The Daily Sign-Out

Aberrant Wnt1 and beta-catenin expression in NSCLC

This month's April 2011 Journal of Thoracic Oncology has an interesting paper by Xu and colleagues (abstract) that examines Wnt1 expression in non-small cell lung cancer (NSCLC) in relation to downstream Wnt signaling molecules, including beta-catenin, and correlates different marker expression with traditional clinicopathological parameters.

This is an immunohistochemical study of a tissue microarray composed of 262 NSCLC resected specimens.    The authors define aberrant beta-catenin expression as: 1) decreased membranous pattern in less than 70% of tumor cells, 2) cytoplasmic pattern of expression, or 3) nuclear pattern of expression.  As expected, the majority of patients are stage 1 or 2 but nearly 36% of the study population are never-smokers--over 50% in the adenocarcinoma histology group (!).  The authors found that Wnt1 overexpression was significantly associated with never-smoker status and ADC histology; aberrant beta-catenin expression was associated with SQC histology.  While each Wnt1 overexpression and aberrant beta-catenin expression were independently associated with decreased OS regardless of TNM stage, patients that demonstrated both had worse OS than other combinations of Wnt1/beta-catenin expression.  Further, Wnt1/beta-catenin expression was also an independent factor adjusted by stage, pleural invasion, lymphatic invasion, ECOG PS or stage, age, gender, histology, and p53 expression.

A few comments about this paper.  One significant caveat to the findings is the over-representation of never smokers (35%) in this Asian study population, especially nearly 54% of never smokers in the ADC group, compared to a more typical American population in a community hospital setting (about 10%).  Overexpression of Wnt1 was significantly associated with never-smoking and as well as ADC histology, but, somewhat paradoxically, Wnt1 overexpression was associated with significantly shorter overall survival.  Unfortunately, the authors do not break this further down into Wnt1 overexpression and OS with respect to histological type--perhaps some SQCs showing Wnt1 overexpression and dismal OS are skewing the data here since one would expect never-smokers with ADC to have a better prognosis in general.  Another is the usual issues with using TMAs to represent complex heterogenous tumors.  The authors do not sufficiently describe from where they target obtaining cores for constructing the TMA--are they targeting the invasive margin, the central tumor, wherever?  what about mixed histology tumors?  These are critical methodological issues--especially when extrapolating the data concerning marker expression.  One admirable feature of this paper is the clear definition, supported by appropriate photomicrographs, of "aberrant" beta-catenin expression.  As I am currently working on projects that are similarly assessing beta-catenin expression in NSCLC, I found this very helpful in assessing this paper's findings.

Optimal panel for differentiating NSCLC in small specimens

There has been a plethora of papers, abstracts, and marketing materials in the last 12 to 18 months regarding the "optimal" panel for differentiating adenocarcinoma (ADC) from squamous cell carcinoma (SQC) in non-small cell lung cancer (NSCLC).  The December 2010 American Journal of Surgical Pathology features a paper from Terry, Leung, Laskin et al. that evaluates an optimal panel and, perhaps, more importantly, a testing algorithm for differentiating ADC from SQC in small biopsy specimens.

Caveat: one of the authors is the Medical Director/Chief Pathologist for a commercial pathology reference lab specializing in IHC diagnostics (PhenoPath Laboratories).

This study makes several practical points that can be nicely summarized as bullets:

• For ADC the most sensitive marker and best negative predictor is CK7: 93% sensitivity, 91% NPV.
• For ADC the most specific marker and best positive predictor is Napsin A: 94% specificity, 90% PPV.
• TTF-1 has similar specificity as Napsin A for ADC.
• For SQC the most sensitive marker and best negative predictor is p63: 84% sensitivity, 86% NPV.
• For SQC the most spepcific marker and best positive predictor is NTKR2 (a neurotrophic tyrosine kinase receptor, rarely used outside of research labs) but CK5/6 has a nearly identical specificity and PPV: about 95% specificity, about 95% PPV.
• No single marker is BOTH highly sensitive and specific for either ADC or SQC.

The authors identified a 6-marker panel that includes p63, TTF-1, CK5/6, CK7, Napsin A, and mucicarmine has demonstrating the best AUC in ROC analysis.  Since a 6-marker panel may not be feasible (or even possible) due to various factors with small specimens with limited diagnostic tumor material, the authors develped a binary scheme using a sequential addition of markers (Fig. 3, 1809).

