Human papillomavirus infection in women undergoing in-vitro fertilization: effects on embryo development kinetics and live birth rate.

BACKGROUD: Several studies showed that human papillomavirus (HPV) affects male fertility, but its impact on female fertility and in vitro fertilization (IVF) outcome is not yet clear. METHODS: Objective of this observational, prospective, cohort study was to evaluate the prevalence of HPV infection in women candidate to IVF, and the effects of HPV infection on the kinetic of embryonic development and on IVF outcome. A total number of 457 women candidate to IVF were submitted to HR-HPV test; among them, 326 underwent their first IVF cycle and were included in the analysis on IVF results. RESULTS: 8.9% of women candidate to IVF were HPV-positive, HPV16 being the most prevalent genotype. Among the infertility causes, endometriosis was significantly more frequent in HPV-positive than in negative women (31.6% vs. 10.1%; p < 0.01). Granulosa and endometrial cells resulted HPV-positive in 61% and 48% of the women having HPV-positive cervical swab, respectively. Comparing HPV-positive and negative women at their first IVF cycle, no significant difference was observed in the responsiveness to controlled ovarian stimulation (COS) in terms of number and maturity of retrieved oocytes, and of fertilization rate. The mean morphological embryo score was comparable in the two groups; embryos of HPV-positive women showed a quicker development in the early stages, with a significantly shorter interval between the appearance of pronuclei and their fusion. In the following days, embryo kinetic was comparable in the two groups until the early blastocyst stage, when embryos of HPV-positive women became significantly slower than those of HPV-negative women. Overall, these differences did not affect live birth rate/started cycle, that was comparable in HPV-positive and negative women (22.2 and 28.1%, respectively). CONCLUSIONS: (a) the prevalence of HPV infection in women candidate to IVF is similar to that observed in the general female population of the same age range; (b) HPV infection migrates along the female genital apparatus, involving also the endometrium and the ovary, and perhaps participates in the genesis of pelvic endometriosis; (c) HPV slightly affects the developmental kinetic of in vitro-produced embryos, but does not exert an effect on live birth rate.

Gene status in HER2 equivocal breast carcinomas: impact of distinct recommendations and contribution of a polymerase chain reaction-based method.

BACKGROUND: The primary objectives of this study on carcinomas with equivocal HER2 expression were to assess the impact of distinct recommendations with regard to identifying patients eligible for anti-HER2 agents by fluorescence in situ hybridization (FISH) and to elucidate whether multiplex ligation-dependent probe amplification (MLPA) may be of support in assessing HER2 gene status. METHODS: A cohort of 957 immunohistochemistry-evaluated HER2-equivocal cases was analyzed by dual-color FISH. The results were assessed according to U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines and American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) 2007 and 2013 guidelines for dual- and single-signal in situ hybridization (ISH) assays. A subgroup of 112 cases was subjected to MLPA. RESULTS: HER2 amplification varied from 15% (ASCO/CAP 2007 HER2/CEP17 ratio) to 29.5% (FDA/EMA HER2 copy number). According to the ASCO/CAP 2013 interpretation of the dual-signal HER2 assay, ISH-positive carcinomas accounted for 19.7%. In contrast with the ASCO/CAP 2007 ratio, this approach labeled as positive all 32 cases (3.34%) with a HER2/CEP17 ratio <2 and an average HER2 copy number ≥6.0 signals per cell. In contrast, only one case showing a HER2 copy number <4 but a ratio ≥2 was diagnosed as positive. MLPA data correlated poorly with FISH results because of the presence of heterogeneous HER2 amplification in 33.9% of all amplified carcinomas; however, MLPA ruled out HER2 amplification in 75% of ISH-evaluated HER2-equivocal carcinomas. CONCLUSION: The ASCO/CAP 2013 guidelines seem to improve the identification of HER2-positive carcinomas. Polymerase chain reaction-based methods such as MLPA can be of help, provided that heterogeneous amplification has been ruled out by ISH.

Validation of full-remote reporting for cervicovaginal cytology: the Caltagirone-Acireale distributed lab.

