Breast cancer is the most common cancer among urban Indian women with a CIR of 29.4/100,000 in the MMTR. Nearly 50% of the patients present with locally advanced cancers to the Institute. The Institute has pioneered the use of concurrent chemo-radiation for Head and Neck cancers, especially oral cancer and in breast cancer (Shanta and Krishnamurthy, 1976; Shanta et al., 1985; Shanta and Krsihnamurthy,1991). However, world over the current practice is to use neo-adjuvant chemotherapy only followed by surgery and then radiation. The use of neo-adjuvant treatment helps to downstage the tumour making it suitable for surgery ( mastectomy and at some centres breast conservation is also done) and also assess the response of the tumours to the treatment given. In the Institute, the pathologic complete response (pCR) rates following concurrent chemo-radiotherapy is around 29 % ; while with the use of neo-adjuvant chemotherapy alone, the pCR rates have ranged from 13 – 25% depending on the drugs used. Since most of the patients treated in the Institute are from the lower socio-economic group, use of Taxanes and Trastuzumab (Herceptin), are beyond the reach of most patients.  

Concurrent chemoradiation has its advantages- Spatial co-operation, Radiation sensitization and toxicity intdependence (Steels Principles).One of the reasons for the non-use of neo-adjuvant concurrent chemo-radiotherapy in most centers is the fear of toxicity, especially to the skin. While skin toxicity is known to occur, grade 3 or 4 toxicity, are unusual. The ability to achieve pCR also helps in achieving a better DFS and OS, especially in node positive patients . In a country with limited resources, optimization of the treatment modalities is essential using the proven methods of treatment.

OBJECTIVES :

1. To compare  the clinical efficacy of neo-adjuvant chemotherapy as against neo-adjuvant concurrent  chemo-radiation in Locally Advanced Breast Cancer.

2. To compare the toxicities of the two therapy arms

3. To compare the Pathologic Complete Response in the two arms

4. To compare the Disease Free Survival

5. To assess the Quality of life in the randomized patients

6.  To do a Proteomics profiling of the pre-treatment tumour tissue sample and identify and validate characteristic signatures which can help predict Pathologic Complete Response (pCR). Additionally, the proteins contributing to the peaks will also be identified and validated.

LITERATURE REVIEW

 INTERNATIONAL

There is limited literature available on the use of concurrent chemoradiation in breast cancer. There are few prospective and retrospective comparative studies that have compared use of CMF and RT and Anthracycline based chemotherapy and radiation in the adjuvant setting in early stage Breast cancer(Ismaili et al., 2003). After mastectomy or BCT, the adjuvant treatment based on anthracycline and concurrent RT reduced breast cancer relapse rate, and significantly improved LRFS, EFS and OS in the patients receiving more than 1 cycle of concurrent CT. There were more hematologic and non hematologic toxicities in the anthracycline group.

The concept of using preoperative chemotherapy in patients with operable breast cancer originated from experimental and clinical observations, as well as from theoretical hypotheses on tumor cell growth and dissemination. Results from nonrandomized studies with different chemotherapeutic agents or combination regimens given preoperatively demonstrated substantial clinical response rates but low pathologic tumor response rates. In addition, several such studies were able to show that-by reducing primary breast tumor size-preoperative chemotherapy can lead to an increase in the rate of breast-preserving procedures The National Surgical Adjuvant Breast and Bowel Project (NSABP) B-18 trial was the largest randomized trial that aimed to compare preoperative to postoperative chemotherapy in operable breast cancer.

