Benjamin F. Calvo

Activation of a Novel Calcium-dependent Protein-tyrosine Kinase CORRELATION WITH c-Jun N-TERMINAL KINASE BUT NOT MITOGEN-ACTIVATED PROTEIN KINASE ACTIVATION

Many G protein-coupled receptors (e.g. that of angiotensin II) activate phospholipase Cβ, initially increasing intracellular calcium and activating protein kinase C. In the WB and GN4 rat liver epithelial cell lines, agonist-induced calcium signals also stimulate tyrosine phosphorylation and subsequently increase the activity of c-Jun N-terminal kinase (JNK). We have now purified the major calcium-dependent tyrosine kinase (CADTK), and by peptide and nucleic acid sequencing identified it as a rat homologue of human PYK2. CADTK/PYK2 is most closely related to p125FAK and both enzymes are expressed in WB and GN4 cells. Angiotensin II, which only slightly increases p125FAK tyrosine phosphorylation in GN4 cells, substantially increased CADTK tyrosine autophosphorylation and kinase activity. Agonists for other G protein-coupled receptors (e.g. LPA), or those increasing intracellular calcium (thapsigargin), also stimulated CADTK. In comparing the two rat liver cell lines, GN4 cells exhibited ∼ 5-fold greater angiotensin II- and thapsigargin-dependent CADTK activation than WB cells. Although maximal JNK activation by stress-dependent pathways (e.g. UV and anisomycin) was equivalent in the two cell lines, calcium-dependent JNK activation was 5-fold greater in GN4, correlating with CADTK activation. In contrast to JNK, the thapsigargin-dependent calcium signal did not activate mitogen-activated protein kinase and Ang II-dependent mitogen-activated protein kinase activation was not correlated with CADTK activation. Finally, while some stress-dependent activators of the JNK pathway (NaCl and sorbitol) stimulated CADTK, others (anisomycin, UV, and TNFα) did not. In summary, cells expressing CADTK/PYK2 appear to have two alternative JNK activation pathways: one stress-activated and the other calcium-dependent.

HER4 Mediates Ligand-Dependent Antiproliferative and Differentiation Responses in Human Breast Cancer Cells

ABSTRACT The function of the epidermal growth factor receptor (EGFR) family member HER4 remains unclear because its activating ligand, heregulin, results in either proliferation or differentiation. This variable response may stem from the range of signals generated by HER4 homodimers versus heterodimeric complexes with other EGFR family members. The ratio of homo- and heterodimeric complexes may be influenced both by a cells EGFR family member expression profile and by the ligand or even ligand isoform used. To define the role of HER4 in mediating antiproliferative and differentiation responses, human breast cancer cell lines were screened for responses to heregulin. Only cells that expressed HER4 exhibited heregulin-dependent antiproliferative responses. In-depth studies of one line, SUM44, demonstrated that the antiproliferative and differentiation responses correlated with HER4 activation and were abolished by stable expression of a kinase-inactive HER4. HB-EGF, a HER4-specific ligand in this EGFR-negative cell line, also induced an antiproliferative response. Moreover, introduction and stable expression of HER4 in HER4-negative SUM102 cells resulted in the acquisition of a heregulin-dependent antiproliferative response, associated with increases in markers of differentiation. The role of HER2 in these heregulin-dependent responses was examined through elimination of cell surface HER2 signaling by stable expression of a single-chain anti-HER2 antibody that sequestered HER2 in the endoplasmic reticulum. In the cell lines with either endogenously (SUM44) or exogenously (SUM102) expressed HER4, elimination of HER2 did not alter HER4-dependent decreases in cell growth. These results suggest that HER4 is both necessary and sufficient to trigger an antiproliferative response in human breast cancer cells.