INTRODUCTION: Cervical cancer is the fourth most common cancer in women, and its prevention is based on vaccination and screening. Screening consists of molecular human papillomavirus (HPV) testing and cytologic analysis of cervical smears, which require expensive equipment and the interaction of numerous professionals such as biologists, cytologists, laboratory technicians, and pathologists. MATERIALS AND METHODS: We centralize the cervical samples from more than 51 clinics in 1 main laboratory, where automated HPV testing is performed. HPV-positive cases are collected and used to prepare a liquid-based cytology slide, which is stained and immediately scanned. The resulting whole-slide images (WSIs) are immediately available in a remote laboratory where they are examined by experienced cytologists using virtual microscopy. This setup was validated by making each of the 3 readers independently diagnose 506 specimens in random order, using both conventional light microscopy (CLM) and WSIs, with a minimum wash-out period of 3 weeks and with a final discussion for all cases. RESULTS: Intraobserver agreement among CLM and WSI ranged from 0.71 to 0.79, and interobserver agreement for the 3 readers compared with the consensus diagnosis was similar for the 2 modes of assessment. Readers subjectively felt confident in their WSI diagnosis for inadequate and negative cases, but less so in other cases. The perceived difficulty was slightly higher in WSI readings. CONCLUSIONS: Interobserver agreement in cervicovaginal cytology is moderate and does not vary if the slides are examined conventionally or digitally. Despite higher reported subjective difficulty and lower confidence in the WSI diagnosis, we did not observe a deterioration in diagnostic performance using WSI compared with CLM.

Reliability of whole sentinel lymph node analysis by one-step nucleic acid amplification for intraoperative diagnosis of breast cancer metastases.

OBJECTIVE: To assess the reliability of using the One-Step Nucleic Acid Amplification (OSNA) assay as a single test on whole sentinel lymph nodes (SLN) as a method of intraoperative diagnosis and staging of SLNs in breast cancer. BACKGROUND: Combining histological and molecular assessment of metastasis on the same SLN may not fully reproduce the actual load of cancer cells present in the SLN and create problems in decisions regarding axillary dissection. METHODS: Selection criteria for the whole SLN OSNA test required that the primary tumor expressed CK19 in more than 80% of tumor cells. Imprint cytology analysis of SLNs was performed together with the OSNA. RESULTS: Of the 279 patients enrolled for SLN evaluation, 123 gave consent to the OSNA protocol and 156 to the standard histology. Thirteen patients were excluded from OSNA evaluation because of low CK19 gene expression in the primary tumor; only 2.3% were truly negative. The kappa of concordance between the imprint cytology and OSNA results was 0.52. The rate of macrometastases determined by OSNA was 11% versus 20% determined by histology, whereas the rate of OSNA-micrometastases (18%) was significantly higher than that determined by histology (8%). The rate of SLN-negative cases was similar between the 2 protocols. Macrometastases correlated with the presence of vascular invasion in both protocols. The rate of axillary lymph node metastases was consistent with SLN tumor load. CONCLUSIONS: Intraoperative OSNA assay performed on the whole SLN gave objective and reproducible results that were useful for directing decisions regarding axillary dissection and for accurately defining the SLN stage.

Technical limits of comparison of step-sectioning,immunohistochemistry and RT-PCR on breast cancer sentinel nodes: a study on methacarn-fixed tissue.

The optimal pathological assessment of sentinel nodes (SLNs) in breast cancer is a matter of debate. Currently, multilevel histological evaluation and immunohistochemistry (IHC) are recommended, but alternative RT-PCR procedures have been developed. To assess the reliability of these different procedures, we devised a step-sectioning protocol at 100 micron-intervals of 74 SLNs using methacarn fixation. mRNA was extracted from sections collected from levels 4 to 5. Mammaglobin, CEA and CK19 were used for RT-PCR. mRNA extraction was successful in 69 SLNs. Of these, 7 showed macrometastases (>2mm), 2 showed micrometastases (<2 mm) and 7 showed isolated tumour cells (ITC) by IHC. RT-PCR was positive for the three markers in 6 of 7 macrometastases and in 1 of 2 micrometastases. In the 2 RT-PCR negative cases, metastases were detected only on sections distant from those analysed by RT-PCR. CEA and/or CK19 were positive by RT-PCR in 3 of 7 ITC and in 23 morphologically negative SLNs. In conclusion, the main goal of our study was to show that the use of alternate sections of the same sample for different procedures is the key reason for the discrepancies between molecular and morphological analyses of SLN. We believe that only prospective studies with quantitative mRNA analysis of specific metastatic markers on the whole lymph node can elucidate the utility of molecular assessments of SLN.

