Assessment of response to therapy in multiple myeloma by multiparameter flow cytometry. Usefulness of an eight-color single tube with monoclonal antibodies in dried formulation.

OBJECTIVE: Multiparameter flow cytometry is considered the gold standard to evaluate minimal residual disease in multiple myeloma (MM) and patients in complete remission can achieve "Flow MRD-negative" status (i.e. immunophenotypically abnormal plasma cells not detectable). In the current study we report the usefulness of an eight-color flow cytometric method with a 10-5 sensitivity, using monoclonal antibodies in dried formulation. MATERIALS AND METHODS: Forty-six patients with MM were treated with bortezomib-based regimens and, when eligible, with autologous stem cell transplantation. Response to therapy was assessed according to the criteria validated by the International Myeloma Working Group. Multiparameter flow cytometry was carried out with an 8-color panel validated by the Euroflow Consortium. A commercially available single 8-color tube in dried formulation was used and almost 2,000,000 events were acquired, in order to obtain a 10-5 sensitivity. RESULT: Sixteen patients achieved stringent complete remission and another three patients achieved complete remission. In these groups of patients, the "Flow MRD-negative" status was achieved in sixteen cases. In patients who had a different degree of response (very good partial response, partial response, minimal response) immunophenotypically abnormal plasma cells were always detected. CONCLUSION: Using a single eight-color tube in dried formulation, and an acquisition strategy able to obtain a 10-5 sensitivity, not only is it possible to detect a deep response to modern therapy in patients who obtained at least complete remission, but it is also always possible to detect minimal residual disease in patients with either complete remission or stringent complete remission.

Unusual onset of a case of chronic recurrent multifocal osteomyelitis.

BACKGROUND: Chronic recurrent multifocal osteomyelitis (CRMO) is a rare condition that commonly affects the clavicle and pelvis. CASE PRESENTATION: We report here a case a 12 years old girl with CRMO arising with recurrent episodes of left supraorbital headache, followed by the appearance of a periorbital dyschromia. Magnetic resonance imaging (MRI) of the skull and orbits revealed an important subacute inflammatory process. Few months after, the child presented a painful swelling of the left clavicle; the histological examination of the related biopsy allowed to establish the diagnosis of CRMO. CONCLUSION: CRMO presenting as acute headache involving neurocranium is rare; to our knowledge this is the first recognized case in the world literature. This pathological condition is frequently misdiagnosed as infection or neoplasm and needs a deep investigation for the differential diagnosis. The physical, laboratoristic and instrumental diagnostic investigations of the patient and the treatment employed are described in detail.

Multiparameter immunophenotyping by flow cytometry as a diagnostic tool in multiple myeloma and monoclonal gammopathy of undetermined significance.

BACKGROUND: Immunophenotyping by multiparameter flow cytometry (MFC) provides relevant information about prognosis and minimal residual disease detection in multiple myeloma (MM) and might be used to distinguish MM from monoclonal gammopathies of undetermined significance (MGUS). MATERIALS AND METHODS: We evaluated a possible usage of MFC to predict the differential diagnosis between MM and MGUS. One hundred consecutive patients were studied at diagnosis and underwent conventional diagnostic procedures. We carried out a double-blind study. Immunophenotyping was performed on samples from myeloaspirates before establishing diagnosis, while the final clinical diagnosis was established independently from MFC results. A five- or six-color method was carried out by means of monoclonal antibody combinations able to identify abnormal plasma cells (CD19-) and the most relevant immunophenotypic aberrations (loss of CD27; overexpression of CD117, CD56, CD28; asynchronous expression of CD20). MFC was applied following the indications of the European Myeloma Network. When abnormal plasma cells were /= 3.1%, MGUS was predicted. RESULTS: MFC results predicted 63 cases of MM and 37 cases of MGUS. At the end of our study, 61 cases of MM and 39 cases of MGUS were diagnosed. Therefore, 4% of patients were misdiagnosed by MFC parameters alone, with sensitivity and specificity of 0.983 and 0.92, respectively. CONCLUSIONS: Only a small proportion of patients with MM and MGUS were misdiagnosed by MFC alone and a possible systematic application of MFC in all patient with MM and MGUS at diagnosis might be proposed. Novel additional criteria could be necessary to improve the diagnostic impact of MFC in monoclonal gammopathies.

