Morphogenomics and morphoproteomics: a role for anatomic pathology in personalized medicine.
Article Type: Report
Subject: Cancer (Care and treatment)
Cancer (Genetic aspects)
Cancer (Research)
Enzyme inhibitors (Health aspects)
Enzyme inhibitors (Research)
Immunohistochemistry (Usage)
Proteomics (Research)
Author: Brown, Robert E.
Pub Date: 04/01/2009
Publication: Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2009 College of American Pathologists ISSN: 1543-2165
Issue: Date: April, 2009 Source Volume: 133 Source Issue: 4
Topic: Event Code: 310 Science & research
Product: Product Code: 2834345 Enzyme Inhibitors NAICS Code: 325412 Pharmaceutical Preparation Manufacturing SIC Code: 2834 Pharmaceutical preparations
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 230246988
Full Text: I would like to discuss a practice we have implemented in our department (Department of Pathology, University of Texas Health Science Center, Houston) and something that was born at the Geisinger Medical Health System (Danville, Pennsylvania) in 2003. Here, I discuss the application of morphogenomics and morphoproteomics (1) as a role for anatomic pathology in personalized medicine.


The list of immunohistochemical probes and their sources for morphoproteomics and the fluorescence in situ hybridization probe for morphogenomics are contained in Table 1.


* Morphogenomics, as it relates to medicine, incorporates morphology plus genomics.

* Morphoproteomics comprises morphology plus proteomics.

* Personalized medicine is therapy customized for the individual patient.

Morphoproteomics: Evolution of Concept and Historical Perspective

Most anatomic pathologists are familiar with applying a probe to detect the presence or absence of estrogen receptor (ER) [alpha]. We have been practicing this process routinely for some time in our laboratories, looking for the presence of ER-positive breast cancer tumors versus ER-negative tumors, so that we can provide some guidance to the clinician with respect to the role of antiestrogenic therapy, such as tamoxifen. Then along came immunohistochemical testing for human epidermal growth factor receptor 2 (HER2/neu), c-erb-B2, and many of us have been applying this to assess the level of plasmalemmal (cell membrane) expression of HER2 total protein as one guide for the application of trastuzumab therapy. More recently, our clinical colleagues requested that we assess the plasmalemmal expression level of epidermal growth factor receptor (EGFR) in carcinoma of the colon as a guide to therapy in terms of cetuximab (an antibody that is directed against the ectodomain of EGFR).

However, we have to put the predictive value of these latter assessments into perspective. If we identify 3+ (strong) plasmalemmal expression of HER2/neu, what is the therapeutic implication? Even if we apply trastuzumab, as a single agent, to those cases, what is the response rate? It is approximately 15%. So, there is something else missing here, basically, in terms of its utility in predicting response to trastuzumab, used alone, in a given patient. The story is even a little more disturbing with regard to EGFR. As we heard yesterday, just a little more than 1% of the tumor cells have to show any plasmalemmal expression of EGFR to be considered a positive result.

The sorry truth is that if we have 1+ expression, 6% of the patients will respond to cetuximab. If we have 2+ expression, 13% of patients respond, and if we have 3+ expression, no patients respond. So, there is something wrong with this picture. Even patients with "no expression" can respond. We need to do a little better.


Many are familiar with the fluorescence in situ hybridization probe, which directs a probe for DNA hybridization to the HER2/ neu gene on chromosome 17. This results in a red fluorescent signal for the HER2 /neu gene. There is also an internal control, where the probe to the centromere on chromosome 17 is used, which results in a green fluorescent signal. We score each cell and determine the ratio of red to green. Nonamplified expression is defined as a ratio of less than 1.8:1, whereas a ratio of HER2/ neu (red) to centromere on chromosome 17 (green) signals of more than 2.2:1 is amplified. The level of amplification, as determined by such numerical ratios, and its predictive value in terms of responsiveness for an individual patient's tumor to trastuzumab therapy are currently under study.


When focusing on morphoproteomics in guiding targeted cancer therapies, we can talk about concepts and proofs of concept and then application. But first, how do we define the clinical application of morphoproteomics?

Concept and Definition

The application of morphoproteomics, in its most complete sense, involves the immunohistochemical assessment of the activation of metabolic pathways in cancer cells, and actually, in any diseased cells, to predict susceptibility to small-molecule inhibitors, specific chemotherapeutic agents, and possibly, differentiating agents. What is the morphologic component of that? Well, the morphologic component is essential; it is a sine qua non. By using morphologic analysis, we can assess translocation of a protein from one subcellular compartment to another. For example, has the protein been translocated from the cytosolic compartment to the plasmalemmal compartment? That is a sign of the activation of certain proteins, such as protein kinase C [alpha] (PKC-[alpha]), and of phospholipase D1, particularly if it is coexpressed with PKC-[alpha].