Columnar cell lesions associated with breast calcifications on vacuum-assisted core biopsies: clinical, radiographic, and histological correlations.

Columnar cell lesions of the breast are increasingly recognized at mammography for their tendency to calcify. We studied 392 vacuum-assisted core biopsies performed solely for calcifications to evaluate the frequency of columnar cell lesions, their relationship with radiological risk, appearance of calcifications, and clinical data. Management and follow-up of columnar cell lesions without and with atypia (flat epithelial atypia) was analyzed. Cases with architectural atypia (cribriform spaces and/or micropapillae) were excluded from flat epithelial atypia. Calcifications were within the lumen of acini affected by columnar cell lesions in 137 out of 156 biopsies diagnosed with some columnar cell lesions. These represented 37% of vacuum-assisted core biopsies and 62% of low radiological risk (BI-RADS3) calcifications. High-risk (BI-RADS5) calcifications were never associated with columnar cell lesions. Age and menopausal status were comparable in columnar and in not-columnar cell lesions. Atypia was associated with long-term hormone replacement therapy in both lesions. Surgical biopsy was recommended for all cases with atypia. Flat epithelial atypia, as the only histological findings on vacuum-assisted core biopsies, was never associated with malignancy at surgery. In conclusion, we suggest that surgical excision is not mandatory when flat epithelial atypia is found as the most advanced lesion on vacuum-assisted core biopsy performed for low radiological risk calcifications, and that women should be advised of the possible hormone dependency of this entity.

Radical resection of a giant, invasive and symptomatic malignant Solitary Fibrous Tumour (SFT) of the pleura.

Solitary Fibrous Tumours (SFTs) of the pleura are rare neoplasms, with unpredictable biological behaviour. Although usually benign, malignant SFTs are described, and they are often associated with large, necrotic and locally invasive tumours. Radical resection represents the treatment of choice in all cases; recurrences are uncommon, and redo-surgery should be considered. The case of a giant, invasive, radically resected malignant SFT, is described. The role of postoperative radiotherapy, to reduce the risk of recurrence, is also discussed.

Merits of the PMiT (papillary microtumor) terminology in the definition of a subset of incidental papillary microcarcinomas of the thyroid.

An exponential increase in the detection of papillary thyroid microcarcinomas (PTMCs) has been observed in recent times, possibly because of recent improvements in the management of thyroid lesions and extensive histological examination. However, no definitive treatment guideline has been developed for PTMC, resulting in patients undergoing overtreatment. In 2003, the term papillary microtumor of the thyroid (PMiT) was proposed for small (< or =1 cm) intrathyroidal tumors with excellent prognostic prospects along with strict definition criteria. Since then, the term PMiT has been adopted by clinicians and surgeons. In this article, the authors report a series of 50 consecutive cases of PMiT collected and treated at the University Hospital of Turin, Italy. From the authors' experience, this terminology, which demarks a subset of PTMC, should be widely adopted as it is biologically sound, well accepted by both clinicians and patients, decreases the danger of overtreatment, minimizes the psychological anxiety engendered by a diagnosis of carcinoma, and maintains the patient's eligibility for health insurance.

Gefitinib (ZD1839): therapy in selected patients with non-small cell lung cancer (NSCLC)?

PURPOSE: To evaluate response rate, toxicity and epidermal growth factor (EGFR) mutations and gene copy number as outcome predictive factors in Italian patients with non-small cell lung cancer (NSCLC) treated with gefitinib (Iressa) in an expanded access program (EAP). PATIENTS AND METHODS: A total of 137 patients with advanced NSCLC received gefitinib as first line treatment or after failure of chemotherapy. In 43 cases, tissue specimens were available for EGFR status evaluation: immunohistochemical (IHC) for EGFR, fluorescence in situ hybridisation (FISH) or Chromogenic in situ hybridisation (CISH)-(ISH) analysis for EGFR and HER2 gene copy number, and PCR-DNA sequencing for mutational analysis of EGFR were performed. RESULTS: In the study population, response rate (PR) was 13%; disease stabilization (DS) 26%; overall disease control rate 39%; median survival 6.3 months and time to progression 2.7 months. Toxicity was mild (G3 skin toxicity in 3% and G3 liver toxicity in 4% of patients). An EGFR-mutation was detected in 9/43 patients: Eight deletions in exon 19 and 1 missense mutation in exon 21. Increased gene copy number for EGFR and/or HER2 was detected in 17/43 patients. Response rate was significantly higher in women, non-smokers, in mutation carriers than in wild type carriers, in EGFR-trisomy/polysomy carriers and HER2-trisomy/polysomy carriers. CONCLUSIONS: In this study, response rate and toxicity to gefitinib treatment were consistent with previously reported data for whites. Female gender, absence of smoking history, EGFR-mutations, EGFR and HER2-polysomy were significantly associated with response to gefitinib therapy in NSCLC patients.

