Anna Sapino

Meta-analysis of non-sentinel node metastases associated with micrometastatic sentinel nodes in breast cancer.

The need for further axillary treatment in patients with breast cancer with low‐volume sentinel node (SN) involvement (micrometastases or smaller) is controversial.

Down-regulation of caspase 3 in breast cancer: A possible mechanism for chemoresistance

Caspase-3 is a member of the cysteine protease family, which plays a crucial role in apoptotic pathways by cleaving a variety of key cellular proteins. Caspase-3 can be activated by diverse death-inducing signals, including the chemotherapeutic agents. The purpose of this study was to determine the levels of caspase-3 expression in breast tumor samples and to determine whether alterations in its expression can affect their ability to undergo apoptosis. Primary breast tumor and normal breast parenchyma samples were obtained from patients undergoing breast surgery and the expression of caspases-3 was studied. Similarly, normal mammary epithelial cells and several established mammary cancer cell lines were studied for caspases-3 expression by reverse transcriptase-polymerase chain reaction, Northern blot analysis, and Western blot analysis. Approximately 75% of the tumor as well as morphologically normal peritumoral tissue samples lacked the caspase-3 transcript and caspase-3 protein expression. In addition, the caspases-3 mRNA levels in commercially available total RNA samples from breast, ovarian, and cervical tumors were either undetectable (breast and cervical) or substantially decreased (ovarian). Despite the complete loss of caspase-3, the expression levels of other caspases, such as caspase-8 and caspase-9, were normal in all of the tumor samples studied. The sensitivity of caspase-3-deficient breast cancer (MCF-7) cells to undergo apoptosis in response to doxorubicin and other apoptotic stimuli could be augmented by reconstituting caspase-3 expression. These results suggest that the loss of caspases-3 expression may represent an important cell survival mechanism in breast cancer patients.

Pathological work-up of sentinel lymph nodes in breast cancer. Review of current data to be considered for the formulation of guidelines.

Controversies and inconsistencies regarding the pathological work-up of sentinel lymph nodes (SNs) led the European Working Group for Breast Screening Pathology (EWGBSP) to review published data and current evidence that can promote the formulation of European guidelines for the pathological work-up of SNs. After an evaluation of the accuracy of SN biopsy as a staging procedure, the yields of different sectioning methods and the immunohistochemical detection of metastatic cells are reviewed. Currently published data do not allow the significance of micrometastases or isolated tumour cells to be established, but it is suggested that approximately 18% of the cases may be associated with further nodal (non-SN) metastases, i.e. approximately 2% of all patients initially staged by SN biopsy. The methods for the intraoperative and molecular assessment of SNs are also surveyed.

Discrepancies in current practice of pathological evaluation of sentinel lymph nodes in breast cancer. Results of a questionnaire based survey by the European Working Group for Breast Screening Pathology.

Aims: To evaluate aspects of the current practice of sentinel lymph node (SLN) pathology in breast cancer via a questionnaire based survey, to recognise major issues that the European guidelines for mammography screening should address in the next revision. Methods: A questionnaire was circulated by mail or electronically by the authors in their respective countries. Replies from pathology units dealing with SLN specimens were evaluated further. Results: Of the 382 respondents, 240 European pathology units were dealing with SLN specimens. Sixty per cent of these units carried out intraoperative assessment, most commonly consisting of frozen sections. Most units slice larger SLNs into pieces and only 12% assess these slices on a single haematoxylin and eosin (HE) stained slide. Seventy one per cent of the units routinely use immunohistochemistry in all cases negative by HE. The terms micrometastasis, submicrometastasis, and isolated tumour cells (ITCs) are used in 93%, 22%, and 71% of units, respectively, but have a rather heterogeneous interpretation. Molecular SLN staging was reported by only 10 units (4%). Most institutions have their own guidelines for SLN processing, but some countries also have well recognised national guidelines. Conclusions: Pathological examination of SLNs throughout Europe varies considerably and is not standardised. The European guidelines should focus on standardising examination. They should recommend techniques that identify metastases > 2 mm as a minimum standard. Uniform reporting of additional findings may also be important, because micrometastases and ITCs may in the future be shown to have clinical relevance.