Long-Term Outcome of Neoadjuvant Therapy for Locally Advanced Breast Carcinoma: Effective Clinical Downstaging Allows Breast Preservation and Predicts Outstanding Local Control and Survival

ObjectiveTo review the long-term follow-up data from the authors’ institutional experience of 62 patients with locally advanced breast cancer (LABC) treated with a uniform multimodality regimen. The authors determined the rate of breast preservation, the disease-free and overall survival, and the factors associated with locoregional and distant recurrent disease. Summary Background DataIt remains a challenge to achieve local and distant control of LABC. Over the last decade, preoperative or neoadjuvant chemotherapy has emerged as the standard of care for these patients. Successful tumor downstaging has been associated with increased rates of breast-conserving therapy (BCT), but the overall effect on long-term survival remains to be seen. MethodsThis study examines a cohort of 62 patients with LABC treated at the authors’ institution from 1992 to 1998. The uniform treatment regimen consisted of neoadjuvant doxorubicin (Adriamycin), followed by operation (BCT if sufficient clinical downstaging), followed by non-cross-resistant cyclophosphamide/methotrexate/5-fluorouracil, followed by radiation therapy. Treatment was both dose-intensive and time-intensive, with a total treatment time of 32 to 35 weeks. ResultsIn this patient population, the median age was 44 years, with approximately two thirds white patients and one third African American. Eighty-two percent of patients were clinical stage III at presentation, 13 patients had T4d inflammatory cancers, and 3 patients were stage IV at diagnosis. Eighty-four percent of patients demonstrated a significant clinical response to doxorubicin. Twenty-eight patients had sufficient clinical downstaging to attempt BCT, and 22 (45%) of 49 noninflammatory patients underwent successful BCT. Pathologic complete response was seen in 15% of patients. Median follow-up for the cohort was 70 months. The local recurrence rate was 14%, including two ipsilateral breast tumor recurrences (10%) in the BCT patients. Seven (12%) patients developed a new primary cancer in the contralateral breast. Distant metastases occurred in 18 (31%) patients, and the 5-year overall survival rate for the cohort was 76%. Furthermore, in the patients who underwent an attempt at BCT, the survival rate was 96% at 5 years. ConclusionsDose-intensive and time-intensive multimodality neoadjuvant therapy was successfully administered to a mixed racial group over shortened times. Patients who had sufficient clinical downstaging to allow BCT have the best long-term outcome. Patients who required mastectomy are at a higher risk of relapse, as well as the development of new contralateral cancers, yet have 5-year survival rates of over 50%.

A six-gene signature predicts survival of patients with localized pancreatic ductal adenocarcinoma.

Jen Jen Yeh and colleagues developed and validated a six-gene signature in patients with pancreatic ductal adenocarcinoma that may be used to better stage the disease in these patients and assist in treatment decisions.

Preoperative tyrosine kinase inhibition as an adjunct to debulking nephrectomy.

OBJECTIVES Since the introduction of tyrosine kinase inhibitors (TKI), treatment of metastatic renal cell carcinoma (RCC) has undergone dramatic changes. However, the use of TKI therapy in adjunctive settings remains to be defined. We present a single-institution experience of patients who received preoperative TKI before nephrectomy for metastatic or unresectable disease. METHODS The records of 9 patients with locally advanced or metastatic RCC treated with TKI therapy before nephrectomy at the University of North Carolina were reviewed. All procedures and radiographic images were performed at 1 institution. The cases were surveyed for the effect of TKI on tumor burden and surgical approach and timing. RESULTS The patients received systemic therapy with either sorafenib or sunitinib before proceeding to nephrectomy on clinical trials for metastatic disease or as the standard of care. The surgery was well tolerated by all patients, without an apparent effect from TKI therapy on the surgical technique or complications. Responses were observed in the primary tumor, as well as in the metastatic sites. CONCLUSIONS Neoadjuvant TKI therapy can induce responses in the primary tumor and has the potential advantage of cytoreduction when administered before nephrectomy for RCC. This setting also potentially provides an opportunity to evaluate the TKI responsiveness of patients with metastatic disease. However, prospective trials evaluating adjunctive surgical approaches to locally advanced and metastatic RCC are needed to determine the significant benefits of TKI therapy and to define the optimal agent, timing of therapy, and disease stage to derive benefit for preoperative therapy.

Extracapsular extension of the sentinel lymph node metastasis: a predictor of nonsentinel node tumor burden.

