RAS mutations were not detected in plasma, potentially explained by low tumor burden or ctDNA shedding. Prediction of treatment benefit in patients receiving anti-EGFR plus irinotecan in second- or third-line was equivalent if tested with SoC PCR and ctDNA. Forty-eight percent of the patients showed mutant allele fractions in plasma below 1%. CONCLUSIONS: Plasma RAS determination showed high overall agreement and captured a mCRC population responsive to anti-EGFR therapy with the same predictive level as SoC tissue testing. The feasibility and practicality of ctDNA analysis may translate into an alternative tool for anti-EGFR treatment selection.

Single dose bioavailability and pharmacokinetic study of a innovative formulation of α-lipoic acid (ALA600) in healthy volunteers.

AIM: α-Lipoic acid is an important micronutrient with several pharmacological as well as antioxidant properties. The present study was aimed to examine the human bioavailability, pharmacokinetics (PK) and tolerability of an innovative oral formulation (ALA600) containing racemic α-lipoic acid 600 mg. METHODS: After a single 600-mg oral administration in healthy volunteers, blood samples were collected up to 8 hours post dosing, and plasma α-lipoic acid concentrations were determined by Liquid Chromatography-Mass Spectrometry (LC-MS) detection. RESULTS: The PK data revealed a short time to reach plasma peak oncentrations (50.8± 4.2 min) with a C(max) of 6.86±1.29 µg/mL. The C(max) implying that the new pharmaceutical form positively influences absorption and absorption time. The AUC value of 5.65±0.79 µg/mL*h is the more reliable measure of new formulation bioavailability. The half-life and MRT values further show that new formulation is absorbed consistently and rapidly and is eliminated efficiently. These PK data appear to promote further refinement of present formulation. Should the authors compare the obtained data with the recent published data, the new formulation of α-lipoic acid tends to show an improvement of C(max) value (2.5-5.4 times) and AUC (1.8 times). CONCLUSION: ALA600 formulation is characterized by rapid absorption, high bioavailability, brief half-life and low toxicity. These PK parameters could significantly increase clinical use of lipoic acid with improvement of the therapeutic effects at the cellular level and might also prove to be the most suitable formulation for chronic administration such as peripheral neuropathies.

Sebaceous lymphadenoma of salivary gland: a case report and a review of the literature.

The unusual case is described of a benign parotid gland neoplasm with intermingled sebaceous and lymphoid tissue, synchronous to breast cancer. In the past, the patient had undergone a simple surgical procedure for a cystic parotid gland lesion in that same gland. Secondary neoplasms have only occasionally been reported, since there are few cases for corroborating the strong correlation between salivary neoplasms and other carcinomas as in Muir-Torre syndrome; the previous cystic lesion showed the origin of the neoplasm from a sebaceous inclusion in the lymph node as a postulate of Warthin tumour.

Spermatic cord liposarcoma: a report of four cases.

Spermatic cord liposarcoma is a rare pathology (1-4); currently about one hundred cases are documented. The therapy of choice is surgery, followed sometimes by radiotherapy. We herein describe our experience of 4 cases between 1995 and 2000, with median follow-up of 34 months (mean 48 months, range 28-95 months), in order to stress the role of orchifuniculectomy, even when mass-ablation first procedure may seem radical.

Mitochondrial DNA mutations and mitochondrial abnormalities in dilated cardiomyopathy.