Randomised clinical trial: twice daily esomeprazole 40 mg vs. pantoprazole 40 mg in Barrett's oesophagus for 1 year.

BACKGROUND: Barrett's oesophagus is regarded as the most important risk factor for development of oesophageal adenocarcinoma. According to current guidelines, treatment should be limited to symptomatic Barrett's oesophagus. AIM: To evaluate the expression of Ki67, cyclooxygenase-2 (COX-2) and apoptosis in Barrett's oesophagus after 12 months of double-dose proton pump inhibitor therapy. The effectiveness of esomeprazole and pantoprazole was also compared. METHODS: Seventy-seven nondysplastic Barrett's oesophagus patients underwent baseline upper endoscopy. Patients were then randomised into two groups: one group was allocated to receive esomeprazole 40 mg b.d. and the other group pantoprazole 40 mg b.d. for 12 months. A follow-up endoscopy was performed at the end of treatment. Sixty-five of 77 patients agreed to undergo oesophageal manometry and 24-h pH-metry. Barrett's oesophagus biopsies, obtained at baseline and after treatment, were analysed using immunohistochemistry to assess Ki67 and COX-2 expression; apoptosis was evaluated using TUNEL. RESULTS: In the esomeprazole group, a significant decrease in Ki67 and COX-2 expression, as well as an increase in apoptosis, were observed (P < 0.05). By contrast, in the pantoprazole group Ki67, COX-2 and apoptosis did not vary significantly from baseline. By 24-h oesophageal pH-monitoring, a normal acid exposure time was recorded in patients treated with esomeprazole, while those allocated to pantoprazole displayed abnormal acid exposure (P < 0.05). CONCLUSIONS: Treatment of Barrett's oesophagus patients with high-dose esomeprazole, but not pantoprazole, promoted a decrease in proliferative markers, concomitantly with a decrease in apoptotic cell death. Moreover, esomeprazole allowed a better oesophageal acid control than pantoprazole.

Vascular endothelial growth factor polymorphisms in mantle cell lymphoma.

In this study, we determined the allele and genotype frequencies of vascular endothelial growth factor (VEGF) G+405C, C-460T, C+936T and C-2578A single nucleotide polymorphisms (SNPs) in 32 patients affected by mantle cell lymphoma (MCL) and 58 healthy controls. Real-time PCR combined with melting curve analysis was used for the determination of SNP alleles. A significant difference in the allele frequency of VEGFC-460T and C+936T SNPs in MCL and healthy cases was not observed. On the contrary, VEGF G+405C and C-2578A SNP allele distribution was significantly lower in the patient group than among normal controls (p = 0.014, p = 0.001). This observation suggests that further investigation is warranted, both in vitro and in a larger series of patients, to further examine the role of VEGF polymorphisms in the pathogenesis of MCL. In addition, the use of quantitative real-time PCR combined with a melting curve analysis method in the detection of the 4 VEGF SNPs may have the potential to replace older and more time-consuming PCR-RFLP methods and bears further investigation.

Leiomyosarcoma of the parotid gland in an HIV-positive patient: therapeutic approach, clinical course and review of the literature.

Leiomyosarcoma of the parotid gland is a rare tumor with only six cases reported in the english literature. To date, the association of this rare tumor with HIV infection has never been reported. We report the first case of a 19-year-old Caribbean woman affected by leiomyosarcoma of the parotid gland and HIV infection. Surgery, radiotherapy and chemotherapy used in this patient did not provide a good result in terms of overall survival. Intercurrent disease, opportunistic infection and chemotherapy cross-reaction have not been reported during this treatment regimen. The ability to use combined modality interventions in patients with secondary malignancies and immunosuppression requires further study with focus on both tolerance and efficacy.

Contrast-enhanced ultrasonography in nodular splenomegaly associated with type B Niemann-Pick disease: an atypical hemangioma enhancement pattern.