The authors do point out, however, that the "best" sequential order may vary by laboratory according to the actual senstitivities and specificities acheived in individual labs and this is important to remember if your lab does not perform all of the stains.

I think one important caution, however, is to note that this study was performed using tissue microarrays derived from resected lung cancer specimens and did not study or validate the performance of these markers on "real" small biopsy specimens.  The authors claim that the results are "similar" to earlier studies using actual patient biopsy material.  Given their careful statement above regarding the "best" sequential order, I think that this is somewhat disingenuous elision over important details regarding significant differences between resected tumors and small biopsy specimens.  Notwithstanding this major issue, there are some good quantitative data here that warrants your consideration in evaluating your own panels at least for comparing your own sensitivity and specificity for individual markers.

Detecting EML4-ALK Fusion Gene Mutation in Endobronchial Aspiration Specimens

The October 15, 2010 issue of Clinical Cancer Research has an intriguing article that could have major implications for staging and prognostication in lung cancer.  Sakairi and colleagues from Chiba Cancer Center in Japan show the feasibility of using endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) to obtain specimens suitable for testing for EML4-ALK fusion gene in NSCLC lung cancer patients with proven hilar and/or mediastinal lymph node metastasis.

The protocol for this study involved chest CT followed by EBUS-TBNA for lymph nodes >5 mm.  They obtained a histologic core using a 22-gauge needle and divided the specimen into two parts with one part placed in 20% (!) formalin and the other half placed in a cryopreservative solution and then frozen at −80℃. Histological examination was performed in all cases.  The authors present a triage for EML4-ALK fusion gene mutation including screening by IHC, followed by FISH, and then final confirmation with RT-PCR.  They also tested for EGFR gene mutation in all samples.

The IHC procedure is of interest to pathologists because they used a intercalated antibody-enhanced polymer (iAEP) technique (Takeuchi K et al. Clin Cancer Res 2009;15: 3143–9) which incorporates an intercalating antibody between the primary antibody to ALK and dextran polymer–based detection reagents. They used a primary antibody specific for the intracellular region of ALK (5A4, Abcam) which is not a ALK fusion mutation specific antibody.

Adenocarcinoma was identified in 75% of cases and 20% of patients were never smokers.  A mean of 2.1 lymph nodes were sampled per patient and the median size of the sampled nodes was 12 mm.  Six out of 109 cases examined by IHC were ALK immunopositive; 17 were categorized as "suspicious," including 1 "probably positive" case.  FISH confirmed the presence of the ALK fusion gene in all size cases but the "probably positive" case had too few cells for FISH analysis.  However, RT-PCR confirmed the presence of EML4-ALK in all six IHC-positive cases as well as the "probably positive" case for a total of 7 cases positive for the ALK fusion gene (6.4% of all cases).  EGFR mutation was detected in 25 cases (22.4%).

All ALK-positive cases had an adenocarcinoma histology and lacked EGFR gene mutations. Six out of the seven ALK-positive cases were either never-smokers or light smokers. No significant difference in gender was observed between ALK-positive and ALK-negative patients.  Interestingly, the mean primary tumor diameter of ALK-positive cases was significantly smaller than that of ALK-negative cases (28.6 mm versus 41.9 mm; P < 0.05).

If these methods and findings can be replicated, EBUS-TBNA with multiplex gene mutation testing could have a major effect on the diagnosis, staging and management of lung cancer.  In the more short-term future, the use of immunohistochemistry with iAEP technique that would include using a highly sensitive mutation-specific antibody would have a major impact on testing for ALK since FISH testing is technically difficult due to the close proximity of the two fusion gene components.

 

ALK Inhibition in Non-Small Cell Lung Cancer and Inflammatory Myofibroblastic Tumor

Great stuff in this week's NEJM!

Today's (October 28, 2010) New England Journal of Medicine has no less than three articles reporting the use of small molecule ALK tyrosine kinase inhibitor crizotinib (PF-02341066, Pfizer) in non-small-cell lung cancers harboring the oncogenic fusion gene EML4-ALK and in a patient with ALK-rearrangement positive inflammatory myofibroblastic tumor, a distinctive but uncommon soft tissue neoplasm.  

