Giancarlo Pruneri

The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014

BACKGROUND The morphological evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer (BC) is gaining momentum as evidence strengthens for the clinical relevance of this immunological biomarker. Accumulating evidence suggests that the extent of lymphocytic infiltration in tumor tissue can be assessed as a major parameter by evaluation of hematoxylin and eosin (H&E)-stained tumor sections. TILs have been shown to provide prognostic and potentially predictive value, particularly in triple-negative and human epidermal growth factor receptor 2-overexpressing BC. DESIGN A standardized methodology for evaluating TILs is now needed as a prerequisite for integrating this parameter in standard histopathological practice, in a research setting as well as in clinical trials. This article reviews current data on the clinical validity and utility of TILs in BC in an effort to foster better knowledge and insight in this rapidly evolving field, and to develop a standardized methodology for visual assessment on H&E sections, acknowledging the future potential of molecular/multiplexed approaches. CONCLUSIONS The methodology provided is sufficiently detailed to offer a uniformly applied, pragmatic starting point and improve consistency and reproducibility in the measurement of TILs for future studies.

Chemotherapy Is More Effective in Patients with Breast Cancer Not Expressing Steroid Hormone Receptors A Study of Preoperative Treatment

Purpose: The purpose of this research was to identify factors predicting response to preoperative chemotherapy. Experimental Design: In a large volume laboratory using standard immunohistochemical methods, we reviewed the pretreatment biopsies and histologic specimens at final surgery of 399 patients with large or locally advanced breast cancer (cT2-T4, N0–2, M0) who were treated with preoperative chemotherapy. The incidence of pathological complete remission and the incidence of node-negative status at final surgery were assessed with respect to initial pathological and clinical findings. Menopausal status, estrogen receptor status, progesterone receptor status [absent (0% of the cells positive) versus expressed], clinical tumor size, histologic grade, Ki-67, Her-2/neu expression, and type and route of chemotherapy were considered. Results: High rates of pathological complete remission were associated with absence of estrogen receptor and progesterone receptor expression (P < 0.0001), and grade 3 (P = 0.001). Significant predictors of node-negative status at surgery were absence of estrogen receptor and progesterone receptor expression (P < 0.0001), clinical tumor size <5 cm (P < 0.001), and use of infusional regimens (P = 0.003). The chance of obtaining pathological complete remission or node-negative status for patients with endocrine nonresponsive tumors compared with those having some estrogen receptor or progesterone receptor expression was 4.22 (95% confidence interval, 2.20–8.09, 33.3% versus 7.5%) and 3.47 (95% confidence interval, 2.09–5.76, 42.9% versus 21.7%), respectively. Despite the significantly higher incidence of pathological complete remission and node-negative status achieved by preoperative chemotherapy for patients with estrogen receptor and progesterone receptor absent disease, the disease-free survival was significantly worse for this cohort compared with the low/positive expression cohort (4-year disease-free survival %: 41% versus 74%; hazard ratio 3.22; 95% confidence interval, 2.28–4.54; P < 0.0001). Conclusions: Response to preoperative chemotherapy is significantly higher for patients with endocrine nonresponsive tumors. New chemotherapy regimens or combinations should be explored in this cohort of patients with poor outcome. For patients with endocrine responsive disease, the role of preoperative endocrine therapies should be studied.

Predicting the Risk for Additional Axillary Metastases in Patients With Breast Carcinoma and Positive Sentinel Lymph Node Biopsy

