the four fractions (Figure 3B) that are highly unlikely to arise from tailing. A Venn diagram (Figure 4) has been used to summarize the observed distribution of the proteins over the four sucrose gradient fractions as determined by the hierarchical clustering. A notable characteristic for the normal data set is that only 844 of the 2184 proteins (38.6%) were uniquely found in a single fraction. A further 296 proteins (13.6%) were found to be ubiquitously distributed over all fractions. The remaining 1044 proteins (47.8%) were consistent with intermediate distribution over multiple, but not all, subcellular locations. Of these 1044 proteins, 248 (11.4% of total proteins) were distributed over two fractions (e.g., cytosol and mitochondria, Figure 3C) or over three fractions (e.g., cytosol, membrane proteins and mitochondria, Figure 3B) in a "bimodal" manner that is inconsistent with

inclusion in a single subcellular organelle and "tailing" over the sucrose gradient.

Fig. 3. Hierarchical clustering and heat map across the four fractions. Individual proteins are represented by a single row, each fraction is represented by a single column, and each cell represents the abundance of a single protein in a single fraction. The color scale is for normalized relative abundance from 6.0 (red) to 1.0 (yellow) to 0.0 (blue, not detected). The expansions show typical regions of the heat map corresponding to: (a) proteins observed uniquely in cytosol, (b) "bimodal" proteins (see text) observed in fractions cytosol (CT), plasma membrane (PM), mitochondria (MT), and (c) "bimodal" proteins observed in

fractions cytosol and mitochondria.

Fig. 4. Four-way Venn diagrams summarizing the distribution of the 2184 proteins over different combinations of the sucrose gradient fractions.

#### **2.1.3 The data represent a good sampling of the distribution over multiple subcellular locations for the observed proteins**

Inspection of the distribution of the proteins between primary and secondary locations revealed that they are well dispersed over the regions compatible with a primary location and 1**-**3 secondary locations (Figure 5). Thus, for example, proteins for which we detected a primary location and a single secondary location must lie on the line from (0.5, 0.5) to (1.0, 0.0) (green plus signs in Figure 5), but are well dispersed along that line. For 2**-**3 secondary

Fig. 5. Distribution of proteins with a primary location and 1 (green), 2 (blue), or 3 (red) secondary locations over compatible areas of a plot of primary mole fractions vs secondary mole fractions. For each protein, the spectral counts observed in a specific gradient fraction were expressed as mole fractions of the total number of spectral counts observed in all four gradient fractions.

Quantitative Organelle Proteomics of Protein Distribution in Breast Cancer MCF-7 Cells 211

*a* Proteins where the name is shown in bold correspond to proteins which exemplify general

Table 2. Test for overlap of proteins between sucrose gradient fractions

*b* Normalized abundances were calculated from the Spectral Abundance Factor using GeneSpring, that is, the abundances have been normalized using a correction for the differing number of amino acids in the proteins. For all proteins, the normalized abundances ranged from 0.018 to 22.25. A dash indicates

*c* Selection criteria. A filter to select non detected proteins was applied to a chosen fraction (ND). In an adjacent fraction in the sucrose gradient, the proteins were sorted according to abundance and the

characteristics of the data that are noted in the text.

seven most abundant proteins (top) are shown.

the protein was not detected.

locations, the initial data set shows better sampling near the edges of the compatible regions, for example, there are more data points at large values of the primary mole fraction and at very small values of the secondary mole fractions. Many of these data points arise from proteins corresponding to sequencing of only one peptide and only 1**-**2 spectral counts in a specific fraction. This is a consequence of the sampling properties of spectral counting. The dispersion of the data points in Figure 5 over the compatible areas of the plot is a strong indication that the data represent a good sampling of the distribution over multiple subcellular locations for the observed proteins.

#### **2.1.4 Spurious tailing of proteins in the sucrose gradient does not make any major contributions to the observed multiplicity of locations**

A more quantitative evaluation of the possibility of tailing in the gradient was obtained by looking for proteins with high abundance in a given gradient fraction, but with no detectable abundance in the adjacent fractions. For the most abundant proteins, the MS detection method was capable of detecting as little as about 0.2% of the protein in an adjacent fraction. Because the proteins may correspond to different subcellular organelles, tailing between two fractions need not be symmetrical, e.g. tailing from CT to PM may not be the same as tailing from PM to CT. This leads to the six tests for the possibility of tailing shown in Table 2. For all the fractions there are many highly abundant proteins which do not tail into the adjacent fraction (Table 2). The highly abundant proteins also reveal some characteristics which are common in the data set. Some very abundant proteins were found uniquely in a single fraction (e.g., see hepatoma-derived growth factor and Protein S100-A9 in Table 2). Other proteins were detected in only two fractions, but with a bimodal distribution over the fractions (e.g., see sialic acid synthase and pyridoxal kinase in Table 2). Many proteins were distributed over several fractions, with substantial proportions of the protein present in different fractions (e.g., see ATP-citrate synthase in Table 2). Some proteins were primarily present in a single fraction, but small amounts of the protein were found in other fractions (see e.g. Rho GDP-dissociation inhibitor 1 and nucleophosmin in Table 2). We conclude from the data in Table 2 that spurious tailing of proteins in the sucrose gradient does not make any major contributions to the observed multiplicity of locations.

#### **2.1.5 Annotations of subcellular location**

We have used previous subcellular location annotations in the UniProtKB database (in the keyword "subcellular location" field and the ontology "subcellular component" field) and in the Locate Subcellular Location database to compare three aspects of the present work with earlier work: (1) the degree to which the individual sucrose gradient fractions are enriched with proteins corresponding to specific subcellular organelles; (2) the extent to which the multiplicity of subcellular locations observed here is reflected in current annotations of subcellular locations; and, (3) the extent to which there are discrepancies between this work and previous annotations of subcellular locations.

In evaluating these comparisons, it is important to keep in mind that there is not an exact mesh between our experimental strategy and the ontological descriptions of subcellular location used in the databases. The top level of our experimental design matches the levels (extracellular region, plasma membrane, cytoplasm, nucleus) in the GO classification scheme, but the experiment excludes the extracellular region and the nucleus. At a lower level we only tried to obtain an approximate resolution of the cytoplasm as (cytosol,

locations, the initial data set shows better sampling near the edges of the compatible regions, for example, there are more data points at large values of the primary mole fraction and at very small values of the secondary mole fractions. Many of these data points arise from proteins corresponding to sequencing of only one peptide and only 1**-**2 spectral counts in a specific fraction. This is a consequence of the sampling properties of spectral counting. The dispersion of the data points in Figure 5 over the compatible areas of the plot is a strong indication that the data represent a good sampling of the distribution over multiple

**2.1.4 Spurious tailing of proteins in the sucrose gradient does not make any major**

A more quantitative evaluation of the possibility of tailing in the gradient was obtained by looking for proteins with high abundance in a given gradient fraction, but with no detectable abundance in the adjacent fractions. For the most abundant proteins, the MS detection method was capable of detecting as little as about 0.2% of the protein in an adjacent fraction. Because the proteins may correspond to different subcellular organelles, tailing between two fractions need not be symmetrical, e.g. tailing from CT to PM may not be the same as tailing from PM to CT. This leads to the six tests for the possibility of tailing shown in Table 2. For all the fractions there are many highly abundant proteins which do not tail into the adjacent fraction (Table 2). The highly abundant proteins also reveal some characteristics which are common in the data set. Some very abundant proteins were found uniquely in a single fraction (e.g., see hepatoma-derived growth factor and Protein S100-A9 in Table 2). Other proteins were detected in only two fractions, but with a bimodal distribution over the fractions (e.g., see sialic acid synthase and pyridoxal kinase in Table 2). Many proteins were distributed over several fractions, with substantial proportions of the protein present in different fractions (e.g., see ATP-citrate synthase in Table 2). Some proteins were primarily present in a single fraction, but small amounts of the protein were found in other fractions (see e.g. Rho GDP-dissociation inhibitor 1 and nucleophosmin in Table 2). We conclude from the data in Table 2 that spurious tailing of proteins in the sucrose gradient does not make any major contributions to the observed multiplicity of

We have used previous subcellular location annotations in the UniProtKB database (in the keyword "subcellular location" field and the ontology "subcellular component" field) and in the Locate Subcellular Location database to compare three aspects of the present work with earlier work: (1) the degree to which the individual sucrose gradient fractions are enriched with proteins corresponding to specific subcellular organelles; (2) the extent to which the multiplicity of subcellular locations observed here is reflected in current annotations of subcellular locations; and, (3) the extent to which there are discrepancies between this work

In evaluating these comparisons, it is important to keep in mind that there is not an exact mesh between our experimental strategy and the ontological descriptions of subcellular location used in the databases. The top level of our experimental design matches the levels (extracellular region, plasma membrane, cytoplasm, nucleus) in the GO classification scheme, but the experiment excludes the extracellular region and the nucleus. At a lower level we only tried to obtain an approximate resolution of the cytoplasm as (cytosol,

subcellular locations for the observed proteins.

**2.1.5 Annotations of subcellular location**

and previous annotations of subcellular locations.

locations.

**contributions to the observed multiplicity of locations**

*a* Proteins where the name is shown in bold correspond to proteins which exemplify general characteristics of the data that are noted in the text.

*b* Normalized abundances were calculated from the Spectral Abundance Factor using GeneSpring, that is, the abundances have been normalized using a correction for the differing number of amino acids in the proteins. For all proteins, the normalized abundances ranged from 0.018 to 22.25. A dash indicates the protein was not detected.

*c* Selection criteria. A filter to select non detected proteins was applied to a chosen fraction (ND). In an adjacent fraction in the sucrose gradient, the proteins were sorted according to abundance and the seven most abundant proteins (top) are shown.

Table 2. Test for overlap of proteins between sucrose gradient fractions

Quantitative Organelle Proteomics of Protein Distribution in Breast Cancer MCF-7 Cells 213

fraction, 59 (36.2%) of the bimodal proteins only had annotation at the level (nucleus, extracellular region, no annotation). Furthermore, only 22 (13.5%) of the proteins show multiple locations at the annotation level (cytoplasm/cytosol, plasma membrane, endoplasmic reticulum, Golgi apparatus, mitochondrion). In general these results are consistent with the conclusion that current database annotations of subcellular location are

Fig. 6. Distribution of current subcellular location annotations in the UniProtKB database over the proteins observed solely in a single sucrose gradient fraction in the initial data set.

compatible with our experimental strategy (upper: extracellular region, plasma membrane, cytoplasm, nucleus; lower: cytosol/cytoplasm, endoplasmic reticulum, Golgi apparatus, mitochondrion). A small number of proteins had multiple lower level annotations and are shown in the region color coded as multiple. Proteins that had multiple annotations that included other locations different from the color code are indicated by the radial letters. The

The annotations are color coded (legend) according to the GO classification levels

heavy white lines delineate slice regions that have different compatibility with the

experimental data.

sparse and skewed toward single locations for proteins.

endoplasmic reticulum, mitochondria), while the databases typically use (cytoplasm/ cytosol, endoplasmic reticulum, mitochondrion, Golgi apparatus). Overall, relative to the UniProtKB subcellular locations, 271 proteins had no annotations, 1388 had annotations at the top level and 525 had annotations at the lower level. For the 481 (22.0%) proteins in the initial data set that were observed in only a single fraction, we compared their locations with previous experimental information about subcellular location in the UniProtKB database. Figure 6 summarizes the proportion of these "unique" proteins which were previously assigned to various subcellular locations. This data provides an overview of the enrichment of the four fractions with cytosolic, plasma membrane, endoplasmic reticulum and mitochondrial proteins respectively. First, all four fractions show a substantial proportion of proteins either for which there is no previous annotation of subcellular location, or for which the previous annotation is only nucleus or extracellular region (from 5 (19%) of proteins in ER to 83 (40%) of proteins in MT). These annotations are compatible with the enrichment of the fractions with their various types of proteins and the present results constitute new annotation information for these proteins. Fraction CT shows three other major slices: (1) proteins which are fully compatible with cytosolic proteins, (2) proteins which have previously been assigned to cytoplasm, but also to other subcellular locations, and (3) proteins which have been previously assigned to other subcellular locations, but not to cytoplasm or cytosol. There is some ambiguity in the second and third groups since cytosol is not distinguished in many experimental strategies and the assigned locations are daughters of cytoplasm (but not of cytosol) in the GO ontology. Overall for the 127 proteins observed only in fraction CT, 119 (93.7%) have annotations that are compatible with enrichment of this fraction with cytosolic proteins. Only 8 proteins (6.3%) appear to be discrepancies that have other, incompatible locations. Of the 119 compatible proteins, 16 proteins have previous annotations that deviate from observation uniquely in fraction CT. For the other sucrose gradient fractions the cytoplasm/cytosol distinction also leads to some ambiguity, but overall the number/proportion of proteins compatible with enrichment of fraction PM (plasma membrane), fraction ER (endoplasmic reticulum) and fraction MT (mitochondrion) with the respective protein types are 94 (78.3%), 18 (67.0%), and 184 (88.9%) respectively. Because there is some inconsistency between the different subcellular location annotation sources , these numbers vary somewhat if the UniProtKB subcellular components or the Subcellular Location database are used, but do not change the overall conclusion. Within the limitations of such comparisons, we conclude that the previous annotations are largely consistent with enrichment of the fractions with the expected protein types. Apparent experimental/ database annotation discrepancies for all 2184 proteins are considered in more detail below. Is the apparent multiplicity of protein subcellular locations observed in our experiments captured in current database annotations? To address this question, we used the set of 163 proteins in the initial data set that showed bimodal, nonadjacent distributions over the sucrose gradient fractions (includes proteins observed only in combinations of non-adjacent fractions CT**-**ER, CT**-**MT, PM**-**MT, CT**-**PM**-**MT, and CT**-**ER**-**MT, i.e. proteins that clearly have multiple locations) and which also had at least 8 spectral counts. The latter condition ensures that the classification of these proteins as bimodal is not unduly influenced by the dynamic range limitations of MS/MS spectral counting. This set of proteins was compared with (merged) subcellular location annotations from the UniProtKB and LOCATE Subcellular Location databases. Figure 7 shows the distribution over the bimodal combinations of fractions and the annotations of subcellular location for all 163 proteins. As seen above with the proteins identified in only a single