Predicting gefitinib responsiveness in lung cancer by fluorescence in situ hybridization/chromogenic in situ hybridization analysis of EGFR and HER2 in biopsy and cytology specimens.

In non-small cell lung cancer (NSCLC), epidermal growth factor receptor (EGFR) mutational analysis is an excellent predictor of responsiveness to treatment with tyrosine kinase inhibitors, such as gefitinib. In up to 80% of NSCLCs, cytologic samples or endoscopic biopsies are the only specimens available for molecular analysis, but PCR amplification of DNA from small fixed and paraffin-embedded samples may create artifactual mutations. Fluorescence in situ hybridization (FISH) of EGFR and HER2 has been proposed as an alternative method of analysis. This project aimed to determine the optimal scoring method for FISH or chromogenic in situ hybridization (CISH) assays when analyzing small NSCLC samples to predict response. FISH or CISH analysis of EGFR and HER2 genes was done on 42 small samples derived from NSCLC patients treated with gefitinib. EGFR mutational analysis was done after quantity and quality controls of DNA. In seven of seven cases, a balanced increase in EGFR gene and chromosome 7 number was found to correlate with the presence of specific EGFR mutations. In addition, seven of seven cases with balanced EGFR/HER2 polysomy and two of three cases with balanced EGFR/HER2 trisomy responded to gefitinib (75% of responders). Instead, the EGFR mutations predicted only 7 of 12 (58%) of gefitinib-responsive patients. When only endoscopic biopsies or cytologic specimens are available, we propose using FISH/CISH for EGFR and HER2 as the test of choice for selecting patients for treatment with gefitinib and to consider as negative predictive factor the absence of EGFR/HER2 gene gain.

Late recurrence of Wilms' tumour with exclusive skeletal muscle phenotype 23 years after primary diagnosis.

A late recurrence of Wilms' tumour (WT) 23 years after the primary diagnosis is described. The primary tumour occurred in a 10-month-old girl and showed various degrees of differentiation, including skeletal muscle phenotype. A postoperative chemotherapy was performed. Twenty-three years after the surgery, the tumour relapsed: the lesion was exclusively composed of mature skeletal muscle elements (diffuse and intense desmin reactivity) derived from the primary tumour as confirmed by WT1 immunoreactivity. Chemotherapy and radiotherapy have been reported previously to ablate the immature components of WT; especially, chemotherapy can modify the histological type, reducing the immature elements while leaving mature cells unaffected. We can hypothesise that both morphological and molecular features of the tumour as well as the effect of therapy can influence a tumour relapse in WT. The latter results in a high degree of differentiation and a long disease-free interval after the first diagnosis.

The impact of malignant nipple discharge cytology (NDc) in surgical management of breast cancer patients.

BACKGROUND: The role of nipple discharge cytology (NDc) in the surgical management of breast cancer patients is unclear. We aimed: (i) to evaluate the effect of malignant NDc on the surgical approach to the nipple-areola complex, and (ii) to verify the association between malignant NDc and nipple malignancy. METHODS: We retrospectively analyzed a case series of 139 patients with NDc who underwent breast surgery. The clinical and histological findings, types of surgery with emphasis on nipple-areola complex amputation, immunohistochemical phenotypes of the carcinomas and measurements of the tumor-nipple distance were recorded. Additionally, in patients who showed HER2-positive lesions on definitive surgery, we evaluated the HER2 immunocytochemistry of the NDc smears. RESULTS: Thirty-two malignant and 107 benign/borderline NDc diagnoses were identified. All 32 malignant-NDc cases were histologically confirmed as malignant. Thirty borderline/benign-NDc cases were histologically diagnosed as malignant (sensitivity 58%). The majority of the patients with malignant NDc were treated with nipple-areola complex amputations in both the mastectomy and conservative surgery groups (P<0.001, χ251.77). Nipple involvement was strongly associated with HER2-positive ductal carcinoma in-situ (P<0.001, χ211.98). HER2 immunocytochemistry on the NDc revealed a 100% correlation with the immunocytochemistry performed on the surgical tissues. CONCLUSIONS: Malignant NDc influenced surgical management. The association of malignant NDc with nipple involvement is highly related to ductal carcinoma in-situ with HER2 overexpression. In case of HER2 positive NDc, nipple-areola complex involvement is more likely than in HER2 negative cases.