Mitochondrial (mt)DNA defects, both deletions and tRNA point mutations, have been associated with cardiomyopathies. The aim of the study was to determine the prevalence of pathological mtDNA mutations and to assess associated defects of mitochondrial enzyme activity in dilated cardiomyopathy (DCM) patients with ultrastructural abnormalities of cardiac mitochondria. In a large cohort of 601 DCM patients we performed conventional light and electron microscopy on endomyocardial biopsy samples. Cases with giant organelles, angulated, tubular, and concentric cristae, and crystalloid or osmiophilic inclusion bodies were selected for mtDNA analysis. Mutation screening techniques, automated DNA sequencing, restriction enzyme digestion, and densitometric assays were performed to identify mtDNA mutations, assess heteroplasmy, and quantify the amount of mutant in myocardial and blood DNA. Of 601 patients (16 to 63 years; mean, 43.5 +/- 12.7 years), 85 had ultrastructural evidence of giant organelles, with abnormal cristae and inclusion bodies; 19 of 85 (22.35%) had heteroplasmic mtDNA mutations (9 tRNA, 5 rRNA, and 4 missense, one in two patients) that were not found in 111 normal controls and in 32 DCM patients without the above ultrastructural mitochondrial abnormalities. In all cases, the amount of mutant was higher in heart than in blood. In hearts of patients that later underwent transplantation, cytochrome c oxidase (Cox) activity was significantly lower in cases with mutations than in those without or controls (P = 0.0008). NADH dehydrogenase activity was only slightly reduced in cases with mutations (P = 0.0388), whereas succinic dehydrogenase activity did not significantly differ between DCM patients with mtDNA mutations and those without or controls. The present study represents the first attempt to detect a morphological, easily identifiable marker to guide mtDNA mutation screening. Pathological mtDNA mutations are associated with ultrastructurally abnormal mitochondria, and reduced Cox activity in a small subgroup of non-otherwise-defined, idiopathic DCMs, in which mtDNA defects may constitute the basis for, or contribute to, the development of congestive heart failure.

Evidence that amyloidogenic light chains undergo antigen-driven selection.

AL amyloidosis is characterized by fibrillar tissue deposits (amyloid) composed of monoclonal light chains secreted by small numbers of indolent bone marrow plasma cells whose ontogenesis is unknown. To address this issue and to provide insights into the processes that accompanied pathogenic light chain formation, we isolated the complete variable (V) regions of 14 light (VL) and 3 heavy (VH) chains secreted by amyloid clones at diagnosis (10 Bence Jones and 4 with complete Igs, 9 lambda and 5 kappa) by using an inverse polymerase chain reaction-based approach free of primer-induced biases. Amyloid V regions were found to be highly mutated compared with the closest germline genes in the databases or those isolated from the patients' DNA, and mutations were not associated with intraclonal diversification. Apparently high usage of the lambdaIII family germline gene V lambdaIII.1 was observed (4 of 9 lambda light chains). Analysis of the nature and distribution of somatic mutations in amyloid V regions showed that there was statistical evidence of antigen selection in 8 of 14 clones (7 in VL and 1 in VH). These results indicate that a substantial proportion of the amyloid clones developed from B cells selected for improved antigen binding properties and that pathogenic light chains show evidence of this selection.

Restrictive cardiomyopathy, atrioventricular block and mild to subclinical myopathy in patients with desmin-immunoreactive material deposits.

OBJECTIVES: We present clinical data and heart and skeletal muscle biopsy findings from a series of patients with ultrastructural accumulations of granulofilamentous material identified as desmin. BACKGROUND: Desmin cardiomyopathy is a poorly understood disease characterized by abnormal desmin deposits in cardiac and skeletal muscle. METHODS: Clinical evaluation, endomyocardial and skeletal muscle biopsy, light and electron microscopy and immunohistochemistry were used to establish the presence of desmin cardiomyopathy. RESULTS: Six hundred thirty-one patients with primary cardiomyopathy underwent endomyocardial biopsy (EMB). Ultrastructural accumulations of granulofilamentous material were found in 5 of 12 biopsy samples from patients with idiopathic restrictive cardiomyopathy and demonstrated specific immunoreactivity with anti-desmin antibodies by immunoelectron microscopy. Immunohistochemical findings on light microscopy were nonspecific because of a diffuse intracellular distribution of desmin. All five patients had atrioventricular (AV) block and mild or subclinical myopathy. Granulofilamentous material was present in skeletal muscle biopsy samples in all five patients, and unlike the heart biopsy samples, light microscopic immunohistochemical analysis demonstrated characteristic subsarcolemmal desmin deposits. Two patients were first-degree relatives (mother and son); another son with first-degree AV block but without myopathy or cardiomyopathy demonstrated similar light and ultrastructural findings in skeletal muscle. Electrophoretic studies demonstrated two isoforms of desmin--one of normal and another of lower molecular weight--in cardiac and skeletal muscle of the familial cases. CONCLUSIONS: Desmin cardiomyopathy must be considered in the differential diagnosis of restrictive cardiomyopathy, especially in patients with AV block and myopathy. Diagnosis depends on ultrastructural examination of EMB samples or light microscopic immunohistochemical studies of skeletal muscle biopsy samples. Familial desminopathy may manifest as subclinical disease and may be associated with abnormal isoforms of desmin.