INTRODUCTION: Niemann-Pick disease (NPD) types A and B are lipid storage disorders. NPD type A is a fatal disorder of infancy. Type B is a non-neuronopathic form observed in children and adults. It is associated with enlargement of the liver, spleen, or both, and nodular splenomegaly may be detected with ultrasound. METHODS: A 21-year-old female was admitted to the Emergency Room with fever, pharyngitis, and left upper quadrant abdominal pain. Labwork revealed anemia, thrombocytopenia, increased levels of AST, ALT, GGT, AF, LDH, triglycerides, and total cholesterol and low levels of HDL-cholesterol. PCR blood assays for CMV and EBV were both negative. Chest X-ray was unremarkable. Transabdominal B-mode ultrasound (US) revealed splenomegaly (long axis: >22 cm), an irregular subcapsular hypoechoic lesion in the superior pole that was consistent with splenic infarction, and multiple round highly echogenic nodes measuring 1-5 cm in diameter. Contrast-enhanced ultrasonography (CEUS) was performed using SonoVue(®) (Bracco). RESULTS: The presence of a splenic infarction was confirmed. The nodular lesions showed arterial-phase enhancement with late parenchymal phase wash-out. (18)F-FDG-PET revealed splenic nodular uptake. Primary splenic lymphoma was suspected, and the patient underwent open splenectomy. The diagnosis was type B NPD with splenic hemangiomas. DISCUSSION: CEUS confirmed the diagnosis and extent of splenic infarction, but the nodular atypical enhancement pattern together with nodular (18)F-FDG-PET uptake was misleading, suggesting as it did lymphoproliferative involvement of the spleen.

Gamma-glutamyltransferase in fine-needle liver biopsies of subjects with chronic hepatitis C.

Serum gamma-glutamyltransferase (GGT) is considered as a sensitive but rather nonspecific marker of hepatobiliary disease, including chronic hepatitis C virus (HCV) infection. Although its increase in HCV infection is associated with poor response to interferon-alpha (IFN-alpha) and poor prognosis, there is little knowledge of the reasons of its increase during disease. Immunohistochemistry and enzyme histochemistry were performed on fine-needle biopsies of subjects with HCV infection. GGT was detected in the lumen of bile ducts and in bile canaliculi. Furthermore, in subjects with elevated serum GGT, immunoreactive and catalytically active GGT was also detected on the sinusoidal surface of hepatocytes and diffuse cytoplasmic positivity appeared in isolated hepatocytes and hepatocellular foci. Antigen unmasking procedures showed the presence of GGT in the cytoplasm of mature and immature bile cells and of inflammatory cells. These results suggest that during chronic HCV infection there is a general enhancement of GGT activity within the liver. As the activity of several inflammatory mediators, such as leukotrienes, nitric oxide, and interleukins is modulated by GGT activity, the present findings suggest a direct relationship between serum GGT, enhanced intrahepatic GGT activity and prognosis and therapeutic outcome of chronic HCV infection.

SV40 can be reproducibly detected in paraffin-embedded mesothelioma samples.

We studied tissue sections from 18 paraffin embedded mesothelioma specimens diagnosed by the Pathology Department of S. Chiara Hospital of Pisa. Using PCR analysis and Southern blot hybridization we examined the specimens for the DNA regulatory region of the virus. 10/18 (55.5%) of the samples tested contained SV40 DNA regulatory sequences, and of these positive samples, 80% were found to contain Tag sequences by PCR and Southern Blot hybridization. These results confirm that SV40 can be amplified and detected in paraffin embedded mesothelioma samples.

[Allergic fungal sinusitis: is this rare disease an allergy or infection?]. / Sinusite Allergica Micotica: una rara affezione allergica o infettiva?