endoplasmic reticulum, mitochondria), while the databases typically use (cytoplasm/ cytosol, endoplasmic reticulum, mitochondrion, Golgi apparatus). Overall, relative to the UniProtKB subcellular locations, 271 proteins had no annotations, 1388 had annotations at the top level and 525 had annotations at the lower level. For the 481 (22.0%) proteins in the initial data set that were observed in only a single fraction, we compared their locations with previous experimental information about subcellular location in the UniProtKB database. Figure 6 summarizes the proportion of these "unique" proteins which were previously assigned to various subcellular locations. This data provides an overview of the enrichment of the four fractions with cytosolic, plasma membrane, endoplasmic reticulum and mitochondrial proteins respectively. First, all four fractions show a substantial proportion of proteins either for which there is no previous annotation of subcellular location, or for which the previous annotation is only nucleus or extracellular region (from 5 (19%) of proteins in ER to 83 (40%) of proteins in MT). These annotations are compatible with the enrichment of the fractions with their various types of proteins and the present results constitute new annotation information for these proteins. Fraction CT shows three other major slices: (1) proteins which are fully compatible with cytosolic proteins, (2) proteins which have previously been assigned to cytoplasm, but also to other subcellular locations, and (3) proteins which have been previously assigned to other subcellular locations, but not to cytoplasm or cytosol. There is some ambiguity in the second and third groups since cytosol is not distinguished in many experimental strategies and the assigned locations are daughters of cytoplasm (but not of cytosol) in the GO ontology. Overall for the 127 proteins observed only in fraction CT, 119 (93.7%) have annotations that are compatible with enrichment of this fraction with cytosolic proteins. Only 8 proteins (6.3%) appear to be discrepancies that have other, incompatible locations. Of the 119 compatible proteins, 16 proteins have previous annotations that deviate from observation uniquely in fraction CT. For the other sucrose gradient fractions the cytoplasm/cytosol distinction also leads to some ambiguity, but overall the number/proportion of proteins compatible with enrichment of fraction PM (plasma membrane), fraction ER (endoplasmic reticulum) and fraction MT (mitochondrion) with the respective protein types are 94 (78.3%), 18 (67.0%), and 184 (88.9%) respectively. Because there is some inconsistency between the different subcellular location annotation sources , these numbers vary somewhat if the UniProtKB subcellular components or the Subcellular Location database are used, but do not change the overall conclusion. Within the limitations of such comparisons, we conclude that the previous annotations are largely consistent with enrichment of the fractions with the expected protein types. Apparent experimental/ database annotation discrepancies for all 2184 proteins are considered in more detail below. Is the apparent multiplicity of protein subcellular locations observed in our experiments captured in current database annotations? To address this question, we used the set of 163 proteins in the initial data set that showed bimodal, nonadjacent distributions over the sucrose gradient fractions (includes proteins observed only in combinations of non-adjacent fractions CT**-**ER, CT**-**MT, PM**-**MT, CT**-**PM**-**MT, and CT**-**ER**-**MT, i.e. proteins that clearly have multiple locations) and which also had at least 8 spectral counts. The latter condition ensures that the classification of these proteins as bimodal is not unduly influenced by the dynamic range limitations of MS/MS spectral counting. This set of proteins was compared with (merged) subcellular location annotations from the UniProtKB and LOCATE Subcellular Location databases. Figure 7 shows the distribution over the bimodal combinations of fractions and the annotations of subcellular location for all 163 proteins. As seen above with the proteins identified in only a single fraction, 59 (36.2%) of the bimodal proteins only had annotation at the level (nucleus, extracellular region, no annotation). Furthermore, only 22 (13.5%) of the proteins show multiple locations at the annotation level (cytoplasm/cytosol, plasma membrane, endoplasmic reticulum, Golgi apparatus, mitochondrion). In general these results are consistent with the conclusion that current database annotations of subcellular location are sparse and skewed toward single locations for proteins.

Fig. 6. Distribution of current subcellular location annotations in the UniProtKB database over the proteins observed solely in a single sucrose gradient fraction in the initial data set. The annotations are color coded (legend) according to the GO classification levels compatible with our experimental strategy (upper: extracellular region, plasma membrane, cytoplasm, nucleus; lower: cytosol/cytoplasm, endoplasmic reticulum, Golgi apparatus, mitochondrion). A small number of proteins had multiple lower level annotations and are shown in the region color coded as multiple. Proteins that had multiple annotations that included other locations different from the color code are indicated by the radial letters. The heavy white lines delineate slice regions that have different compatibility with the experimental data.

Quantitative Organelle Proteomics of Protein Distribution in Breast Cancer MCF-7 Cells 215

proteins), mitochondrion (168) and endoplasmic reticulum (243) proteins. There is rather little concordance between the annotation sets, which presumably must reflect the inclusion

Fig. 8. Venn diagrams comparing the status of annotations of subcellular locations in the UniProtKB subcellular location components, UniProtKB cellular component GO terms and LOCATE Subcellular Location database for proteins observed in this work. 443 proteins annotated as plasma membrane. 168 proteins annotated as mitochondrion. 243 proteins

Only 8 of the 443 proteins with annotations of plasma membrane were so annotated in all three data sets! For the proteins annotated to plasma membrane, endoplasmic reticulum and mitochondrion that we did not observe in the corresponding gradient fractions, our data would suggest different primary locations for these 120 proteins, but does not exclude their presence in the annotated subcellular locations as secondary locations which could not be detected at our sensitivity limits. We believe that some occurrences of apparent discrepancies are almost inevitable for three reasons. First, there is still very little information about whether subcellular distributions of proteins are the same in different cell types or under different cellular conditions. Second, many experiments do not distinguish between different isoforms of the same protein, which may have different subcellular distributions. Indeed, the present data set includes these proteins, which in part show different distributions over subcellular locations for isoforms of the same protein. This data will be analyzed in a separate paper. Third, the databases attempt to aggregate data from experimental strategies with very different sensitivity, selectivity, dynamic range, and coverage of proteins. Targeted searches for individual proteins in purified subcellular fractions with antibody methods probably have the highest sensitivity for detecting trace amounts of proteins in any specified location, even if the trace is a tiny proportion of the total protein abundance. Conversely, some high throughput methods may have limited

of very different experimental data sets.

annotated as endoplasmic reticulum.

Fig. 7. Comparison of the present data on subcellular location of bimodal proteins with (merged) subcellular location annotations in the UniProtKB subcellular location comments, UniProtKB subcellular component GO terms and LOCATE Subcellular Location database. (left) Distribution of the 299 bimodal proteins in the initial data set over different combinations of sucrose gradient fractions. The indicated combinations of fractions can only arise for proteins with at least two different subcellular locations. (right) Summary of the (merged) subcellular location annotations for all 299 bimodal proteins. The annotations are color coded (legend) according to the GO classification levels compatible with our experimental strategy (upper: extracellular region, plasma membrane, cytoplasm, nucleus; lower: cytosol/cytoplasm, endoplasmic reticulum, Golgi apparatus, mitochondrion). A small number of proteins had multiple lower level annotations and are shown in the region color coded as multiple. Proteins that had multiple annotations that included other locations different from the color code are indicated by the radial letters. Slices that have color coded radial lettering are those corresponding to proteins whose annotations indicate multiple subcellular locations within the GO classification levels of our experimental design.

Over all of the 2184 proteins, the annotations at the subcellular level in the examined databases tend to be to single locations. Given that many previous proteomics studies were biased against detection of proteins in multiple locations (e.g., studies of purified organelles) and that annotations at sub-cytoplasmic levels are clearly still very sparse, we consider that the previously available annotations of experimental data are not inconsistent with the proposal that many, probably a sizable majority, of the proteins have multiple subcellular locations. Using the initial data set of (protein, fraction) pairs, there were a relatively small number of discrepancies between our data and previous annotations of subcellular location in the two databases. Of the 1441 proteins identified in fraction ER, there were a total of 33 proteins previously annotated to endoplasmic reticulum that we did not observe in fraction ER. Similarly for fractions PM (1611 proteins) and MT (1610 proteins), there were a total of 58 and 29 proteins previously annotated to plasma membrane and mitochondrion respectively that we did not observe in the corresponding gradient fraction.

Inconsistencies in the databases might contribute to the apparent discrepancies. For the 2184 proteins identified here, Figure 8 shows the status of annotations of plasma membrane (443

Fig. 7. Comparison of the present data on subcellular location of bimodal proteins with (merged) subcellular location annotations in the UniProtKB subcellular location comments, UniProtKB subcellular component GO terms and LOCATE Subcellular Location database.

combinations of sucrose gradient fractions. The indicated combinations of fractions can only arise for proteins with at least two different subcellular locations. (right) Summary of the (merged) subcellular location annotations for all 299 bimodal proteins. The annotations are

experimental strategy (upper: extracellular region, plasma membrane, cytoplasm, nucleus; lower: cytosol/cytoplasm, endoplasmic reticulum, Golgi apparatus, mitochondrion). A small number of proteins had multiple lower level annotations and are shown in the region color coded as multiple. Proteins that had multiple annotations that included other locations different from the color code are indicated by the radial letters. Slices that have color coded radial lettering are those corresponding to proteins whose annotations indicate multiple subcellular locations within the GO classification levels of our experimental design.

Over all of the 2184 proteins, the annotations at the subcellular level in the examined databases tend to be to single locations. Given that many previous proteomics studies were biased against detection of proteins in multiple locations (e.g., studies of purified organelles) and that annotations at sub-cytoplasmic levels are clearly still very sparse, we consider that the previously available annotations of experimental data are not inconsistent with the proposal that many, probably a sizable majority, of the proteins have multiple subcellular locations. Using the initial data set of (protein, fraction) pairs, there were a relatively small number of discrepancies between our data and previous annotations of subcellular location in the two databases. Of the 1441 proteins identified in fraction ER, there were a total of 33 proteins previously annotated to endoplasmic reticulum that we did not observe in fraction ER. Similarly for fractions PM (1611 proteins) and MT (1610 proteins), there were a total of 58 and 29 proteins previously annotated to plasma membrane and mitochondrion

Inconsistencies in the databases might contribute to the apparent discrepancies. For the 2184 proteins identified here, Figure 8 shows the status of annotations of plasma membrane (443

(left) Distribution of the 299 bimodal proteins in the initial data set over different

color coded (legend) according to the GO classification levels compatible with our

respectively that we did not observe in the corresponding gradient fraction.

proteins), mitochondrion (168) and endoplasmic reticulum (243) proteins. There is rather little concordance between the annotation sets, which presumably must reflect the inclusion of very different experimental data sets.

Fig. 8. Venn diagrams comparing the status of annotations of subcellular locations in the UniProtKB subcellular location components, UniProtKB cellular component GO terms and LOCATE Subcellular Location database for proteins observed in this work. 443 proteins annotated as plasma membrane. 168 proteins annotated as mitochondrion. 243 proteins annotated as endoplasmic reticulum.