Interobserver reproducibility of cytologic p16INK4a /Ki-67 dual immunostaining in human papillomavirus-positive women.

BACKGROUND: The accumulation of cyclin-dependent kinase inhibitor 2A (p16ink4a ) protein in a cell is associated with neoplastic progression in precancerous cervical lesions. Dual staining for p16ink4a and Ki-67 has been proposed as a triage test in cervical cancer screening for women who test positive for human papillomavirus DNA. In this study, interobserver reproducibility of the interpretation of this test was assessed. METHODS: Forty-two immunostained, liquid-based cytology slides were divided into 2 sets and were interpreted by 17 to 21 readers from 9 different laboratories, yielding a total of 816 reports. Immunostaining results were classified as positive, negative, inconclusive, or inadequate. After evaluation of the first set of slides and before circulation of the second set, the results were discussed in a plenary meeting. The 10 slides with the most discordant results were evaluated again by selected expert cytopathologists. RESULTS: The overall κ value was 0.612 (95% confidence interval [CI], 0.523-0.701), it was higher for the positive and negative categories (κ = 0.692 and κ = 0.641, respectively), and it was almost null for the inconclusive category (κ = 0.058). Considering only readers from laboratories with documented experience, the κ value was higher (κ = 0.747; 95% CI, 0.643-0.839) compared with nonexperienced centers (κ = 0.498; 95% CI, 0.388-0.616). The results were similar in both sets of slides (κ = 0.505 [95% CI, 0.358-0.642] and κ = 0.521 [95% CI, 0.240-0.698] for the first and second sets, respectively). Reinterpretation of the slides with the most discordant results did not provide any improvement (first evaluation, κ = 0.616 [95% CI, 0.384-0.866]; second evaluation, κ = 0.403 [95% CI, 0.182-0.643]). CONCLUSIONS: Dual staining for p16 ink4a and Ki-67 demonstrated good reproducibility, confirming its robustness, which is a necessary prerequisite for its adoption as a triage test in cervical cancer screening programs that use human papillomavirus DNA as a primary test. Cancer Cytopathol 2017;125:212-220. © 2016 American Cancer Society.

Routine assessment of prognostic factors in breast cancer using a multicore tissue microarray procedure.

We propose multicore tissue microarray (TMA) as an alternative to whole section for routine assessment of prognostic factors in breast cancer. Since 2004, we introduced the multicore TMA for testing estrogen (ER) and progesterone receptors (PR), proliferation activity by Ki67, and HER2 overexpression and amplification in routine work. At least four tumor foci were selected on the whole section, and a dedicated technician used a stereomicroscope for accurate sampling of the selected areas. To identify a specific case in the TMA, a separate file and a computerized reporting form with the TMA map were created. A preliminary pilot study comparing the TMA results with those obtained on whole sections showed the specificity of the procedure. Moreover, in everyday diagnosis, hormone receptors were repeated on full section when negative in TMA, without significant discrepancy. Retrospective analysis of the 237 breast carcinomas studied by TMA showed the expected correspondence of tumor-grade differentiation with the hormone receptor pattern, the proliferation activity, and HER2 immunohistochemical and FISH values. In conclusion, multicore TMA may be an efficient approach in the routine study of prognostic factors in breast cancer, significantly reducing costs, time, and burden of slides necessary to accomplish these mandatory tests.

Flexible lab-tailored cut-offs for suitability of formalin-fixed tumor samples for diagnostic mutational analyses.