Allergic Fungal Sinusitis (AFS) is a newly recognized form of benign, non invasive sinusitis the histopathologic features of which are similar to those of allergic bronchopulmonary aspergillosis. AFS is a rare condition. However, because treatment and prognosis vary widely, it is important that this disorder be recognized and differentiated from chronic bacterial sinusitis and other forms of fungal sinusitis. AFS does not discriminate by age although it is primarily found in young adults. AFS patients are usually atopic, often having a history of asthma and nasal polyposis. Many have suffered from the symptoms of chronic sinusitis for years while others have had multiple sinus surgery. Radiographs reveal the involvement of multiple sinuses, often with bone destruction. Laboratory findings support an allergic state with a marked increase in eosinophilia and total IgE. At times RAST testing proves positive for fungi and immediate cutaneous reactivity to fungi is also present. Histologic review of the sinus contents reveals characteristic "allergic mucin", with numerous eosinophiles, Charcot-Leyden crystals and fungal hyphae, without any fungi tissue invasion. A wide variety of fungal agents has been implicated, although the majority belong the Dematiacee family. Those patients with allergic mucin but no documented fungi are indicated as having AFS-like syndrome. The pathogenesis of AFS is uncertain. There is controversy in the literature as to what role hypersensitivity (Gell and Coombs type I and type III responses) in infection play. To date current therapeutic recommendations include complete exenteration of all allergic mucin. Adjunctive, short-term systemic steroids often prove useful and nasal steroid spray should be continued for long term. Systemic antifungal agents are not recommended in AFS. Recurrence is common and thus close clinical, endoscopic and radiographic follow-up is important. The clinicopathologic features of one patient with AFS are reported and etiopathogenetic problems are discussed. The presented case showed a positive culture with negative immunological testing (RAST-positive and immediate cutaneous reactivity to fungal antigen), thus confirming the pathogenetic hypothesis of the saprophytic fungal growth in an atopic patient.

[Human subcutaneous dirofilariasis: 8 more cases in Italy]. / Dirofilariasi umana sottocutanea: 8 ulteriori casi in Italia.

Eight new cases of Human Subcutaneous Dirofilariasis due to Dirofilaria repens in subjects aged 14 to 83 years, 5 men and 3 women, are recorded. The patients were living in Piedmont (5 cases), Tuscany (1), Friuli-Venetia Julia (1) and Apulia (1). The nematodes were immature in 6 cases (2 males and 4 females) and adults in 2 (both females). Microfilariae in the uterus of one of them were detected.

Molecular biology studies on mesothelioma tumor samples: preliminary data on H-ras, p21, and SV40.

The ras gene is one of the oncogenes most commonly detected in human cancers; this protooncogene is converted to active oncogene by point mutations occurring at either codon 12, 13, or 61. SV40 is a DNA-transforming simian virus, a 105 bp sequence of which has been shown recently to be present in a significant fraction of human mesothelioma cells. Eleven human malignant mesotheliomas were examined for H-ras gene mutations at codon 12, 13, and 61 and for the presence of SV40-like sequences. DNA prepared from formalin-fixed and paraffin-embedded tissue was amplified by means of PCR and analyzed using designed restriction fragment length polymorphism. No mutation with respect to H-ras was found in any tumor sample, but the majority of mesothelioma cells contained SV40-like sequences.

Treatment of hepatocellular carcinoma with percutaneous ethanol injection: evaluation with contrast-enhanced MR imaging.

OBJECTIVE: The aim of this study was to investigate the usefulness of unenhanced and enhanced MR imaging in evaluating the response of hepatocellular carcinoma to percutaneous injection of ethanol. SUBJECTS AND METHODS: Thirty-one patients with 40 hepatocellular carcinomas less than 5 cm in diameter were examined with MR imaging before and after percutaneous injection of ethanol. Unenhanced T1- and T2-weighted and contrast-enhanced T1-weighted spin-echo images were obtained. CT and percutaneous biopsy were performed 1 month after the final injection of ethanol and repeated at 6-month intervals to establish the outcome of treatment: complete tumor necrosis was shown in 36 lesions and incomplete tumor necrosis was seen in four lesions. RESULTS: Of the 36 proved necrotic lesions, 31 showed homogeneously low signal intensity on T2-weighted MR images obtained after treatment, owing to coagulative necrosis of the tumor. In the remaining five necrotic lesions, hypointense and hyperintense areas coexisted; the hyperintense areas were caused by liquefactive necrotic material in two cases and by chronic inflammatory tissue along the boundary of the necrotic area in three cases. None of the 36 necrotic lesions showed contrast enhancement on T1-weighted images obtained after IV injection of gadopentetate dimeglumine. In the four lesions with incomplete necrosis, the viable portion of the tumor was identified as a hyperintense area on T2-weighted images and as an enhancing area on contrast-enhanced T1-weighted images. No correlation was found between lesion features on unenhanced T1-weighted images and outcome of therapy. CONCLUSION: Contrast-enhanced T1-weighted MR images allow a reliable evaluation of the effectiveness of treatment and are more accurate than unenhanced MR studies. Contrast-enhanced MR imaging may be considered a valuable alternative to contrast-enhanced CT in the follow-up of hepatocellular carcinomas treated with percutaneous injection of ethanol.

