Amy S. Duffield

Radical prostatectomy findings in patients in whom active surveillance of prostate cancer fails.

PURPOSE Little data are available on radical prostatectomy findings in men who experience disease progression following active surveillance. MATERIALS AND METHODS A total of 470 men in our active surveillance program underwent annual repeat needle biopsies to look for progression defined as any Gleason pattern grade 4/5, more than 50% cancer on any core or cancer in more than 2 cores. Slides were available for review in 48 of 51 radical prostatectomies with progression. RESULTS The average time between the first prostate biopsy and radical prostatectomy was 29.5 months (range 13 to 70), with 44% and 75% of the patients showing progression by the second and third biopsy, respectively. There were 31 (65%) organ confined cases, of which 25 (52%) were Gleason score 6. Of 48 cases 17 (35%) had extraprostatic extension, 3 had seminal vesicle/lymph node involvement and 7 (15%) had positive margins. Mean total tumor volume was 1.3 cm(3) (range 0.02 to 10.8). Of the 48 tumors 13 (27%) were potentially clinically insignificant (organ confined, dominant nodule less than 0.5 cm(3), no Gleason pattern 4/5) and 19% (5 of 26) of the radical prostatectomies with a dominant tumor nodule less than 0.5 cm(3) demonstrated extraprostatic extension, 4 with Gleason pattern 4. All 10 tumors with a dominant nodule greater than 1 cm(3) were located predominantly anteriorly. CONCLUSIONS Most progression after active surveillance occurs 1 to 2 years after diagnosis suggesting undersampling of more aggressive tumor rather than progression of indolent tumor. Even with progression most tumors have favorable pathology (27% potentially insignificant). A small percentage of men have advanced stage disease (pT3b or N1). The anterior region should be sampled in men on active surveillance.

The tetraspanin CD63 enhances the internalization of the H,K-ATPase β-subunit

The tetraspanin CD63 resides in late endosomes, lysosomes, secretory vesicles, and at the plasma membrane, and it moves among these compartments. We find that CD63 is present also in tubulovesicular elements, the intracellular compartments that contain the H,K-ATPase in unstimulated gastric parietal cells. The H,K-ATPase β-subunit and CD63 colocalize in parietal cells and form a complex that can be coprecipitated. The β-subunit and CD63 also interact when they are coexpressed in COS-7 cells. Furthermore, expression with CD63 induces the redistribution of the β-subunit from the cell surface to CD63+ intracellular compartments. Immunofluorescence and biochemical experiments reveal that this redistribution occurs by enhanced endocytosis of H,K-ATPase β-subunit complexed with CD63. Coexpression of the β-subunit with mutant CD63 polypeptides demonstrates that the enhanced internalization of the β-subunit depends on the capacity of CD63 to interact with adaptor protein complexes 2 and 3. These data indicate that CD63 serves as an adaptor protein that links its interaction partners to the endocytic machinery of the cell and suggest a previously uncharacterized protein-trafficking role for the tetraspanins.

Long Interspersed Element-1 Protein Expression Is a Hallmark of Many Human Cancers

Cancers comprise a heterogeneous group of human diseases. Unifying characteristics include unchecked abilities of tumor cells to proliferate and spread anatomically, and the presence of clonal advantageous genetic changes. However, universal and highly specific tumor markers are unknown. Herein, we report widespread long interspersed element-1 (LINE-1) repeat expression in human cancers. We show that nearly half of all human cancers are immunoreactive for a LINE-1-encoded protein. LINE-1 protein expression is a common feature of many types of high-grade malignant cancers, is rarely detected in early stages of tumorigenesis, and is absent from normal somatic tissues. Studies have shown that LINE-1 contributes to genetic changes in cancers, with somatic LINE-1 insertions seen in selected types of human cancers, particularly colon cancer. We sought to correlate this observation with expression of the LINE-1-encoded protein, open reading frame 1 protein, and found that LINE-1 open reading frame 1 protein is a surprisingly broad, yet highly tumor-specific, antigen.

MAL decreases the internalization of the aquaporin-2 water channel.