We look for phosphorylation of a protein analyte by using immunohistochemical probes that are phosphospecific probes and directed against sites of putative activation of a given molecule. For example, in the Akt protein, a serine (Ser)/threonine (Thr) kinase, serine 473, is one of the activation sites. In p70S6kinase, threonine 389 is one of the activation sites. In nuclear factor (NF)-[kappa]Bp65, it is serine 536. In short, these putative sites of activation of signaling molecules can be detected using bright-field microscopy and phosphospecific immunohistochemical probes. To make it all come together, we look for correlative expression of the proteins, upstream and downstream along signal transduction pathways, and we can determine that expression in the same tumor and in the same types of cells.

We also look at the morphoproteomic analysis in the context of what is happening in the companionate cell population. What is happening in the endothelial cell? What is happening in the stromal cell? All of these analyses give us very important information about how we might direct therapies.

Proofs of Concept: Outline and Objectives

In terms of the proofs of concept, I am going to give you some examples just to build your confidence in this type of approach. We will look at preclinical and clinical laboratory studies and clinical outcomes data that validate the methodology. In other words, to some degree, they validate using the light microscope and some component of morphoproteomics in terms of its predictive value.

But before we can take these tools and apply them to the patient, we have certain issues that we must address and certain things that we must keep in mind. We have to overcome the concept of "magic bullets." We have to consider everything that our clinical colleagues have done, during the course of the years, using cytotoxic therapies, and we have to take advantage of that wealth of knowledge, and ask how can we integrate cytotoxic agents with differentiating agents and signal transduction inhibitors and apply it to a particular patient's tumor? Then, we have to consider the shifting paradigm in tumorigenesis, which is really an exciting area in which we, as anatomic pathologists, can play a role.

I am going to illustrate the predictive value of morphoproteomic makers in clinical trials, provide an example of clinical success using morphoproteomics as a guide to therapy, and finally, give an overview of Consultative Proteomics (University of Texas Medical School, Houston) with a case study to illustrate and outline the process and our interactions with our oncologist colleagues.

Preclinical and Clinical Laboratory Studies

In terms of preclinical and clinical laboratory studies, my colleagues and I at Geisinger Medical Center looked at the morphoproteomic and pharmacoproteomic correlates in a hormone receptor-negative breast cancer cell line. Basically, what we showed was an association between the immunohistochemical expression of signal transduction markers (protein analytes) and in vitro inhibition by pharmaceutical agents. We also noted that there are correlations between the sites of action of the pharmaceutical agents and the downstream expression of proteins in hormone receptor-negative breast cancer cells. We published this in the Annals of Clinical and Laboratory Science. (2)

Fan Lin, MD, and I, along with several other colleagues, (3) looked at the morphoproteomic and molecular concomitants of an overexpressed and activated mammalian target of rapamycin (mTOR) pathway in renal cell carcinoma. We applied phosphospecific probes and noted the nuclear translocation of phosphorylated (p)-p70S6K (Thr 389) and also the plasmalemmal and, to some extent, the nuclear expression of phosphorylation-mammalian target of rapamycin (p-mTOR; Ser 2448). When we compared these expressions with that in nonneoplastic kidney, we saw an obvious difference in the level of expression for these analytes. In general, the signals for each analyte appeared more diffuse and stronger in the tumors in comparison with the somewhat microanatomic compartmentalization of p-mTOR and p-p70S6K in the distal tubular/collecting system of the nonneoplastic kidneys.

When we went to Western blot to confirm this, we found that, in the nonneoplastic kidney, we had very little band density for p-mTOR and p-p70S6K but strong expression of both by Western blot in the samples of clearcell renal cell carcinomas. Moreover, the average band density was increased for p-mTOR (Ser 2448) and p-p70S6K (Thr 389) and, to a lesser extent, for p-Akt (Ser 473) in all of the sampled carcinomas vis-a-vis their nonneoplastic counterparts. In short, what we determined by morphoproteomic analysis, through our semiquantitative analyses with combined immunostaining scores and through our impressions by bright-field microscopy, was confirmed and reaffirmed by Western blot analysis.

Clinical Outcomes

What about clinical outcomes? Ping L. Zhang, MD, and I, along with several other colleagues, (4) reported on the overexpression of p-NF-[kappa]Bp65 (Ser 536) in tonsillar squamous cell carcinoma and high-grade dysplasia, and we found that it was associated with poor prognosis in terms of high recurrence and poor survival. Furthermore, when we looked at the relative expression levels and, specifically, the expression of p-NF-[kappa]Bp65 translocated to the nucleus, and compared those patients with 1+ scores with those with 3+ scores, we found that the prognosis was worse in those with 3+ , according to our Kaplan-Meier curves.

In another outcomes study, Shikha Bose, MD, et al (5) looked at the Akt pathway in a tissue array-based analysis of human breast cancer, using immunohistochemical probes. They found that the cancers with p-mTOR overexpression showed a 3 times greater risk for disease recurrence.

Another example is provided by the work of Julie G. Izzo, MD, and coauthors, (6) who showed that esophageal cancers with activated NF-[kappa]B have aggressive clinical biology and poor treatment outcomes. In their study, they used an immunohistochemical probe to the unphosphorylated total NF-[kappa]B, but they relied on nuclear translocation as a marker for activation.