Pathologists should be familiar with these articles--at least so you can suggest appropriate testing for ALK rearrangement but also that you might "shine" at your next Tumor Conference ; )  BTW, the article by Kwak et al. presents in paper form the data that rocked this past summer's ASCO Annual Meeting regarding crizotinib.

Of interest to pathologists is how the EML4-ALK rearrangements were identified.  Importantly, Kwak et al prospectively evaluated about 1500 NSCLC formalin-fixed, paraffin-embedded tumor samples by FISH using an ALK break-apart probe.  The study also included a retrospective analysis on the FISH-positive cases using RT-PCR for specific EML4-ALK fusions and immunohistochemistry using anti-ALK antibodies.  Note that the authors did not use ALK-rearrangement-specific antibodies that have recently become available.  Mino-Kenudson and colleagues recently reported their experience in Clinical Cancer Research using a highly sensitive antibody for ALK-rearrangement by IHC.  The availability of these mutation-specific antibodies could make IHC a preferred (and cheaper, more widely available) method for screening to replace FISH.

In this same issue, Choi and colleagues report report the discovery of two secondary mutations within the kinase domain of EML4-ALK in tumor cells isolated from a patient during the relapse phase of treatment with an ALK inhibitor that appear to confer resistance against two different ALK inhibitors (!).  Curiously, each mutation developed independently in subclones of the tumor.

Finally, Butrynski and colleagues report a sustained partial response to the crizotinib in a patient with an ALK-translocated inflammatory myofibroblastic tumor (IMT) indicating the biological role for ALK signaling in tumors with ALK rearrangement and a therapeutic strategy for crizotinib in ALK-rearranged IMTs.

Drs. Bengt Hallberg and Ruth Palmer provide an insightful editorial which should also be read (at a minimum).

Follow-up on Adenocarcinoma with BAC Features

My last post was a "Digital Case Challenge" presenting a case of pulmonary adenocarcinoma with non-mucinous bronchioloalveolar (BAC) features.

Here's my synopsis of 3 recent papers on bronchioloalveolar carcinoma that focus on the clinicopathological, histological, and molecular differences between the two subtypes, non-mucinous and mucinous, as well as the practical importance in distinguishing these two subtypes.

1.  Wislez et al, in a recently published paper in Lung Cancer (2010;68:185-191), studied 50 cases of non-resectable adenocarcinoma with bronchioloalveolar (ADC-BAC) features who were enrolled in a multicenter study to evaluate gefitinib as a first-line therapy for non-resectable ADC-BAC.  Previous trials (SWOG S0126 and IFCT0401) have shown that the mucinous subtype was associated with a lower response rate and was predictive for shorter survival after treatment with gefitinib.  The purpose of this study was to identify markers that would better characterize each subtype.  They first classified each tumor by routine H&E staining using conventional criteria.  They found that three variables enabled segregation of 96% of cases (48/50 patients): diastase-resistant PAS staining, IHC staining for TTF-1, and EGFR mutation/genomic gain.

dr-PAS: 80% non-mucinous ADC-BAC tumors showed <5% positive cells; 83% mucinous ADC-BAC >5%

TTF-1: 95% non-mucinous ADC-BAC express TTF-1 >20% of cells; only 27% mucinous ADC-BAC

EGFR mutations (exons 18-21): only 3/25 (12%) in non-mucinous ADC-BAC but none in mucinous ADC-BAC

The last findings are lower than previously published rates for non-mucinous ADC-BAC in both Asian and U.S./European studies.  Moreover, EGFR exon 18-21 mutations have been reported in mucinous ADC-BAC, albeit at a much lower rate compared to non-mucinous subtype.  Which leads me to a second recent report I recently reviewed.

2.  Casali et al (J Thorac Oncol 2010;5:830:836) presented a single-institution retrospective study from Italy of 40 cases of BAC based on strict application of the 2004 WHO histologic classification of BAC (although 6 cases were not completely sampled for histology due to large size/pneumonic involvement).  Their purpose was to compare previous studies of BAC in  primarily Japanese/East Asian studies with their exclusively European Caucasian patient population.  Their findings are similar but not identical to these previous studies.  Non-mucinous BAC presents more frequently as a solitary mass and early stage (cf. mucinous subtype), whilst mucinous BAC more frequently presents as multifocal tumor or a pneumonic pattern of lung involvement and in more advanced stage.