Coexistence of mitochondrial DNA and beta myosin heavy chain mutations in hypertrophic cardiomyopathy with late congestive heart failure.

OBJECTIVE: To investigate the possible coexistence of mitochondrial DNA (mtDNA) mutations in patients with beta myosin heavy chain (beta MHC) linked hypertrophic cardiomyopathy (HCM) who develop congestive heart failure. DESIGN: Molecular analysis of beta MHC and mtDNA gene defects in patients with HCM. SETTING: Cardiovascular molecular diagnostic and heart transplantation reference centre in north Italy. PATIENTS: Four patients with HCM who underwent heart transplantation for end stage heart failure, and after pedigree analysis of 60 relatives, eight additional affected patients and 27 unaffected relatives. A total of 111 unrelated healthy adult volunteers served as controls. Disease controls included an additional 27 patients with HCM and 102 with dilated cardiomyopathy. INTERVENTION: Molecular analysis of DNA from myocardial and skeletal muscle tissue and from peripheral blood specimens. MAIN OUTCOME MEASURES: Screening for mutations in beta MHC (exons 3-23) and mtDNA tRNA (n = 22) genes with denaturing gradient gel electrophoresis or single strand conformational polymorphism followed by automated DNA sequencing. RESULTS: One proband (kindred A) (plus seven affected relatives) had arginine 249 glutamine (Arg249Gln) beta MHC and heteroplasmic mtDNA tRNAIle A4300G mutations. Another unrelated patient (kindred B) with sporadic HCM had identical mutations. The remaining two patients (kindred C), a mother and son, had a novel beta MHC mutation (lysine 450 glutamic acid) (Lys450Glu) and a heteroplasmic missense (T9957C, phenylalanine (Phe)-->leucine (Leu)) mtDNA mutation in subunit III of the cytochrome C oxidase gene. The amount of mutant mtDNA was higher in the myocardium than in skeletal muscle or peripheral blood and in affected patients than in asymptomatic relatives. Mutations were absent in the controls. Pathological and biochemical characteristics of patients with mutations Arg249Gln plus A4300G (kindreds A and B) were identical, but different from those of the two patients with Lys450Glu plus T9957C(Phe-->Leu) mutations (kindred C). Cytochrome C oxidase activity and histoenzymatic staining were severely decreased in the two patients in kindreds A and B, but were unaffected in the two in kindred C. CONCLUSIONS: beta MHC gene and mtDNA mutations may coexist in patients with HCM and end stage congestive heart failure. Although beta MHC gene mutations seem to be the true determinants of HCM, both mtDNA mutations in these patients have known prerequisites for pathogenicity. Coexistence of other genetic abnormalities in beta MHC linked HCM, such as mtDNA mutations, may contribute to variable phenotypic expression and explain the heterogeneous behaviour of HCM.

Enteroviral RNA and virus-like particles in the skeletal muscle of patients with idiopathic dilated cardiomyopathy.

The role of chronic viral infection in the etiopathogenesis of idiopathic dilated cardiomyopathy (IDC) has generated considerable research. Enteroviruses were the favorite candidates as etiologic agents of IDC. However, enteroviruses were rarely demonstrated in affected hearts. We investigated whether enteroviral infection persists in the heart and in extracardiac sites, particularly in skeletal muscle, in patients with IDC. Blood and myocardial and skeletal muscle samples were collected at cardiac transplantation from 31 IDC patients, 24 non-IDC heart disease patients, and 3 heart donors. Samples underwent ultrastructural studies and ribonucleic acid (RNA) extraction. RNA was reverse-transcribed, and 2 nested fragments (bps 179 and 126) were amplified in the highly conserved 5' noncoding region of enteroviral genomic RNA. Enteroviral RNA was found in the skeletal muscle of 12 cases, whereas only 4 hearts (2 of which with positive skeletal muscle) were positive. Of the 24 controls, 2 were positive (1 muscle and heart, 1 muscle only). Automated sequencing confirmed the enteroviral nature of the amplified products. Ultrastructural study showed enterovirus-like particles in 4 of the enterovirus-positive muscles, and myopathic changes in all enterovirus-positive cases. Skeletal muscle hosts chronic enteroviral infection in more than one third of patients with sporadic IDC. Two hypotheses may explain this link. Myocardial damage may derive directly from recurrent subclinical heart infections caused by enteroviruses harbored in skeletal muscle. Alternatively, enterovirus-related myopathy may trigger an autoimmune response to antigens shared by muscle and myocardium. Further studies are needed to assess the importance of these, non-mutually exclusive mechanisms in IDC pathogenesis.