Immunohistochemical detection of p185 product, p21 product, and proliferating cell nuclear antigen (PCNA) in formalin-fixed, paraffin-embedded tissues from ovarian carcinomas. Preliminary data.

Immunohistochemical techniques for the detection of oncogene products and the assessment of cell kinetics can represent promising investigational tools in clinical oncology. In the present paper the immunohistochemical expression of p185, p21 and proliferating cell nuclear antigen (PCNA) was retrospectively assessed in formalin-fixed, paraffin-embedded tissue samples taken from 28 primary ovarian carcinomas at first surgery. Positive immunostaining for p185 was found in 0% of 6 Stage I and 23% of 22 Stage III-IV tumors. Positive immunostaining for p21 was observed in 0% of early and 41% of advanced carcinomas; this immunohistochemical finding correlated significantly with histologic grade (G3 vs G1-2 = 47% vs 9%, p = 0.042). Elevated PCNA immunoreactivity was detected in 33% of Stage I and 50% of Stage III-IV tumors. Among the 20 patients with advanced carcinoma who underwent cisplatin or carboplatin based chemotherapy followed by second-look laparotomy, the pathologic complete response (pCR) rate was 36% for patients with low PCNA expression and 0% for those with elevated PCNA expression. A tendency towards a higher pCR rate was also found for patients with negative immunostaining for p185 or for p21. The prognostic value of the immunohistochemical detection of p185, p21, and PCNA in ovarian carcinoma deserves to be further investigated.

[Post-operative examination of 2930 thyroid glands: observations on primary carcinoma. Incidental carcinoma and the preoperative diagnostic assessment of thyroidectomy for cancer]. / L'esame post-operatorio di 2930 ghiandole tiroidee: osservazioni sul carcinoma primitivo. Il carcinoma incidentale e l'iter diagnostico pre-operatorio delle tiroidectomie per cancro.

The histological examinations performed in our Institute in the years 1985-1991 and concerning thyroid diseases pertain to 2930 patients. These were considered clinically benign in 2463 cases and malignant in 467. After histological control, the actually benign cases resulted to be 2335, whereas carcinomas were 588 (495 papillary, 61 follicular, 17 anaplastic, 15 medullary). Six lymphomas and 1 fibrosarcoma have been also observed. A comparison between clinical and morphological diagnosis for thyroids thought to be clinically benign has shown a diagnostic overlapping of 93.8%, with absolute agreement for Basedow's diseases, whereas thyroiditis is the disease most often not diagnosed preoperatively. Incidental carcinomas were 132, with an attendance related directly to the number of samples randomly taken from the surgical specimen. In 538 cases of carcinoma it has been possible to evaluate the intraglandular spread of the tumor: bilateral multifocal cases were 18.4%, with an incidence of latero-cervical metastases (41.4%) nearly twice as that observed in monolateral cases (24.2%). Post-surgical examination of the whole gland in 381 patients treated with a total thyroidectomy for carcinoma has discovered 20 false positive cases: 11 were operated on only according to clinical and instrumental examinations, 2 on the basis of just the cytological results, and 7 on the basis of cytologically positive evidence though refuted by intraoperative frozen sections. In the 381 cases taken into account, intraoperative frozen sections have been done 256 times: the results have been confirmed by the histological post-surgical examination of the whole gland in 94.5% of the cases. No false positive cases did occur. False negative cases were 7, due to inadequate sampling (in the operating room or in the pathology lab). Delayed diagnoses were 4, whereas truly false negative cases have been in all 3 (1.2%). The greater part of our data has been disaggregated in two different periods (1985-1988 and 1989-1991) to point out any change with time in the parameters under observation. The most relevant reflections relating to the time were the following: (1) per cent increase of bilateral multifocal carcinomas (13.9 vs. 21.8%), (2) per cent decrease of latero-cervical metastases in monolateral carcinomas (31.8 vs 17.7%), (3) frank decrease in the median value of incidental carcinomas' diameters (2.2 cm in the first vs. 0.6 cm in the last year), (4) frank decrease of false positive cases among patients operated on without prior cytological or histological verification (6/21 vs. 1/27), (5) remarkable improvement of the rate of correlation between intraoperative frozen section diagnosis and post-surgical histological examination (92.1 vs. 97.4%).(ABSTRACT TRUNCATED AT 400 WORDS)