Objective:To assess whether the risk for nonsentinel node metastases may be predicted, thus sparing a subgroup of patients with breast carcinoma and a positive sentinel lymph node (SLN) biopsy completion axillary lymph node dissection (ALND). Summary Background Data:The SLN is the only involved axillary lymph node in the majority of the patients undergoing ALND for a positive SLN biopsy. A model to predict the status of nonsentinel axillary lymph nodes could help tailor surgical therapy to those patients most likely to benefit from completion ALND. Methods:All the axillary sentinel and nonsentinel lymph nodes of 1228 patients were reviewed histologically and reclassified according to the current TNM classification of malignant tumors as bearing isolated tumor cells only, micrometastases, or (macro)metastases. The prevalence of metastases in nonsentinel lymph nodes was correlated to the type of SLN involvement and the size of the metastasis, the number of affected SLNs, and the prospectively collected clinicopathologic variables of the primary tumors. Results:In multivariate analysis, further axillary involvement was significantly associated with the type and size of SLN metastases, the number of affected SLNs, and the occurrence of peritumoral vascular invasion in the primary tumor. A predictive model based on the characteristics most strongly associated with nonsentinel node metastases was able to identify subgroups of patients at significantly different risk for further axillary involvement. Conclusions:Patients with the most favorable combination of predictive factors still have no less than 13% risk for nonsentinel lymph node metastases and should be offered completion ALND outside of clinical trials of SLN biopsy without back-up axillary clearing.

Multidisciplinary Meeting on Male Breast Cancer: Summary and Research Recommendations

Male breast cancer is a rare disease, accounting for less than 1% of all breast cancer diagnoses worldwide. Most data on male breast cancer comes from small single-institution studies, and because of the paucity of data, the optimal treatment for male breast cancer is not known. This article summarizes a multidisciplinary international meeting on male breast cancer, sponsored by the National Institutes of Health Office of Rare Diseases and the National Cancer Institute Divisions of Cancer Epidemiology and Genetics and Cancer Treatment and Diagnosis. The meeting included representatives from the fields of epidemiology, genetics, pathology and molecular biology, health services research, and clinical oncology and the advocacy community, with a comprehensive review of the data. Presentations focused on highlighting differences and similarities between breast cancer in males and females. To enhance our understanding of male breast cancer, international consortia are necessary. Therefore, the Breast International Group and North American Breast Cancer Group have joined efforts to develop an International Male Breast Cancer Program and to pool epidemiologic data, clinical information, and tumor specimens. This international collaboration will also facilitate the future planning of clinical trials that can address essential questions in the treatment of male breast cancer.

Clinical Activity of Rituximab in Gastric Marginal Zone Non-Hodgkin's Lymphoma Resistant to or Not Eligible for Anti–Helicobacter Pylori Therapy

PURPOSE Preliminary results using rituximab in extranodal marginal zone (MALT) non-Hodgkins lymphoma (NHL) patients seem to indicate a relevant clinical activity. Aim of the present study is to investigate the efficacy of conventional weekly treatment using rituximab in gastric MALT NHL patients resistant/refractory or not suitable for eradication treatment, and to evaluate the relevance of the t(11; 18)(q21; q21) translocation and its possible role as a predictive criteria of response. PATIENTS AND METHODS Twenty-seven patients presenting with gastric MALT NHL at any stage, relapsed/refractory to initial treatment or not suitable for eradication were treated with rituximab in a weekly conventional schedule and evaluated for response and relapse. Flourescence in situ hybridization (FISH) analysis for the presence of 18q21 translocation was performed in 21 patients and was evaluated with clinical outcome. RESULTS Among the 26 evaluated patients, 20 (77%) achieved an objective response. Twelve patients (46%) had a pathological and clinical complete remission, and eight (31%) had a partial response. With a median follow-up of 33 months, only two patients relapsed at 26 and 14 months, respectively. No correlation was founded between FISH analysis and response or relapse. CONCLUSION Our experience seems to confirm the clinical activity of rituximab in gastric MALT NHL patients resistant/refractory to antibiotics treatment or not presenting with clinical evidence of Helicobacter pylori infection. The t(11; 18)(q21; q21) translocation seems not to be a predictive marker to response or to subsequent relapse.