The use of neo-adjuvant chemotherapy has been increasingly used in the last decade following the results of the NSABP- 18 and other similar trials. Approximately 13% of primary breast carcinoma cases exhibited both a clinical complete response (cCR) and a pathologic complete response (absence of invasive tumor [pCR]) to preop AC. An additional 7% of patients exhibited a pCR in the absence of a cCR. A pCR occurred in 38% of those patients determined to have achieved a cCR. Poor nuclear grade of the tumor cells in the pre-entry FNA and/or TC specimens significantly predicted a pCR. Patients with the latter exhibited a better OS and DFS compared with those with a pathologic partial response (presence of sparse invasive tumor [pPR]) or no pathologic response (pNR)

Pathological CR has been shown to be an independent predictor of prolonged DFS/OS but very few patients achieve pCR and that too is dependent upon the type of chemotherapy. Earlier trials testing anthracyclines as NACT produced pCR rates of 2-13% and there was no difference in DFS/OS. Subsequently, taxanes have been used either alone or with anthracyclines and it has shown to improve pCR rates. Single agent docetaxel has produced pCR rates of 16-20% and near complete responses of 18% to more than 25%. In combination with anthracyclines pCR rates of 10% to >20% are reported depending upon the stages of tumor included in that particular study.

Historically, NACT has not led to increased DFS/OS but achieving pCR is associated with longer DFS/OS. In a recent update of NSABP B-18 and B-27 trials, there was no survival advantage with NACT and addition of taxane to preoperative chemo did not translate into higher survivals although pCR rates were higher.

NATIONAL

At the Institute the use of neoadjuvant therapy has its origins dating back to the 1980 s when we used Radiation alone or Radiation along with MeFu . The results were seen to be better with the concurrent use of RT and MeFU – this was not randomized. Having found the results with this schedule being better concurrent  chemoradiation became the protocol therapy for the LABC patients. Initially we used CMF and RT . Later anthracycline based chemotherapy( FAC initially and then FEC ) with RT  became more frequently used. Taxanes- alone or with anthracycline  were used only  occasionally with RT

WORK PLAN:

A. CLINICAL TRIAL                            

B. PREDICTIVE MARKER ASSESSMENT

A. Clinical Trial

Study Design: A randomized controlled clinical 2 arm study.

Sample size

With an expectation of 10% difference in pCR% and 10-15% difference in 5-year disease free survival between PCR achieved and not achieved cases, with the least failure rate of 21%, a sample size of 250 cases in each arm is required at 85% power and significance level of 5% for two sided testing of null hypothesis that “effects of Arm I is no different from the effects of Arm II” and various power constraints. We plan to include 275 in each arm, in order to make a provision for 10% lost to follow-up. We assume that the entire period of the study – recruiting and completion of therapy will be about 4-5 years.

Arms Of The Study

Arm 1: Neoadjuvant concurrent chemoradiation with FEC-60 , followed by surgery and adjuvant chemotherapy with Docetaxel  , with/without Radiation

Arm 2: Neoadjuvant chemotherapy with FEC-90 , followed by surgery, adjuvant chemotherapy with 4 cycles Docetaxel and sequential Radiation

Primary outcomes to be studied

1. Pathological complete response (PCR)

    

    

   Secondary Outcomes to be studied

1.5-year Disease free survival

1.  5-year Overall survival

2. Toxicity

3. Quality of Life (QOL)

Randomization

All consecutive cases accepted for treatment from a pre-specified index date will be screened for eligibility criteria. The eligible cases will then be asked for informed consent.Patients with operable Locally Advanced Breast Cancer will also be given an option of initial Mastectomy followed by adjvant therapy. Those giving consent   for the study  will be randomized to one of the arms (Arm I or Arm II). Randomization is done by generating random number tables through computer programs for the proposed total number of cases in the study. A document is then prepared giving the allocation of all the subjects to the two arms in chronological order. This information is kept confidential at the statistics department and provided only to the administering clinician on requisition during every physical allocation of cases to the treatment arms.