Interference of thyroperoxidase on immuno-cytochemical determination of steroid receptors in thyroid tissue.

The presence of sexual steroid receptor proteins in thyroid tissue has been previously demonstrated by biochemical means. The aim of this study was to determine the estrogen (ER) and progesterone (PR) receptors in malignant (12 papillary and 1 follicular carcinoma) and nonmalignant (19 multinodular goiters, 1 Graves' disease, 1 Hashimoto's thyroiditis) thyroid diseases using immunocytochemical assay employing monoclonal anti-ER and anti-PR antibodies and the peroxidase-antiperoxidase technique. Positive results were obtained in 24/34 (70%) for ER (ER-ICA+) and 22/34 (64%) for PR (PR-ICA+). To evaluate the possible interference of thyroperoxidase in the immunostaining, in consecutive sections of a positive specimen, primary antibody or primary antibody plus bridging antibody or PAP complex was omitted. Using these modified procedures, staining distribution was similar to that obtained by the standard procedure: in contrast, no staining was found in the positive control, i.e. a breast cancer specimen. The inhibition of the endogenous peroxidase caused a loss of staining in both the standard and modified procedures on thyroid specimens; no staining modification was obtained in the positive control. These results suggest that the staining observed in thyroid tissue is not specific and related to the activity of thyroperoxidase on chromogen solution. The complete loss of staining after peroxidase inhibition appears to be in contrast with the results obtained by biochemical method, and different antigenicity of thyroid receptors in comparison with breast receptors may explain this discrepancy.

The clinical, serological, pathological and immunocytochemical features of a case of primary carcinoma of the fallopian tube.

We report the case of a patient with Stage I fallopian tube carcinoma who underwent bilateral salpingo-oophorectomy, total hysterectomy, omentectomy and appendicectomy followed by cisplatin based chemotherapy. CA 125 antigen was immunocytochemically detected in more than 50% of neoplastic cells, and serum CA 125 assay correlated with the clinical course of disease. Since CA 125 levels progressively increased during chemotherapy, an immunoscintigraphy with 111 In labeled CA 125 monoclonal antibody was performed and revealed a 5 cm area of abnormal captation placed in the posterior abdominal wall next the left kidney. A CAT scan detected an enlargement of left para-aortic lymph nodes, without cleavage plane with the left psoas muscle. The present case confirms that serum CA 125 assay is a useful noninvasive tool in the management of fallopian tube carcinoma.

[Chronic calculous cholecystitis: elementary and dysplastic lesions, echographic correlations and immunohistochemical approach]. / La colecistite cronica calcolosa: lesioni elementari e displastiche, correlazioni ecografiche e approccio immunoistochimico.

The morphological analysis of 223 cases of chronic calculous cholecystitis has drawn attention to the following outstanding facts: (a) elementary lesions are numerous (above all ulceration, mucosal erosion, antral and intestinal metaplasia) and put together a complex sight; (b) mild, moderate and severe dysplasias have a surprising high incidence (67%), even in the age-group under 40; (c) dysplasia is more common in the gallbladders bearing parallel hyperplastic and metaplastic mucosal changes, if compared with gallbladders where these changes are lacking; (d) thickness of gallbladder's wall and incidence of dysplasia are in direct relation; (e) echographic preoperative assessment of wall's thickness has predictive value for presence of dysplasia; (f) CEA and CA 19-9 have a progressively increasing expression in the various degrees of dysplasia.