Does chromosome 17 centromere copy number predict polysomy in breast cancer? A fluorescence in situ hybridization and microarray-based CGH analysis

Approximately 8% of breast cancers show increased copy numbers of chromosome 17 centromere (CEP17) by fluorescence in situ hybridization (FISH) (ie average CEP17 >3.0 per nucleus). Currently, this pattern is believed to represent polysomy of chromosome 17. HER2‐amplified cancers have been shown to harbour complex patterns of genetic aberrations of chromosome 17, in particular involving its long arm. We hypothesized that aberrant copy numbers of CEP17 in FISH assays may not necessarily represent true chromosome 17 polysomy. Eighteen randomly selected CEP17 polysomic cases and a control group of ten CEP17 disomic cases, as defined by dual‐colour FISH, were studied by microarray‐based comparative genomic hybridization (aCGH), which was performed on microdissected samples using a 32K tiling‐path bacterial artificial chromosome microarray platform. Additional FISH probes were employed for SMS (17p11.2) and RARA (17q21.2) genes, as references for chromosome 17 copy number. Microarray‐based comparative genomic hybridization revealed that 11 out of the 18 polysomic cases harboured gains of 17q with involvement of the centromere, one displayed 17q gain sparing the centromeric region, and only one could be defined as polysomic. The remaining five cases displayed amplification of the centromeric region. Among these, one case, showing score 2+ by immunohistochemistry and 8.5 HER2 mean copy number, was classified as not amplified by HER2/CEP17 ratio and as amplified by HER2/SMS ratio. Our results suggest that true chromosome 17 polysomy is likely to be a rare event in breast cancer and that CEP17 copy number greater than 3.0 in FISH analysis is frequently related to gain or amplification of the centromeric region. Larger studies investigating the genetic profiles of CEP17 polysomic cases are warranted. Copyright

Ultrasonographically-guided fine-needle aspiration of axillary lymph nodes: role in breast cancer management

The knowledge of the status of axillary lymph nodes (LN) of patients with breast cancer is a fundamental prerequisite in the therapeutic decision. In the present work, we evaluated the impact of fine-needle aspiration cytology (FNAC) of ultrasonographically (US) selected axillary LN in the diagnosis of LN metastases and subsequently in the treatment of patients with breast cancer. Axillary US was performed in 298 patients with diagnosed breast cancer (267 invasive carcinomas and 31 ductal carcinoma in situ DCIS), and in 95 patients it was followed by FNAC of US suspicious LN. Smears were examined by routine cytological staining. Cases of uncertain diagnosis were stained in immunocytochemistry (ICC) with a combination of anticytokeratin and anti-HMFG2 antibodies. Eighty-five FNAC were informative (49 LN were positive for metastases, 36 were negative). In 49 of 267 patients with invasive breast carcinoma (18%), a preoperative diagnosis of metastatic LN in the axilla could be confirmed. These patients could proceed directly to axillary dissection. In addition, US-guided FNAC presurgically scored 49 out of 88 (55%) metastatic LN. Of all others, with nonsuspicious LN on US (203 cases including 31 DCIS), in which no FNAC examination was performed, 28 invasive carcinomas (16%) turned out to be LN positive on histological examination. Based on these data, US examination should be performed in all patients with breast cancer adding ICC-supported FNAC only on US-suspect LN. This presurgical protocol is reliable for screening patients with LN metastases that should proceed directly to axillary dissection or adjuvant chemotherapy, thus avoiding sentinel lymph node biopsy.

Genomic and immunophenotypical characterization of pure micropapillary carcinomas of the breast

Pure invasive micropapillary carcinoma (MPC) is a special histological type that accounts for 0.7–3% of all breast cancers. MPC has a distinctive growth pattern and a more aggressive clinical behaviour than invasive ductal carcinomas of no special type (IDC‐NSTs). To define the molecular characteristics of MPCs, we profiled a series of 12 MPCs and 24 grade and oestrogen receptor (ER)‐matched IDC‐NSTs using high‐resolution microarray comparative genomic hybridization (aCGH). In addition, we generated a tissue microarray containing a series of 24 MPCs and performed immunohistochemical analysis with ER, PR, Ki‐67, HER2, CK5/6, CK14, CK17, EGFR, topoisomerase‐IIα, cyclin D1, caveolin‐1, E‐cadherin, and β‐catenin antibodies. In situ hybridization probes were employed to evaluate the prevalence of amplification of HER2, TOP2A, EGFR, CCND1, MYC, ESR1, and FGFR1 genes. aCGH analysis demonstrated that MPCs significantly differed from IDC‐NSTs at the genomic level. Gains of 1q, 2q, 4p, 6p, 6q23.2–q27, 7p, 7q, 8p, 8q, 9p, 10p, 11q, 12p, 12q, 16p, 17p, 17q, 19p, 20p, 20q, and 21q, and losses of 1p, 2p, 6q11.1–q16.3, 6q21–q22.1, 9p, 11p, 15q, and 19q were more prevalent in MPCs. High‐level gains/amplifications of 8p12–p11, 8q12, 8q13, 8q21, 8q23, 8q24, 17q21, 17q23, and 20q13 were significantly associated with MPCs. A comparison between 24 MPCs and a series of 48 grade and ER‐matched IDC‐NSTs revealed that high cyclin D1 expression, high proliferation rates, and MYC (8q24) amplification were significantly associated with MPCs. Our results demonstrate that MPCs have distinct histological features and molecular genetic profiles supporting the contention that they constitute a distinct pathological entity. Copyright

