Enzo Mammano

Multiplexed cell signaling analysis of human breast cancer applications for personalized therapy.

Phosphoprotein driven cellular signaling events represent most of the new molecular targets for cancer treatment. Application of reverse-phase protein microarray technology for the study of ongoing signaling activity within breast tumor specimens holds great potential for elucidating and profiling signaling activity in real-time for patient-tailored therapy. Analysis of laser capture microdissection primary human breast tumors and metastatic lesions reveals pathway specific profiles and a new way to classify cancer based on functional signaling portraits. Moreover, the data demonstrate the requirement of laser capture microdissection for analysis and reveal the metastasis-specific changes that occur within a new microenvironment. Analysis of biopsy material from clinical trials for targeted therapeutics demonstrates the feasibility and utility of comprehensive signal pathway activation profiling for molecular analysis.

Metastatic transcriptional pattern revealed by gene expression profiling in primary colorectal carcinoma

Metastatic spread to the liver is the major contributor to mortality in patients with colorectal carcinoma (CRC). In order to seek for gene expression patterns associated with metastatic potential in primary CRC, we compared the transcriptional profiles of 10 radically resected primary CRCs from patients who did not develop distant metastases within a 5‐year follow‐up period with those of 10 primary/metastatic tumor pairs from patients with synchronous liver metastases. To focus selectively on neoplastic cells, the study was conducted on laser‐microdissected bioptic tissues. Arrays of 7,864 human cDNAs were utilized. While a striking transcriptional similarity was observed between the primary tumors and their distant metastases, the nonmetastasizing primary tumors were clearly distinct from the primary/metastatic tumor pairs. Of 37 gene expression differences found between the 2 groups of primary tumors, 29 also distinguished nonmetastasizing tumors from metastases. The gene encoding for mannosyl (α‐1,3‐)‐glycoprotein β‐1,4‐N‐acetyl‐glucosaminyl‐transferase (GnT‐IV) became significantly upregulated in primary/metastatic tumor pairs (p < 0.001). GnT‐IV upregulation was confirmed by RT‐PCR. These data support the existence of a specific transcriptional signature distinguishing primary colon adenocarcinomas with different metastatic potential, the further pursuit of which may lead to relevant clinical and therapeutic applications.

Serum proteomic analysis identifies a highly sensitive and specific discriminatory pattern in stage 1 breast cancer

BackgroundMass spectrometry (MS)-based profiling was used to determine whether ion fingerprints could distinguish women with stage 1 breast cancer from women without breast cancer.MethodsThe initial study population consisted of 310 subjects: 155 women with yearly negative breast examination and negative mammography findings for at least 4 years, and 155 women undergoing surgery for pathology-proven stage 1 invasive ductal carcinoma. High-resolution SELDI-TOF (surface-enhanced laser desorption ionization–time of flight) analysis was performed on serum obtained from blood samples collected before mammography in controls, and before surgery in patients with breast cancer. Samples were divided into a training (109 controls and 109 cancers) and blinded (46 controls and 46 cancers) testing set; each group had similar age demographics. In addition, an independent study set of 46 serum samples was analyzed 14 months after the initial study to validate the robustness of the classifier.ResultsA discriminatory profile consisting of seven ion peaks found in the training set, when applied to the blinded test set, achieved a sensitivity and specificity of 95.6% and 86.5%, respectively. This same seven-peak profile achieved a 96.5% sensitivity and 85.7% specificity, with correct identification of all of 17 T1a tumors when applied to the validation study set.ConclusionsMass spectrometry profiling of human serum generated a robust classifier composed of seven low-molecular-weight ions that yielded a highly sensitive and specific diagnostic procedure for the discrimination of women with stage 1 breast cancer compared with women without breast cancer in this research study set.

Multiplexed cell signaling analysis of metastatic and nonmetastatic colorectal cancer reveals COX2-EGFR signaling activation as a potential prognostic pathway biomarker

The identification of prognostic determinants of colorectal cancer (CRC), including prediction of occult metastasis, is of urgent consideration, based on the tremendous differences in outcome and survival between patients who present with metastasis or develop metastasis versus those patients with organ-confined or nonrecurrent disease. Currently, a great deal of attention has been focused on using gene expression profiles of tumor specimens as a launch point for prognostic biomarker discovery. In our study, we chose to focus on functional protein-based pathway biomarkers as a new information archive because it is these proteins that form the functional signaling networks that control cell growth, motility, apoptosis, survival, and differentiation. We used reverse-phase protein microarray analysis of laser capture microdissected CRC tumor specimens to profile broad cell signaling pathways from patients who presented with liver metastasis versus patients who remained recurrence free after follow-up. Our results indicate that members of the EGFR and COX2 signaling pathways appear differentially activated in the primary tumors of patients with synchronous metastatic disease. If validated in larger study sets, this pathway defect might be useful as a prognostic clinical tool as well as a guide to potential therapeutic intervention strategies that target occult disease and/or preventative measure.