ObjectiveTo identify predictors of nonsentinel node (NSN) tumor involvement in patients with a tumor-involved sentinel node (SN). Summary Background DataFor many breast cancer patients who undergo intraoperative lymphatic mapping and sentinel lymphadenectomy (LM/SL), the SN is the only tumor-involved axillary node. Associations between NSN tumor involvement and several clinical and histopathologic factors have been identified. The authors hypothesize that extracapsular extension (ECE) of the SN metastasis is highly predictive of NSN tumor involvement. MethodsBetween May 1998 and December 2001, 260 patients (263 cases) with clinical T1 or T2 (<5.0 cm) breast cancer underwent LM/SL at the University of North Carolina, using a combined blue dye and technetium sulfur colloid technique. In all cases with a tumor-involved SN, axillary lymph node dissection (ALND) was recommended. Statistical analysis, with Pearson chi-square tests, Fisher exact test, and multiple logistic regression, was performed. ResultsThe SN contained tumor in 74 (28.1%) cases. ALND was performed in 70 of the 74 cases. ECE of the SN metastasis was present in 18 (25.7%) of the 70 cases. Patients with ECE of the SN metastasis were more likely to have NSN tumor involvement and had a greater total number of tumor-involved nodes than patients without ECE of the SN metastasis. Increasing size of the SN metastasis and increasing size of the primary tumor, examined as continuous variables, were associated with an increased likelihood of NSN tumor involvement on univariate analysis. However, only ECE of the SN metastasis was associated with NSN tumor involvement on multivariate analysis. ConclusionsECE of the SN metastasis is a strong predictor of NSN tumor involvement. All patients with ECE of the SN metastasis should undergo mandatory completion ALND.

Cholangiocarcinoma: Spectrum of appearances on mr images using current techniques

This study describes the spectrum of appearances of cholangiocarcinoma on magnetic resonance (MR) sequences, including gadolinium-enhanced, fat-suppressed spoiled gradient echo images and MR cholangiography. Fifteen patients were included in the study. Histologic diagnosis was established in 11 patients by surgical resection (6 patients), percutaneous biopsy (4 patients), and open liver biopsy (1 patient). The final diagnosis was determined by correlation of the MR findings with cholangiographic studies and laboratory studies in 4 patients. MR studies were performed at 1.5 T, and the following sequences were obtained: T1-weighted spoiled gradient echo (SGE), T1-weighted fat-suppressed spin echo or SGE, T2-weighted fat-suppressed conventional or turbo spin echo, MR cholangiography, and gadolinium-enhanced T1-weighted fat-suppressed SGE images. The following determinations were made: tumor location, tumor extent, ductal dilatation, ductal wall thickness, signal intensity, enhancement pattern, and associated findings. Mass-like neoplasms were peripheral (6 patients), hilar (1 patient), and extrahepatic (2 patients). Circumferential tumors were hilar (2 patients) and extrahepatic (4 patients). All peripheral tumors were multifocal. Mass-like tumors were well-defined, rounded, and ranged from 1 to 14 cm in diameter. Circumferential tumors had less well-defined margins and measured from 3 to 15 mm in thickness. All mass-like tumors were moderately hypointense on T1-weighted images and mildly to moderately hyperintense on T2-weighted images. The circumferential tumors were iso- to moderately hypointense on T1-weighted images and iso- to mildly hyperintense on T2-weighted images. Mass-like tumors were generally well shown on non-contrast and immediate gadolinium-enhanced images, whereas circumferential tumors were poorly seen on non-contrast images and best shown on gadolinium-enhanced T1-weighted fat-suppressed images. The degree of enhancement ranged from minimal to intense on immediate gadolinium-enhanced images, with all tumors becoming more homogeneous in signal intensity on images obtained between 1 and 5 min following contrast administration. Tumor-containing lymph nodes greater than or equal to 1 cm in diameter were demonstrated in 11 out of 15 patients (73.3%). These were best shown on T2-weighted fat-suppressed images and gadolinium-enhanced fat-suppressed SGE images. MR cholangiography demonstrated the level of obstruction and degree of dilatation of the proximal biliary system in 5 out of 6 patients who underwent MR cholangiography. The spectrum of appearances of cholangiocarcinoma is demonstrable on MR images. Mass-like tumors are well shown on both pre- and post-gadolinium sequences. Circumferential tumors may cause minimally increased duct wall thickness and are most clearly shown on gadolinium-enhanced fat-suppressed SGE images obtained 1 to 5 min following gadolinium administration.