Expression of apocrine differentiation markers in neuroendocrine breast carcinomas of aged women

Neuroendocrine (NE) breast carcinomas are a rare entity in young women; however, their frequency increases in aged patients. The present work demonstrates that NE breast carcinomas in elderly women can also express an apocrine immunophenotype and analyzes the histological and clinical aspects of such differentiation. A selected series of 50 NE tumors (positive for NE markers in ≥50% of the cells) was tested for the immunocytochemical expression of gross cystic disease fluid protein-15 (GCDFP-15). The results demonstrated that about 50% of moderately (G2) and well-differentiated (G1) NE breast carcinomas (mucinous, solid papillary, and solid cohesive histotypes) coexpressed the apocrine marker. In these cases, specific mRNA for GCDFP-15 (PIP) and for chromogranin A (ChA) was demonstrated using in situ hybridization (ISH). Carcinomas of the alveolar subtype (G2) and poorly differentiated carcinomas (G3), including one case of atypical carcinoid, were pure NE carcinomas, devoid of apocrine differentiation. The steroid receptor status of these lesions was evaluated to test a possible involvement of androgen receptors in apocrine differentiation. We demonstrated that the level of AR and the mean age of patients at diagnosis were significantly higher in apocrine than in nonapocrine differentiated tumors. The histological grade and the expression of estrogen receptor (ER) significantly influenced the prognosis of these NE carcinomas, either pure or NE-apocrine differentiated. The most original result of our study is therefore the demonstration of a possible divergent apocrine differentiation of NE breast carcinomas that might be regulated by the activation of androgen receptors in elder patients. In addition, the possibility for using Chs or GCDFP-15 serum values in the follow-up of these patients, as demonstrated in two cases of the present series, can justify the immunophenotyping of the tumors.

Colectomy rate in steroid-refractory colitis initially responsive to cyclosporin: a long-term retrospective cohort study

BackgroundThere is consistent evidence that 50% of patients with acute, steroid-resistant flare of ulcerative colitis (UC) may achieve remission and avoid colectomy if treated with cyclosporin (CsA). However, follow-up of the responders has shown that most of them relapse and need surgery shortly after the response. We compared the records of our CsA-treated patients with those of other groups in order to help clarify this matter.MethodsAll patients admitted consecutively to our Unit with an attack of UC and treated with CsA between January 1991 and December 1999 were studied. Patients were begun on continuously-infused CsA at 2 mg/kg/day (1991–1996), or on NEORAL at an initial dose of 5 mg/kg/day (1996–1999). The maintenance treatment included oral CsA for 3–6 months with or without azathioprine (AZA). CsA failure was defined as a relapse requiring steroids with or without progression to colectomy; the cumulative probability of relapse/colectomy was assessed by Fishers exact tests and Kaplan-Meier analysis.ResultsAmong the patients, 39/61 (63%) initially responded. These 39 included a fatality and 4 drop-outs (unrelated to the side-effects of CsA), leaving 34 patients for the study. Of these, 61% and 35% were colectomy-free at 1 and 7 years, respectively; the corresponding figures were 80 and 60% respectively in the subset treated with AZA, but 47% and 15% in the AZA-untreated subgroup (p= 0.0007 at 7 years). Among the 34 patients, 44% were relapse-free at 1 year, but all had relapsed at 7 years (p = 0.0635). The overall resort to colectomy was 72%, while 19% of the patients remained colectomy-free.ConclusionSixty percent of a cohort of patients with steroid-refractory colitis responded to CsA and 60% of these responders retained the colon after 1 year. These figures fell to 35% at 7 years but improved to 60% on AZA. The overall need for colectomy remains high in these patients and toxicity must be monitored.

Mammographically detected in situ lobular carcinomas of the breast.

Abstract We present ten cases of mammographically detected lobular carcinoma in situ (LCIS), involving a single area of variable size (up to a quadrant) in seven cases and the entire gland in three cases. Histologically, calcifications were associated with necrotic central areas within the in situ carcinomatous foci. Multiple foci of LCIS were observed in all five cases in which mastectomy had been performed. Cytologically, the lesions were characterized by a solid proliferation of round noncohesive cells with nuclei of intermediate size. Immunocytochemically, all cases were E-cadherin and p53 negative, and c-ErbB-2, GCDFP-15 and estrogen receptor positive. The proliferation index, evaluated with Ki67, was in the low range. Four cases were associated with foci of infiltrating lobular carcinoma (ILC). These findings contradict the commonly held opinion that LCIS is not mammographically detectable because of its lack of necrosis and calcification. This study documents the existence of a variant of LCIS exhibiting the mammographic features and central necrosis classically associated with ductal carcinoma in situ (DCIS), while retaining the spatial distribution, cytological composition and immunocytochemical features of lobular carcinoma.