In their Kaplan-Meier curves, they6 demonstrated that the outcome in terms of disease-free survival was worse and that the outcome in terms of overall survival was worse in the NF-[kappa]B-positive cases. To reiterate, it is important to note that an assessment of the compartmentalization of the immunohistochemical signal for NF-[kappa]B guided their interpretation.

Daniela Opel, MD, and colleagues, (7) found that the activation of Akt predicts poor outcome in neuroblastoma. They used immunohistochemistry with phosphospecific probes to various putative sites of activation on the molecule. They then showed that those that overexpressed p-Akt had a worse outcome in terms of event-free survival (they had a control in terms of phosphorylated extracellular signal-regulated kinase (p-ERK), which didn't show any statistically significant difference in terms of outcome).

An article by W. Weichert, MD, et al (8) demonstrated that high cytoplasmic and nuclear expression of RelA/p65 had negative prognostic effect in pancreatic cancer. They showed the 2-year survival without cytoplasmic or nuclear RelA positivity was 41% and 40% versus 22% and 20% in those with strong cytoplasmic or nuclear RelA/p65 expressions. The authors also showed that high RelA expression was correlated to increased expression of NF-[kappa]B target genes. In essence, they showed some genomic correlates, and their conclusion was "Based on our findings, this subgroup could be identified by applying simple immunohistochemical techniques." (8)

Morphoproteomic Applications in Guiding Therapy

What about morphoproteomics and applications in guiding therapy? We can use morphoproteomics in retrospect to explain why some drugs work. This is a study that I did (9) on Langerhans cell histiocytosis (LCH), trying to make some sense out of why some of these various agents have therapeutic efficacy in osteolytic LCH work.

For example, it is known that interferon a has some therapeutic efficacy in LCH, and interferon a relies, at least in part, on PKC-[alpha] expression to effect a therapeutic response. We demonstrated that PKC-[alpha] was not only expressed but also translocated to the plasmalemmal aspect of the cell, and, therefore, that translocation is one potential explanation for why interferon [alpha] can be effective in this disease. Pamidronate, an aminobisphosphonate, has some efficacy in LCH. It targets the farnesylation pathway upstream at the level of farnesyl diphosphate synthase. Because we were able to demonstrate the immunohistochemical expression of the [alpha] subunit common to both farnesyl and geranyl/geranyl transferase (which lies downstream from the site of inhibition of pamidronate) in both LCH histiocytes and, to some extent, in osteoclasts, we believe that this explains why bisphosphonates work in the osteolytic form of LCH.

It has also been shown that indomethacin has some efficacy in LCH, and, I believe, we were the first to demonstrate the cyclooxygenase 2 expression is present in osteolytic LCH using an immunohistochemical probe.

In a Letter to the Editor, (10) I responded to some work of Albrecht Reichle, MD, and colleagues, (11) reported in The British Journal of Hematology. They published on the successful application of anti-inflammatory and angiostatic therapies in recurrent and refractory LCH. In my follow-up communication, I provided morphoproteomic evidence of the constitutive activation of the NF-[kappa]B pathway in LCH histiocytes and discussed the role of 2 agents in their therapeutic regimen, pioglitazone and rofecoxib, in inhibiting this pathway.

Available Therapeutic Antibodies and Small Molecule Inhibitors

Morphoproteomics can help explain or, at least, give some insight into why certain drugs might be applicable in a given patient's tumor. However, if we, as pathologists, are going to play a role in guiding targeted therapy, we have to understand the therapeutic molecules that are available. What is available? The list is growing. As one might imagine, there are a whole host of pharmaceutical agents and therapeutic antibodies, such as trastuzumab. The list includes the following examples: cetuximab to the EGFR ectodomain; tyrosine (Tyr) kinase inhibitors, like gefitinib and erlotinib; imatinib mesylate; the proteasome and NF-[kappa]B pathway inhibitor, bortezomib; rapalogs (temsirolimus, sirolimus), as mTOR pathway inhibitors; histone deacetylase (HDAC) inhibitors, as differentiating agents for tumor-initiating stem cells and as antiangiogenic agents; and so forth. We have to be familiar with the pharmaceutical agent and the molecular target (a more complete listing of agents applicable to morphoproteomics is found in Table 2).

Overcoming Concept of Magic Bullets

One of our issues to keep in mind is the concept of the magic bullet. We have to convince our clinical colleagues that there are no such things, except on rare occasion.

By way of example, of a modern day magic bullet, we have the article by Brian J. Druker, MD, et al, (12) reporting on the efficacy and safety of imatinib mesylate, actually, a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. Now, I am not going to diminish the success of this report because it is very successful in this clinical setting. I think in 87% to about 90% of the cases, imatinib mesylate shows therapeutic efficacy in chronic myeloid leukemia. So, imatinib mesylate is truly a form of a magic bullet. But when we try to apply this to other situations, like a gastrointestinal stromal tumor, for example, which has a c-kit gene mutation in approximately 90% of the cases and a platelet-derived growth factor receptor a gene mutation in approximately 10% of the cases, we find that the therapeutic efficacy wanes. Why is that? Efficacy wanes because we get secondary mutations in the c-kit gene. We get secondary mutations in the platelet-derived growth factor receptor [alpha] gene, and, in addition, there are 59 receptor tyrosine kinases for upstream signaling. We do not assess every one of those. These could be collaborating in bypassing the effect of the specific tyrosine kinase inhibitor that we employ.