The overall incidence of EGFR mutation was similar to the study by Wislez et al (17%) which is also less frequent than that reported in primarily Japanese reports; they did confirm a strong correlation with never-smoking status and female gender.  Also, they found a significant difference in mutation profile between subtypes: 6/14 non-mucinous BAC versus none in mucinous BAC.  Conversely, KRAS mutation was found in 12/13 mucinous BAC versus 3/20 non-mucinous BAC.

Although this is a small, single-institution study, there is helpful and interesting survival data that shows significantly increased OS and RFS for non-mucinous versus mucinous/mixed BAC in early stage (IA and IB) tumors.  They also note the raw numbers for the small subgroup of patients with multifocal tumor (N=6) and pneumonic involvement (N=7) which suggest a dismal survival for these patterns of involvement.  Four out of six patients with multifocal tumor died of disease; 5 of 7 with pneumonic involvement died of disease--all 5 of whom had mucinous BAC type.

Finally, they confirmed by multivariate analysis that stage (p=0.004) and histologic subtype (p=0.035) were independent prognostic factors for overall survival.  It would be nice to have this data presented as hazard ratios to assess the relative effect of each of these factors.

3.  Finally, Hata et al (J Thorac Oncol 2010;5:1197-1200) recently reported a study of EGFR and KRAS mutations in Japanese patients with mucinous BAC/adenocarcinoma with mucinous BAC subtype features (MBAC/AWBF).  They analyzed 20 cases of MBAC/AWBF for EGFR and KRAS mutations and compared them with 24 cases of N-MBAC/AWBF.  Briefly, they found in MBAC/AWBF there is a low incidence of EGFR mutations (3/20, 15%) while there is a frequent incidence of KRAS mutations (12/20, 70%).  Conversely, in N-MBAC/AWBF there is a higher incidence of EGFR mutations (14/24, 58%) but less frequent incidence of KRAS mutations (7/24, 29%).  Note that in contrast to the studies above, these authors did find some EGFR mutations in MBAC/AWBF tumors.  Moreover, the authors identified 3 patients with MBAC/AWBF with concomitant EGFR and KRAS mutations--thus showing that these mutations are not always necessarily mutually exclusive.  The reasons for these discrepancies may be methodological becasue of different mutation detection techniques or may be due to racial/ethnic differences.  Further work to examine differences across populations would definitely be interesting.  But, more importantly from a practical standpoint,  I think this is significant because it illustrates that we cannot simply use histology as a perfect triage for mutation testing.  It would be quite interesting to see if any of the 3 patients with MBAC/AWBF with EGFR mutations were treated with EGFR-TKIs and what the response was in comparison to other histologic types and subtypes.

Bottom line?  Pathologists should use the above information to try and differentiate mucinous versus non-mucinous subtypes in BAC/ACA with BAC features, especially in early stage tumors as this has prognostic and, possibly, therapeutic significance.  Also, although histological subtype can predict the likelihood of an EGFR mutation being present, it alone should not be used as a criterion for mutational testing, particularly for EGFR mutations.

Grading system for lung adenocarcinoma still elusive

The August 2010 issue of American Journal of Surgical Pathology features an article from the Memorial Sloan Kettering Cancer Center group proposing a grading system for lung adenocarcinoma in stage 1 cancer.

Sica G, Yoshizawa A, Sima CS, et al.  A grading system of lung adenocarcinoma based on histologic pattern is predictive of disease recurrence in stage I tumors.  Am J Surg Pathol 2010;34:1155-1162.

The authors articulate the frustration pathologists experience in dealing with the histological heterogeneity of lung adenocarcinoma (ACa) since the vast majority are mixed subtype as well as the lack of an objective, clinically relevant grading system for lung ACa.  Recent literature has shown that mixed subtype tumors with solid or micropapillary components appear to have worse outcomes compared with those that do not, while tumors with a predominantly bronchioloalveolar (BAC) component appear to have a better prognosis.