Body water homeostasis depends critically on the hormonally regulated trafficking of aquaporin-2 (AQP2) water channels in renal collecting duct epithelial cells. Several types of posttranslational modifications are clearly involved in controlling the distribution of AQP2 between intracellular vesicles and the apical plasma membrane. Little is known, however, about the protein interactions that govern the trafficking of AQP2 between these organelles. MAL is a detergent-resistant membrane-associated protein implicated in apical sorting events. We wondered, therefore, whether MAL plays a role in the regulated trafficking of AQP2 between intracellular vesicles and the apical surface. We find that AQP2 and MAL are coexpressed in epithelial cells of the kidney collecting duct. These two proteins interact, both in the native kidney and when expressed by transfection in cultured cells. The S256-phosphorylated form of AQP2 appears to interact more extensively with MAL than does the water channel protein not phosphorylated at this serine. We find that MAL is not involved in detergent-resistant membrane association or apical delivery of AQP2 in LLC-PK1 renal epithelial cells. Instead, MAL increases the S256 phosphorylation and apical surface expression of AQP2. Furthermore, internalization experiments show that MAL induces surface expression of AQP2 by attenuating its internalization. Thus, the involvement of MAL in the cell surface retention of apical membrane proteins could play an important role in regulated absorption and secretion in transporting epithelia.

Knock-in of a FLT3/ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model

Constitutive activation of FLT3 by internal tandem duplication (ITD) is one of the most common molecular alterations in acute myeloid leukemia (AML). FLT3/ITD mutations have also been observed in myelodysplastic syndrome patients both before and during progression to AML. Previous work has shown that insertion of an FLT3/ITD mutation into the murine Flt3 gene induces a myeloproliferative neoplasm, but not progression to acute leukemia, suggesting that additional cooperating events are required. We therefore combined the FLT3/ITD mutation with a model of myelodysplastic syndrome involving transgenic expression of the Nup98-HoxD13 (NHD13) fusion gene. Mice expressing both the FLT3/ITD and NHD13 transgene developed AML with 100% penetrance and short latency. These leukemias were driven by mutant FLT3 expression and were susceptible to treatment with FLT3 tyrosine kinase inhibitors. We also observed a spontaneous loss of the wild-type Flt3 allele in these AMLs, further modeling the loss of the heterozygosity phenomenon that is seen in human AML with FLT3-activating mutations. Because resistance to FLT3 inhibitors remains an important clinical issue, this model may help identify new molecular targets in collaborative signaling pathways.

Sorting of H,K‐ATPase β‐Subunit in MDCK and LLC‐PK1 Cells is Independent of μ1B Adaptin Expression

The cytoplasmic tail of the H,K‐ATPase β‐subunit contains a putative tyrosine‐based motif that directs the β‐subunits basolateral sorting when it is expressed in Madin‐Darby Canine Kidney (MDCK) cells. When expressed in LLC‐PK1 cells, however, the β‐subunit is localized to the apical membrane. Several proteins that contain tyrosine‐based motifs, including the low‐density lipoprotein and transferrin receptors, show a similar sorting ‘defect’ when expressed in LLC‐PK1 cells. For low‐density lipoprotein and transferrin receptors, this behavior is due to the differential expression of the μ1B subunit of the AP‐1B clathrin adaptor complex. μ1B is expressed by MDCK cells, but not LLC‐PK1 cells, and transfection of μ1B into LLC‐PK1 cells restores basolateral localization of low‐density lipoprotein and transferrin receptors. For the β‐subunit, however, μ1B expression in LLC‐PK1 cells does not induce its basolateral expression. We found that the β‐subunit interacts with both μ1B and μ1A in vitro and in vivo. The capacity to participate in a μ1B interaction therefore is not sufficient to program the β‐subunits basolateral localization in MDCK cells. Our data suggest that the H,K‐ATPase β‐subunits basolateral sorting signal is either masked in certain epithelial cells, or requires an interaction with sorting machinery other than AP‐1B for delivery to the basolateral plasma membrane.

Chapter 4 Protein Trafficking in Polarized Cells

Epithelial cells line the lumens of organs and thus constitute the interface between the bodys interior and exterior surfaces. This position endows these cells with the important task of regulating what enters and what is exported from the body. In order to accomplish this function, epithelia must have structurally and functionally distinct membrane surfaces: the apical surface exposed to the lumen, and the basolateral surface in contact with the laterally adjacent epithelial cells, and the connective tissue and capillary network below the epithelia. The specific lipid and protein contents of the apical and basolateral membrane surfaces are determined by a number of sorting and retention mechanisms. Many of these sorting and retention mechanisms are shared with other polarized cell types including neurons and certain cells of the immune system. This chapter focuses on recent advances in understanding how these various mechanisms facilitate the generation, maintenance, and dynamic regulation of protein and lipid trafficking within epithelial cells.