Combinatorial Therapies: Integrating Cytotoxic Agents With Differentiating Agents and Signal Transduction Inhibitors

What about combinatorial therapies integrating cytotoxic agents and differentiating agents and signal transduction inhibitors? We have learned one thing in trying to practice morphoproteomics to guide therapy and taking into account these signal transductions. We found out, in working with our clinical colleagues, that many of these agents are cytostatic. Therefore, we need to have a working knowledge of the cytotoxic agent so that we can devise combinatorial therapies. To do this, we have to know the tumoral phenotype and the cell cycle in a given case and be familiar with where cytotoxic agents do or do not work in the cell cycle or whether they are cell cycle independent. We devised an algorithm (Figure 1) to look at tumoral phenotype-specific cytotoxic therapies with historical efficacy and where they might act. Do they work best in the G1 phase, the S phase, the G2/M phase, or the mitotic phase; in non-cell-cycle-dependent processes; or in combinations of the above? We can assess their appropriateness in an individual patient's tumor by looking at Ki-67 (a G1, S, G2, and M phase protein marker), Skp2 (which is an S phase kinase-associated protein), and the mitotic index. We can look at other cell cycle-related proteins that might contribute to our understanding of why a tumor is in a particular phase or what other therapeutic targets are available (eg, topoisomerase IIa expression as a target for etoposide therapy). We have that capability with immunohistochemistry by bright-field microscopy.

We can then follow the algorithm down and put a patient's analysis into the configuration and come out with what agents might work, but we must also look at it within the context of what those agents are doing. Many of the agents are up-regulating the NF-[kappa]B pathway. In short, they will produce or enhance the constitutive activation of the NF-[kappa]B pathway, potentially leading to more chemo-resistance or radioresistance. Finally, we have to look at the potential therapeutic agents in the context of the NFKB pathway and what other chemoresistant factors are already present and decide how to minimize this adaptive response using combinatorial therapies.

Shifting Paradigm in Tumorigenesis

Another very important point is the shifting paradigm in tumorigenesis. Malcolm R. Alison, PhD, DSc, FRCPath, and colleagues, (13) writing on the stem cell theory of cancer, reasoned that if drugs are employed that kill cancer stem cells or make them differentiate, they will make the tumor shrink with no ability for self-renewal, and we get a cure.

If, however, a drug kills most of the tumor cells but spares the cancer stem cell, the tumor shrinks, and we get a temporary remission. The cancer grows back. We have to be mindful, therefore, that the cancer stem cell may be a component of all of this when we consider the therapeutic approach, and we have to be able to assess the cancer stem cell component of any given tumor. This can be done by morphoproteomics.


Medulloblastoma and Tumor-Initiating Stem Cells

Figure 2 (A and B) is an example of a medulloblastoma. CD133, a putative neural precursor or progenitor marker, is also a cancer stem cell marker that is evident in a subpopulation of tumor cells showing brown (chromogenic) expression on their plasmalemmal aspect and with some showing a perivascular distribution (Figure 2, A). We also detected a similar expression of CD117 (c-kit) in this tumor (Figure 2, B) in a perivascular distribution (a perivascular niche). CD117 is a known stem cell in the right setting. Furthermore, in our laboratory, we have shown, by morphoproteomic analysis of bone marrow-derived [CD133.sup.+] stem cells, that CD117 and nestin are coexpressed in this neural precursor cell population.

Predictive Value of Morphoproteomic Markers in Clinical Trials

What is the predictive value of morphoproteomic markers in clinical trials? Evanthia Galanis, MD, DSc, and coworkers14 showed that high levels of p-p70S6K in baseline tumor samples predicted a patient population more likely to derive benefit from temsirolimus treatment. That is to say, those with a high nuclear stain index for p-p70S6K had a good response, and the poor responders had very little expression (low staining index).

In another study, Carter Van Waes, MD, et al, (15) showed that if they used bortezomib, which is an NF-[kappa]B pathway inhibitor, with reirradiation, it produced detectable differences in cellular NF-[kappa]B phospho-p65 localization in apoptosis and in NF-[kappa]B-modulated gene expression and cytokines. These effects seemed to coincide with some therapeutic efficacy in the form of tumor reduction.