INCLUSION CRITERIA

1. Patients with Locally Advanced Breast Cancer  - stages IIB , IIIA and IIIB-operable and inoperable

2. Patients  PS I and II

3. Patients fit for anthracycline based chemotherapy – FEC and single agent Docetaxel

4. No previous chemotherapy or hormone therapy

EXCLUSION CRITERIA

1. PS III and PS IV

2. Age > 65 yrs

3. Stage III C- ipsilateral supraclavicular  node positive

4. Inflammatory Breast  Cancer

5. Patients with co-morbidities that preclude the use of anthracylines such as cardiac disease, uncontrolled diabetes

6. Patients with liver dysfunction (s.bilirubin < 1.5 mg/dl, SGOT,  SGPT and serum  alkaline phosphatase should be less than twice  normal)

7. Patients with fixed axillary nodes, multiple cutaneous nodules (> 3) in the skin overlying the breast, ipsilateral arm lymphedema.

8. Patients with large and pendulous breasts on whom  concurrent chemoradiation will have more skin toxicity

END POINTS

 Primary – Pathologic Complete Response (pCR)

 Secondary – Disease Free Survival, Overall Survival, Toxicity Evaluation, QOL

Criteria for taking Patients off the study will include

1. Patients who progress on Neoadjuvant chemotherapy alone

2. Patients who continue to be inoperable after concurrent chemo-radiation

3. Life threatening toxicity

Statistical Methods

Descriptive statistics will be generated in the form of two-way tables giving the proportion of cases in Arm I and Arm II pertaining to different factors related to the patient, disease, treatment and outcomes studied. The comparison of these proportions between Arm I and Arm II would be carried out using Chi-square test (for nominal scale data) and Student-t test (for interval scale data) for statistical significance. Survival probability will be estimated by actuarial method. Log rank test would be employed to test for differences in survival. Cox proportional hazard model would be utilized to elicit the independent effect of the study arm on survival after adjusting for other prognostic factors.

STUDY PROTOCOL

Pre-Treatment Investigations

All patients will have a core needle biopsy for histopathology and for Proteomic study. 15ml blood sample will be drawn from the patients after their inclusion in the study. This blood sample will be stored after separating the cells and the plasma for future studies (genomic and proteomic). Along with histopathological studies, immunohistochemistry will be done for the following markers – Estrogen receptor (ER), Progesterone Receptor (PR), HER2, EGFR, CK5/6, Ki67.

In addition, in an equal number of patients in both arms (n=50), it is proposed to draw blood samples during the chemotherapy for estimation of serum drug and metabolite levels using HPLC and ESI MS/MS. This will be done only after obtaining informed consent. 5 ml blood sample prior to administration of chemotherapy, and at 30 minutes, 60 minutes, 120 minutes, 6 hours, 12 hours, 24 hours, 48 hours and 72 hours. In a proportion of the women, who participate in the study, tru-cut biopsy 24 hours after the chemotherapy is given will be done, if they are willing and provide an informed consent. Frozen section confirmation of the tumour with at least 70% tumour cells, will be done. These samples will then be processed immediately for quantification of the drugs and their metabolites using HPLC – ESI - MS. The genotyping will be done once funding is obtained.

The staging evaluation includes a Chest X-Ray, CT chest, Ultrasound of the liver and the pelvis, contralateral mammogram and a Bone Scan. . A MRI / PET CT scan may be requested in those patients who have an abnormality on the Bone Scan. A   2-D ECHO  is done for all the patients prior to randomization.

 All patients fulfilling the Inclusion criteria and those who have given written Informed Consent will be included in the Trial.  Randomization is done after obtaining the informed consent in the Dept. of Epidemiology.

Management of patients taken off the trial

Those patients who have progressed during chemotherapy are taken off the study and are considered for the addition of radiation.

Patients who still have Inoperable LABC will have documentation of disease (FNAC) and taken off the study. They can be considered for alternate chemotherapy, if willing.

Patients taken off the study due to life threatening toxicity, will be considered for CMF chemotherapy, if willing.

ARM 1 – CONCURRENT CHEMO-RADIATION ARM

Patients in this arm will receive I FEC on Day 1.