Clinical and Pathologic Features of Secondary Acute Promyelocytic Leukemia

Acute promyelocytic leukemia (APL) is a relatively common form of acute myeloid leukemia (AML) that has an excellent prognosis. In contrast, secondary acute myeloid leukemias, including therapy-related AML and AML with myelodysplasia-related changes, have a relatively poor prognosis. We identified 9 cases of APL at our institution in which there was a history of chemotherapy, radiotherapy, chronic immunosuppression, or antecedent myelodysplastic syndrome. The clinical and pathologic findings in these cases of secondary APL were compared with the clinical and pathologic findings in cases of de novo APL. We found that secondary and de novo APL had abnormal promyelocytes with similar morphologic and immunophenotypic features, comparable cytogenetic findings, comparable rates of FMS-like tyrosine kinase mutations, and similar rates of recurrent disease and death. These data suggest that secondary APL is similar to de novo APL and, thus, should be considered distinct from other secondary acute myeloid neoplasms.

Detection of Cancer in Radical Prostatectomy Specimens With no Residual Carcinoma in the Initial Review of Slides

Background Radical prostatectomy (RP) specimens occasionally contain no carcinoma in the initial slides of an entirely submitted specimen, but no protocol has been established to assess for carcinoma in the remainder of the specimen. Design We evaluated 34 cases with no carcinoma in the initial slide review of the entirely submitted RP over a 2-year interval out of 2200 RPs. Our sequential protocol for cases with no initial tumor is (1) review the biopsy; (2) do immunostains on suspicious foci; (3) perform levels on blocks with high-grade prostatic intraepithelial neoplasia; (4) perform 3 levels on the posterior sextant and adjacent sextant region where cancer was identified on biopsy; and (5) flip the blocks in these regions and perform 3 additional levels. Results The mean age was 58.1 years (41 to 69 y) with a mean prostate-specific antigen level of 5.9 ng/mL (0.8 to 19 ng/mL). On review, all of the biopsies had carcinoma with a Gleason score (GS) of 3+3=6. The number of positive cores was 1 [n=29 (85%)], 2 (n=3), 3 (n=1), and 4 (n=1). Fifty-nine percent (20/34) of the biopsies had immunohistochemistry (IHC) for basal cells and/or α-methylacyl CoA racemase. RPs on average weighed 73.6 g (36 to 155 g). Of the 34 cases with no initial cancer, cancer was found in 26 (76%), and 8 (24%) had no residual carcinoma despite extensive leveling in all cases and IHC in 1 case. IHC was performed on 12 of the 34 RP cases. Of 26 RP cases with cancer, 22 had cancer on only 1 slide, and 4 had cancer on 2 slides. All of the cancers in the radical prostatectomies were GS 6, and the GS agreed with the corresponding biopsy in all cases. In 83% (20/24) of cases that specified laterality in the biopsy, RP carcinoma was ipsilateral to carcinoma in the biopsy. In 93% (14/15) of the cases that specified sextant site in the biopsy, the location of the carcinoma in the RP was in the same or the adjacent inferior-superior sextant site. Of the 29 cases that required leveling, in 7 cases cancer was found only after flipping the blocks and doing additional levels. Of the 8 cases with no cancer, all biopsies had only 1 positive core with 6/8 having <10% of the core involved. Conclusions In about 1.5% of RP cases no tumor will be seen in the initially entirely submitted specimen. A methodical limited targeted approach to identifying cancer can identify cancer in 73% of the cases with no initial cancer, yet there will still be 0.4% of all RPs where cancer is not been identified. As cancer was seen in areas away from the biopsy site in some of our cases with minute tumor, leveling all the blocks may have identified cancer in some of the cases in which we found no tumor with our protocol.

Ion Pump‐Interacting Proteins: Promising New Partners

Abstract: The sorting and regulation of the Na,K and H,K‐ATPases requires that the pump proteins must associate, at least transiently, with kinases, phosphatases, scaffolding molecules, and components of the cellular trafficking machinery. The identities of these interacting proteins and the nature of their associations with the pump polypeptides have yet to be elucidated. We have begun a series of yeast two‐hybrid screens employing structurally defined segments of pump polypeptides as baits in order to gain insight into the nature and function of these interacting proteins.