An Example of Success Using Morphoproteomics as a Guide to Therapy

One example of a published success using morphoproteomics as a guide to therapy involved a patient with recurrent acute lymphoblastic leukemia. The patient had relapsed multiple times. The pediatric oncologist asked, "What can we do?" I said, "We'll profile it." We found that p-Akt was expressed in the relapsed leukemic cells and so were p-NF-[kappa]Band bcl-2. We created a schematic, and said, "Look, if you add bortezomib and dexamethasone in combination, it might work." This combinatorial approach resulted in measurable reduction in both the chest wall lesion and the kidney lesion as assessed by computerized tomography, before and after the therapy. It was reported in the Annals of Clinical and Laboratory Science. (16)

Consultative Proteomics Service Line

Now, we are at our current state of the art. To offer this service, we need to know and receive various things from our clinical colleagues, including a formal request for analysis by morphoproteomics, the body site of the biopsy, a copy of the pathology report, and most important, clinical data regarding therapies and response or lack thereof and the results of corresponding imaging studies, when available. Clinical data are absolutely essential to practicing morphoproteomics and to Consultative Proteomics. In general, we probe for an upstream signal transducer that might be applicable to that tumor, based on our experience and a review of the literature. We look at downstream effectors and, specifically, at the pathways of convergence, namely, the Ras/Raf kinase/ERK pathway, the PI3'-K/ Akt/mTOR pathway, the NF-[kappa]B pathway, and the PKC-[alpha] pathway. We look at the expression levels of the protein analytes in these pathways and score them; we quantify the cell cycle, that is, we look at the cell cycle impact. We probe for antiapoptotic, tumorigenic, angiogenic, and chemoresistance factors. We also probe for proapoptotic factors and growth inhibitors and, finally, for stem cell markers that might be important in tumorigenesis. These data are reported in tabular form on page 1 of the report (Figure 3).

We provide a narrative interpretation that tries to take into account and correlate all of those findings that we have scored in the front tabular portion, and we provide clinical scientific literature in the form of references to back it up. An example of this follows.


This confirms and expands upon our preliminary report transmitted via e-mail on April 25, 2008 to Drs xxxxxx and xxxxxx and the subsequent discussion with, and follow-up response to, Dr xxxxxx on April 28, 2008.

An archival paraffin-embedded block of the patient's mediastinal mass and the corresponding surgical pathology report were received from xxxxx'x xxxxxxx; xxxxx (their No. MSxx-xxx; our No. CP08-xxUT). These had been sent at the request of Drs xxxxx xxxxxx and xxxxxx xxxxxx, the patient's pediatric oncologists for morphoproteomic analysis.

By way of clarification, Consultative Proteomics incorporates the following with respect to the protein analytes in lesional and companionate cells; their immunohistochemical detection; quantification of their intensity and/or percentage of the DAB chromogenic (brown) signal by visual analysis on a 0 to 3+ scale or by an automated cellular imaging system (in the case of cell cycle-related protein analytes): their site of subcellular compartmentalization; and an assessment of their state of molecular activation to include phosphorylation (p) and functional grouping (morphoproteomics). (1) In general, this approach is intended for those patients who have failed conventional therapy or for those tumors for which there is no established protocol. In this patient, the intent is to create a profile of the tumoral and companionate cells in his midline carcinoma with NUT rearrangement for the purpose of raising therapeutic options customized for this patient.

The results of the morphoproteomic analysis are summarized in the table shown in Figure 3 and in the composite graphic in Figure 4, A through O. Additionally, the following narrative is provided to note aspects in the patient's tumor that are relevant to signal transduction pathways that could be contributing to the tumor's integrity, potential for growth, metastasis and recurrence, and chemoradioresistance:

* There is constitutive activation of the mTOR pathway as evidenced by (1) phosphorylation (p) of mTOR on its putative site of activation, namely serine 2448 with relatively strong nuclear expression in the tumor cells of p-mTOR (Ser 2448); (2) correlative activation of Akt on serine 473 and also with nuclear translocation of p-Akt (Ser 473), consistent with its role in signaling the activation of mTOR; and (3) constitutive activation of p70S6K, a downstream effector in the mTOR pathway, at threonine 389 with nuclear translocation of p-p70S6K (Thr 389). (1,17-32) Additionally, the activation of PKC-[alpha] in the form of plasmalemmal translocation in some of the tumor cells and the coexpression of plasmalemmal phospholipase D1 provides another independent pathway for the activation of p70S6K. That is to say, PKC-[alpha] translocated to the plasmalemmal compartment activates plasmalemmal phospholipase D1, (33) which, in turn, leads to the release of phosphatidic acid, the product of phospholipase D enzymatic activity. Phosphatidic acid binds to, and activates, p70S6K, resulting in an increase in its kinase activity associated with phosphorylation on Thr 389 (independent of mTOR). (34)

* Consistent with the expression of p-p70S6K (Thr 389) is the evidence of cell cycle activity (35) in the patient's tumor as reflected in the following: a moderate proliferation index (Ki-67 at 27% [which comprises G1, S, and G2/M phases]); a relatively high S phase kinase-associated protein (Skp) 2 at 26%, which helps define the S phase (36); expression of nuclear cyclin D1 (also at 27%), which could serve to facilitate progression from the G1 phase into the S phase; and a high mitotic index at 21/10 high-power fields as determined with the aid of p-histone H3 (Ser 28) immunohistochemical labeling. (37) The G1 to S and S to G2/M progression may be being facilitated by a mutant type p53 (detection of p53 in 30% of tumoral nuclei) and by topoisomerase IIa in 93% of tumoral nuclei, respectively.