The dosage for the FEC is as follows

5-FU 600 mg/m²                                  - intravenous        Day 1

Epirubicin 60 mg/m²                            - intravenous       Day 1

Cyclosphosphamide 600mg/m²          - intravenous       Day 1

 The dosage of Epirubicin has been kept at 60 mg/m². Our current protocol at the Institute uses Epirubicin at 60mg/m² and at this dosage we have fairly acceptable toxicity -  Skin toxicity   is Gr III – 25 % and Gr  IV less than 5 %.

The incidence of Febrile Neutropenia is 5% .

We do not wish to give Epirubicin at a dosage of 75mg/m2 as we feel that the skin and haematological toxicity will be higher.

Radiation will be started within the first week of starting chemotherapy.

Radiation is given to the breast, axilla and supraclavicular regions to a total dose of 40 Gy. Radiation is delivered using 6 Mev from a Linear Accelerator. The Internal Mammary nodal region is included in the field, if feasible. During the radiation the patient will receive the II cycle of FEC on day 22 and then continue the radiation .  On the day of chemotherapy radiation is not given. The third cycle of chemotherapy is given on day 43. During radiation, if the patient develops severe Grade III or Grade IV reactions then the chemotherapy is delayed by a few days till the reaction settles down.

ARM 2 - NEOADJUVANT CHEMOTHERAPY ARM

Patients will receive the 3 cycles FEC , once in 21 days.

5-FU  600 mg/m²                                 - intravenous   Day 1

Epirubicin 90 mg/m²                            - intravenous   Day 1

Cyclosphosphamide 600mg/m²          - intravenous   Day 1

Patients are reassessed 1 week after the 3 rd chemotherapy and planned for Surgery which will be Modified Radical Mastectomy.

 Prophylactic Growth factors are used in all the patients as per the NCCN guidelines

Patients are reassessed by the Surgical Oncologist and planned for the Surgery about 4 weeks from the completion of Radiation and about 3 weeks from the last chemotherapy.

POST-OPERATIVE TREATMENT

After the surgery the next cycle chemotherapy is given after the wound has healed well and the sutures and drains are removed. In both the arms we plan to use single agent Docetaxel for    4 adjuvant chemotherapy cycles.

Adjuvant Chemotherapy Schedule- same for both arms

Inj Docetaxel    75mg/ sq m   I.V, Once in 3 weeks, 4 cycles

In patients who are not considered fit for Docetaxel – like uncontrolled diabetes we plan to continue the same chemotherapy

In  ARM II, the radiation to the chest wall and the draining regions is given 3 weeks after the last cycle of chemotherapy.

In the  ARM I  the radiation to the Internal Mammary region is given 3 weeks after the 6^th cycle – if it was not given earlier.

All receptor positive post-menopausal patients, on completion of the chemotherapy, will be given Letrozole for 5 years. In receptor positive pre-menopausal women, laparoscopic BSO will be done if they continue to menstruate after  the completion  of chemotherapy.They will then receive Letrazole for 5 years. Patients who achieve chemo induced amenorrhoea will receive Tamoxifen for 5 years.

HISTOPATHOLOGICAL ASSESSMENT

The modified radical mastectomy specimen will be assessed by several sections of the tumour bearing area and its surroundings (15 – 30 sections, median – 18 sections ) depending on the size of the tumour, excluding sections taken from other quadrants, resected margins and axillary nodes. The presence of residual tumour, its size, grade and the pattern of response, whether localized or diffuse/patchy as well as other morphological changes in the breast parenchyma and nodal involvement will be recorded.

RESPONSE CRITERIA

A pathological complete response (pCR) will be defined as the absence of all microscopic evidence of tumour. A pathological partial response (pPR) will be defined as the presence of tumour either localized/focal or diffuse and patchy surrounded by fibrous stroma. Pathological no response (pNR) will be the persistence of tumour with no response to treatment.