The selection of proper tissues from formalin-fixed and paraffin-embedded tumors before diagnostic molecular testing is responsibility of the pathologist and represents a crucial step to produce reliable test results. The international guidelines suggest two cut-offs, one for the percentage and one for the number of tumor cells, in order to enrich the tumor content before DNA extraction. The aim of the present work was two-fold: to evaluate to what extent a low percentage or absolute number of tumor cells can be qualified for somatic mutation testing; and to determine how assay sensitivities can guide pathologists towards a better definition of morphology-based adequacy cut-offs. We tested 1797 tumor specimens from melanomas, colorectal and lung adenocarcinomas. Respectively, their BRAF, K-RAS and EGFR genes were analyzed at specific exons by mutation-enriched PCR, pyrosequencing, direct sequencing and real-time PCR methods. We demonstrate that poorly cellular specimens do not modify the frequency distribution of either mutated or wild-type DNA samples nor that of specific mutations. This observation suggests that currently recommended cut-offs for adequacy of specimens to be processed for molecular assays seem to be too much stringent in a laboratory context that performs highly sensitive routine analytical methods. In conclusion, new cut-offs are needed based on test sensitivities and documented tumor heterogeneity.

Interpretation of p16(INK4a) /Ki-67 dual immunostaining for the triage of human papillomavirus-positive women by experts and nonexperts in cervical cytology.

BACKGROUND: The triage of human papillomavirus (HPV)-positive women is needed to avoid overreferral to colposcopy. p16(INK4a) immunostaining is an efficient triage method. p16(INK4a) /Ki-67 dual staining was introduced mainly to increase reproducibility and specificity compared with stand-alone p16(INK4a) staining. METHODS: Within a pilot project, HPV-positive women were referred to colposcopy if cytology was abnormal or unsatisfactory or HPV testing was still positive after 1 year. For 500 consecutive women, a slide obtained during colposcopy was immunostained for p16(INK4a) /Ki-67 and independently interpreted by 7 readers without previous experience with dual staining. Four of these readers were experts in cervical pathology and 3 were not. Kappa values for multiple raters, sensitivity, and specificity for cervical intraepithelial neoplasia type 2-positive histology were computed. Because women with normal cytology were underrepresented, estimates for all HPV-positive women were obtained as weighted means of cytology-specific estimates. RESULTS: The overall kappa for HPV-positive women was 0.70 (95% confidence interval [95% CI], 0.60-0.77). Kappa values were not found to be significantly different between expert and nonexpert readers with regard to cervical cytology but were significantly increased (P =. 0066) after consensus discussion. The overall specificity estimate for HPV-positive women was 64.0% (95% CI, 57.4%-70.2%): 66.7% (95% CI, 59.8%-73.0%) for experts and 60.5% (95% CI, 59.8%-73.0%) for nonexperts. Among women with abnormal cytology, the sensitivity was 85.5% (95% CI, 77.9%-90.8%): 85.8% (95% CI, 77.9%-91.2%) for experts and 85.1% (95% CI, 76.6%-90.9%) for nonexperts. CONCLUSIONS: p16(INK4a) /Ki-67 immunostaining demonstrated good reproducibility and specificity when triaging HPV-positive women. Dual-staining interpretation can be performed, after short training, even by staff who are not experts in cervical cytology. This allows HPV-based screening with triage to be performed in settings in which such expert staff is not available.

Detection procedures for neuroblastoma cells metastatic to blood and bone marrow: blinded comparison of chromogranin A heminested reverse transcription polymerase chain reaction to tyrosine hydroxylase nested reverse transcription polymerase chain reaction and to anti-GD2 immunocytology.

Specific and sensitive tumor cell detection is becoming increasingly important for diagnosing and staging as well as for the therapeutic management of neuroblastoma patients. We propose a chromogranin A heminested reverse transcription polymerase chain reaction (CgA hn RT-PCR) procedure for the detection of neuroblastoma minimal residual disease in peripheral blood and bone marrow samples. The results were checked in comparison with the presently available procedures (i.e., with the tyrosine hydroxylase nested RT-PCR [TH n RT-PCR] and with the immunocytochemical approach using anti-GD2 antibodies). Controls from healthy patients or from people with unrelated disease (12 samples of bone marrow and 23 samples of peripheral blood) and serial dilution experiments using neuroblastoma cell lines (SKNLP, SKNFI, STA6, STA8) showed CgA hn RT-PCR full specificity and sensitivity ranging from 10(3) to 10(6) (depending on the cell line). The results compared favorably with those obtained using TH n RT-PCR. Preliminary data obtained analyzing bone marrow and peripheral blood specimens from stage IV neuroblastomas showed substantially overlapping results between CgA and TH n RT-PCR procedures. Our data support the potential usefulness of CgA heminested RT-PCR as a specific and sensitive procedure for minimal disease detection in neuroblastoma. A prospective evaluation of this tool in clinical studies might be warranted.