Protein pathway biomarker analysis of human cancer reveals requirement for upfront cellular-enrichment processing.

Tissues are complex structures composed of different cell types, each of which present specific functions and characteristics. To better understand and measure the effect of tumor cell enrichment on protein pathway profiling and drug target activation measurements, the signaling activation portraits of laser capture microdissected (LCM) cancer epithelium and tumor stroma were compared with patient-matched whole-tissue specimens from 53 primary colorectal cancer samples. Microdissected material and whole-tissue lysate from contiguous cryostat sections were subjected to reverse-phase protein microarray analysis to determine the level of phopshorylation and expression of 75 different proteins known to be involved in cancer progression. The results revealed distinct differences in the protein activation portraits of cancer epithelium and stroma. Moreover, we found that the signaling activation profiles of the undissected whole-tissue specimens are profoundly different from the matched LCM material. Attempts to rescale the undissected pathway information based on percent endogenous tumor epithelium content were unsuccessful in recapitulating the LCM tumor epithelial signatures. Analysis of epidermal growth factor receptor phosphorylation and COX2 expression in these same sample sets revealed wholesale differences in the rank ordering of patient determination when LCM was compared with undissected samples. On the basis of these data, we conclude that accurate protein pathway activation status, which is under evaluation as a basis for patient selection and stratification for personalized therapy, must include upfront cellular-enrichment techniques such as LCM to generate accurate drug target activation status.

Gene Expression Profile of Primary Gastric Cancer: Towards the Prediction of Lymph Node Status

BackgroundThe identification of gastric tumors associated with a higher risk of lymph node metastasis could help surgeons select patients who may benefit from extended lymph node dissection. The aim of this study was to screen the genome in the search of primary gastric cancer gene expression profiles that might predict lymph node status.MethodsThe gene expression profile was evaluated in frozen tumor samples obtained from 32 patients with primary gastric adenocarcinomas. The array consisted of a duplicated spot panel of 5,541 human genes. To classify node-positive (N+) and node-negative (N−) cases, a logistic regression model was fitted optimizing the Akaike Information Criteria after a stepwise gene selection. The accuracy was evaluated by means of leave-one-out cross validation.ResultsAll patients underwent radical gastrectomy and extended lymphadenectomy. Of all the cases, 21 were N+ and 11 demonstrated no lymph node involvement (N−). After quality filtering, the analysis of variance selected a set of 136 genes potentially correlated with nodal involvement (P value <.05). Of these 136 genes, 5 were differentially expressed (adjusted P value <.05). After a stepwise gene selection, only three genes (Bik, aurora kinase B, eIF5A2) were retained in the logistic model, which could correctly predict lymph node status in 30 of 32 cases.ConclusionsIf our findings were confirmed, the identified gene pattern might be used to tailor the extent of lymph node dissection on a single patient basis.

Association of p53 polymorphisms and colorectal cancer: Modulation of risk and progression.

OBJECTIVES p53 Gene variants BstUI RFLP at codon 72 in exon 4, 16bp tandem repeat in intron 3 and MspI RFLP in intron 6, which code for two functionally different protein isoforms, have been shown to modulate susceptibility to different types of human neoplasms. METHODS p53 genotype was assessed in 90 CRC patients, 321 age-matched controls and 322 centenarians. RESULTS The p53 codon 72 arginine, the p53 16bp deletion, and the MspI RFLP were significantly more frequent in CRC patients in comparison to the controls and to the centenarians (odd ratio 1.44 and 1.93). In the CRC group, the BstUI RFLP polymorphism was the more frequent combination (62.2%), and it was significantly associated with highly infiltrating (p<0.01), poorly differentiated (p<0.01), and metastatic (p<0.05) tumours. Our findings indicate that the p53 codon 72 polymorphisms are associated with a higher risk of CRC and are associated with more advanced and undifferentiated tumours.