Genomic and expression profiling identifies the B‐cell associated tyrosine kinase Syk as a possible therapeutic target in mantle cell lymphoma

Among B‐cell lymphomas mantle cell lymphoma (MCL) has the worst prognosis. By using a combination of genomic and expression profiling (Affymetrix GeneChip Mapping 10k Xba131 and U133 set), we analysed 26 MCL samples to identify genes relevant to MCL pathogenesis and that could represent new therapeutic targets. Recurrent genomic deletions and gains were detected. Genes were identified as overexpressed in regions of DNA gain on 3q, 6p, 8q, 9q, 16p and 18q, including the cancer genes BCL2 and MYC. Among the transcripts with high correlation between DNA and RNA, we identified SYK, a tyrosine kinase involved in B‐cell receptor signalling. SYK was amplified at DNA level, as validated by fluorescence in situ hybridisation (FISH) analysis, and overexpressed at both RNA and protein levels in the JeKo‐1 cell line. Low‐level amplification, with protein overexpression of Syk was demonstrated by FISH in a small subset of clinical samples. After treatment with low doses of the Syk inhibitor piceatannol, cell proliferation arrest and apoptosis were induced in the cell line overexpressing Syk, while cells expressing low levels of Syk were much less sensitive. A combination of genomic and expression profiling suggested Syk inhibition as a new therapeutic strategy to be explored in lymphomas.

Size of Breast Cancer Metastases in Axillary Lymph Nodes: Clinical Relevance of Minimal Lymph Node Involvement

BACKGROUND Overt ipsilateral axillary lymph node metastases of breast cancer are the most significant prognostic indicators for women who have undergone surgery, yet the clinical relevance of minimal involvement (isolated tumor cells and micrometastases) of these nodes is uncertain. PATIENTS AND METHODS We evaluated biologic features, adjuvant treatment recommendations, and prognosis for 1,959 consecutive patients with pT1-3, pN0, minimal lymph node involvement (pN1mi or pN0i+), or pN1a (single positive node) and M0, who were operated on and counseled for medical therapy from April 1997 to December 2000. RESULTS Patients with pN1a and pN1mi/pN0i+, when compared with patients with pN0 disease, were more often prescribed anthracycline-containing chemotherapy (39.1% v 33.2% v 6.1%, respectively; P < .0001) and were less likely to receive endocrine therapy alone (9.8% v 19.4% v 41.9%, respectively; P < .0001). At the multivariate analysis, a statistically significant difference in disease-free survival (DFS) and in the risk of distant metastases was observed for patients with pN1a versus pN0 disease (hazard ratio [HR] = 2.04; 95% CI, 1.46 to 2.86; P < .0001 for DFS; HR = 2.32; 95% CI, 1.42 to 3.80; P = .0007 for distant metastases) and for patients with pN1mi/pN0i+ versus pN0 disease (HR = 1.58; 95% CI, 1.01 to 2.47; P = .047 for DFS; HR = 1.94; 95% CI, 1.04 to 3.64; P = .037 for distant metastases). CONCLUSION Even minimal involvement of a single axillary node in breast cancer significantly correlates with worse prognosis compared with no axillary node involvement. Further studies are required before widespread modification of clinical practice.

Dual Effect of Metformin on Breast Cancer Proliferation in a Randomized Presurgical Trial

PURPOSE Metformin is associated with reduced breast cancer risk in observational studies in patients with diabetes, but clinical evidence for antitumor activity is unclear. The change in Ki-67 between pretreatment biopsy and post-treatment surgical specimen has prognostic value and may predict antitumor activity in breast cancer. PATIENTS AND METHODS After tumor biopsy, we randomly allocated 200 nondiabetic women with operable breast cancer to either metformin 850 mg/twice per day (n = 100) or placebo (n = 100). The primary outcome measure was the difference between arms in Ki-67 after 4 weeks adjusted for baseline values. RESULTS Overall, the metformin effect on Ki-67 change relative to placebo was not statistically significant, with a mean proportional increase of 4.0% (95% CI, -5.6% to 14.4%) 4 weeks apart. However, there was a different drug effect depending on insulin resistance (homeostasis model assessment [HOMA] index > 2.8, fasting glucose [mmol/L] × insulin [mU/L]/22.5; P(interaction) = .045), with a nonsignificant mean proportional decrease in Ki-67 of 10.5% (95% CI, -26.1% to 8.4%) in women with HOMA more than 2.8 and a nonsignificant increase of 11.1% (95% CI, -0.6% to 24.2%) with HOMA less than or equal to 2.8. A different effect of metformin according to HOMA index was noted also in luminal B tumors (P(interaction) = .05). Similar trends to drug effect modifications were observed according to body mass index (P = .143), waist/hip girth-ratio (P = .058), moderate alcohol consumption (P = .005), and C-reactive protein (P = .080). CONCLUSION Metformin before surgery did not significantly affect Ki-67 overall, but showed significantly different effects according to insulin resistance, particularly in luminal B tumors. Our findings warrant further studies of metformin in breast cancer with careful consideration to the metabolic characteristics of the study population.