Only 8 of the 443 proteins with annotations of plasma membrane were so annotated in all three data sets! For the proteins annotated to plasma membrane, endoplasmic reticulum and mitochondrion that we did not observe in the corresponding gradient fractions, our data would suggest different primary locations for these 120 proteins, but does not exclude their presence in the annotated subcellular locations as secondary locations which could not be detected at our sensitivity limits. We believe that some occurrences of apparent discrepancies are almost inevitable for three reasons. First, there is still very little information about whether subcellular distributions of proteins are the same in different cell types or under different cellular conditions. Second, many experiments do not distinguish between different isoforms of the same protein, which may have different subcellular distributions. Indeed, the present data set includes these proteins, which in part show different distributions over subcellular locations for isoforms of the same protein. This data will be analyzed in a separate paper. Third, the databases attempt to aggregate data from experimental strategies with very different sensitivity, selectivity, dynamic range, and coverage of proteins. Targeted searches for individual proteins in purified subcellular fractions with antibody methods probably have the highest sensitivity for detecting trace amounts of proteins in any specified location, even if the trace is a tiny proportion of the total protein abundance. Conversely, some high throughput methods may have limited

Quantitative Organelle Proteomics of Protein Distribution in Breast Cancer MCF-7 Cells 217

(ranging from 54.8% for cell interaction to 66.5% for protein processing). Furthermore, the latter characteristic was most pronounced for the 93 proteins that were involved in more

Fig. 9. Four-way Venn diagrams summarizing the distribution of the breast-cancer-related set of 519 proteins over the subcellular locations for the cellular processes: signaling (131 proteins), cell growth (127), protein processing (230), apoptosis (68 proteins), and cell interaction (62), as well as for proteins involved in more than one of these cellular processes (93). The shaded regions of the diagrams correspond to proteins with 3 or 4 locations.

Large scale and quantitative proteomics analysis of subcellular organelles revealed 268 nuclear proteins and 22 extracellular region proteins that were found in various sucrose gradient fractions, but which had previously only been annotated experimentally to the nucleus and extracellular region respectively. Another 271 proteins that we detected had no prior annotation at either the upper level (cytoplasm, plasma membrane) or lower level (cytosol, endoplasmic reticulum, mitochondria) of our experimental strategy. The present experiments were not designed to obtain specific annotations at the lower level, e.g. to mitochondrion. Hence, observation of a protein in a Fraction MT that is enriched in mitochondrial proteins should presently only be taken as an indication and not as proof of its presence in mitochondria. Nonetheless, the present experiments gave several hundred

than one of the high level cellular processes (68.8%)

**3. Conclusion**

resolution for some subcellular locations, for example, distinguishing cytosol from cytoplasm, and may have insufficient sensitivity and dynamic range to detect trace amounts of proteins in specific locations. Aggregating subcellular location information from many cell types and conditions obtained with very different experimental strategies, many of which do not distinguish protein isoforms, then becomes a very tricky task which seems likely to produce some discrepancies with any specific experimental method/data set. Although only a few of the fractions from the sucrose density gradient have been analyzed, the normal data set provides clear evidence that a minimum of 543 of the 2184 proteins (24.9%) show multiple locations. The minimum estimate is based on those proteins that are either present in all fractions or show bimodal distributions with abundance peaks in nonadjacent fractions of the sucrose gradient (Figure 3B, C). For the 321 proteins (14.7%) that were found only in adjacent fractions of the gradient (i.e., CT**-**PM, PM**-**ER, and ER**-**MT), the present experiments are insufficient to exclude that this might be due to the presence of a single organelle that occupies an intermediate position between the two fractions. On the other hand, we intentionally spaced the analyzed fractions widely in the sucrose gradient and for the 476 proteins (21.8%) that were found in three adjacent fractions (i.e., CT**-**PM**-**ER, or PM**-**ER**-**MT), it is improbable that these proteins have single subcellular locations. Especially since other proteins demonstrated lack of overlap (e.g., proteins in fractions CT**-**ER or PM**-**MT) and lack of tailing in the sucrose gradient (Table 2). Furthermore, in most cases the relative abundances for the proteins observed in three adjacent fractions were substantial and did not correspond to trace proportions. Thus, the normal data set provides evidence indicating that 38.6% of the proteins may have unique locations, 24.9% certainly have multiple locations, 21.8% most likely have multiple locations and 14.7% may have either unique or multiple locations. We have used the observed set of proteins to examine possible connections between subcellular location and function as related to breast cancer. Many of the proteins observed in our experiments have previously been annotated with functional information.

#### **2.1.6 Breast cancer related proteins**

Biological process annotations for 1673 proteins, molecular function annotations for 1980 proteins, Reactome Pathway annotations for 176 proteins and posttranslational modification annotations for 1653 proteins were available in the UniProtKB database. We used the BioBase Biological Databases, BIOBASE Knowledge Library (BKL) and ExPlainTM 2.3 platform to identify 94 proteins in our data set that are known or suspected to be implicated in breast cancer via disease molecular mechanism, diagnostic marker and therapeutic target association. These proteins were examined for common Gene Ontology (http://www.geneontology.org) biological process and molecular function terms and for common Reactome Pathway (http://www.reactome.org) terms, which were then used as lures to obtain the set of proteins identified in this study that share the same terms. A majority of the proteins implicated in breast cancer were related to five high level cellular processes that involved a subset of 519 proteins observed in our experiments: apoptosis (68 proteins), cell growth (127), signaling (131), cell interaction (62), and protein processing (230). 93 proteins were involved in more than one of the five processes. Figure 9 shows how the proteins associated with each cellular process are distributed over the subcellular locations using the initial data set. The striking features are that each process is distributed over all four locations, as might be anticipated for regulated processes, and that for all of the cellular processes there is an appreciable majority of proteins with 3**-**4 subcellular locations (ranging from 54.8% for cell interaction to 66.5% for protein processing). Furthermore, the latter characteristic was most pronounced for the 93 proteins that were involved in more than one of the high level cellular processes (68.8%)

Fig. 9. Four-way Venn diagrams summarizing the distribution of the breast-cancer-related set of 519 proteins over the subcellular locations for the cellular processes: signaling (131 proteins), cell growth (127), protein processing (230), apoptosis (68 proteins), and cell interaction (62), as well as for proteins involved in more than one of these cellular processes (93). The shaded regions of the diagrams correspond to proteins with 3 or 4 locations.

### **3. Conclusion**

216 Breast Cancer – Recent Advances in Biology, Imaging and Therapeutics

resolution for some subcellular locations, for example, distinguishing cytosol from cytoplasm, and may have insufficient sensitivity and dynamic range to detect trace amounts of proteins in specific locations. Aggregating subcellular location information from many cell types and conditions obtained with very different experimental strategies, many of which do not distinguish protein isoforms, then becomes a very tricky task which seems likely to produce some discrepancies with any specific experimental method/data set. Although only a few of the fractions from the sucrose density gradient have been analyzed, the normal data set provides clear evidence that a minimum of 543 of the 2184 proteins (24.9%) show multiple locations. The minimum estimate is based on those proteins that are either present in all fractions or show bimodal distributions with abundance peaks in nonadjacent fractions of the sucrose gradient (Figure 3B, C). For the 321 proteins (14.7%) that were found only in adjacent fractions of the gradient (i.e., CT**-**PM, PM**-**ER, and ER**-**MT), the present experiments are insufficient to exclude that this might be due to the presence of a single organelle that occupies an intermediate position between the two fractions. On the other hand, we intentionally spaced the analyzed fractions widely in the sucrose gradient and for the 476 proteins (21.8%) that were found in three adjacent fractions (i.e., CT**-**PM**-**ER, or PM**-**ER**-**MT), it is improbable that these proteins have single subcellular locations. Especially since other proteins demonstrated lack of overlap (e.g., proteins in fractions CT**-**ER or PM**-**MT) and lack of tailing in the sucrose gradient (Table 2). Furthermore, in most cases the relative abundances for the proteins observed in three adjacent fractions were substantial and did not correspond to trace proportions. Thus, the normal data set provides evidence indicating that 38.6% of the proteins may have unique locations, 24.9% certainly have multiple locations, 21.8% most likely have multiple locations and 14.7% may have either unique or multiple locations. We have used the observed set of proteins to examine possible connections between subcellular location and function as related to breast cancer. Many of the proteins observed in our experiments have previously been annotated with

Biological process annotations for 1673 proteins, molecular function annotations for 1980 proteins, Reactome Pathway annotations for 176 proteins and posttranslational modification annotations for 1653 proteins were available in the UniProtKB database. We used the BioBase Biological Databases, BIOBASE Knowledge Library (BKL) and ExPlainTM 2.3 platform to identify 94 proteins in our data set that are known or suspected to be implicated in breast cancer via disease molecular mechanism, diagnostic marker and therapeutic target association. These proteins were examined for common Gene Ontology (http://www.geneontology.org) biological process and molecular function terms and for common Reactome Pathway (http://www.reactome.org) terms, which were then used as lures to obtain the set of proteins identified in this study that share the same terms. A majority of the proteins implicated in breast cancer were related to five high level cellular processes that involved a subset of 519 proteins observed in our experiments: apoptosis (68 proteins), cell growth (127), signaling (131), cell interaction (62), and protein processing (230). 93 proteins were involved in more than one of the five processes. Figure 9 shows how the proteins associated with each cellular process are distributed over the subcellular locations using the initial data set. The striking features are that each process is distributed over all four locations, as might be anticipated for regulated processes, and that for all of the cellular processes there is an appreciable majority of proteins with 3**-**4 subcellular locations

functional information.

**2.1.6 Breast cancer related proteins**

Large scale and quantitative proteomics analysis of subcellular organelles revealed 268 nuclear proteins and 22 extracellular region proteins that were found in various sucrose gradient fractions, but which had previously only been annotated experimentally to the nucleus and extracellular region respectively. Another 271 proteins that we detected had no prior annotation at either the upper level (cytoplasm, plasma membrane) or lower level (cytosol, endoplasmic reticulum, mitochondria) of our experimental strategy. The present experiments were not designed to obtain specific annotations at the lower level, e.g. to mitochondrion. Hence, observation of a protein in a Fraction MT that is enriched in mitochondrial proteins should presently only be taken as an indication and not as proof of its presence in mitochondria. Nonetheless, the present experiments gave several hundred

Quantitative Organelle Proteomics of Protein Distribution in Breast Cancer MCF-7 Cells 219

over multiple subcellular locations can be important to their functional roles, and that large numbers of (protein, location) pairs deserving of further investigation of functional/regulatory roles can be delineated. We are still very far from having good static descriptions of the spatial distributions of cellular proteins, let alone dynamic information on relationships between spatio/temporal distribution and function. However, highthroughput proteomics in combination with other experimental methods seems to offer

We would like to thank Professor Samia M. A. Al-Amoudi (King Abdulaziz University, Jeddah, SA) for generous help and discussions and to the Wellcome Trust, UK, King Faisal Foundation, Riyadh, SA, Sheikh Mohammed Hussien Al-Amoudi Centre Scientific Chair for Breast Cancer Researches, Jeddah, SA, Susan G. Komen for the Cure Breast Cancer Foundation, USA, King Faisal Specialist Hospital and Research Center, Riyadh, SA for their

Figs, tables and part of the text reprinted and adapted with permission from Qattan et al

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Hanahan, D. & R. A. Weinberg (2011) Hallmarks of cancer: the next generation. *Cell,* 144,

Imyanitov, E. & K. Hanson (2004) Mechanisms of breast cancer. *Drug Discovery Today:*

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ways forward.

**4. Acknowledgment**

continuous support.

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new location annotations at the level (plasma membrane, cytoplasm). There are several ways the limits on MS detection sensitivity may influence the number of locations in which the proteins were observed. In particular, for the highest abundance proteins, the sensitivity and dynamic range of the MS spectral counting methods are such that trace amounts as small as about 0.2% of a protein in a secondary location could be detected. As shown above for the normal data set, trace amounts of abundant proteins in secondary locations do not strongly influence estimates of the proportion of proteins with multiple subcellular locations. Conversely, the proportion of a protein which must be present in a secondary location to be detectable increases as the overall abundance of the proteins decreases, for example, for the lowest abundance proteins, only the highest abundance, primary location falls within the detection limits of the MS methods. Furthermore, for lower abundance proteins or for trace proportions of proteins in specific fractions, the sampling constraints on spectral counting that result from MS/MS sequencing of only the more abundant peptides means that only one peptide may be counted in some fractions. For example, there were 847 (38.8%) proteins classified as "unique" (observed in a single fraction) in the normal data set, but only 481 (22.0%) in the initial data set. This difference corresponds to proteins in various gradient fractions that were only counted with a single peptide and 1 or 2 spectral counts. This means that estimations of multiple locations based on the normal data set are very conservative and certainly underestimate, probably strongly, the proportion of proteins with multiple subcellular locations. Given that estimates based on the normal data set provide evidence for multiple locations of at least 46.7% of the observed proteins, we conclude that a substantial majority of the proteins observed have multiple subcellular locations. Given that only 22% of proteins were seen solely in a single fraction in the initial data set, perhaps as much as 75% of the proteins have multiple locations. We noted above that 120 proteins had annotations to subcellular locations that we did not observe in the corresponding sucrose gradient fractions (33 to endoplasmic reticulum, 58 to plasma membrane and 29 to mitochondrion). We suggested that these discrepancies were not inconsistent with our data if the annotations corresponded to secondary locations. On the basis of the observed spectral counts, there are 39 of these proteins for which our data suggest that the previous annotations correspond to proteins with functional significance in a secondary location, but that **>**80% of the protein is in a different primary location. This kind of analysis can be extended to many other proteins where the functional activity and the measured mole fractions indicate functional roles at secondary locations. Indeed, some of the proteins that we detected at trace amounts (**<**3%) in secondary locations already have known functions at those locations. The present experiments thus indicate numerous proteins with primary locations which probably differ from current function/location annotations and for which confirmation of the primary location (and potentially of other functional activities) might be profitably sought. The present experiments suggest 1383 (protein, location, function) data points for 519 proteins involved in five major cellular functional processes for which investigation of functional roles might further elucidate mechanisms involved in breast cancer. This is a very promising situation for experiments aimed at investigating dynamic changes in the spatio/temporal location/form of proteins in breast cancer cells, their potential roles in regulation and their potential importance in breast cancer disease. Finally, in summary, we have found evidence that strongly suggests a majority of the detected proteins have multiple subcellular locations in the breast cancer model MCF-7 cells, that even with a fairly simple experiment a wealth of new annotation data can be obtained, that available evidence suggests that for many proteins distribution over multiple subcellular locations can be important to their functional roles, and that large numbers of (protein, location) pairs deserving of further investigation of functional/regulatory roles can be delineated. We are still very far from having good static descriptions of the spatial distributions of cellular proteins, let alone dynamic information on relationships between spatio/temporal distribution and function. However, highthroughput proteomics in combination with other experimental methods seems to offer ways forward.