Efficiency of a preoperative axillary ultrasound and fine-needle aspiration cytology to detect patients with extensive axillary lymph node involvement.

BACKGROUND: Recent studies have demonstrated that axillary lymph node dissection (ALND) does not affect patient survival, even in those with one or two positive sentinel lymph nodes (SLNs). On the other hand, patients with 3 or more metastatic lymph nodes are eligible for chemotherapy. Therefore, it is crucial to identify a priori patients at risk of having a high number of metastatic axillary lymph nodes for their surgical and/or clinical management. Ultrasound (US) guided Fine-Needle Aspiration (FNA) has been proven to be a useful and highly specific method for detecting metastatic axillary lymph nodes. However, only one recent study has evaluated the efficiency of this method in identifying patients with high metastatic nodal involvement. Our aim was to validate US-guided FNA as a reliable method to discriminate a priori patients with >3 metastatic lymph nodes. METHODS: A retrospective series of 1287 breast cancer patients who underwent a simultaneous preoperative breast and axillary US to stage their axilla was collected. A total of 365 patients, with either positive SLNs (278) or positive axillary lymph nodes detected via US-guided FNA (87), underwent ALND. In these two subgroups, we compared the number of metastatic lymph nodes in the axilla. RESULTS: The number of metastatic axillary lymph nodes in patients who underwent US-guided FNA was significantly higher (63% had >3 metastatic lymph nodes) than that in patients with SLNs positive for micro- or macrometastases (3% and 27%, respectively) (P<0.001, χ(2) = 117.897). CONCLUSIONS: Preoperative axillary US-guided FNA could act as a reliable tool in identifying breast cancer patients with extensive nodal involvement.

High-throughput molecular analysis from leftover of fine needle aspiration cytology of mammographically detected breast cancer.

We investigated whether residual material from diagnostic smears of fine needle aspirations (FNAs) of mammographically detected breast lesions can be successfully used to extract RNA for reliable gene expression analysis. Twenty-eight patients underwent FNA of breast lesions under ultrasonographic guidance. After smearing slides for cytology, residual cells were rinsed with TRIzol to recover RNA. RNA yield ranged from 0.78 to 88.40 µg per sample. FNA leftovers from 23 nonpalpable breast cancers were selected for gene expression profiling using oligonucleotide microarrays. Clusters generated by global expression profiles partitioned samples in well-distinguished subgroups that overlapped with clusters obtained using "biologic scores" (cytohistologic variables) and differed from clusters based on "technical scores" (RNA/complementary RNA/microarray quality). Microarray profiling used to measure the grade of differentiation and estrogen receptor and ERBB2/HER2 status reflected the results obtained by histology and immunohistochemistry. Given that proliferative status in the FNA material is not always assessable, we designed and performed on FNA leftover a multiprobe genomic signature for proliferation genes that strongly correlated with the Ki67 index examined on histologic material. These findings show that cells residual to cytologic smears of FNA are suitable for obtaining high-quality RNA for high-throughput analysis even when taken from small nonpalpable breast lesions.

Metastasis of malignant peritoneal epithelioid mesothelioma in endoscopic gastric biopsy: a diagnostic pitfall.

AIM: To describe a case of metastasis of malignant peritoneal epithelioid mesothelioma in gastric antral mucosa in a patient with a cryptogenic liver cirrhosis associated with esophageal varices, abdominal pain and distension, ascites, and weight loss. MATERIALS AND METHOD: The patient underwent esophageal gastric endoscopy for varices, and a biopsy of a polypoid antral lesion was performed. The latter revealed a proliferation of polygonal cells with moderately atypical nuclei and pale eosinophilic, peripherally condensed cytoplasm infiltrating into the lamina propria between the normal mucosal glands of the antrum. The tumor cells were diffusely positive to anticalretinin antibody, whereas anti-claudin 4 and anti-CEA antisera were negative. CONCLUSIONS: Metastases of malignant peritoneal mesotheliomas are unusual, and a predominantly gastrointestinal localization is rare. Pathologists should be aware of this possibility to avoid misdiagnosis, particularly in small biopsy specimens.