IMMUNOHISTOCHEMICAL ANALYSIS

Immunohistochemistry will be performed on formalin-fixed, paraffin embedded sections. Less than 5 micron sections on APES coated slides will be obtained. Sections will be deparaffinized and rehydrated. The sections will then be subject to wet pressure antigen retrieval for half an hour. After cooling, the sections will be washed in TRIS buffer saline (TBS) (pH 7.4)) and incubated with 0.03% hydrogen peroxide in TBS for 30 mins at room temperature. This will be followed by incubation with primary antibody overnight followed by incubation with secondary antibody for ½ hour the following day. Next step will be incubation with DAB for 2-5 minutes followed by counterstaining with haematoxylin, dehydration, clearing and mounting. Positive and negative control slides will be processed under the same conditions as above omitting the primary antibody in negative control. The slides will then be viewed under a microscope and analyzed.

Quality of Life Assessment

Methodology – Cancer patients with locally advanced breast cancer receiving chemotherapy or chemo-radiation who satisfy the selection criteria will be assessed for QOL, Distress, Fatigue and patient satisfaction at 4 different time periods. Psycho-oncologists who conduct the assessment will be blind to the study.

Assessment Schedule:

TOOLS

FOLLOW-UP

Patients will be followed up once every 3 months with a thorough clinical examination. If clinically warranted investigations such as Xray chest etc will be done.

Investigations are done 1 year after the completion of therapy. Investigations include a contralateral mammogram  and 2-D ECHO.

If a patient defaults in follow-up, the Institute’s established method of trying to get the patient back will be done.

All patients will be followed up for their life time.

B. Proteomics Study

The core needle biopsy will be assessed for percentage tumour cells by frozen section analysis and only samples with at least more than 60% tumour cells will be included in the proteomics study. The flash frozen tissue samples will be homogenized in CHAPS lysis buffer with added protease and phosphatase inhibitors using a FastPrep® homogenizer at 4^oC in a cold room. Following centrifugation the supernatant from the homogenized sample is collected and will be quantitated using bicinchoninic acid (BCA) based protein assay. The quality of proteins isolated will be assessed by SDS PAGE analysis.

Biomarker discovery and analysis by MALDI-TOF MS based system- CLINPROT

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS) is a powerful method for the detection of large numbers of peptides and proteins and therefore can be used for acquisition of proteomic profile spectra from complex protein mixtures. The work comprises of initial fractionation by superparamagnetic microparticles with functional surfaces designed to undergo specific interactions with peptides and proteins. This is followed by co-crystallization in a surplus of matrix molecules on a MALDI plate target. The analyte is evaporated into the gas phase using pulsed laser and the spectral data is acquired in the designated mass/charge (m/z) range. The spectra will be anaysed using ClinProTools, a data interpretation software for biomarker analysis.

5-10 µg of protein lysate will be fractionated using magnetic beads exhibiting biochemical functionalities like hydrophobic interaction (HIC8), ionic exchange (WCX), and metal affinity (IMAC resin). The elutes from these fractionations will be co-crystallized using a matrix solution composed of α-cyano-4-hydroxycinnamic acid (HCCA 0.3 g/l in ethanol:acetone 2:1)in a Anchor Chip MALDI plate. The ratio of sample to matrix solution will be fixed at a ratio of 1:1. Each sample will be spotted in four replicate spots, CLINPROT standards are also spotted to calibrate the MALDI mass spectrometer before data acquisition. The data will be acquired for a total of 450-550 shots fired at 15 to 18 locations in the spot at 30 shots per location. Mass spectra of all the samples generated using MALDI–TOF/TOF will be smoothed and baseline-subtracted using FlexAnalysis software (version 2.2). Three workflows involved in ClinProTools ’Peak Statistic Calculation’, ’Model Generation’ and ’Classification’ will be performed. For Peak Statistic Calculation the spectra will be recalibrated and average spectra calculated, peak picking, peak calculation and statistic calculation will be assessed in the Peak Statistic report. Next, Model will be generated using the spectra from the training data set using four different Model generating algorithms Genetic Algorithm (GA), Support Vector Machine (SVM), Supervised Neural Network (SNN), QuickClassifier (QC). The samples used for the test set will be obtained blinded to the clinical response details. Classification of the test data will be performed using the models generated and presented as classification report. The spectral peaks which represent the maximum differences between the classes denoted by their high ROC curve values will be taken up for further identification.