#### **4. Acknowledgment**

218 Breast Cancer – Recent Advances in Biology, Imaging and Therapeutics

new location annotations at the level (plasma membrane, cytoplasm). There are several ways the limits on MS detection sensitivity may influence the number of locations in which the proteins were observed. In particular, for the highest abundance proteins, the sensitivity and dynamic range of the MS spectral counting methods are such that trace amounts as small as about 0.2% of a protein in a secondary location could be detected. As shown above for the normal data set, trace amounts of abundant proteins in secondary locations do not strongly influence estimates of the proportion of proteins with multiple subcellular locations. Conversely, the proportion of a protein which must be present in a secondary location to be detectable increases as the overall abundance of the proteins decreases, for example, for the lowest abundance proteins, only the highest abundance, primary location falls within the detection limits of the MS methods. Furthermore, for lower abundance proteins or for trace proportions of proteins in specific fractions, the sampling constraints on spectral counting that result from MS/MS sequencing of only the more abundant peptides means that only one peptide may be counted in some fractions. For example, there were 847 (38.8%) proteins classified as "unique" (observed in a single fraction) in the normal data set, but only 481 (22.0%) in the initial data set. This difference corresponds to proteins in various gradient fractions that were only counted with a single peptide and 1 or 2 spectral counts. This means that estimations of multiple locations based on the normal data set are very conservative and certainly underestimate, probably strongly, the proportion of proteins with multiple subcellular locations. Given that estimates based on the normal data set provide evidence for multiple locations of at least 46.7% of the observed proteins, we conclude that a substantial majority of the proteins observed have multiple subcellular locations. Given that only 22% of proteins were seen solely in a single fraction in the initial data set, perhaps as much as 75% of the proteins have multiple locations. We noted above that 120 proteins had annotations to subcellular locations that we did not observe in the corresponding sucrose gradient fractions (33 to endoplasmic reticulum, 58 to plasma membrane and 29 to mitochondrion). We suggested that these discrepancies were not inconsistent with our data if the annotations corresponded to secondary locations. On the basis of the observed spectral counts, there are 39 of these proteins for which our data suggest that the previous annotations correspond to proteins with functional significance in a secondary location, but that **>**80% of the protein is in a different primary location. This kind of analysis can be extended to many other proteins where the functional activity and the measured mole fractions indicate functional roles at secondary locations. Indeed, some of the proteins that we detected at trace amounts (**<**3%) in secondary locations already have known functions at those locations. The present experiments thus indicate numerous proteins with primary locations which probably differ from current function/location annotations and for which confirmation of the primary location (and potentially of other functional activities) might be profitably sought. The present experiments suggest 1383 (protein, location, function) data points for 519 proteins involved in five major cellular functional processes for which investigation of functional roles might further elucidate mechanisms involved in breast cancer. This is a very promising situation for experiments aimed at investigating dynamic changes in the spatio/temporal location/form of proteins in breast cancer cells, their potential roles in regulation and their potential importance in breast cancer disease. Finally, in summary, we have found evidence that strongly suggests a majority of the detected proteins have multiple subcellular locations in the breast cancer model MCF-7 cells, that even with a fairly simple experiment a wealth of new annotation data can be obtained, that available evidence suggests that for many proteins distribution

We would like to thank Professor Samia M. A. Al-Amoudi (King Abdulaziz University, Jeddah, SA) for generous help and discussions and to the Wellcome Trust, UK, King Faisal Foundation, Riyadh, SA, Sheikh Mohammed Hussien Al-Amoudi Centre Scientific Chair for Breast Cancer Researches, Jeddah, SA, Susan G. Komen for the Cure Breast Cancer Foundation, USA, King Faisal Specialist Hospital and Research Center, Riyadh, SA for their continuous support.

Figs, tables and part of the text reprinted and adapted with permission from Qattan et al (2010), J Proteom Res 9, 459-508. Copyright (2011) American Chemical Society.

### **5. References**

http://www.ncbi.nlm.nih.gov

Hanahan, D. & R. A. Weinberg (2000) The hallmarks of cancer. *Cell,* 100, 57-70.

**Part 3**

**Diagnosis and Imaging**

**Part 3**

**Diagnosis and Imaging**

220 Breast Cancer – Recent Advances in Biology, Imaging and Therapeutics

Qattan, A. T., C. Mulvey, M. Crawford, D. A. Natale & J. Godovac-Zimmermann (2010)

Roberts, G. C. & C. W. Smith (2002) Alternative splicing: combinatorial output from the

Rogers, L. D. & L. J. Foster (2007) The dynamic phagosomal proteome and the contribution of the endoplasmic reticulum. *Proc Natl Acad Sci U S A,* 104, 18520-5. Sebastian, T. & P. F. Johnson (2006) Stop and go: anti-proliferative and mitogenic functions

Simpson, J. C. & R. Pepperkok (2006) The subcellular localization of the mammalian

Sorlie, T., C. M. Perou, R. Tibshirani, T. Aas, S. Geisler, H. Johnsen, T. Hastie, M. B. Eisen, M.

Soule, H. D., J. Vazguez, A. Long, S. Albert & M. Brennan (1973) A human cell line from

Usaite, R., J. Wohlschlegel, J. D. Venable, S. K. Park, J. Nielsen, L. Olsson & J. R. Yates Iii

van de Vijver, M. J., Y. D. He, L. J. van't Veer, H. Dai, A. A. Hart, D. W. Voskuil, G. J.

Xu, P., M. Crawford, M. Way, J. Godovac-Zimmermann, A. W. Segal & M. Radulovic (2009) Subproteome analysis of the neutrophil cytoskeleton. *Proteomics,* 9, 2037-49. Yates, J. R., 3rd, A. Gilchrist, K. E. Howell & J. J. Bergeron (2005) Proteomics of organelles

comparison of two quantitative methods. *J Proteome Res,* 7, 266-75.

predictor of survival in breast cancer. *N Engl J Med,* 347, 1999-2009.

and large cellular structures. *Nat Rev Mol Cell Biol,* 6, 702-14.

van de Rijn, S. S. Jeffrey, T. Thorsen, H. Quist, J. C. Matese, P. O. Brown, D. Botstein, P. Eystein Lonning & A. L. Borresen-Dale (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. *Proc*

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(2008) Characterization of global yeast quantitative proteome data generated from the wild-type and glucose repression saccharomyces cerevisiae strains: the

Schreiber, J. L. Peterse, C. Roberts, M. J. Marton, M. Parrish, D. Atsma, A. Witteveen, A. Glas, L. Delahaye, T. van der Velde, H. Bartelink, S. Rodenhuis, E. T. Rutgers, S. H. Friend & R. Bernards (2002) A gene-expression signature as a

of the transcription factor C/EBPbeta. *Cell Cycle,* 5, 953-7.

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Quantitative organelle proteomics of MCF-7 breast cancer cells reveals multiple subcellular locations for proteins in cellular functional processes. *J Proteome Res,* 9,

**12**

Tadaharu Matsunaga

*Nagumo Clinic*

*Japan*

**Intraductal Breast Cytology and Biopsy**

Detection of breast cancer at an early stage is the most effective approach to decrease mortality, and bloody nipple discharge is an important clue to detect ductal cancer.  The intraductal approach does not destroy the ductal structure and does not cause needle tract dissemination.  Mammary duct endoscopy (MS) has been employed to detect intraductal lesions and MS yields both an optical image and biopsy. Intraductal breast biopsy (IDBB) and direct ductal lavage cytology were used to obtain pathological and cytological diagnoses in intraductal lesions with nipple discharge. The usefulness of MS, IDBB and direct ductal lavage cytology for intraductal lesions, with particular regard to differential diagnosis of ductal carcinoma

Four hundred and sixty two MS procedures were performed in 348 patients who had nipple discharge but no overt mass between October 1993 and September 2009 at Tokyo Metropolitan Cancer Detection Center (October 1993 to December 2002), Minamiaoyama Breastopia Clinic (April 2003 to June 2006), and Tokyo Medical University Hachioji Medical Center (November 2006 to Sep 2009). In these 348 cases, nipple discharge was the primary diagnostic finding and there were no specific findings on mammography or sonography. Of these 348 patients, 81 patients with breast cancer and 163 patients of intraductal papilloma (IDP) were diagnosed pathologically. The other 104 patients were diagnosed as benign with

In these 348 patients, mammography, ultrasound and cytology of nipple discharge were performed routinely. Nipple discharge was examined with a glass slide touching the nipple directly and immediately immersed into 90% alcohol as a routine cytology method (touch method). The presence of a cluster of ductal cells was taken to indicate the presence of an intraductal papillary lesion. Nipple discharge was collected in a narrow vessel containing

**1. Introduction**

and intraductal papilloma.

**2. Materials and methods**

cytology and followed clinically for more than 5 years.

**to the Detection and Treatment**

**of Intraductal Lesions of the Breast**

### **Intraductal Breast Cytology and Biopsy to the Detection and Treatment of Intraductal Lesions of the Breast**

Tadaharu Matsunaga *Nagumo Clinic Japan*

#### **1. Introduction**

Detection of breast cancer at an early stage is the most effective approach to decrease mortality, and bloody nipple discharge is an important clue to detect ductal cancer.  The intraductal approach does not destroy the ductal structure and does not cause needle tract dissemination.  Mammary duct endoscopy (MS) has been employed to detect intraductal lesions and MS yields both an optical image and biopsy. Intraductal breast biopsy (IDBB) and direct ductal lavage cytology were used to obtain pathological and cytological diagnoses in intraductal lesions with nipple discharge. The usefulness of MS, IDBB and direct ductal lavage cytology for intraductal lesions, with particular regard to differential diagnosis of ductal carcinoma and intraductal papilloma.

#### **2. Materials and methods**

Four hundred and sixty two MS procedures were performed in 348 patients who had nipple discharge but no overt mass between October 1993 and September 2009 at Tokyo Metropolitan Cancer Detection Center (October 1993 to December 2002), Minamiaoyama Breastopia Clinic (April 2003 to June 2006), and Tokyo Medical University Hachioji Medical Center (November 2006 to Sep 2009). In these 348 cases, nipple discharge was the primary diagnostic finding and there were no specific findings on mammography or sonography. Of these 348 patients, 81 patients with breast cancer and 163 patients of intraductal papilloma (IDP) were diagnosed pathologically. The other 104 patients were diagnosed as benign with cytology and followed clinically for more than 5 years.

In these 348 patients, mammography, ultrasound and cytology of nipple discharge were performed routinely. Nipple discharge was examined with a glass slide touching the nipple directly and immediately immersed into 90% alcohol as a routine cytology method (touch method). The presence of a cluster of ductal cells was taken to indicate the presence of an intraductal papillary lesion. Nipple discharge was collected in a narrow vessel containing

Intraductal Breast Cytology and Biopsy

**2.2 Assessment of ductal cytology and IDBB**

**3. Result**

followed for more than 5 years (consisting of a total of 78 ducts).

**3.1.1 Visualization of intraductal lesions with MS**

to the Detection and Treatment of Intraductal Lesions of the Breast 225

Fig. 2. Method of intraductal breast biopsy (IDBB) 1)The MS is covered with a metalic tube with a side aperture near the tip and the MS with a metalic tube is inserted into the mammary duct. 2) The metallic tube is advanced along the outside of the mammoscope to the intraductal mass and the mass is entrapped in the tube through a side aperture under observation by the MS. 3)Then the metallic tube was turned to cut off the intraductal mass. 4) Afterwards, the MS is removed and then the tissue piece within a metallic tube is collected by manually applied negative pressure. The specimen was diagnosed as IDP.