Microsatellite Instability Testing in Colorectal Carcinoma: Choice of Markers Affects Sensitivity of Detection of Mismatch Repair-Deficient Tumors

Purpose: Microsatellite instability (MSI) is found in 10% to 15% of sporadic colorectal tumors and is usually caused by defects in DNA mismatch repair (MMR). In 1997, a panel of microsatellite markers including mononucleotide and dinucleotide repeats was recommended by a National Cancer Institute workshop on MSI. We investigated the relationship between instability of these markers and MMR protein expression in a cohort of sporadic colorectal cancer patients. Experimental Design: Paraffin sections of normal and tumor tissue from 262 colorectal cancer patients were examined for MSI status by PCR amplification and for MMR protein expression using antibodies against hMLH1, hPMS2, hMSH2, and hMSH6. Results: Twenty-six (10%) of the patients studied had tumors with a high level of MSI (MSI-H). The frequencies of MSI were the same in African-American and Caucasian patients. Each of the MSI-H tumors had mutations in both mononucleotide and dinucleotide repeats and had loss of MMR protein expression, as did two tumors that had low levels of MSI (MSI-L). These two MSI-L tumors exhibited mutations in mononucleotide repeats only, whereas eight of the other nine MSI-L tumors had mutations in just a single dinucleotide repeat. There was not a statistically significant difference in outcomes between patients whose tumors were MMR-positive or MMR-negative, although there was a slight trend toward improved survival among those with MMR-deficient tumors. Conclusions: The choice of microsatellite markers is important for MSI testing. Examination of mononucleotide repeats is sufficient for detection of tumors with MMR defects, whereas instability only in dinucleotides is characteristic of MSI-L/MMR-positive tumors.

Cytokeratin immunohistochemical validation of the sentinel node hypothesis in patients with breast cancer

No standard method for handling and histopathologic examination of the sentinel node (SN) exists. We hypothesized that a focused examination of all nodes with serial sectioning and cytokeratin immunohistochemical staining would confirm the SN as the node most likely to harbor metastasis. Intraoperative lymphatic mapping and sentinel lymphadenectomy using blue dye and (99m)technetium-labeled sulfur colloid were performed. All nodes were stained with H&E. All tumor-free nodes underwent additional sectioning and staining with H&E and an immunohistochemical stain. Routine H&E examination detected SN metastases in 27.6% of cases. Occult SN metastases were identified in 12.7% of cases. None of the 724 non-SNs examined contained occult metastases. The SN false-negative rate was zero. This study confirms histopathologically that the SN has biologic significance as the axillary node most likely to harbor metastatic tumor Standardization of the handling, sectioning, and staining of the SN is necessary as lymphatic mapping and sentinel lymphadenectomy become integrated into the care of patients with breast cancer

Size of Residual Lymph Node Metastasis After Neoadjuvant Chemotherapy in Locally Advanced Breast Cancer Patients Is Prognostic

BackgroundThe prognostic significance of micrometastasis after neoadjuvant chemotherapy for locally advanced breast cancer is unknown. We examined the residual lymph node metastasis size in patients after treatment with neoadjuvant chemotherapy to determine the relevance of metastasis size on outcome.MethodsStage II/III breast cancer patients treated with neoadjuvant chemotherapy at our institution from 1991 to 2002 were included. We examined the relationship of postneoadjuvant chemotherapy lymph node metastasis size and number with distant disease-free survival (DDFS) and overall survival (OS).ResultsIn 122 patients with a median follow-up of 5.4 years, we found not only that patients with an increasing number of residual positive nodes had progressively worse DDFS and OS (P < .0001 for both) compared with patients with negative nodes, but also that the size of the largest lymph node metastasis was associated with worse DDFS and OS (P < .0001 for both) in both univariate and multivariate analysis. Compared with negative nodes, even lymph node micrometastasis (<2 mm) was associated with worsened DDFS and OS (adjusted P = .02 and P = .005, respectively).ConclusionsResidual micrometastatic disease in the axillary lymph nodes after neoadjuvant chemotherapy is predictive of worse prognosis than negative nodes. In this study, the lymph node metastasis size and the number of involved lymph nodes were independent powerful predictors of DDFS and OS.