Identification of Biomarker by Top-Down Sequencing (TDS)

The fractionation which yielded the candidate biomarker will be performed again, however the reaction size will be increased 10 fold. The eluted fraction will be separated on a reverse phase (RP) C18 capillary column and fractions of the eluent are transferred to a 384-well pre- spotted MALDI target by the Bruker Proteineer FC II fraction collector. To determine the fraction which contains the biomarker candidate all the 384 fractions will be analysed by MALDI TOF/TOF in the linear mode. The target fractions containing proteins with the expected molecular weight of the biomarker will be taken up for MALDI-TDS. The MALDI-TDS spectra generated by in-source decay in reflector mode for the proteins are obtained and Proteins are identified from MALDI-TDS spectra using Mascot.

Quantitative Analysis of Proteins In Core Needle Biopsies of Breast Cancer

Protein expression in the Biopsies of Breast Cancer will be assessed using Tandem Mass Tags (TMT) set which enables multiplexing up to six samples and Nano LC-ESI-MS/MS. Tandem Mass Tags (TMT) comprises of isobaric tagging reagent which within a set has the same nominal parent (precursor) mass and is composed of an amine-reactive NHS-ester group, a spacer arm and an unique MS/MS reporter. These reporter ions that are in the low mass region of the MS/MS spectrum are used to report relative protein expression levels during peptide fragmentation. The protein samples will labeled with TMT tags prior to trypsin digestion since combining labeled samples earlier in the sample process will reduce sample variability. The labeled proteins are desalted and excess TMT tags are removed from the solution. Using strong cation exchange ion chromatography the samples will be fractionated prior to performing Nano LC-MS/MS with a C18 reverse phase (RP) column.

For the protocol 100µg of the protein lysate will be used. 45μL of 100mM Triethyl ammonium bicarbonate (TEAB) is added to the sample and the final volume is adjusted to 100 μL. 5μL of the 200mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) is added and the sample is incubated at 55^oC for 1hr. Following which 5μL 375mM iodoacetamide prepared in TEAB is added and incubation continued for another 30 minutes. For labeling with TMT label reagents the reduced and alkylated protein is transferred to TMT Reagent vial containing 24μL of the TMT Label Reagent for each 100μg of protein sample. The reaction is incubated at room temperature for 1 hour following which, 8μL of 5% hydroxylamine is added to the sample and incubate for 15 minutes to quench the reaction. The reaction is desalted and cleared of the unlabelled TMT tags by using 10K spin filter cartridge.  The desalted and concentrated proteins are re-suspended in 100μL of 100mM TEAB 2.5μg of trypsin per 100μg of protein is added and the reaction incubated overnight at 37°C. The trypsin digested proteins will be fractionated using strong cation exchange chromatography using PolySULFOETHYL Aspartamideâ„¢ (1.0-mm i.d.) HPLC columns. Initially the peptides will be eluted using shallow gradient to about 180 mM salt which would form most of the gradient time, this is followed by a steep gradient to ~ 0.5 M salt. Approximately over 90 fractions will be collected. The fractions will be pooled with each pool comprising of 5 fractions which would result in ~18 combined fractions. These fractions will be anlysed by  Nano LC-ESI-MS/MS using a C18 RP column (15 cm) connected to a ESI ion source. The fragmentation method used would be electron transfer dissociation (ETD) to generate the reporter ions. The relative quantitation of reporter ions released from labeled peptides, and protein identification will be performed using ProteinScape 2.1.

VALIDATION OF THE PROTEINS

Once the differentially expressed proteins are identified between the pCR and non-pCR tumours, an independent and blinded set of 200 tumors will be assessed by IHC.