The cytologic findings were classified into 4 categories; cancer, suspected cancer, suspected benign papillary lesion with the presence of a ductal cells cluster without atypia, and considered normal without any cluster. Cancer and suspected cancer categories were defined as positive findings. The diagnostic accuracy of these three methods , i.e. touch, collection, and directed ductal lavage were compared to IDBB in detecting malignancy. The therapeutic effect of IDBB for intraductal papillomas was also studied in 73 patients

The normal mammary duct wall is white or light pink in color. The wall is smooth and lustrous with discharge and capillary veins might be observed. Of 81 breast cancer lesions, intraductal lesions were observed in 50 (61.7%) by MS. Then feature of MS were classified into three types; hemispheric, papillary and flat, irregular ductal wall. The hemispheric and papillary shape were most common in intraductal papilloma and the flat irregular pattern was most common in ductal carcinoma. In the other 26 lesions, bloody secretion emanating

cell fixation liquid three times a day for three days at home in 70 breast cancer patients (collection method). This collection method was performed to obtain much cytologic specimen. Galactography was done if a papillary lesion was suggested by cytology or if there was an obvious bloody background. MS was indicated based on the galactographic findings and the cytological findings

#### **2.1 Intraductal approach to the detection of intraductal lesions**

At the time of the MS procedure, 4 % procaine was sprayed on the nipple. After injecting 1% procaine into the duct, the nipple orifice and ductal sphincter were dilated with a tear duct bougie. MS measuring 0.72 mm in outer diameter (Fiber Tech Company, Tokyo, Japan) were used. Intraductal breast biopsy (IDBB) under mammoscopic observation was performed in 226 intraductal papillomas (in a total of 128 patients) and in 38 ductal carcinomas (in a total of 35 patients). In most cases, IDBB was performed using a metallic tube with a side aperture near the tip (JN Biopsy Needle, Hakko Company, Tokyo, Japan). In directed ductal lavage cytolology, an epidural tube was used. After MS, the tube was inserted and advanced into the ductal branch where the papillary lesion was observed. When the lesion could not be observed by MS, emanation of bloody discharge from periphery suggested the direction of the lesion.We usually insufflated air to observe the ductal lumen by MS. Since after MS the lumen of the duct was enlarged, making it easy to selectively advance an epidural tube into an abnormal branch. When the lesion could not be observed by MS, emanation of bloody discharge from periphery suggested the direction of the lesion. An epidural tube, a 1 ml syringe of saline, and a 20 ml syringe were connected with a three-way valve. First the valve was turned to a 1-ml saline syringe and negative pressure was applied using a 20-ml syringe. Then the valve was turned to a-20 ml syringe and saline was sucked into the duct using the negative intraductal pressure. Then the valve was turned to a 1-ml syringe and intraductal fluid was collected into a 20-ml syringe. These procedures were repeated several times while changing the saline solution. Directed ductal lavage cytology was performed in this way in 38 patients. The cytologic findings were assessed by both cytology technician and a pathologist. These MS, IDBB and lavage procedures were performed in outpatient clinics..

Fig. 1. Mammary duct endscopy (MS) and biopsy needle

Fig. 2. Method of intraductal breast biopsy (IDBB) 1)The MS is covered with a metalic tube with a side aperture near the tip and the MS with a metalic tube is inserted into the mammary duct. 2) The metallic tube is advanced along the outside of the mammoscope to the intraductal mass and the mass is entrapped in the tube through a side aperture under observation by the MS. 3)Then the metallic tube was turned to cut off the intraductal mass. 4) Afterwards, the MS is removed and then the tissue piece within a metallic tube is collected by manually applied negative pressure. The specimen was diagnosed as IDP.

#### **2.2 Assessment of ductal cytology and IDBB**

The cytologic findings were classified into 4 categories; cancer, suspected cancer, suspected benign papillary lesion with the presence of a ductal cells cluster without atypia, and considered normal without any cluster. Cancer and suspected cancer categories were defined as positive findings. The diagnostic accuracy of these three methods , i.e. touch, collection, and directed ductal lavage were compared to IDBB in detecting malignancy. The therapeutic effect of IDBB for intraductal papillomas was also studied in 73 patients followed for more than 5 years (consisting of a total of 78 ducts).

#### **3. Result**

224 Breast Cancer – Recent Advances in Biology, Imaging and Therapeutics

cell fixation liquid three times a day for three days at home in 70 breast cancer patients (collection method). This collection method was performed to obtain much cytologic specimen. Galactography was done if a papillary lesion was suggested by cytology or if there was an obvious bloody background. MS was indicated based on the galactographic

At the time of the MS procedure, 4 % procaine was sprayed on the nipple. After injecting 1% procaine into the duct, the nipple orifice and ductal sphincter were dilated with a tear duct bougie. MS measuring 0.72 mm in outer diameter (Fiber Tech Company, Tokyo, Japan) were used. Intraductal breast biopsy (IDBB) under mammoscopic observation was performed in 226 intraductal papillomas (in a total of 128 patients) and in 38 ductal carcinomas (in a total of 35 patients). In most cases, IDBB was performed using a metallic tube with a side aperture near the tip (JN Biopsy Needle, Hakko Company, Tokyo, Japan). In directed ductal lavage cytolology, an epidural tube was used. After MS, the tube was inserted and advanced into the ductal branch where the papillary lesion was observed. When the lesion could not be observed by MS, emanation of bloody discharge from periphery suggested the direction of the lesion.We usually insufflated air to observe the ductal lumen by MS. Since after MS the lumen of the duct was enlarged, making it easy to selectively advance an epidural tube into an abnormal branch. When the lesion could not be observed by MS, emanation of bloody discharge from periphery suggested the direction of the lesion. An epidural tube, a 1 ml syringe of saline, and a 20 ml syringe were connected with a three-way valve. First the valve was turned to a 1-ml saline syringe and negative pressure was applied using a 20-ml syringe. Then the valve was turned to a-20 ml syringe and saline was sucked into the duct using the negative intraductal pressure. Then the valve was turned to a 1-ml syringe and intraductal fluid was collected into a 20-ml syringe. These procedures were repeated several times while changing the saline solution. Directed ductal lavage cytology was performed in this way in 38 patients. The cytologic findings were assessed by both cytology technician and a pathologist. These MS, IDBB and lavage procedures were performed in outpatient

**2.1 Intraductal approach to the detection of intraductal lesions**

Fig. 1. Mammary duct endscopy (MS) and biopsy needle

findings and the cytological findings

clinics..

#### **3.1.1 Visualization of intraductal lesions with MS**

The normal mammary duct wall is white or light pink in color. The wall is smooth and lustrous with discharge and capillary veins might be observed. Of 81 breast cancer lesions, intraductal lesions were observed in 50 (61.7%) by MS. Then feature of MS were classified into three types; hemispheric, papillary and flat, irregular ductal wall. The hemispheric and papillary shape were most common in intraductal papilloma and the flat irregular pattern was most common in ductal carcinoma. In the other 26 lesions, bloody secretion emanating

Intraductal Breast Cytology and Biopsy

to the Detection and Treatment of Intraductal Lesions of the Breast 227

method to obtain a lot of cells must be considered. Collection method and direct ductal

Fig. 4.  1) Galactography showed multiple filling defects in proximal brunches. 2) MS views corresponding to galactography were shown. There was intraductal papillary masses at the third bifurcation. 3) IDBB was performed to the papillary lesion at the third bifurcation and presence of large stalks and biphasic structure with mioepithelial cells were revealed. These character indicated IDP.

lavage method were performed to obtain much cytologic specimen.

from the periphery suggested the presence of ductal carcinoma were observed. So the lesional ductal brunches were recognized in 76/81 cases (93.8%). Five carcinoma lesions could not be recognized by MS because the MS could not be advanced into the peripheral duct.

Fig. 3. Shape of intraductal lesions observed with MS. 1) Left; carcinoma, Right; IDP 2) Left; IDP, Right; cancer 3) Both cancer

Table 1. Recognition of intraductal lesions of cancer with MS

#### **3.1.2 Ability of IDBB in obtaining specimen**

IDBB was performed 38 times in 35 of 47 cancer cases in which intraductal lesions were observed. Of the 35 cases, IDBB yielded diagnostic specimens in 29 cases (82.9%) but in the remaining 6 there was insufficient material for diagnosis. With IDBB it was difficult to collect diagnostic specimens in some carcinoma cases because the lesion located periphery and low tissue cohesiveness compared with intraductal papilloma. IDBB provided a definite diagnosis of carcinoma in 15 of the 35 (42.9%), while 9 lesions were in the carcinoma suspected category (atypical papillary lesion; 25.7%) and 5 were diagnosed as intraductal papilloma (14.3%). In 4 of 5 cases which were misdiagnosed as intraductal papilloma, coexistence of intraductal papilloma and carcinoma were confirmed on the resected specimen. In these 35 cases, the sensitivity of IDBB was 68.6% (24/35), directed ductal lavage cytology was 82.8% (29/35), and conventional touch cytology was 37.1% (13/35). The sensitivity of IDBB was 82.8% limited in successful IDBB cases (24/29) and it was equal to directed ductal lavage cytology. IDBB was performed in 226 IDP lesions from 128 patients. The diagnostic specimen was obtained in 197 of 226 IDBB (87.1%) and the successful IDBB yielded IDP in all 197 cases.

#### **3.1.3 Comparison in detecting cells cluster between each method of cytology with regard to diagnostic cell cluster**

Touch cytology yielded no false positive result among the 348 patients. Twenty eight of 81 cancer cases were diagnosed as cancer or cancer suspected category. The sensitivity of touch cytology was 34.6% (28/81) and specificity was 100%. The presence of a cluster of ductal cells was the clue to take diagnostic procedure to detect intraductal papillary lesions and the

from the periphery suggested the presence of ductal carcinoma were observed. So the lesional ductal brunches were recognized in 76/81 cases (93.8%). Five carcinoma lesions could not be recognized by MS because the MS could not be advanced into the peripheral

Fig. 3. Shape of intraductal lesions observed with MS. 1) Left; carcinoma, Right; IDP 2) Left;

**Shape total solitary multiple hemispheric 6 4 2 papillary 6 4 2 flat irregular and mixed 38 16 22**

IDBB was performed 38 times in 35 of 47 cancer cases in which intraductal lesions were observed. Of the 35 cases, IDBB yielded diagnostic specimens in 29 cases (82.9%) but in the remaining 6 there was insufficient material for diagnosis. With IDBB it was difficult to collect diagnostic specimens in some carcinoma cases because the lesion located periphery and low tissue cohesiveness compared with intraductal papilloma. IDBB provided a definite diagnosis of carcinoma in 15 of the 35 (42.9%), while 9 lesions were in the carcinoma suspected category (atypical papillary lesion; 25.7%) and 5 were diagnosed as intraductal papilloma (14.3%). In 4 of 5 cases which were misdiagnosed as intraductal papilloma, coexistence of intraductal papilloma and carcinoma were confirmed on the resected specimen. In these 35 cases, the sensitivity of IDBB was 68.6% (24/35), directed ductal lavage cytology was 82.8% (29/35), and conventional touch cytology was 37.1% (13/35). The sensitivity of IDBB was 82.8% limited in successful IDBB cases (24/29) and it was equal to directed ductal lavage cytology. IDBB was performed in 226 IDP lesions from 128 patients. The diagnostic specimen was obtained in 197 of 226 IDBB (87.1%) and the successful IDBB

**3.1.3 Comparison in detecting cells cluster between each method of cytology with**

Touch cytology yielded no false positive result among the 348 patients. Twenty eight of 81 cancer cases were diagnosed as cancer or cancer suspected category. The sensitivity of touch cytology was 34.6% (28/81) and specificity was 100%. The presence of a cluster of ductal cells was the clue to take diagnostic procedure to detect intraductal papillary lesions and the

Table 1. Recognition of intraductal lesions of cancer with MS

**3.1.2 Ability of IDBB in obtaining specimen**

duct.

IDP, Right; cancer 3) Both cancer

yielded IDP in all 197 cases.

**regard to diagnostic cell cluster**

method to obtain a lot of cells must be considered. Collection method and direct ductal lavage method were performed to obtain much cytologic specimen.

Fig. 4.  1) Galactography showed multiple filling defects in proximal brunches. 2) MS views corresponding to galactography were shown. There was intraductal papillary masses at the third bifurcation. 3) IDBB was performed to the papillary lesion at the third bifurcation and presence of large stalks and biphasic structure with mioepithelial cells were revealed. These character indicated IDP.

Intraductal Breast Cytology and Biopsy

recognized.

to the Detection and Treatment of Intraductal Lesions of the Breast 229

Fig. 6. Magnified view of directed lavage cytology. High NC ratio atypical cells, uneven distribution of nucleus, intranuclear enclosed body (arrow), and lack of cohesiveness were

Fig. 7.  1) Galactography showed irregularity and defect at second branch (arrowhesd). 2) Hemispheric intraductal mass and flat irregulariry were observed with MS. 3) IDBB suggested atypical papillary lesion. 4) Ductal lavage cytology suggested atypical papillary lesion. Excisional biopsy was

recommended for definite diagnosis but was denied. After 3 years, breast lump occurred at

this area and it was proven to be cancer.