* The presence of a constitutively activated nuclear factor (NF)-[kappa]B in the form of p-NF-[kappa]Bp65 (phosphorylated on serine 536), with nuclear translocation is noted. (1) Correlative downstream expression of Bcl-2 protein and glutathione S-transferase pi coincides with the activation of this pathway. (38-40) Contributing pathways in the activation of NF-[kappa]Bp65 in this tumor likely include signaling from p-Akt, PKC-[alpha], and p-p38 mitogen-activated protein kinase (p-p38MAPK; Thr 180/tyrosine 182). (41-45) The expression of p-NF-[kappa]Bp65 (Ser 536) could also be promoting cell cycle progression at G2/M phase. (46)

* A relatively low expression level of p-extracellular signal-regulated kinase (p-ERK) 1/2 (Thr 202/Tyr 204) in tumoral nuclei but with high expression levels in endothelial cell nuclei suggests that the PKC and PI3'-K/Akt pathways are dominant in this tumor. (1) Moreover, this finding has potential therapeutic implications that are discussed latter (vide infra).

* Potential proapoptotic factors include expression of estrogen receptor (ER) [beta] in the nuclei of the tumor in the absence of ER-[alpha]. (47-50)

* Finally, a tumoral stem cell lineage in the histogenesis of his midline carcinoma is suggested by CD133 expression on the plasmalemmal aspect of a portion of the tumor cells, by heterogeneous nestin expression, and by a minor population of [CD34.sup.+] tumor cells. (51-54)

* A computer-assisted search of the National Library of Medicine's MEDLINE database revealed virtually no information on midline carcinoma with NUT rearrangement and the pathways of convergence defined in this morphoproteomic analysis.


Next, we review, discuss, and illustrate the findings in our cell cycle analysis of the patient's tumor. Then we offer various therapeutic options in the context of the morphoproteomic findings for the patient's oncologist to consider with the patient and provide references that support the therapeutic considerations as follows.

Therapeutic Implications and Considerations

The molecular pathways (circuitry) created by the functional groupings of these proteins provide not only for a pathogenic sequence leading to the sustained integrity and renewal of the patient's midline carcinoma with NUT rearrangement [as confirmed by cytogenetics as t(15;19)(q13;q13.2) with a breakpoint at the NUT location on chromosome 15 by fluorescence in situ hybridization analysis] but also opportunities for therapeutic intervention at one of several points along tumorigenic signal-transduction pathways (see blue coloration in the diagram [Figure 5], the minus [-] symbol indicating inhibition). Moreover, when these morphoproteomic data are considered in the context of phenotype-specific and cell cycle-dependent and cell cycle-independent therapies, it is possible to create an analysis leading to recommendations for specific combinatorial therapies for your consideration. A discussion of such opportunities is listed below as follows:

Therapeutic Options Designed to Effect Cytotoxic Reduction of Residual Tumor

* A G1 to S and/or an S phase-active agent and/or an S phase to G2/M phase and/or an M phase-active agent would seem to be appropriate given the cell cycle data. Specifically, topotecan is reported to block cells in S phase and to lead to apoptosis, and would seem to be a logical choice. Similarly, the relatively high percentage of topoisomerase IIa expressing tumoral cell nuclei also raises the question of using a topoisomerase II inhibitor (eg, etoposide). Docetaxel, a microtubule stabilizing agent, has been reported to interfere with spindle microtubule dynamics causing mitotic cell cycle arrest and apoptosis (one of its effects is to cause phosphorylative inactivation of Bcl-2, which is antiapoptotic protein highly expressed in his tumor). (55-58) Anecdotally, docetaxel has been reported to have efficacy in a case of midline carcinoma. (59) Although gemcitabine is also an S phase-active agent, it appears to require an activated ERK pathway, and tumor cells in which the ERK pathway has been inhibited become more resistant to gemcitabine. (60-61) This patient's tumor cells, per se, show only occasional cells with p-ERK 1/2 expression and nuclear translocation and may be relatively chemoresistant to gemcitabine.

* Combinatorial therapy with an NF-[kappa]B pathway inhibitor, such as bortezomib (Velcade), would seem to be indicated given the constitutive expression of p-NF-[kappa]Bp65 in his tumor and the concomitant expression of downstream effectors of said pathway, such as Bcl-2 and glutathione S-transferase Pi (GST-pi). If a topoisomerase inhibitor, such as topotecan or etoposide, were used in a combinatorial fashion with bortezomib, sequencing would be important. That is to say, it appears that PS-341 (bortezomib [Velcade]) can enhance the DNA damage-induced apoptosis, if given in tandem (shortly [probably within a few hours] after the topoisomerase-targeting agent is given). (62) Other agents, such as carboplatin, should be given either concurrently or following bortezomib. (63-64) Bortezomib should probably not be used as a combinatorial agent with docetaxel. (65)

* Combinatorial therapy with rapamycin or a rapamycin analog might be useful with docetaxel, if given in a sequential fashion. That is to say, oral rapamycin given for several weeks and then stopped 48 hours before docetaxel could synchronize the rush of the tumor cells into the cell cycle, making them a target for docetaxel and then after docetaxel, resumed. This is similar to the approach used by Merimsky (66) for attempted synchronization and would also take advantage of rapamycin's reported effect of enhancing the antitumoral activity of taxanes, such as paclitaxel, in preclinical studies, particularly if the cytotoxic (paclitaxel) is administered before the rapamycin. (67,68)



Finally, the use of fluorodeoxyglucose-positron emission tomography, to assess the amount of residual tumor, may be useful, given the constitutive activation of the mTOR pathway in the patient's midline carcinoma. That is to say, fluorodeoxyglucosepositron emission tomography scintigraphy has been viewed as a surrogate for the demonstration of the mTOR pathway (69) and, therefore, should be useful in assessing residual tumor in this patient.