Protein pathway activation mapping of colorectal metastatic progression reveals metastasis-specific network alterations.

The mechanism by which tissue microecology influences invasion and metastasis is largely unknown. Recent studies have indicated differences in the molecular architecture of the metastatic lesion compared to the primary tumor, however, systemic analysis of the alterations within the activated protein signaling network has not been described. Using laser capture microdissection, protein microarray technology, and a unique specimen collection of 34 matched primary colorectal cancers (CRC) and synchronous hepatic metastasis, the quantitative measurement of the total and activated/phosphorylated levels of 86 key signaling proteins was performed. Activation of the EGFR–PDGFR-cKIT network, in addition to PI3K/AKT pathway, was found uniquely activated in the hepatic metastatic lesions compared to the matched primary tumors. If validated in larger study sets, these findings may have potential clinical relevance since many of these activated signaling proteins are current targets for molecularly targeted therapeutics. Thus, these findings could lead to liver metastasis specific molecular therapies for CRC.

A Pilot Characterization of Human Lung NSCLC by Protein Pathway Activation Mapping.

Background: An understanding of the activated protein signaling architecture in non–small-cell lung cancer (NSCLC) is of critical importance to the development of new therapeutic approaches and identification of predictive and prognostic biomarkers for patient stratification. Methods: We used reverse-phase protein microarrays to map the activated protein signaling networks of 47 NSCLC tumors, 28 of which were node negative, which were subjected to tumor cellular enrichment using laser capture microdissection. The phosphorylation/cleavage levels of 111 key signaling proteins and total levels of 17 proteins were measured for broadscale signaling analysis. Results: Pathway activation mapping of NSCLC revealed distinct subgroups composed of epidermal growth factor receptor (ERBB1), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ERBB3), v-erb-a erythroblastic leukemia viral oncogene homolog 4 (ERBB4), v-akt murine thymoma viral oncogene homolog 1- mammalian target of rapamycin (AKT-mTOR), protein kinase, AMP-activated, alpha 2 catalytic subunit (AMPK), and autophagy-related signaling, along with transforming growth factor-beta-signaling protein 1 (SMAD), insulin-line growth factor receptor (IGFR), rearranged during transfection proto-oncogene (RET), and activated CDC42-associated kinase (ACK) activation. Investigation of epidermal growth factor receptor (EGFR)-driven signaling identified a unique cohort of tumors with low EGFR protein expression yet high relative levels of phosphorylated EGFR and high EGFR total protein with low relative levels of phosphorylation. Last, mapping analysis of patients with NSCLC with N0 disease revealed a pilot pathway activation signature composed of linked epidermal growth factor receptor family (HER)-AMPK-AKT-mTOR signaling network along with focal adhesion kinase- LIM domain kinase-1 (FAK-LIMK) and janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathways that correlated with short-term survival and aggressive disease. Conclusions: Functional protein pathway activation mapping of NSCLC reveals distinct activation subgroups that are underpinned by important therapeutic targets and that patients with early-stage node negative disease and poor prognosis may be identified by activation of defined, biochemically linked protein signaling events. Such findings, if confirmed in larger study sets, could help select and stratify patients for personalized targeted therapies.

Primary Great Saphenous Vein Leiomyosarcoma: Report of a Case

Leiomyosarcomas rarely arise in primary veins, especially the great saphenous vein. We have found only 20 case reports of leiomyosarcoma arising in the great saphenous vein, most of which manifested as nonspecific symptoms of advanced disease, such as a palpable mass, swelling, and back or abdominal pain. We report the case of greater saphenous vein leiomyosarcoma diagnosed in a 48-year-old man with a 4-month history of an inguinal mass. Ultrasonography and computed tomography showed a 6-cm mass attached to the right superficial femoral vein. Fine-needle aspiration biopsy confirmed that it was a vascular sarcoma. At the time of surgery there was no evidence of distant metastasis; therefore, we removed the tumor en bloc along with the sartorius muscle, inguinal lymph nodes, and 10 cm of the common femoral vein, and replaced the femoral vein with a polytetrafluoroethylene graft. A pathological examination revealed poorly differentiated leiomyosarcoma of the great saphenous vein, involving the deep femoral vein, without lymph node involvement. During follow-up, a thrombosis of the prosthesis developed, followed by proximal stenosis, which was treated successfully with percutaneous transluminal angioplasty. The patient was found to have lung metastases 25 months after surgery and he died about 5 months later.