Fig. 5.  1) Galactography showed irregularity and defect of duct (arrow heads). 2) Excisional biopsy revealed DCIS, extended 8 mm in maxumum dimater with comedo~solid intraductal component. 3) Only a few cells clusters without atypia were recognized in touch cytology and diagnosed as benign. 4) MS could not be advanced into the lesion because of the narrow ductal caliber and directed ductal lavage cytology was performed. A diagnostic large papillary cells clusters were obtained by directed ductal lavage cytology and could be diagnosed as cancer.

Fig. 5.  1) Galactography showed irregularity and defect of duct (arrow heads). 2) Excisional biopsy revealed DCIS, extended 8 mm in maxumum dimater with comedo~solid intraductal component. 3) Only a few cells clusters without atypia were recognized in touch cytology and diagnosed as benign. 4) MS could not be advanced into the lesion because of the narrow ductal caliber and directed ductal lavage cytology was performed. A diagnostic large papillary cells clusters were

obtained by directed ductal lavage cytology and could be diagnosed as cancer.

Fig. 6. Magnified view of directed lavage cytology. High NC ratio atypical cells, uneven distribution of nucleus, intranuclear enclosed body (arrow), and lack of cohesiveness were recognized.

Fig. 7.  1) Galactography showed irregularity and defect at second branch (arrowhesd). 2) Hemispheric intraductal mass and flat irregulariry were observed with MS. 3) IDBB suggested atypical papillary lesion. 4) Ductal lavage cytology suggested atypical papillary lesion. Excisional biopsy was recommended for definite diagnosis but was denied. After 3 years, breast lump occurred at this area and it was proven to be cancer.

Intraductal Breast Cytology and Biopsy

**3.2.3 Therapeutic effect of IDBB**

months and finally atypia was diagnosed (Fig. 10,11).

to the Detection and Treatment of Intraductal Lesions of the Breast 231

benign and malignant papillary lesions beyond the first bifurcation were recognized in 11 cases. In these 15 cases, 3 cases were positive by touch cytology (20.0%). Collection cytology was performed in 14 cases and 5 cases were positive (35.7%). Directed ductal lavage cytology was performed in 11 cases and 5 cases were positive (45.5%). IDBB was performed in 14 of these 15 cases. The coexistence of papilloma and carcinoma was confirmed by the first IDBB in a case (Fig. 9). Of these cases, the diagnosis obtained by IDBB was definitely cancer in 2, atypical papillary lesions in 4, intraductal papilloma in 5, and insufficient material in 3. In a case of a 31-year-old woman, IDBB was performed 4 times during 8

**diagnosis of cytology**

**diagnosis of cytology**

**cancer atypia benign**

**cancer atypia benign**

**Bifurcation cluster+ cluster-0 5 6 3****1 1 4 3 9 4 2 3 3 9 2 3~ 4 0 16 9 Total 16 12 37 16**

**Bifurcation cluster+ cluster-0 3 3 1 0 1 4 2 2 0 2 4 4 2 1 3~ 10 2 0 0 Total number 21 11 5 1**  Table 4. Localization of intraductal lesions and sensitivity of ductal lavage cytology

Of 218 IDBB for 128 IDP patients, 197 specimens were diagnostic materials. There were 73 patients with IDP (composing a combined total of 78 lesions) who were followed for more than 5 years. Of these 73 patients, 39 experienced cessation of nipple discharge after the first IDBB and another 13 experienced cessation of nipple discharge after a subsequent IDBB. Thus the nipple discharge was halted in a total of 52 patients (71.2%). In another 5 patients, the bloody background and papillary clusters of ductal cells were not recognized after IDBB and intraductal masses could not be detected on either ductography or MS. Thus the therapeutic effectiveness of IDBB was recognized in 57 of 73 intraductal papilloma patients (78.1%). There was a tendency for lesions in the effective IDBB to be located in a major duct and hemispheric or papillary shape. There was less therapeutic efficacy of IDBB in flat

lesions and in multiple nipple discharge orifices and intraductal lesions.

Table 3. Localization of intraductal lesions and sensitivity of touch cytology

#### **3.2.1 Comparison of the accuracy in detecting malignancy**

The sensitivities of the cytologic methods differed somewhat. The collection method was applied in 243 benign cases and only one case was converted from the benign category on touch cytology to the cancer suspected category by the collection method. The rate of atypia (involving the suspicious category) increased from 35.0% in touch cytology to 37.1% in collection cytology and 84.2% in directed ductal lavage cytology. Collection and directed ductal lavage cytology were not performed when cancer was already diagnosed by a prior modality. The presence of a cluster of ductal cells indicated the presence of papillary lesion. Of 38 cases of cancer lesions, papillary clusters were detected in 37 by directed ductal lavage, (sensitivity in 97.4%). The remaining case had a mucinous intraductal component and the lesion was located beyond second bifurcation and about 4 cm length from nipple (Table2). In 35 cases who underwent IDBB, the rate of atypia was 68.6% (24/35) in IDBB, 82.8% (29/35) in directed ductal lavage cytology, and 37.1% (13/35) in conventional touch cytology.

Table 2. Sensitivity of cytology and IDBB in detecting cancer

#### **3.2.2 Sensitivity of cytology and localization of the lesions**

The locations of the lesions were described as in the thoraco-bronchial tree, as the number of divergences of ductal branches approached from the nipple orifice. When cancer was recognized in a major duct (nondivergent), sensitivity of touch cytology was 73.3% (11/15). The sensitivity was 35.0% (7/20) in the first divergent branch, 35.3% in the second branch and 35.8% in the third branch and beyond. The sensitivity was extremely high only when cancer was present in a major duct (Table 3). The sensitivity of directed ductal lavage cytology was also studied in relation to the location of intraductal cancer. The sensitivity rates were 85.7% for lesions in a major duct, 75.0% for those in the first bifurcation, 72.7% beyond the second bifurcation, and 100% for those beyond the third bifurcation . The sensitivity of directed ductal lavage cytology did not depend on the location at the number of divergences of ductal branches (Table 4).

There were 15 cases in which coexistence of benign and malignant papillary lesions in the same duct-lobular unit was confirmed pathologically by sequential section slices at 3 mm of the surgically resected specimen. In these 15 cases, papillary lesions in which severe atypia was not recognized and myoepithelial cells remained in the side of proximal to the nipple orifice and ductal cancer was present in a more internal location. Coexistence of intraductal papilloma in a major duct and ductal cancers separately in a peripheral duct were recognized in 4 cases. In these 4 cases, IDBB revealed IDP in a major duct. Multiple foci of

The sensitivities of the cytologic methods differed somewhat. The collection method was applied in 243 benign cases and only one case was converted from the benign category on touch cytology to the cancer suspected category by the collection method. The rate of atypia (involving the suspicious category) increased from 35.0% in touch cytology to 37.1% in collection cytology and 84.2% in directed ductal lavage cytology. Collection and directed ductal lavage cytology were not performed when cancer was already diagnosed by a prior modality. The presence of a cluster of ductal cells indicated the presence of papillary lesion. Of 38 cases of cancer lesions, papillary clusters were detected in 37 by directed ductal lavage, (sensitivity in 97.4%). The remaining case had a mucinous intraductal component and the lesion was located beyond second bifurcation and about 4 cm length from nipple (Table2). In 35 cases who underwent IDBB, the rate of atypia was 68.6% (24/35) in IDBB, 82.8% (29/35) in directed ductal lavage cytology, and 37.1% (13/35) in conventional touch

**atypical papillary**

The locations of the lesions were described as in the thoraco-bronchial tree, as the number of divergences of ductal branches approached from the nipple orifice. When cancer was recognized in a major duct (nondivergent), sensitivity of touch cytology was 73.3% (11/15). The sensitivity was 35.0% (7/20) in the first divergent branch, 35.3% in the second branch and 35.8% in the third branch and beyond. The sensitivity was extremely high only when cancer was present in a major duct (Table 3). The sensitivity of directed ductal lavage cytology was also studied in relation to the location of intraductal cancer. The sensitivity rates were 85.7% for lesions in a major duct, 75.0% for those in the first bifurcation, 72.7% beyond the second bifurcation, and 100% for those beyond the third bifurcation . The sensitivity of directed ductal lavage cytology did not depend on the location at the number

There were 15 cases in which coexistence of benign and malignant papillary lesions in the same duct-lobular unit was confirmed pathologically by sequential section slices at 3 mm of the surgically resected specimen. In these 15 cases, papillary lesions in which severe atypia was not recognized and myoepithelial cells remained in the side of proximal to the nipple orifice and ductal cancer was present in a more internal location. Coexistence of intraductal papilloma in a major duct and ductal cancers separately in a peripheral duct were recognized in 4 cases. In these 4 cases, IDBB revealed IDP in a major duct. Multiple foci of

**Touch 81 16 12 37 16 19.8% 14.8% 45.7% 12.3% Collecting 70 16 10 24 20 22.8% 14.2% 34.3% 28.6% Lavage 38 21 11 5 1 55.3% 28.9% 13.2% 2.6% IDBB 35 15 9 5 insufficient; 6**

**benign benign papillary normal**

**3.2.1 Comparison of the accuracy in detecting malignancy**

**Method n carcinoma Suspicious**

 **42.9% 31.4% 14.3%**

Table 2. Sensitivity of cytology and IDBB in detecting cancer

**3.2.2 Sensitivity of cytology and localization of the lesions**

of divergences of ductal branches (Table 4).

cytology.

benign and malignant papillary lesions beyond the first bifurcation were recognized in 11 cases. In these 15 cases, 3 cases were positive by touch cytology (20.0%). Collection cytology was performed in 14 cases and 5 cases were positive (35.7%). Directed ductal lavage cytology was performed in 11 cases and 5 cases were positive (45.5%). IDBB was performed in 14 of these 15 cases. The coexistence of papilloma and carcinoma was confirmed by the first IDBB in a case (Fig. 9). Of these cases, the diagnosis obtained by IDBB was definitely cancer in 2, atypical papillary lesions in 4, intraductal papilloma in 5, and insufficient material in 3. In a case of a 31-year-old woman, IDBB was performed 4 times during 8 months and finally atypia was diagnosed (Fig. 10,11).

Table 3. Localization of intraductal lesions and sensitivity of touch cytology

Table 4. Localization of intraductal lesions and sensitivity of ductal lavage cytology

#### **3.2.3 Therapeutic effect of IDBB**

Of 218 IDBB for 128 IDP patients, 197 specimens were diagnostic materials. There were 73 patients with IDP (composing a combined total of 78 lesions) who were followed for more than 5 years. Of these 73 patients, 39 experienced cessation of nipple discharge after the first IDBB and another 13 experienced cessation of nipple discharge after a subsequent IDBB. Thus the nipple discharge was halted in a total of 52 patients (71.2%). In another 5 patients, the bloody background and papillary clusters of ductal cells were not recognized after IDBB and intraductal masses could not be detected on either ductography or MS. Thus the therapeutic effectiveness of IDBB was recognized in 57 of 73 intraductal papilloma patients (78.1%). There was a tendency for lesions in the effective IDBB to be located in a major duct and hemispheric or papillary shape. There was less therapeutic efficacy of IDBB in flat lesions and in multiple nipple discharge orifices and intraductal lesions.

Intraductal Breast Cytology and Biopsy

to the Detection and Treatment of Intraductal Lesions of the Breast 233

Fig. 9. 1&2) Yellow hemispherical and red papillary intraductal masses (A) were recognized in a proximal duct and were diagnosed as IDP by IDBB. A whitish-yellow flat mass coated with bloody discharge (B) was recognized distal to the site shown in A. This was diagnosed as ductal carcinoma by IDBB. 3) The slice section just on the line of the major duct in the direction of the nipple showed intraductal papilloma in the proximal duct (A; side central to the nipple) and ductal carcinoma in the disal duct (B). 4) The epithelium of the intraductal papilloma was abraded and was mechanically exfoliated by IDBB (A; HE stain, X330). Ductal cancer was recognized in the distal duct (B; HE stain, X330). In this case, the IDP was located at the proximal portion and the breast cancer was located at the distal portion to the

IDP. Cancer was overlooked by discharge cytology alone. The value of MS lay in observation of the peripheral lesion by inserting the MS to the periphery beyond the proximal lesion after removing the proximal mass and biopsying the distal lesion and

sampling with directed ductal lavage cytology.

Fig. 8. A case of ductal cacer; Ductography showed defect beyond third bifurcation of the duct (upper, arrows). The hemispheric intraductal mass was detected by MS (middle, left). There was no atypical cell obtained by touch cytology (middle, right) but carcinoma was indicated by direct ductal lavage cytology and IDBB (lower left; IDBB and right; lavage cytology).

Fig. 8. A case of ductal cacer; Ductography showed defect beyond third bifurcation of the duct (upper, arrows). The hemispheric intraductal mass was detected by MS (middle, left). There was no atypical cell obtained by touch cytology (middle, right) but carcinoma was indicated by direct ductal lavage cytology and IDBB (lower left; IDBB and right; lavage

cytology).