Cytostatic Therapies Designed to Reduce Tumor Cell Growth

* Cytostatic therapies with oral rapamycin and curcumin, an inhibitor of phospholipase D signaling, (70) could lead to decreased activation of p-p70S6K (Thr 389) and, thereby, to reduced tumor cell growth. Additionally, curcumin has been used in clinical trials (71) with a relatively low toxicity profile and has the added advantage of inhibiting the Akt/NF-[kappa]B pathway, (41) which could also retard tumor cell growth.

Antitumoral Stem Cell Therapies to Reduce Recurrences and Induce Differentiation

* In the context of the tumoral stem cell markers in this specimen, in light of the work at the Hospital for Sick Children in Toronto by Drs Kristen Smith and David Kaplan regarding tumor initiating stem cells, (72) albeit in neuroblastoma, and in the context of the suggestion to use antiangiogenic therapy to remove the perivascular niche of tumoral stem cells, the following comments are offered: (1) Rapamycin may indeed be a useful adjunct to act as an antitumoral stem cell agent and to reduce the tumor volume and retard its growth, particularly if combined with oral curcumin (70); and (2) An antiangiogenic strategy, employing either rapamycin and vinblastine (73) and/or sorafenib (Nexavar), to target the vascular endothelial growth factor receptor Ras/Raf kinase/ERK pathway, (74) which is highly expressed in the endothelium of the intratumoral vessels in this case, or an histone deacetylase (HDAC) inhibitor, as an antiangiogenic agent. (75)

* Potential differentiating agents that might be applied in this tumor include genistein and HDAC inhibitors. Genistein, which is an isoflavone and phytoestrogen, could be useful as a ligand for ER-[beta] in tumoral nuclei, thereby promoting cell differentiation and apoptosis. (47-50) (As you know, genistein is relatively nontoxic and is currently in clinical trials). (76) Genistein also mediates histone acetylation and demethylation and activates tumor suppressor genes in a preclinical study. (77) Histone deacetylase inhibitors have shown promise as epigenetic modulators to transdifferentiate normal adult stem cells and to cure disorders caused by uncontrolled growth of malignant stem cells and to promote differentiation to a benign phenotype in neuroblastoma. (78-80) (The feedback from Dr xxxxx concerning the sensitivity of midline carcinoma cell lines to HDAC inhibitors in vitro reinforces consideration of their use as both an inducer of differentiation and of cell cycle arrest and apoptosis.)

Finally, the decision to use one or more of the agents and approaches rests with the clinical judgment of the patient's pediatric oncologists, Drs xxxxxx and xxxxxx.

Date: Robert E. Brown, MD

To reiterate, we incorporate into each report the digital images representative of the morphoproteomic findings and a summary schematic incorporating the pathways and the therapeutic options (Figures 4, A through O, and 5). Also, when applicable we include the morphoproteomicguided algorithmic approach (Figure 1).

Finally, our involvement in the case does not end with the rendering of our report. We remain as consultants to our clinical oncology colleagues. We are often asked, generally via e-mail, to provide clarification of the morphoproteomic findings and input with regard to the regimen and therapeutic options that the oncologists, in consultation with the patients or their families, elect to use. We get feedback during the course of therapy. Moreover, we are consulted regarding new therapies and how they might relate to the morphoproteomic analysis. In short, the application of morphogenomics and morphoproteomics has afforded us, as anatomic pathologists, an exciting opportunity to expand our role from diagnosticians to that of clinical scientists and clinical colleagues, instrumental in helping to customize therapy for the individual patient ("personalized medicine").

I thank Richard A. Breckenridge, ASCP, and Pamela K. Johnston, HT, ASCP, for their technical expertise and Bheravi Patel for her secretarial support and graphic skills.


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Robert E. Brown, MD

Accepted for publication November 7, 2008.

From the Department of Pathology, University of Texas Health Science Center, Houston Medical School, Houston, Texas.

The author has no relevant financial interest in the products or companies described in this article.

Presented in part at the College of American Pathologists Futurescape of Pathology Conference, Rosemont, Illinois, June 7-8, 2008.