An important part of this study is the idea of "comprehensive histologic subtyping" per the mothod outlined by Motoi et al (Am J Surg Pathol, 2008).  It is only by essentially submitting the entire tumor for histology that one can accurately assess the histologic heterogeneity of a tumor and semiquantitatively identify the various subtype components.  Another interesting feature of this paper is the use of a "concordance probability estimate" in their statistical analysis.  Although the term seems like more complex statistical jargon, this measure seems useful to me in that it provides an estimate of the ability of each grading system to predict the outcome for an individual case.

To identify patterns of dissemination of different histological subtypes, the authors retrospectively studied 73 stage IIA-IV primary lung ACa and corresponding metastatic tumors.  The majority of these tumors were composed of predominantly acinar subtype in 48%, solid subtype in 19%, and papillary in 19%.  While tumors at each of the metastatic sites also showed a mixture of patterns, the predominant subtype pattern was more likely to be found at the metastatic site.  Of note, even when the solid or micropapillary patterns were a small percentage of the primary tumor, these patterns are disproportionately more often found in the metastatic site.

Based on this work the authors devised a 3-tier grading system based on the mix of histological subtypes: grade 1, predominantly BAC pattern; grade 2, acinar and papillary, and grade 3, solid and micropapillary patterns.  The second part of this study was to test this grading system and a number of different scoring systems in 366 stage 1 ACa for the ability to predict disease-free survival (DFS).

In the stage 1 ACa group, predominantly acinar subtype was seen in 49%, papillary subtype in 26%, solid in 13%, BAC in 9% and micropapillary in 3%--a slightly different distribution (but perhaps expectedly so) compared to the high stage group.  But here is where this study, imho, jumps off the rails.  I refer the reader to Table 2 on page 1159 for more details but I'll try to summarize.

The authors show some value to reporting the most predominant grade which showed nice separation of DFS but only had a "concordance probability estimate" of 0.61--that is, in 2 randomly selected patients, this system would order them correctly with respect to DFS in 61% of cases.  The authors preferred scoring system based on the two most prominent grades only performed marginally better with a concordance probability estimate of 0.65.  In looking at the data, I would have preferred a scoring system of predominant grade + highest grade because this showed the best separation between the three tiers if you look at the 95% CI of the hazard ratios and yet still had a concordance probability estimate of 0.62.  The reality is that none of these systems actually perform well enough to warrent their clinical use.  The authors' own preferred system seems like it would be less reproducible than the others that were evaluated because it completely relies on semiquantitative estimates of proportions of patterns.  I think it could also underestimate the DFS for tumors which have small but significant proportions of solid and micropapillary patterns.

Anyhow, it is a start and much further work is needed before we get something that approaches, for example, the Nottingham scoring system for breast cancer.

 

 

Prognostic factors in T1 colorectal adenocarcinoma

A frequently encountered specimen is the adenocarcinoma with submucosal invasion arising in a dysplastic adenomatous polyp or sessile lesion that has been resected endoscopically.  While much effort is directed at determining margin adequacy and depth of invasion, less attention has been given to other parameters that may be associated with risk of lymph node (LN) metastasis. Measuring the depth of invasion is frequnetly problematic given the morcellated nature of most specimens.  Generally, if the lesion is felt to be adequately excised, a formal colectomy may not necessarily be needed since the risk of LN metastasis in a T1 tumor is felt to be too low to warrent the morbidity of a colectomy.

A recent article in the August 2010 Modern Pathology sought to detect patients at high risk for LN metastasis after endoscopic mucosal resection.

Tateishi Y, Nakamishi Y, Taniguchi H et al.  Pathological prognostic factor spredicting lymph node metastasis in submucosal invasive (T1)colorectal adenocarcinoma.  Mod Pathol 2010;23:1068-1072.

This is a single-institution study that examined 322 consecutive patients treated by colectomy (complete tumor excision plus lymphadenectomy) for T1 colorectal adenocarcinoma.  The entire lesion was examined microscopically in all cases and criteria evaluated for the study included submucosal invasive depth, tumor size, lymphatic invasion, venous invasion, histologic grade, tumor budding at the submucosal invasion front, and status of muscularis mucosae.