Fortuitous diagnosis of cystic fibrosis at laparotomy for acute appendicitis.

Two children aged 11 and 9 years were operated upon for acute appendicitis and were discovered to have cystic fibrosis: the histologic pattern of the removed appendix was typical for this disorder. Appendicitis may thus be the first presenting sign of occult cystic fibrosis.

Ten-year experience with endomyocardial biopsy in myocarditis presenting with congestive heart failure: frequency, pathologic characteristics, treatment and follow-up.

The present study summarizes our ten-year (1985-1995) experience with endomyocardial biopsy (EMB) in patients with idiopathic congestive heart failure (CHF), with specific reference to frequency of myocarditis, treatment policy, relative benefits, and follow-up. Of the 601 patients who constituted our series, 38 were clinically suspected of having myocarditis on the bases of a very recent onset of congestive heart failure and/or of arrhythmias and/or of conduction disturbances, and of a close-to-recent history of flu-like febrile illness. Corresponding EMBs showed myocarditis in 16 of the 38 cases (42.1%). A further 10 EMBs, from patients with a recent onset of congestive heart failure without prior infection episodes, showed myocarditis. Therefore, biopsy-proven myocarditis occurred in 26 of the 601 patients (4.3%). Of the 26 cases, 21 were lymphocytic, 1 was necrotizing granulomatous, 1 was eosinophilic and occurred in a patient who later developed overt zoonosis, 1 had some giant cells within endocardial inflammatory infiltrates, and 2 were borderline forms. In active myocarditis, inflammatory cells mostly constituted of T-lymphocytes (CD45RO+) with sparse macrophages (CD68+) and a few B cells (CD20+). B-lymphocytes and macrophages, along with activated T-lymphocytes, all expressed MHC class II HLA DR molecules, which were also expressed "de novo" by activated endothelial calls of capillaries and of small intramural vessels. HLA DR revealed itself as a very useful marker for the detection of activated inflammatory and endothelial cells. We also noted an increase in the number of perivascular and interstitial mast cells. Ultrastructural study was helpful for the characterization of myocyte damage and of interactions between inflammatory cells and myocytes. In 4 cases (1 of whom was later revealed as HIV positive, and subsequently died of AIDS), we found microreticulotubular structures in endothelial cells of small vessel and capillaries; in 7 cases, there were myocyte changes similar to those described in polymyositis; in 1 case, we observed subplasmalemmal buddings, but no viral particles; in 6 cases, there was extensive myocyte damage with myofibrillar lysis and focal adipous metaplasia; the remaining 6 cases showed myocyte damage of differing extent and severity; in the borderline forms, such damage coexisted with interstitial fibrosis. One of the 21 lymphocytic myocardites was not treated because during hospital screening the patient proved to be HIV positive; of the remaining 20 active myocardites, 11 were treated with a 6-month tapered steroid and azathioprine protocol (one was treated for 24 months), while 9 were not treated. The corresponding follow-up was: 6 deaths (congestive heart failure), 2 cardiac transplants and 3 survivals (1 with pace-maker) in the treated group, and 3 deaths (2 of congestive heart failure and 1 of sudden death), 1 cardiac transplant and 5 survivals (1 on the waiting list for transplantation) in the non-treated group. One of the 2 patients with borderline myocarditis died of congestive heart failure, and 1 is alive. Of the 22 patients with clinical diagnosis of myocarditis and negative biopsy, 7 died of congestive heart failure (2 on the waiting list for transplantation), 4 underwent cardiac transplantation, and 11 are alive (1 is awaiting transplantation). Of the 20 patients currently alive, 1 was originally in NYHA class III, 15 were in class II and 4 were in class I. Of the 20 overall patients who died, 12 were originally in NYHA class IV, 6 in class III, 2 in class II; of the 8 patients who underwent transplantation, 6 were originally in NYHA class IV and 2 in class III. Our overall experience shows that the frequency of myocarditis diagnosed according to Dallas criteria is high in patients with clinical diagnosis of myocarditis, while it is extremely low in dilated cardiomyopathy patients. This finding suggests that, although non-specific, recent onset of symptoms and prior febrile infe