Fig. 9. 1&2) Yellow hemispherical and red papillary intraductal masses (A) were recognized in a proximal duct and were diagnosed as IDP by IDBB. A whitish-yellow flat mass coated with bloody discharge (B) was recognized distal to the site shown in A. This was diagnosed as ductal carcinoma by IDBB. 3) The slice section just on the line of the major duct in the direction of the nipple showed intraductal papilloma in the proximal duct (A; side central to the nipple) and ductal carcinoma in the disal duct (B). 4) The epithelium of the intraductal papilloma was abraded and was mechanically exfoliated by IDBB (A; HE stain, X330). Ductal cancer was recognized in the distal duct (B; HE stain, X330). In this case, the IDP was located at the proximal portion and the breast cancer was located at the distal portion to the IDP. Cancer was overlooked by discharge cytology alone. The value of MS lay in observation of the peripheral lesion by inserting the MS to the periphery beyond the proximal lesion after removing the proximal mass and biopsying the distal lesion and sampling with directed ductal lavage cytology.

Intraductal Breast Cytology and Biopsy

duct could be detected by MS.

peripheral ductal cells by lavage.

**4. Discussion**

to the Detection and Treatment of Intraductal Lesions of the Breast 235

Surgical excision previously was the only method available for pathological diagnosis and treatment for nipple discharge. MS contributes not only to the diagnosis of cases of nipple discharge, but also is of benefit in the treatment of IDP. The development of the MS has opened a new era in the diagnosis and treatment of secreting IDP by IDBB under direct observation. The MS helps to eliminate unnecessary surgical excision for benign lesions. Surgical excision has now been superseded by IDBB under endoscopic observation for the treatment of solitary IDP. In the previous report, neither the amount of specimen collected nor the amount of residual lesion after IDBB had a significant influence on the effect of treatment for nipple discharge. Some morphological changes after MS were also reported. It was speculated that the necrosis of IDP could be caused by the biopsy procedure. One factor related to the poor therapeutic effects of IDBB for IDP is lesion multiplicity. It was suggested that there was a tendency towards multifocal generation of intraductal papillary lesions in cases with multiple nipple orifices. Duct-lobular unit distribution patterns are infinite and all mammary ductal braches could not always be visualized by galactography. When IDP was located in a branch diverging at a sharp angle, only the proximal margin in a major

MS was also effective in cases of coexisting intraductal papilloma and carcinoma in the same duct-lobular unit. In these cases, the IDP is usually located at the proximal portion of the duct-lobular unit, and the breast cancer could be overlooked by discharge cytology and galactography. The value of MS in such cases lay in observation of the peripheral lesion by inserting the MS to the periphery beyond the proximal lesion or removing the proximal mass and biopsying the peripheral lesion. Thus MS finally contributed to the pathological diagnosis of intraductal masses with limited invasiveness compared with excisional biopsy. Although IDBB is a definitive method for cancer diagnosis but the drawback of IDBB is the difficulty of obtaining diagnostic specimen. In addition, pathological diagnosis of intraductal papillary lesions was difficult in IDBB even with sufficient materials. Because of these technical defects of IDBB, the most reliable method in detecting ductal cancer was turned out to be directed ductal lavage cytology. The usefulness of cytologic specimens of nipple aspirate fluid has been reported in the evaluation of prognostic and predictive factors of breast cancer. Fluorescence in situ hybridization of ductal lavage samples was reported to be more effective in identifying malignancy. Ductal lavage was reported to detect intraductal breast cells 3.2 times more frequently than nipple aspiration but the actual cytological materials are very similar to those obtained by nipple aspiration devices. Directed ductal lavage cytology was complementary and helps to obtain most sensitive results, detecting intraductal papillary lesions in 97.4%. In only one case of mucinous intraductal component, was mucous fluid thought to be a negative factor for collecting

At present, the terminal duct-lobular unit from which carcinoma originates cannot be observed with MS. But MS contributes to the diagnosis of intraductal lesions through observation of the proximal portion of intraductal carcinoma and to in obtaining specimens. MS contribute the high sensitivity in cancer detection by directed ductal lavage cytology by selecting the abnormal branch to which the tube is advanced. The MS can be an important adjunct in breast-conservation management. Multiple intraductal lesions in peripheral ducts suggest cancer development. Surgical excision should be performed in patients with atypical papillary lesions revealed by IDBB or directed ductal lavage cytology. There were 2

Fig. 10. 1) Section slice of intraductal lesion. The square surrounded part was the lesion concerned. 2) The slice section of major duct to the pariphery where the intraductal papillary lesion exsisted. 3) Magnification of the area of intraductal lesions in which coexsistenced biphasic (upper area) and lack of biphasic structure (lower area).

Fig. 11. 1) Specimen galactography. A 24 gage cannula was inserted into nipple orifice and the dye (methylene blue) was injected during excisional biosy (microdochectom). After excision, specimen galactography was performed. 2) The slice section just on the line of the major duct in the derection from specimen galactography (; the square surrounded part of the specimen 1). There were multiple intraductal lesions in periphery (; the square surrounded part ). 3) Magnification of the area of intraductal papillary lesions. The myoepithelial cells were seen in the lesion showned with arrow head but were few in the lesion showned with arrow. The structural and cellular atypia were observed in the latter lesion. There was coexsistenced biphasic (arrow head) and lack of biphasic structure (arrow) and diagnosed as ductal cancer.

#### **4. Discussion**

234 Breast Cancer – Recent Advances in Biology, Imaging and Therapeutics

Fig. 10. 1) Section slice of intraductal lesion. The square surrounded part was the lesion concerned. 2) The slice section of major duct to the pariphery where the intraductal papillary lesion exsisted. 3) Magnification of the area of intraductal lesions in which coexsistenced biphasic (upper area) and lack of biphasic structure (lower area).

Fig. 11. 1) Specimen galactography. A 24 gage cannula was inserted into nipple orifice and the dye (methylene blue) was injected during excisional biosy (microdochectom). After excision, specimen galactography was performed. 2) The slice section just on the line of the major duct in the derection from specimen galactography (; the square surrounded part of the specimen 1). There were multiple intraductal lesions in periphery (; the square surrounded part ). 3) Magnification of the area of intraductal papillary lesions. The myoepithelial cells were seen in the lesion showned with arrow head but were few in the lesion showned with arrow. The structural and cellular atypia were observed in the latter lesion. There was coexsistenced biphasic (arrow head) and lack of biphasic structure (arrow) and diagnosed as ductal cancer.

Surgical excision previously was the only method available for pathological diagnosis and treatment for nipple discharge. MS contributes not only to the diagnosis of cases of nipple discharge, but also is of benefit in the treatment of IDP. The development of the MS has opened a new era in the diagnosis and treatment of secreting IDP by IDBB under direct observation. The MS helps to eliminate unnecessary surgical excision for benign lesions. Surgical excision has now been superseded by IDBB under endoscopic observation for the treatment of solitary IDP. In the previous report, neither the amount of specimen collected nor the amount of residual lesion after IDBB had a significant influence on the effect of treatment for nipple discharge. Some morphological changes after MS were also reported. It was speculated that the necrosis of IDP could be caused by the biopsy procedure. One factor related to the poor therapeutic effects of IDBB for IDP is lesion multiplicity. It was suggested that there was a tendency towards multifocal generation of intraductal papillary lesions in cases with multiple nipple orifices. Duct-lobular unit distribution patterns are infinite and all mammary ductal braches could not always be visualized by galactography. When IDP was located in a branch diverging at a sharp angle, only the proximal margin in a major duct could be detected by MS.

MS was also effective in cases of coexisting intraductal papilloma and carcinoma in the same duct-lobular unit. In these cases, the IDP is usually located at the proximal portion of the duct-lobular unit, and the breast cancer could be overlooked by discharge cytology and galactography. The value of MS in such cases lay in observation of the peripheral lesion by inserting the MS to the periphery beyond the proximal lesion or removing the proximal mass and biopsying the peripheral lesion. Thus MS finally contributed to the pathological diagnosis of intraductal masses with limited invasiveness compared with excisional biopsy.

Although IDBB is a definitive method for cancer diagnosis but the drawback of IDBB is the difficulty of obtaining diagnostic specimen. In addition, pathological diagnosis of intraductal papillary lesions was difficult in IDBB even with sufficient materials. Because of these technical defects of IDBB, the most reliable method in detecting ductal cancer was turned out to be directed ductal lavage cytology. The usefulness of cytologic specimens of nipple aspirate fluid has been reported in the evaluation of prognostic and predictive factors of breast cancer. Fluorescence in situ hybridization of ductal lavage samples was reported to be more effective in identifying malignancy. Ductal lavage was reported to detect intraductal breast cells 3.2 times more frequently than nipple aspiration but the actual cytological materials are very similar to those obtained by nipple aspiration devices. Directed ductal lavage cytology was complementary and helps to obtain most sensitive results, detecting intraductal papillary lesions in 97.4%. In only one case of mucinous intraductal component, was mucous fluid thought to be a negative factor for collecting peripheral ductal cells by lavage.

At present, the terminal duct-lobular unit from which carcinoma originates cannot be observed with MS. But MS contributes to the diagnosis of intraductal lesions through observation of the proximal portion of intraductal carcinoma and to in obtaining specimens. MS contribute the high sensitivity in cancer detection by directed ductal lavage cytology by selecting the abnormal branch to which the tube is advanced. The MS can be an important adjunct in breast-conservation management. Multiple intraductal lesions in peripheral ducts suggest cancer development. Surgical excision should be performed in patients with atypical papillary lesions revealed by IDBB or directed ductal lavage cytology. There were 2

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cancer detection. *J Jpn. Assoc. Breast Cancer Screen*. 11 , 2002, 281-288

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patients in whom carcinoma was detected 3 years after IDBB showed no malignancy. Follow-up at a 6-month interval with ultrasound, checking for nipple discharge by palpation and annual mammography is considered appropriate instead of excision in patients with multiple lesions in peripheral ducts, when ductal lavage cytology showed benign result. The patients without surgical resection after IDBB showed no malignancy should be followed up as long as possible in principle (mean follow-up interval was 74.2 months; 36 to 204 months in our survey).

 Even in cases of nipple discharge with no atypia the associated relative risks for breast cancer increased compared with women who have no nipple discharge. Clinical follow-up was thought to be important to the patients with bloody nipple discharge. In previous study, carcinoma was detected in 3 patients during follow-up among 89 patients with negative MS and cytology. In these 3 patients, nipple discharge ceased after MS and carcinomas were detected at the different areas from previous galactography.

#### **5. Conclusion**

Directed ductal lavage cytology was the most sensitive method in detecting malignancy. MS and IDBB were benefit in the treatment of intraductal papilloma.

#### **6. Acknowledgment**

The author owe this work to all those who supported me. The author is indebted to Prof. Masahiko Fujii of the Department of Pathology of Kyorin University School of Health Science, Dr. Hutoshi Akiyama of the Department of Pathology of Cancer Institute Hospital, Dr. Hiromi Serizawa of the Department of Pathology of Tokyo medical University Hachioji Medical Center for pathological diagnosis and the author is grateful to the chairman of the board of directors Dr Yoshinori Nagumo for his understanding to continue this work.

#### **7. References**

patients in whom carcinoma was detected 3 years after IDBB showed no malignancy. Follow-up at a 6-month interval with ultrasound, checking for nipple discharge by palpation and annual mammography is considered appropriate instead of excision in patients with multiple lesions in peripheral ducts, when ductal lavage cytology showed benign result. The patients without surgical resection after IDBB showed no malignancy should be followed up as long as possible in principle (mean follow-up interval was 74.2

Cancer arises more frequently in the duct-lobular unit with bloody nipple discharge and bloody nipple discharge is thought to be an independent risk factor. Even in cases of nipple discharge with no atypia the associated relative risks for breast cancer increased compared with women who have no nipple discharge. Clinical follow-up was thought to be important to the patients with bloody nipple discharge. In previous study, carcinoma was detected in 3 patients during follow-up among 89 patients with negative MS and cytology. In these 3 patients, nipple discharge ceased after MS and carcinomas were detected at the different

Directed ductal lavage cytology was the most sensitive method in detecting malignancy. MS

The author owe this work to all those who supported me. The author is indebted to Prof. Masahiko Fujii of the Department of Pathology of Kyorin University School of Health Science, Dr. Hutoshi Akiyama of the Department of Pathology of Cancer Institute Hospital, Dr. Hiromi Serizawa of the Department of Pathology of Tokyo medical University Hachioji Medical Center for pathological diagnosis and the author is grateful to the chairman of the board of directors Dr Yoshinori Nagumo for his understanding to continue this work.

Teboul, M. A new concept in breast investigation: echo-histological acino-ductal analysis or

Namba, K, et al. Intraductal biopsy of the breast (IDBB) for intraductal lesions: a case of breast cancer diagnosed by IDBB. *Jpn J Breast Cancer* 4, 1989, 253-258 Makita, M, et al. Duct endoscopy and endoscopic biopsy in the evaluation of nipple discharge. *Breast Cancer Res Treat* 18, 1991, 179-188, DOI:10.1007/BF01990034 Okazaki, A, et al. Fiberoptic Ductoscopy of the Breast: a new diagnostic procedure for nipple

Berna J, Garcia-Medina V, Kuni C: Ductoscopy: a new technique ductal exploration. *Eur J*

Nagase, J & Endo, M. Endoscopic biopsy in the intraductal lesions of the breast for the

Love, SM & Barsky, SH. Breast-duct endoscopy to study stages of cancerous breast disease

analytic echography. *Biomed & Pharmacother* 42, 1988, 282-296.