Reprints: Robert E. Brown, MD, Department of Pathology, University of Texas Health Science Center, Houston Medical School, 6431 Fannin St, Houston, TX 77030 (e-mail:
Table 1. Summary of Immunohistochemical Probe in
Morphoproteomics and Fluorescence In Situ
Hybridization (FISH) Probe for

     Vendor            Location         Protein or DNA Target

abcam               Cambridge,        CD133
                      Massachusetts   Nestin
BD Pharmingen       San Jose,         p16INK4a
  (Becton             California      [beta]-catenin
  Dickinson)                          CD34
Biocare Medical,    Walnut Creek,     Cyclin D1
  LLC                 California
BioGenex            San Ramon,        GST-pi
                      California      ER-[beta]
Cell Signaling      Beverly,          p-ERK1/2 (Thr 202/Tyr
  Technology, Inc     Massachusetts     204)
                                      p-Akt (Ser 473)
                                      p-mTOR (Ser 2448)
                                      p-p70S6K (Thr 389)
                                      p-p38MAPK (Thr 180/
                                      p-NF-[kappa]Bp65 (Ser 536)
DakoCytomation      Carpentaria,      EGFRvIII/EGFR
                      California      Ki-67
                                      Topoisomerase IIa
                                      CD117 (c-Kit)
Novocastra          Newcastle upon    p2 7 Kip1
                      Tyne, United    p53
R&D Systems         Minneapolis,      IL8
Santa Cruz          Santa Cruz,       PKC-[alpha]
  Biotechnology       California      Phospholipase D1
  Inc                                 Skp2
                                      TRAIL-R1 ([DR.sub.4])
Vysis (now Abbott   Des Plaines,      HER2/neu DNA probe
  Molecular Inc)      Illinois
Zymed               Carlsbad,         CD56

Abbreviations: ER-[alpha], estrogen receptor [alpha]; ER-[beta],
estrogen receptor [beta]; EGFRvIII-EGFR, epidermal growth factor
receptor; GST-pi, glutathione S-transferase; Her2-neu DNA probe,
epidermal growth factor receptor DNA probe; Her2-neu, human
epidermal growth factor receptor; IL8, interleukin 8; p-ERK1-2,
extracellular signal-regulated kinase; p,  phosphorylated; PKC-[alpha],
protein kinase C [alpha]; p-mTOR, phosphorylated
mammalian target rapamycin; p-p38MAPK, mitogen-activated protein
kinase; PPAR-[gamma], peroxisome proliferator activated receptor
[gamma];  p-NF-[kappa]Bp65, phosphorylated nuclear factor-[kappa]Bp65;
Ser, serine; Skp2, S phase kinase-associated protein
2;Thr, threonine;Tyr, tyrosine; VEGFR-A, vascular endothelial
growth factor receptor A.

Table 2. Therapeutic Agents Applicable to

       Pharmaceutical Agent                Molecular Targets

Trastuzumab (Herceptin) (a)          HER2/neu (ERBB2) receptor
Gefitinib (Iressa) (b), Erlotinib    EGFR tyrosine kinase
  (Tarceva) (a)
Cetuximab (Erbitux) (c)              EGFR ectodomain
Imatinib mesylate (Gleevec) (d)      bcr-abl, PDGFR, and c-Kit
                                        tyrosine kinases
Statins (Lovastatin) (e)             Mevalonate/prenylation path
                                       way and N-glycosylation
                                       of IGFR and EGFR.
Aminobisphosphonates                 Prenylation pathway via
  (pamidronate (e),                    inhibition of farnesyl
  zoledronate (d))                     diphosphate synthase
Zarnestra (e)                        Prenylation pathway via
                                       inhibition of farnesyl
Captopril (e) (ACE inhibitor),       Transactivation of PDGFR
  losartan (e) (AT1R inhibitor)        and EGFR signaling via
                                       the angiotensin system
Sirolimus (f) (rapamycin;            Immunophilins and mTOR
  Rapamune), temsirolimus              pathway signaling
  (CCI-779), everolimus (RAD001)
Bortezomib (Velcade) (e)             NF-[kappa]B signaling via
                                       proteasome inhibition
Geldanamycin (f)                     Hsp90 chaperone molecules
Bevacizumab (Avastin) (a)            VEGFR
Tamoxifen (e)                        ER signaling
Nexavar (g) (BAY 43-9006)            Raf and VEGFR
Sunitinib (h) (SU11248)              VEGFR/PDGFR
HDAC inhibitors                      HDAC

Abbreviations: ACE, angiotensin-converting enzyme; ATIR,
angiotensin type 1 receptor; ER, estrogen receptor; EGFR,
epidermal growth factor receptor; HDAC, histone deacetylase;
HER2-neu (ERBB2), human  epidermal growth factor receptor 2;
Hsp90, heat shock protein 90; IGFR, insulin-like growth factor
receptor; mTOR, mammalian target  rapamycin; NF-[kappa]B, nuclear
factor-[kappa]B; PDGFR, platelet-derived growth  factor receptor;
VEGFR, vascular endothelial growth factor receptor.

(a) Genetech Inc, South San Francisco, California.

(b) Astra Zeneca Pharmaceuticals LP, Wilmington, Delaware.

(c) Merck & Co, Inc, Whitehouse Station, New Jersey.

(d) Novartis, Basel, Switzerland.

(e) LGM Pharmaceuticals, Inc, Boca Raton, Florida.

(f) AG Scientific Inc, San Diego, California.

(g) Bayer Healthcare Pharmaceuticals, Wayne, New Jersey, and Onyx
Pharmaceuticals, Emeryville, California.

(h) Pfizer, New York City, New York.
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