Frequency and characteristics of coronary thrombosis in the epicardial coronary arteries after cardiac transplantation.

We investigated at autopsy or at retransplantation the frequency and characteristics of coronary thrombosis in 76 cardiac allografts: 37 in place for < or = 2 months (early) and 39 in place >2 to 99 months (late). The 76 allografts were inserted in 69 patients: a single 1 in 56 patients and 2 allografts in 13 patients, 7 of whom subsequently died and had an autopsy. An average of 140 sections from 70 5-mm-long segments of 8 epicardial coronary arteries were examined from each of the 76 allografts with both hematoxylin-eosin and Movat pentachrome stains. Thrombus was found in only 1 coronary artery (3%) (the right one) of the 37 early allografts, and in 24 of 39 late allografts (61%). Of the latter 39 grafts, 29 (79%) had allograft vascular disease (AVD) and 24 (83%) of them had coronary thrombosis. Of the 312 epicardial coronary arteries (4 major and 4 minor) examined in the 39 late cases, 66 arteries (21%) contained thrombus. Of the 24 late cases with thrombus in at least 1 artery, thrombus was present in 66 (34%) of the 192 epicardial coronary arteries examined: in 6 of the 8 arteries in 3 patients; in 5 arteries in 2 patients; in 4 arteries in 1 patient; in 3 arteries in 5 patients; in 2 arteries in 6 patients, and in a single artery in 7 patients. In all 66 arteries with thrombus (24 patients) the thrombus was longer than 5 mm. The thrombus in the late cases was entirely nonocclusive (mural) in 51 (77%) of the 66 epicardial coronary arteries containing thrombus and entirely occlusive in 10 arteries (15%). It consisted exclusively of multiluminal channels in 6 arteries (9%) and combinations in 1 artery (2%). Acute myocardial infarcts were present in 3 patients, all of whom had occlusive thrombi. In all 10 arteries with occlusive thrombi, the thrombus was larger than the underlying plaque and no occlusive thrombi were located over ulcerated plaques. These observations demonstrate that thrombus is common in epicardial coronary arteries >2 months after cardiac transplantation.

Polymorphism of angiotensin-converting enzyme gene in sarcoidosis.

Sarcoidosis is the disease in which increased levels of serum Angiotensin-converting enzyme (sACE) are most often detected. It has recently been shown that the deletion (D) or the insertion (I) of a 250bp-DNA fragment in the ACE gene accounts for three main ACE genotypes (i.e., II, ID, and DD) and for 47% of total phenotypic variance in sACE level. The aim of our work was to investigate whether or not patients with sarcoidosis have an increased incidence of those ACE genotypes coding for highest sACE levels and to investigate whether or not sACE level in sarcoidosis is related to ACE genotypes. We studied 61 unrelated patients with sarcoidosis (test group) and 80 unrelated healthy control subjects (control group). The ACE I and D alleles were detected with polymerase chain reaction on genomic DNA. In the control group we found an ACE genotype distribution that agreed with the Hardy-Weinberg proportion. The ACE genotype distribution was not significantly different in the test group. There was no correlation between ACE genotype and roentgenologic stage of sarcoidosis. Plotting the sACE level in the control group against ACE genotype, we found a trend of increasing mean sACE value according to the order II < ID < DD. The same trend for ACE genotype was found in the test group, in which it also paralleled the trend of sACE values plotted against roentgenologic stage, according to the order Stage I < Stage II < Stage III. We conclude that in sarcoidosis the ACE genotype distribution is not altered. The trends for increasing sACE values in sarcoidosis according to both ACE genotype and roentgenologic stage would suggest that both mechanisms play a role in determining sACE level.