*Radiol* 12, 1991. 127-129, DOI:10.1016/0720-48X(91)90112-9

mammary duct fiberscope. *Jpn J Breast Cancer* 10, 1995, 405-409

*Lancet* 348, 1996, 997-999, DOI 10.1016/S0140-6736(96)04145-1

discharge. *Jpn J Clin Oncol* 21, 1991. 188-193

and IDBB were benefit in the treatment of intraductal papilloma.

months; 36 to 204 months in our survey).

areas from previous galactography.

**5. Conclusion**

**7. References**

**6. Acknowledgment**

**13**

*Germany*

**Diagnostic Optical Imaging of Breast Cancer:**

The use of light-driven imaging techniques for the diagnosis of breast cancer dates back to as early as 1929. Transillumination was evaluated for potential differentiation of breast lesions, e.g. to distinguish benign cysts from solid tumours. Cutler summed the technique to be "...a simple procedure and a valuable aid in the interpretation of pathological conditions in the mammary gland. Its use is recommended in the routine examination of the

Fig. 1. Opacity on transillumination of a solid tumour in the breast (Cutler, 1931, reproduced

At the same time, x-ray mammography emerged, routinely used for detection and diagnosis of breast cancer from the 1950s (Leborgne, 1951). Only relatively low sensitivity and specificity of mammography findings in combination with significant x-ray exposure though always triggered the parallel search for alternative diagnostic approaches. Over the following years, the use of near infrared (NIR) light was discussed to further facilitate

**1. Introduction**

breast..."(Cutler, 1929, 1931).

with permission from Wolters & Kluwer).

**From Animal Models to First-in-Men Studies**

Michel Eisenblätter1, Thorsten Persigehl1, Christoph Bremer2 and Carsten Höltke1

*1Dept. of Clinical Radiology, University Hospital Münster 2Dept. of Radiology, St. Franziskus Hospital Münster*

### **Diagnostic Optical Imaging of Breast Cancer: From Animal Models to First-in-Men Studies**

Michel Eisenblätter1, Thorsten Persigehl1, Christoph Bremer2 and Carsten Höltke1 *1Dept. of Clinical Radiology, University Hospital Münster 2Dept. of Radiology, St. Franziskus Hospital Münster Germany*

#### **1. Introduction**

238 Breast Cancer – Recent Advances in Biology, Imaging and Therapeutics

Urban, JA & Egeli, RA. Non-lactational nipple discharge. *CA Cancer J Clin* 28, 1978, 130-140 Van Zee, KJ, et al. Preoperative galactography increased the diagnostic yield of major duct

Locker, AP, et al: Microdochectomy for single-duct discharge from the nipple. *Br J Surg* 75,

Florio MG, Manganaro T, Pollicino A, et al: Surgical approach to nipple discharge: A ten-

Bauer, RL, et al. Ductal carcinoma in situ-associated nipple discharge: A clinical marker for

Ling H, et al. Fiberoptic ductoscopy-guided intraductal biopsy improve the diagnosis of

excision for nipple discharge. *Cancer* 82, 1997, 1874-1880

locally extensive disease. *Ann Surg Oncol* 5, 1998, 452-455

year experience. *J Surg Oncol* 71, 1999, 235-238

nipple discharge. *Breast J* 15(2); 2009, 168-75

1988, 700-701

The use of light-driven imaging techniques for the diagnosis of breast cancer dates back to as early as 1929. Transillumination was evaluated for potential differentiation of breast lesions, e.g. to distinguish benign cysts from solid tumours. Cutler summed the technique to be "...a simple procedure and a valuable aid in the interpretation of pathological conditions in the mammary gland. Its use is recommended in the routine examination of the breast..."(Cutler, 1929, 1931).

Fig. 1. Opacity on transillumination of a solid tumour in the breast (Cutler, 1931, reproduced with permission from Wolters & Kluwer).

At the same time, x-ray mammography emerged, routinely used for detection and diagnosis of breast cancer from the 1950s (Leborgne, 1951). Only relatively low sensitivity and specificity of mammography findings in combination with significant x-ray exposure though always triggered the parallel search for alternative diagnostic approaches. Over the following years, the use of near infrared (NIR) light was discussed to further facilitate

Diagnostic Optical Imaging of Breast Cancer: From Animal Models to First-in-Men Studies 241

650 – 950 nm is absorbed to a much lesser extent than light of lower wavelengths and can thus be detected in deeper tissue sections (Ntziachristos et al., 2003). Moreover, autofluorescence of tissue is minimal in this so called optical window (Fig. 2), resulting in a

higher signal-to-background ratio for near infrared images (Bremer et al., 2001b).

Fig. 2. The optical window between 650 and 950 nm, where absorption by water

For non-enhanced optical imaging approaches, simple opposed lightsource detector pairs are used. Light is applied to the scan volume at a distinct place and the detector is used to collect the non-absorbed photons. Highly absorbing lesions (e.g. tumour lesions) are supposed to delineate as negative contrast. Diffuse optical tomography (DOT) is based on the same principle, creating 3D absorption maps from e.g. the female breast (Ntziachristos et al., 2000; O'Leary et al., 1995). Commercially available optical imaging devices use adapted light for dye excitation; in experimental settings ultrasound was alternatively used successfully for the excitation of fluorophores (Razansky et al., 2008; Yuan & Liu, 2010). Fluorescence-producing proteins, encoded by so-called imaging target genes can either be light-excitable or metabolism-dependent, producing fluorescence upon interaction with exogenous substrates (Chudakov et al., 2010; Lukyanov et al., 2010). All dyes regularly used for targeted *in vivo* imaging require excitation with light of distinct wavelength to produce a specific emission. An imaging unit therefore always consists of an excitation light source on the one hand and a signal recording camera on the other (Fig. 3). The animal is placed either between both for optical tomography (Fluorescence Mediated Tomography – FMT) or on the ground of an imaging chamber with light source and camera both on one side (Fluorescence Reflectance Imaging – FRI) (Mahmood et al., 1999; Weissleder et al., 1999). Reflectance imaging approaches in this context basically resemble traditional photography, allowing for acquisition of 2D image data at video rate with highly sensitive CCD cameras. Regularly, cameras are adapted with filter sets for each particular wavelength, virtually blocking diffuse background fluorescence. The image acquisition is preceded by illumination of the whole field of view with either light of distinctive wavelengths – adapted for the applied dye – or unfiltered, multi-wavelength light (Graves et al., 2004). The latter of course results in excitation not only of the applied dye but of virtually all substances capable of producing fluorescence. This circumstance contributes to the background signal and thus requires more sophisticated filter equipment at the signal recording side. Further increase of signal-specificity can be achieved by using excitation light of discrete wavelengths, using

haemoglobin and deoxyhaemoglobin is minimal.

transillumination of breast tissue (Watmough, 1982a; Watmough, 1982b). Its unique characteristics regarding interaction with tissue - low absorption and reduced scattering – enable NIR light to travel through tissue and thus to reach deep tissue sections, therefore defining a valuable tool in diagnostic optical imaging (OI) techniques (Weissleder & Ntziachristos, 2003). Originating from transillumination, diffuse optical tomography (DOT) was initially thought to be capable of identifying tumour formation in anatomical regions accessible to NIR light, as tumour tissue is characterized by distinct optical properties, based on altered tissue architecture, blood flow and oxygen consumption. However, the limitations of this technique, especially the limited spatial resolution, have been illustrated in several publications. Although the advances on the field of instrumentation were significant, first clinical studies in breast cancer patients proved discriminating lesions regarding their malignant potential reflected by their absorption characteristics still to be challenging (Boas, 1997). To further improve the specificity of OI, fluorescence contrast agents, fluorophores operating in the NIR range of the spectrum, have been used. Three different types of optical probes are currently available for clinical or preclinical (basic research) purposes – unspecific, specific and so-called smart probes. The only clinically approved fluorochrome, indocyanine green (ICG) is an unspecific, perfusion-type contrast agent. First promising results of ICG-driven DOT for detection and differentiation of breast lesions have recently been published, confirming sensitivity and specificity (Ntziachristos et al., 2000). So far restricted to preclinical use only, specifically binding optical contrast agents, targeted on key-structures of cancer cells, allow for disease specific imaging and *in vivo* characterization of lesions down to the molecular level. As state of the art anatomical imaging frequently fails in detecting early effects of modern anti-tumour therapy, contrast agents targeted to different tumour cell epitopes have been described and used to sensitively detect the molecular characteristics of malignant lesions and their alteration under therapy (Achilefu, 2004; Achilefu et al., 2000; Becker et al., 2001). So called smart probes even increase sensitivity and specificity of OI. Fluorescence characteristics of these probes change upon interaction with specific target structures, e.g. proteinases, therefore allowing detection of vital, invasive tumour lesions with unrivalled sensitivity (Bremer et al., 2001c; Bremer et al., 2002). This review gives an overview about the approaches, recently made on the field of optical tracer development and evaluation with a focus on targets relevant for breast cancer diagnosis and characterization. Especially the visualization of angiogenesis as a key process in tumour progression is emphasized. Many factors that are involved in angiogenic events have been evaluated over the last decades and are now well understood. Several growth factors, their receptors and diverse pathways triggering their expression, are crucially involved in cancer progression and metastasis. Moreover some proteinases, especially matrix metalloproteinases (MMPs) and cathepsins, proved to contribute to tumour growth by e.g. degradation of extracellular matrix (ECM) components. Clinical translation of the efforts made in the past recent years will be the main challenge for researchers active on the molecular imaging field.

#### **2. Instrumentation**

Technically, optical imaging is based on the detection of light and the visualization of photon distribution in tissue. While visible light (380-700 nm) is almost completely absorbed, scattered and reflected in the uppermost sections of tissue already, light of the NIR range of the spectrum can be facilitated for *in vivo* imaging approaches. Light of around

transillumination of breast tissue (Watmough, 1982a; Watmough, 1982b). Its unique characteristics regarding interaction with tissue - low absorption and reduced scattering – enable NIR light to travel through tissue and thus to reach deep tissue sections, therefore defining a valuable tool in diagnostic optical imaging (OI) techniques (Weissleder & Ntziachristos, 2003). Originating from transillumination, diffuse optical tomography (DOT) was initially thought to be capable of identifying tumour formation in anatomical regions accessible to NIR light, as tumour tissue is characterized by distinct optical properties, based on altered tissue architecture, blood flow and oxygen consumption. However, the limitations of this technique, especially the limited spatial resolution, have been illustrated in several publications. Although the advances on the field of instrumentation were significant, first clinical studies in breast cancer patients proved discriminating lesions regarding their malignant potential reflected by their absorption characteristics still to be challenging (Boas, 1997). To further improve the specificity of OI, fluorescence contrast agents, fluorophores operating in the NIR range of the spectrum, have been used. Three different types of optical probes are currently available for clinical or preclinical (basic research) purposes – unspecific, specific and so-called smart probes. The only clinically approved fluorochrome, indocyanine green (ICG) is an unspecific, perfusion-type contrast agent. First promising results of ICG-driven DOT for detection and differentiation of breast lesions have recently been published, confirming sensitivity and specificity (Ntziachristos et al., 2000). So far restricted to preclinical use only, specifically binding optical contrast agents, targeted on key-structures of cancer cells, allow for disease specific imaging and *in vivo* characterization of lesions down to the molecular level. As state of the art anatomical imaging frequently fails in detecting early effects of modern anti-tumour therapy, contrast agents targeted to different tumour cell epitopes have been described and used to sensitively detect the molecular characteristics of malignant lesions and their alteration under therapy (Achilefu, 2004; Achilefu et al., 2000; Becker et al., 2001). So called smart probes even increase sensitivity and specificity of OI. Fluorescence characteristics of these probes change upon interaction with specific target structures, e.g. proteinases, therefore allowing detection of vital, invasive tumour lesions with unrivalled sensitivity (Bremer et al., 2001c; Bremer et al., 2002). This review gives an overview about the approaches, recently made on the field of optical tracer development and evaluation with a focus on targets relevant for breast cancer diagnosis and characterization. Especially the visualization of angiogenesis as a key process in tumour progression is emphasized. Many factors that are involved in angiogenic events have been evaluated over the last decades and are now well understood. Several growth factors, their receptors and diverse pathways triggering their expression, are crucially involved in cancer progression and metastasis. Moreover some proteinases, especially matrix metalloproteinases (MMPs) and cathepsins, proved to contribute to tumour growth by e.g. degradation of extracellular matrix (ECM) components. Clinical translation of the efforts made in the past recent years will be the main challenge for

researchers active on the molecular imaging field.