The authors found LN metastases in 14.3% of patients (46/322)--which was a surprising and sobering finding in itself to me.  By univariate analysis, tumor budding, histologic grade, lymphatic invasion, venous invasion, submucosal invasion depth > 1 mm, and completely disrupted (type B) muscularis mucosae were associated with LN metastasis.  By multivariate analysis, however, only tumor budding, high grade tumor differentiation, and lymphatic invasion were associated with LN metastasis.  Moreover, of the 46 patients with LN metastasis, 40 (87%) had at least 1 out of 3 factors by multivariate analysis associated with LN metastasis; the remaining 6 all showed type B muscularis mucosae status.  Using these four criteria, 285 out of 322 patients were positive for at least one criteria and would have undergone colectomy after submucosal resection--for 100% sensitivity but only 13.4% specificity.

Certainly, the decision whether colectomy is the most appropriate definitive treatment following endoscopic resection must be individualized.  But it seems in my experience that this is a gray zone for surgeons and oncologists and these criteria, while needing independent and prospective validation, are worthy of further study and consideration of being adopted for routine use now.  The evaluation of muscularis mucosae status is not familiar in the U.S. and the reader is referred to the original article by Tominga et al in Dis Colon Rectum (2005) for more detail.

The practical points here are 1) that these parameters can be readily assessed in routine H&E sections and 2) using four histologic risk factors--tumor budding, lymphatic invasion, high-grade histologic tumor differentiation (moderately- to poorly-differentiated), and type B muscularis mucosae status--the authors were able to select all patients who were shown to have LN metastasis. 

Use of oncology decision aids--by patients?!

Medscape reported on an article from J Clin Oncol that I thought was most curious:

September 1, 2010 —The use of decision aids by physicians caring for cancer patients is "low", say the authors of a Canadian study.

Only 24% of general surgeons, medical oncologists, and radiation oncologists in Ontario report using the aids, which are information guides that help patients make choices about cancer treatment.

The study, based on a survey of clinicians, was published in the May issue of the Journal of Clinical Oncology (2010;28:2286-2292).

However, in a letter published online August 30 in the journal, a German researcher raises an interesting possibility: that patients might be using decision aids at higher rates.

In other words, patients might be adapting this clinical tool more widely than the doctors who care for them.

I find this fascinating given the availability of such resources as Adjuvant! Online.  Research into the possible reasons for this discrepancy would be interesting.  Do doctors fear more than patients looking at "hard numbers" and probabilities?  or explaining or translating these numbers to patients in a forthright manner?  Or does the use of a decision aid somehow "dehumanize" a particular patient--the one sitting on front of you?  Your thoughts?

Mucinous bronchioloalveolar carcinoma of lung and ALK mutation

Last month I received a generous note from a patient who responded to a previous post on molecular markers in lung cancer from the USCAP 2010 meeting.  I made the statement that "every adenocarcinoma should be tested for EGFR mutation (with the exception of mucinous ACa/BAC which are always KRAS mutants)."  This patient's experience should cause us to not be so categorical about such statements.  Moreover, as we develop testing algorithms, we should consider an approach which will allow us to detect "exceptions to the rule."

The patient was diagnosed with mucinous BAC.  But not only was the tumor wild-type for KRAS mutation (i.e., no mutation was detected) but instead an ALK mutation identified in the tumor.  The patient subsequently received the drug crizotinib, an inhibitor of the fusion protein EML4/ALK, and has had a sustained response to treatment.  

This is the drug that generated so much attention at the recent ASCO 2010 meeting in Chicago.  In an international phase I study of advanced, heavily pretreated NSCLC patients selected for ALK protein expression, crizotinib produced a 57% response rate with a 6-month PFS predicted to be 72%.  This data was presented at the Plenary Session.  Moreover, it was noted that the majority of patients showed some kind of response (63% including objective and unconfirmed partial responses), the response often occurred dramatically within the first 2 weeks of treatment, and the responses were often durable.  In this study, 96% of responders had adenocarcinoma histology--primarily signet ring morphology.

This is surely a very exceptional case since mucinous ACa/BAC is a very distinctive histological subtype and has been also characterized at the molecular level as being associated with KRAS mutation.  The point is that histology is not a perfect predictor of potentially druggable molecular pathologies.  If targeted therapy is being contemplated in NSCLC, we should adopt a systematic approach to all NSCLC regardless of histology.  At this point, while we are identifying patients who may potentially benefit from targeted therapy, we are building a database that will allow a more full appreciation of the relationship between histological type and molecular pathology.

Thanks to the patient who took the time to respond to my blog and for helping to edify us all!