Human acute leukemia cells injected in NOD/LtSz-scid/IL-2Rγ null mice generate a faster and more efficient disease compared to other NOD/scid-related strains

Transplantation of human acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) primary cells and cell lines in different strains of immunodeficient mice has led to preclinical models extensively used to investigate acute leukemia stem cells, biology and drug sensitivity. We studied the engraftment kinetics of AML and ALL cell lines and primary cells in 3 strains of NOD.CB17‐Prkdcscid (NOD/scid, NS)‐related mice (NOD.Cg‐PrkdcscidB2mtm1Unc/J, abbreviated NOD/scid/β2 null, NSB; and NOD.Cg‐PrkdcscidIl2rgtm1Wjll/SzJ, abbreviated NOD/scid/IL‐2Rγ null, NSG). The engraftment of human malignant cells was investigated by means of clinicopathological criteria, flow cytometry, PCR and immunohistochemistry. In NSG mice, we observed a significantly faster development of leukemia‐related symptoms and a higher percentage of leukemia cells in the blood, in the marrow and in the spleen. The leukemia‐related angiogenic switch (measured as the number of circulating endothelial cells and progenitors) was faster in NSG compared to NS and NSB mice. These models will be instrumental to studies on leukemia‐initiating stem cells, leukemia biology, preclinical treatment studies, and to obtain patient‐specific preclinical models to design and investigate patient‐tailored therapies.

Prognosis and adjuvant treatment effects in selected breast cancer subtypes of very young women (<35 years) with operable breast cancer

BACKGROUND There is limited knowledge about prognosis of selected breast cancer subtypes among very young women. PATIENTS AND METHODS We explored patterns of recurrence by age according to four immunohistochemically defined tumor subtypes: Luminal A and Luminal B (estrogen receptor positive and/or progesterone receptor positive and either human epidermal growth factor receptor 2 (HER2) positive and/or high Ki-67), HER2-positive (and) endocrine receptor absent and Triple Negative, in 2970 premenopausal patients with pT1-3, pN0-3 and M0 breast cancer. RESULTS Patients <35 years of age (315, 11%) presented a significantly increased risk of recurrence and death [hazards ratio (HR) = 1.65, 95% confidence interval (CI) 1.30-2.10 and HR = 1.78, 95% CI 1.12-2.85, respectively] when compared with older patients (2655, 89%) with similar characteristics of disease. This was true considering patients with Luminal B [HR = 1.62, 95% CI 1.21-2.18 for disease-free survival (DFS) and HR = 2.09, 95% CI 0.96-4.53 for overall survival (OS)] and with Triple Negative (HR = 2.04, 95% CI 1.11-3.72 for DFS and HR = 2.20, 95% CI 1.10-4.41 for OS) breast cancer, observing the highest risk of recurrence in the younger patients with HER2-positive breast cancer (HR = 2.37, 95% CI 1.12-5.02) when compared with older patients. CONCLUSIONS Very young patients with Triple Negative, Luminal B or HER2-positive breast cancer have a worse prognosis when compared with older patients with similar characteristics of disease.