P53 in tumor-associated fibroblasts in invasive ductal carcinoma

Hasebe et al. have authored an interesting article in a recent Modern Pathology (2010;23:662-672) which builds on their earlier work that showed P53 expression in tumor-stromal fibroblasts (TSF) not forming fibrotic foci to be an important predictor of outcome in patients with invasive ductal carcinoma.

The fibrotic focus has been shown to be a prognostic factor in breast cancer.  Moreover, criteria for fibrotic foci proposed by Van den Eynden et al. in 2007 (Dr. Hasebe was a co-author) appear to both clinically significant and reproducible in daily practice.  I am surprised that the use of this feature has not been adopted for routine use since presenting breast cancer cases with prominent fibrotic foci never fails to elicit curiosity and questions in Tumor Board.  Further, a fibrotic focus has been proposed as a marker for tumor hypoxia and angiogenesis.

The purpose of the current study was to determine whether the combined assessment of P53 expression in tumor-stromal fibroblasts both forming and not forming fibrotic foci serves as a significant predictor of outcome in patients with invasive ductal carcinoma using multivariate analysis with well-established prognostic factors.

The authors studied 1,039 consecutive patients at a single Japanese institution who received surgery including axillary dissection; 873 also received conventional chemotherapy, endocrine therapy, or both.  Using FFPE serial sections of each tumor, the authors evaluated 8 different histological factors, including measuring the fibrotic focus using 8 mm as the cutoff per the Van den Eynden criteria.  Immunohistochemical staining for P53 was evaluated using the Dako mouse monoclonal antibody clone DO7 and staining of tumor-stromal fibroblasts was scored according to modified Allred scoring.  Furthermore, the authors stratified P53 staining in tumor-stromal fibroblasts into 3 groups each for those tumors with and without fibrotic foci.

373 out of 1,039 cases had fibrotic foci.  Notably, in 97 of these 373 (26%) P53 staining could not be evaluated because the tumor tissue sections examined by P53 IHC did not contain a fibrotic focus identified initially at routine examination.

The authors found significantly higher P53 Allred scores in TSFs not forming compared with TSFs forming fibrotic foci.  But P53 Allred scores >4 in TSFs forming fibrotic foci were also associated with fibrotic focus diameter > 8 mm: the size previously established as a cut-off for predicting adverse outcome independent of stage.

Based on Allred scores, the authors segregated P53 expression for TSFs forming and not forming fibrotic foci into risk classes.  Patients in pTNM stages I-III with intermediate to high risk classes of P53 expression showed a significantly higher tumor recurrence rate than patients in the low risk classes.  In addition, stage II and III patients with intermediate to high risk classes of P53 expression showed a significantly higher disease-related mortality rate.

By multivariate analysis, for stage I patients, intermediate to high risk classes of P53 expression in TSFs forming and not forming fibrotic foci and histological grade 3 were significantly associated with tumor recurrence (but not disease-specific death).  For stage II patients, intermediate to high risk classes of P53 expression in TSFs forming and not forming fibrotic foci were associated with increased tumor recurrence and tumor-related death.  Lymphovascular invasion and blood vessel invasion were significantly associated with increased tumor recurrence, whilst pT1 and fibrotic focus > 8 mm were significantly associated with increased tumor-related death.

(Next to) Bottom Line: P53-expression TSFs located both in the inner fibrotic foci an outer invasive front increase the malignant potential of invasive ductal carcinomas across stages I to III.

SO--should we start doing P53 IHC on all invasive ductal carcinomas?  Perhaps not ready yet for prime-time, but this study provides evidence that may, at least in part, explain the prognostic value of fibrotic focus diameter.  Measuring the fibrotic focus, however, may just be ready for prime-time and you may want to consider reviewing the original paper by Van den Eynden et al published in Histopathology 2007;51:440-451.

BUT I do have a few points to consider.  First, this is a single institution paper, so the methodology needs to be independently validated and a larger, multi-insitutional patient cohort needs to be studied to confirm the association between patient outcomes and P53 staining.  Second, other P53 antibodies should be evaluated to optimize the assay.  Third, the number of invasive ductal carcinomas with fibrotic foci that were excluded from the study is disquieting and may be a major source of observational bias.  Finally, the role of image analysis in providing quantitative P53 cut-offs may provide better discrimination between prognostically different groups.